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TRANSCRIPT
1
CH
AP
TE
R O
NE
INT
RO
DU
CT
ION
The
aim
of
this
dis
sert
atio
n is
to
el
uci
dat
e th
e det
ails
beh
ind th
e ty
rosi
ne
phosp
hory
lati
on
of
a pro
tein
in
volv
ed
in
Cdc4
2-m
edia
ted
signal
ing
pat
hw
ays,
SH
3P
X1.
SH
3P
X1,
also
know
n a
s so
rtin
g n
exin
9 (
SN
X9),
has
bee
n i
den
tifi
ed a
s a
subst
rate
of
the
tyro
sine
kin
ase,
ac
tivat
ed C
dc4
2-a
ssoci
ated
kin
ase
-2 (A
CK
2),
an
d
incr
easi
ngly
im
pli
cate
d i
n t
he
sort
ing o
f var
ious
cell
surf
ace
rece
pto
rs i
ncl
udin
g t
he
insu
lin,
EG
F,
and t
ransf
erri
n r
ecep
tors
. D
ue
to t
he
infl
uen
tial
role
of
SH
3P
X1 i
n a
num
ber
of
signal
ing p
athw
ays,
we
sought
to p
rovid
e a
com
ple
te i
llust
rati
on o
f th
e
bio
logic
al s
yst
ems
involv
ed a
nd t
he
signif
ican
ce o
f th
e des
crib
ed p
rote
ins
on c
om
ple
x
cell
ula
r pro
cess
es s
uch
as
rece
pto
r en
docy
tosi
s an
d d
egra
dat
ion.
Cd
c42 a
nd
its
reg
ula
tors
Cdc4
2,
a m
ember
of
the
Rho
fam
ily
of
smal
l G
TP
-bin
din
g
pro
tein
s (G
pro
tein
s),
has
bee
n
impli
cate
d
in
num
erous
cell
ula
r ev
ents
, in
cludin
g
cell
cy
cle
pro
gre
ssio
n [
1],
apopto
sis
[2,
3],
act
in c
yct
osk
eleto
n r
earr
angem
ent
[4,
5],
act
ivat
ion
of
the
stre
ss-r
esponsi
ve
mit
ogen
-act
ivat
ed p
rote
in (
MA
P)
kin
ases
(c-J
un k
inase
, p38)
[6],
intr
acel
lula
r tr
affi
ckin
g [
7],
and e
ndocy
tosi
s [8
].
Lik
e al
l G
pro
tein
s, C
dc4
2 a
cts
as a
mo
lecu
lar
swit
ch,
cycl
ing b
etw
een a
n a
ctiv
e G
TP
-bound s
tate
and a
n i
nac
tive,
or
bas
al G
DP
-bound s
tate
. T
he
acti
vat
ion o
f C
dc4
2 i
s in
itia
ted b
y n
um
erous
cell
surf
ace
rece
pto
rs,
incl
udin
g
pro
teogly
cans
[9],
re
cepto
r ty
rosi
ne
kin
ases
[1
0],
G
pro
tein
-
couple
d r
ecep
tors
(G
PC
Rs)
[11],
cyto
kin
e re
cepto
rs [
12],
and i
nte
gri
ns
[13].
O
nly
in
this
ac
tive
stat
e is
C
dc4
2 ab
le bin
d to
an
d act
ivat
e dow
nst
ream
ef
fect
or
pro
tein
s
(Fig
ure
1.1
).
2
Fig
ure
1.1
C
dc4
2-m
edia
ted
sig
nali
ng p
ath
ways.
Cdc4
2 G
TP
ase
acti
vit
y i
s ac
tivat
ed b
y a
num
ber
of
upst
ream
cel
l su
rfac
e re
cepto
rs
incl
udin
g
pro
teogly
cans,
re
cepto
r ty
rosi
ne
kin
ases
, G
pro
tein
-couple
d
rece
pto
rs,
cyto
kin
e re
cepto
rs an
d in
tegri
ns.
U
pon ac
tivat
ion,
Cdc4
2 is
th
en ab
le to
m
edia
te
signal
ing p
athw
ays
involv
ed i
n m
ult
iple
bio
logic
al f
unct
ions
incl
udin
g c
yto
skel
etal
org
aniz
atio
n,
mit
ogen
esis
, an
d r
ecep
tor
endocy
tosi
s th
rough i
ts d
ow
nst
ream
eff
ecto
r
pro
tein
s.
3
4
Due
to t
he
com
ple
x r
ole
of
smal
l G
pro
tein
s in
sev
eral
sig
nal
tra
nsd
uct
ion
pat
hw
ays,
the
num
ber
of
pro
tein
s im
pli
cate
d i
n t
hei
r re
gula
tion i
s not
surp
risi
ng [
14].
Thes
e re
gula
tors
are
inv
olv
ed i
n p
rom
oti
ng G
pro
tein
act
ivat
ion,
dea
ctiv
atio
n,
and
traf
fick
ing bet
wee
n th
e cy
toso
l an
d th
e pla
sma
mem
bra
ne.
T
hey
in
clude
guan
ine
nucl
eoti
de
exch
ange
fact
ors
(G
EF
s),
GT
Pas
e ac
tivat
ing p
rote
ins
(GA
Ps)
, an
d g
uan
ine
nucl
eoti
de
dis
soci
atio
n i
nhib
itors
(G
DIs
) (F
igure
1.2
).
By
cata
lyzi
ng
the
exch
ang
e of
GT
P
for
GD
P,
GE
Fs
are
resp
onsi
ble
fo
r
acti
vat
ing G
pro
tein
sig
nal
ing.
GE
Fs
that
act
ivat
e th
e R
ho
-subfa
mil
y o
f G
pro
tein
s
oft
en c
onta
in t
andem
Dbl
ho
molo
gy (
DH
) an
d p
leck
stri
n h
om
olo
gy (
PH
) dom
ains.
The
DH
dom
ain i
s la
rgel
y r
esponsi
ble
for
the
cata
lyti
c ac
tivit
y o
f th
e G
EF
[15,
16],
wher
eas
the
PH
dom
ain i
s th
ought
to b
e pri
mar
ily i
nv
olv
ed i
n l
oca
lizi
ng t
he
exch
ange
fact
or
to t
he
pla
sma
mem
bra
ne
thro
ugh t
he
bin
din
g o
f phosp
hat
idyli
nosi
tides
[17,
18].
Des
pit
e th
is w
idel
y a
ccep
ted r
ole
for
the
PH
dom
ain,
incr
easi
ng e
vid
ence
sugges
ts i
t
has
an a
ddit
ional
role
in m
edia
ting t
he a
ctiv
ity o
f th
e D
H d
om
ain o
f a s
ub
set
of
GE
Fs
[19].
E
xch
ange
fact
ors
hav
e bee
n l
argel
y f
ound t
o e
xis
t in
inac
tive
or
par
tial
ly a
ctiv
e
stat
es w
ithin
the c
ell.
T
her
e ap
pea
r to
be a
nu
mber
of
mec
han
ism
s re
sponsi
ble
for
mai
nta
inin
g t
hei
r in
acti
ve
confo
rmat
ions,
incl
udin
g i
ntr
amole
cula
r or
auto
inhib
itory
inte
ract
ions
bet
wee
n t
he
DH
and P
H d
om
ains
[19,
20],
inte
ract
ions
bet
wee
n t
he
DH
or
PH
d
om
ains
and
an
inhib
itory
dom
ain
[21,
22],
oli
gom
eriz
atio
n
thro
ugh
inte
rmole
cula
r in
tera
ctio
ns
bet
wee
n
DH
dom
ains
[23,
24],
an
d
inte
rmole
cula
r
inte
ract
ions
wit
h c
ellu
lar
pro
tein
s [2
5,
26].
T
he
inac
tive
confo
rmat
ion o
f G
EF
s in
viv
o
appea
rs t
o b
e a
pro
tect
ive
mec
han
ism
, pre
ven
ting t
he
aber
rant
or
hyper
-act
ivat
ion o
f
thei
r G
pro
tein
tar
get
s.
The
bin
din
g o
f G
EF
s to
the
GD
P-b
ound G
pro
tein
cau
ses
a
confo
rmat
ional
chan
ge
wit
hin
the
guan
ine
nucl
eoti
de-
bin
din
g p
ock
et,
intr
oduci
ng a
conse
rved
hydro
phobic
res
idue
into
the
Mg
2+ c
oord
inat
ion s
pher
e an
d e
lim
inat
ing t
he
5
Fig
ure
1.2
A
sc
hem
ati
c d
iagra
m of
the
Cd
c42 G
TP
ase
cy
cle
wit
h ass
oci
ate
d
regu
lato
ry p
rote
ins.
6
7
coord
inat
ion of
a cr
itic
al ly
sine
resi
due
wit
h th
e !
-phosp
hat
e of
GD
P [2
7].
T
his
com
bin
atio
n o
f ev
ents
lea
ds
to t
he
rele
ase
of
GD
P a
nd s
tabil
izat
ion o
f th
e nucl
eoti
de-
free
sta
te [
28],
wher
e hig
her
GT
P c
once
ntr
atio
ns
in c
ells
dri
ve
the
G p
rote
in t
o t
he
GT
P-b
ound s
tate
.
G
TP
ase-
acti
vat
ing p
rote
ins
(GA
Ps)
ser
ve
to a
tten
uat
e th
e G
pro
tein
-med
iate
d
signal
, re
turn
ing t
he
G p
rote
in b
ack t
o i
ts i
nac
tive
stat
e by
cat
alyzi
ng
the
hydro
lysi
s of
GT
P.
T
he
cata
lyti
c ac
tivit
y
of
the
GA
P
requir
es
a co
nse
rved
ar
gin
ine
resi
due,
iden
tifi
ed i
n b
ioch
emic
al a
nd s
truct
ura
l st
udie
s as
a k
ey c
om
ponen
t in
sta
bil
izin
g t
he
tran
siti
on s
tate
[29-3
2].
M
ore
spec
ific
ally
, th
e “a
rgin
ine
finger
,” i
nse
rted
by t
he
GA
P
into
the
GT
P-b
indin
g s
ite,
ser
ves
to s
tabil
ize
the
neg
ativ
e ch
arge
of
the "-
phosp
hat
e of
GT
P i
n t
he
tran
siti
on-s
tate
, as
wel
l as
po
siti
on g
luta
min
e 61 t
o h
ydro
gen
bond t
o t
he
nucl
eophil
ic w
ater
mole
cule
res
ponsi
ble
for
atta
ckin
g t
he "-
phosp
hat
e [3
1,
33].
Even
conse
rvat
ive
muta
tions
of
the
argin
ine
finger
lea
d t
o a
sig
nif
ican
t lo
ss o
f ca
taly
tic
acti
vit
y [
29].
H
ow
ever
, th
e ar
gin
ine
finger
can
not
acco
unt
for
all
of
the
GA
P a
ctiv
ity,
as G
AP
muta
nts
dev
oid
of
the
conse
rved
arg
inin
e re
sidue
are
stil
l ab
le t
o e
lici
t an
~10
-
fold
sti
mula
tion o
f G
TP
hydro
lysi
s, s
ugges
ting a
more
com
ple
x r
egula
tory
role
for
the
GA
P [
29,
34].
R
ecen
t st
udie
s su
ggest
that
an a
ddit
ional
role
for
the
GA
P i
nvolv
es
stab
iliz
ing t
he
swit
ch r
egio
ns
of
the
G p
rote
in,
a m
echan
ism
that
pro
per
ly p
osi
tions
resi
dues
in t
he
G p
rote
ins
that
are
cri
tica
l fo
r G
TP
hydro
lysi
s [2
9, 34].
G
uan
ine
nucl
eoti
de
dis
soci
atio
n i
nhib
itors
(G
DIs
) co
mpri
se t
he
thir
d c
lass
of
pro
tein
s in
volv
ed i
n t
he
regula
tion o
f R
ho
-fam
ily G
pro
tein
s.
The
Rho-f
amil
y G
DIs
(RhoG
DIs
) hav
e bee
n sh
ow
n to
b
e in
volv
ed in
at
le
ast
thre
e dis
tinct
bio
chem
ical
acti
vit
ies.
T
he
firs
t of
thes
e in
volv
es t
he
inhib
itio
n o
f G
DP
dis
soci
atio
n,
the
acti
vit
y
for
whic
h t
hey
wer
e ori
gin
ally
nam
ed [
35,
36].
R
hoG
DIs
are
als
o a
ble
to i
nhib
it G
AP
-
cata
lyze
d G
TP
hydro
lysi
s [3
7].
In
addit
ion,
GD
Is h
ave
also
bee
n s
how
n t
o s
tim
ula
te
the
rem
oval
of
post
-tra
nsl
atio
nal
ly
modif
ied
G
pro
tein
s fr
om
m
embra
nes
.
This
8
acti
vit
y h
as l
ed t
o t
he s
uggest
ion t
hat
GD
Is s
hutt
le t
hei
r G
pro
tein
tar
get
s bet
wee
n
dif
fere
nt
mem
bra
ne
loca
tio
ns.
T
he
bin
din
g o
f R
hoG
DI
to t
he
pre
nyla
ted C
-ter
min
us
of
Rho f
amil
y G
pro
tein
s hel
ps
to m
ask t
hei
r hy
dro
phobic
tai
l fr
om
the
hydro
phil
ic
cyto
pla
smic
envir
onm
ent
[38,
39].
Bio
logic
al
Ro
les
for
Cd
c42
Per
hap
s th
e m
ost
w
idel
y ac
cepte
d ro
le of
Cdc4
2 an
d oth
er R
ho-f
amil
y G
pro
tein
s is
in
the
org
aniz
atio
n o
f th
e act
in c
yto
skel
eton.
T
he
gen
e en
codin
g C
dc4
2
was
fir
st i
den
tifi
ed i
n t
his
conte
xt
in S
acc
haro
myce
s ce
rvis
iae,
wher
e pola
riza
tion o
f
the
acti
n c
yto
skel
eton i
s cr
itic
al f
or
bud f
orm
atio
n [
40].
In
thes
e as
says,
tem
per
ature
-
sensi
tive
(Ts)
muta
nts
def
ecti
ve
in t
he C
DC
42 g
ene
conti
nued
to g
row
at
a re
stri
cted
tem
per
ature
, but
fail
ed
to
bud,
resu
ltin
g
in
larg
e unbudded
ce
lls
[40].
Imm
unofl
uore
scen
ce s
tudie
s fu
rther
confi
rmed
this
role
for
Cdc4
2,
as
it w
as l
oca
lize
d
to t
he
tips
and s
ides
of
enla
rgin
g b
uds
of
S. ce
rvis
iae
[41].
In a
ddit
ion t
o a
ctin
cyto
skel
etal
rea
rran
gem
ent,
a n
um
ber
of
addit
ional
role
s
for
Cdc4
2,
incl
udin
g t
he
contr
ol
of
cell
gro
wth
, hav
e now
com
e to
be
appre
ciat
ed.
Init
ial
insi
ghts
into
a r
ole
for
Cdc4
2 i
n c
ell
gro
wth
cam
e fr
om
the
iden
tifi
cati
on o
f th
e
Dbl
onco
gen
e as
a G
EF
for
Cdc4
2 [
42].
D
bl
was
ori
gin
ally
iso
late
d f
rom
a h
um
an
dif
fuse
B-c
ell
lym
phom
a, a
nd w
as s
how
n t
o t
ransf
orm
NIH
3T
3 c
ells
and t
o c
ause
tum
ors
in n
ude
mic
e [4
3].
T
he
hyper
-act
ivat
ion o
f C
dc4
2 a
nd
rel
ated
Rho-f
amil
y
pro
tein
s has
bee
n s
how
n t
o b
e re
sponsi
ble
for
the
tran
sform
ing p
roper
ties
of
Dbl
[44].
Ear
ly s
tudie
s of
the
effe
cts
of
Rho-f
amil
y p
rote
ins
on c
ell
gro
wth
focu
sed o
n
GT
Pas
e-def
ecti
ve
muta
nts
, bas
ed o
n t
he
tran
sform
ing p
roper
ties
of
the
Ras
-Q61L
an
d
Ras
-G12V
muta
nts
. H
ow
ever
, m
ixed
res
ult
s hav
e bee
n r
eport
ed i
n t
he l
iter
ature
for
the
effe
cts
resu
ltin
g f
rom
GT
Pas
e-def
ecti
ve
muta
nts
of
Cdc4
2,
incl
udin
g t
he
inhib
itio
n
of
cell
gro
wth
rep
ort
ed b
y o
ur
labora
tory
and o
ther
s.
This
led
to t
he
hypoth
esis
that
9
the
cycl
ing o
f C
dc4
2 b
etw
een i
ts G
DP
- an
d G
TP
-bound s
tate
s w
as n
eces
sary
for
its
abil
ity t
o t
ransf
orm
cel
ls.
Indee
d,
Cdc4
2-F
28L
, a
muta
nt
whic
h e
xhib
its
an i
ncr
ease
d
capac
ity t
o e
xch
ange
nucl
eoti
de i
n t
he
abse
nce
of
GE
Fs
wit
hout
com
pro
mis
ing i
ts
abil
ity
to
under
go
GT
P
hydro
lysi
s,
tran
sform
s ce
lls.
More
sp
ecif
ical
ly,
stab
le
expre
ssio
n o
f C
dc4
2-F
28L
enab
les
cel
ls t
o g
row
in l
ow
ser
um
, as
wel
l as
ach
ieve
a
hig
h d
ensi
ty,
dem
onst
rati
ng a
loss
of
conta
ct i
nhib
itio
n [
44,
45].
A
ddit
ional
stu
die
s in
soft
agar
hav
e li
nked
Cdc4
2 t
o a
nch
ora
ge-
indep
enden
t gro
wth
[44].
Muta
tion o
f phen
yla
lanin
e 28 i
n R
as
and r
elat
ed p
rote
ins
to l
euci
ne
has
bee
n
show
n t
o i
ncr
ease
th
e ra
te o
f G
DP
dis
soci
atio
n b
y 1
50 f
old
, ac
tivat
ing t
he
GT
Pas
es i
n
vivo
[4
6].
This
m
uta
tion is
th
ought
to
stab
iliz
e th
e guan
ine
ring
moie
ty
of
the
nucl
eoti
de
thro
ugh p
i-pi
orb
ital
inte
ract
ions
contr
oll
ing G
DP
-GT
P e
xch
ange
[47-4
9].
Cdc4
2-F
28L
under
goes
sponta
neo
us
nucl
eoti
de
exch
ange,
wit
h r
ates
com
par
able
to
those
for
Dbl-
cata
lyze
d e
xch
ange o
n w
ild-t
ype
Cd
c42 [
45],
whil
e th
e G
TP
ase
acti
vit
y
rem
ains
esse
nti
ally
unch
anged
. T
he
inse
rt r
egio
n (
resi
dues
120
-139 i
n C
dc4
2),
un
ique
to
Rho-s
ubfa
mil
y
pro
tein
s,
has
bee
n
iden
tifi
ed
as
an
esse
nti
al
elem
ent
for
the
tran
sform
ing ac
tivit
y of
Cdc4
2-F
28L
. T
he
del
etio
n of
this
in
sert
re
gio
n does
not
affe
ct t
he
abil
ity o
f C
dc4
2-F
28L
to t
rigger
act
in c
yto
skel
etal
rea
rran
gem
ents
nor
to
acti
vat
e m
any d
ow
nst
ream
eff
ecto
rs.
How
ever
, re
moval
of
the
inse
rt p
reven
ts b
indin
g
to t
he
exch
ange
fact
or,
Cool-
1/!
-Pix
, an
d i
ts b
indin
g p
artn
er,
Cbl,
an E
3 u
biq
uit
in
ligas
e.
This
appea
rs t
o a
ccount
for
why C
dc4
2 m
uta
nts
lac
kin
g t
he
inse
rt r
egio
n a
re
tran
sform
atio
n-d
efec
tive
[50].
N
ot
only
do C
dc4
2 a
nd
Ras
shar
e st
ruct
ura
l an
d b
iolo
gic
al s
imil
arit
ies,
but
Cdc4
2 h
as a
lso b
een
found t
o b
e es
senti
al f
or
Ras-
induce
d t
ransf
orm
atio
n.
Expre
ssio
n
of
the
dom
inan
t-neg
ativ
e m
uta
nt,
C
dc4
2-T
17N
, has
bee
n
show
n
to
inhib
it
focu
s
form
atio
n b
y R
as i
n N
IH3T
3 c
ells
[51],
sig
nif
ican
tly d
ecre
ase
Ras-
induce
d a
nch
ora
ge-
10
indep
enden
t gro
wth
, an
d r
ever
t th
e tr
ansf
orm
ed p
hen
oty
pe
obse
rved
in
H-R
as-V
12
cell
lin
es [
51].
T
he
Cdc4
2-D
118N
m
uta
nt
has
also
bee
n
show
n
to
poss
ess
the
abil
ity
to
tran
sform
cel
ls.
This
muta
tio
n o
ccurs
at
a co
nse
rved
moti
f in
volv
ed i
n s
tabil
izin
g t
he
guan
ine
ring
of
the
nucl
eoti
de
thro
ugh
hydro
gen
bondin
g
inte
ract
ions
bet
wee
n
a
conse
rved
asp
arti
c ac
id r
esid
ue,
D118,
and t
he
ring n
itro
gen
(N
7)
[52].
T
he
D118N
muta
tion r
esult
s in
an i
ncr
ease
d r
ate o
f G
DP
-GT
P e
xch
ange
over
that
of
wil
d-t
ype
Cdc4
2,
alth
ough i
t is
slo
w r
elat
ive
to t
he
intr
insi
c nucl
eoti
de
exch
ange
rate
for
the
Cdc4
2-F
28L
muta
nt.
S
urp
risi
ngly
, th
e C
dc4
2-D
118N
muta
nt
has
pro
ven
to b
e m
ore
pote
nt
in
tran
sform
ing
NIH
3T
3
cell
s th
an
the
F28L
m
uta
nt.
The
ampli
fied
tran
sform
ing pro
per
ties
of
D118N
over
F
28L
hav
e bee
n at
trib
ute
d to
th
e D
118
N
muta
nt
bei
ng r
esis
tant
to c
asp
ase
clea
vag
e.
Giv
en th
e fu
nct
ion o
f C
dc4
2 in
m
itogen
ic pat
hw
ays,
a
role
fo
r C
dc4
2 in
infl
uen
cing
signal
ing
by
gro
wth
fa
ctor
rece
pto
rs
was
not
surp
risi
ng.
C
dc4
2
has
num
erous
dow
nst
ream
eff
ecto
rs,
incl
udin
g t
he
Co
ol-
1/!
-Pix
pro
tein
. C
ool-
1/!
-Pix
is
an i
nte
rest
ing p
rote
in i
n t
hat
it
oper
ates
both
as
an u
pst
ream
act
ivat
or
and d
ow
nst
ream
targ
et o
f R
ho-f
amil
y G
pro
tein
s.
Cool-
1 h
as b
een s
how
n t
o a
ct a
s an
exch
ang
e fa
ctor
for
Cdc4
2 in
it
s phosp
hory
late
d st
ate
[Fen
g et
al.
, in
pre
ss]
and ad
dit
ional
ly as
a
scaf
fold
li
nkin
g C
dc4
2 to
th
e E
3 ubiq
uit
in li
gas
e, C
bl.
T
hro
ugh th
is in
tera
ctio
n,
Cdc4
2 h
as
bee
n t
ied t
o C
bl,
involv
ed i
n ca
taly
zing t
he
ubiq
uit
inat
ion o
f th
e E
GF
rece
pto
r at
th
e cel
l su
rfac
e.
T
his
ubiq
uit
inat
ion ev
ent
is nec
ess
ary fo
r th
e E
GF
rece
pto
r to
be
sort
ed t
o v
ario
us
cel
lula
r co
mpar
tmen
ts f
or
deg
radat
ion.
How
ever
,
when
C
dc4
2
form
s a
tern
ary
com
ple
x
wit
h
p85C
ool-
1/!
-Pix
an
d
Cbl,
C
bl
is
seques
tere
d a
way
fro
m t
he
EG
F r
ecep
tor,
and i
s no l
onger
able
to b
ind t
o a
nd b
ecom
e
phosp
hory
late
d b
y t
he
rece
pto
r, a
nec
essa
ry s
tep i
n a
ctiv
atin
g i
ts E
3 u
biq
uit
in l
igas
e
acti
vit
y [
53].
T
he
inhib
itio
n o
f E
GF
rec
epto
r ubiq
uit
inat
ion d
isru
pts
norm
al r
ecep
tor
11
pro
cess
ing,
resu
ltin
g i
n i
ts a
ccum
ula
tion a
t th
e ce
ll s
urf
ace
and e
xce
ssiv
e m
itogen
ic
signal
ing [
54,
55][
Fen
g e
t al
., i
n p
ress
].
This
is
one
exam
ple
of
Cdc4
2 a
nd a
ssoci
ated
pro
tein
s in
volv
emen
t in
gro
wth
fac
tor
rece
pto
r re
gula
tion.
Addit
ional
exam
ple
s w
ill
be
dis
cuss
ed b
elow
.
Erb
B F
am
ily o
f G
row
th F
act
or
Rec
epto
rs
Mem
ber
s of
the
EG
F r
ecep
tor
fam
ily,
also
cal
led E
rbB
or
HE
R r
ecep
tors
, ar
e
involv
ed i
n t
he
init
iati
on o
f var
ious
signal
ing p
athw
ays
resu
ltin
g i
n d
iver
se b
iolo
gic
al
resp
onse
s in
cludin
g
pro
life
rati
on,
dif
fere
nti
atio
n,
mig
rati
on,
and
apopto
sis.
E
rbB
rece
pto
rs s
har
e a
conse
rved
str
uct
ure
consi
stin
g o
f tw
o c
yst
eine-
rich
reg
ions
in t
he
extr
acel
lula
r dom
ain a
nd a
tyro
sine
kin
ase
dom
ain w
ith t
yro
sine
auto
phosp
hory
lati
on
site
s w
ithin
the
cyto
pla
smic
port
ion o
f th
e pro
tein
s.
Thes
e tw
o d
om
ains
are
connec
ted
by a
sin
gle
alp
ha
hel
ix s
pan
nin
g t
he
pla
sma
mem
bra
ne.
E
rbB
rec
epto
rs a
re a
ctiv
ated
by l
igan
d b
indin
g t
o t
he
extr
acel
lula
r dom
ain,
lead
ing t
o r
ecep
tor
dim
eriz
atio
n a
nd
tran
sphosp
hory
lati
on o
f sp
ecif
ic c
yto
pla
smic
tyro
sine
resi
dues
(F
igure
1.3
).
Thes
e
tran
sphosp
hory
lati
on e
ven
ts p
rovid
e re
cognit
ion s
ites
for
the
recr
uit
men
t of
var
ious
pro
tein
s in
volv
ed i
n d
ow
nst
ream
sig
nal
ing c
ascades
. T
he
com
bin
atio
n o
f pat
hw
ays
acti
vat
ed
ult
imat
ely
resu
lts
in
chan
ges
in
gen
e ex
pre
ssio
n,
thus
trig
ger
ing
the
appro
pri
ate
bio
logic
al r
esponse
s to
the
extr
acel
lula
r cu
es.
The
abil
ity of
the
Erb
B re
cepto
rs to
hom
o-
and het
erodim
eriz
e off
ers
the
pote
nti
al f
or
signif
ican
t si
gnal
div
ersi
fica
tion,
as r
efle
cted
by t
he
var
iety
of
dif
fere
nt
ligan
ds
that
can
act
ivat
e th
ese
rece
pto
rs a
nd t
he
man
y d
iffe
rent
signal
ing o
utp
uts
that
they
gen
erat
e.
In t
he
case
of
the
EG
F r
ecep
tor,
a s
ingle
EG
F m
ole
cule
bin
ds
to e
ach
acti
vat
ed r
ecep
tor
[56].
Up
on
gro
wth
fac
tor
bin
din
g,
the
EG
F r
ecep
tor
is t
hough
t to
under
go a
dra
mat
ic c
onfo
rmat
ional
chan
ge
alte
ring t
he
posi
tion o
f th
e dim
eriz
atio
n
12
Fig
ure
1.3
A
rep
rese
nta
tion
of
epid
erm
al
gro
wth
fa
ctor
(EG
F)
rece
pto
r
act
ivati
on
an
d d
ow
nst
ream
sig
nali
ng.
Ad
ap
ted
fro
m B
ioch
emis
try (
Str
yer
), f
ifth
edit
ion
[1
50].
B
indin
g of
EG
F by E
GF
re
cepto
r re
sult
s in
th
e dim
eriz
atio
n an
d
subse
quen
t tr
ans-
phosp
hory
lati
on
of
cyto
pla
smic
ty
rosi
ne
resi
dues
.
This
phosp
hory
lati
on e
ven
t re
cruit
s th
e a
dap
tor
pro
tein
, G
rb2,
to t
he
pla
sma
mem
bra
ne
wher
e it
act
ivat
es
the
Ras
exch
ang
e fa
ctor,
son o
f se
ven
less
(S
os)
. S
os
then
cat
alyze
s
the
exch
ange
of
GT
P f
or
GD
P,
thus
acti
vat
ing R
as,
whic
h u
ltim
atel
y si
gnal
ing t
o
mit
ogen
act
ivat
ed p
rote
in (
MA
P)
kin
ase,
res
ult
ing i
n c
ell
pro
life
rati
on.
13
14
arm
, a
smal
l lo
op
mad
e up
of
amin
o
acid
s 2
42-2
59
involv
ed
in
rece
pto
r dim
er
form
atio
n, sh
ifti
ng f
rom
its
bu
ried
posi
tion i
n t
he i
nac
tive
confo
rmat
ion t
o a
n e
xte
nded
posi
tion i
n t
he
act
ivat
ed s
tate
[57].
D
elet
ions
or
muta
tions
in t
his
dim
eriz
atio
n a
rm
pre
ven
t li
gan
d-i
nduce
d E
GF
rec
epto
r ac
tivat
ion [
58,
59].
The
four
mem
ber
s of
the
Erb
B f
amil
y,
evolu
tionar
ily t
ied t
o t
he
C.
eleg
ans
rece
pto
r ty
rosi
ne
kin
ase
ort
holo
gue,
L
et-2
3
[60],
hav
e under
gone
consi
der
able
div
ersi
fica
tion.
L
igan
d
bin
din
g
contr
ols
th
e ac
tivit
ies
of
the
EG
F
rece
pto
r (a
lso
som
etim
es r
efer
red t
o a
s E
rbB
-1)
and E
rbB
-4,
unli
ke
Erb
B-2
(HE
R2/N
eu)
for
whic
h
no know
n li
gan
d ex
ists
[6
1]
(Fig
ure
1.4
). E
rbB
-3 bin
ds
only
to
m
ember
s of
the
neu
reguli
n f
amil
y (
also
cal
led h
ereg
uli
n)
and l
ack
s kin
ase a
ctiv
ity b
ecau
se o
f se
ver
al
crit
ical
muta
tions
in i
ts a
ctiv
e si
te.
Con
sequen
tly
, nei
ther
Erb
B-2
nor
Erb
B-3
is
able
to
signal
in
dep
enden
tly,
yet
both
re
cepto
rs
signif
ican
tly
enhan
ce
the
acti
vit
y
and
div
ersi
fica
tion of
the
oth
er tw
o m
ember
s [6
2,
63].
In
fa
ct,
Erb
B-2
ex
ists
as
th
e
pre
ferr
ed b
indin
g p
artn
er f
or
the
EG
F r
ecep
tor,
as
wel
l as
for
Erb
B-3
and E
rbB
-4,
once
thes
e re
cepto
rs h
ave
bee
n a
ctiv
ated
by t
hei
r re
spec
tive
ligan
ds.
T
he
bas
is f
or
this
obse
rved
pro
mis
cuit
y o
f E
rbB
-2 a
ppea
rs t
o l
ie i
n t
he
posi
tion o
f it
s dim
eriz
atio
n l
oop.
Wher
e oth
er E
rbB
rec
epto
rs m
ust
bin
d a
lig
and
in o
rder
to e
xte
nd t
he
dim
eriz
atio
n
loop,
the
corr
espondin
g d
imer
izat
ion l
oop o
f E
rbB
-2 i
s pre
-exte
nded
, th
ereb
y p
ois
ed
for
het
erodim
er f
orm
atio
n [
58].
T
he
tran
sform
ing c
apab
ilit
ies
of
Erb
B-2
lik
ely r
esult
s
from
th
e te
nden
cy of
Erb
B-2
het
erodim
ers
to under
go re
cycl
ing bac
k to
th
e ce
ll
surf
ace
rath
er t
han
deg
radat
ion i
n t
he
lyso
zom
e [6
4,
65].
In
gen
eral
, hom
odim
eric
rece
pto
r co
mbin
atio
ns
are
less
m
itogen
ic an
d tr
ansf
orm
ing th
an th
e co
rres
pondin
g
het
erodim
eric
com
bin
atio
ns
[62,
63].
F
or
exam
ple
, E
GF
R-E
rbB
-2 h
eter
odim
ers
are
asso
ciat
ed
wit
h
a m
ore
ro
bust
si
gnal
th
an
EG
F
rece
pto
r hom
odim
ers
[66].
Surp
risi
ngly
, th
e m
ost
tra
nsf
orm
ing o
f th
e het
erodim
eric
com
bin
atio
ns
occ
urs
when
15
Fig
ure
1.4
A
rep
rese
nta
tion
of
the
Erb
B f
am
ily o
f re
cep
tor
tyro
sin
e k
inase
s.
Ad
ap
ted
fro
m M
ose
sson
an
d Y
ard
en [
151].
16
17
the
ligan
d-f
ree
Erb
B-2
rec
epto
r co
mple
xes
wit
h t
he
kin
ase-
def
icie
nt
Erb
B-3
rec
epto
r,
resu
ltin
g f
rom
an i
nit
ial
bin
din
g e
ven
t bet
wee
n h
ereg
uli
n a
nd E
rbB
-3 [
62, 67, 68].
All
pai
rwis
e co
mbin
atio
ns
of
the
Erb
B
rece
pto
r fa
mil
y
mem
ber
s,
four
hom
odim
eric
an
d si
x het
erodim
eric
st
ates
, ca
n b
e ac
tivat
ed by th
e el
even
sp
ecif
ic
Erb
B l
igan
ds
char
acte
rize
d t
o d
ate.
A
ll h
igh
-aff
init
y l
igan
ds
are
com
pri
sed
of
an
EG
F-l
ike
dom
ain a
nd t
hre
e dis
ulf
ide-
bonded
intr
amole
cula
r lo
ops
[69].
T
hes
e pep
tide
ligan
ds
are
expre
ssed
in t
he
extr
acel
lula
r dom
ain o
f tr
ansm
embra
ne
pro
tein
s an
d a
re
gen
erat
ed b
y r
egula
ted p
rote
oly
sis
to y
ield
gro
wth
fac
tors
that
conta
in 4
9-8
5 a
min
o
acid
s.
T
he
gen
erat
ed
gro
wth
fa
ctors
dem
onst
rate
dif
fere
nt
spec
ific
itie
s fo
r th
e
rece
pto
r fa
mil
y m
ember
s.
Fo
r ex
ample
, E
GF
and T
GF
-! h
ave
a hig
h a
ffin
ity f
or
the
EG
F
rece
pto
r,
whil
e neu
reguli
ns
bin
d
to
Erb
B-3
an
d
Erb
B-4
re
cepto
rs.
Over
expre
ssio
n of
Erb
B-2
, an
d th
e su
bse
quen
t het
erodim
eriz
atio
n of
the
rece
pto
r,
seem
s to
bro
aden
lig
and s
pec
ific
ity,
wher
e li
gan
ds
that
are
bet
ter
at r
ecru
itin
g t
his
co-
rece
pto
r ca
n c
om
pet
itiv
ely r
educe
the
bin
din
g o
f le
ss e
ffec
tive-
ligan
ds.
The
bin
din
g o
f var
ious
ligan
ds
and t
he
abil
ity o
f E
rbB
rec
epto
rs t
o h
om
o-
and
het
erodim
eriz
e en
han
ce t
he
var
iati
on o
f ty
rosi
ne
phosp
hory
lati
on e
ven
ts t
hat
can
occ
ur
as a
n o
utc
om
e of
tran
sphosp
hory
lati
on.
In a
ddit
ion t
o t
he
phosp
ho-t
yro
sin
e re
sidues
gen
erat
ed by au
tophosp
hory
lati
on,
non-r
ecep
tor
tyro
sine
kin
ases
, su
ch as
S
rc,
can
phosp
hory
late
the
cyto
pla
smic
dom
ain o
f E
rbB
rec
epto
rs t
o f
urt
her
div
ersi
fy s
ignal
tran
sduct
ion p
athw
ays.
T
he
spec
ific
phosp
ho-t
yro
sine
resi
dues
det
erm
ine
the
sub
set
of
cyto
pla
smic
tar
get
s re
cruit
ed t
o t
he
cell
surf
ace
for
acti
vat
ion. T
hes
e ta
rget
pro
tein
s
gen
eral
ly f
all
into
the
cate
gory
of
Src
hom
olo
gy 2
(S
H2)-
or
phosp
ho-t
yro
sine
bin
din
g
(PT
B)
dom
ain-c
onta
inin
g p
rote
ins.
S
H2 d
om
ains
are
com
pose
d o
f ap
pro
xim
atel
y 1
00
amin
o a
cids
that
rec
ogniz
e phosp
hory
late
d t
yro
sine
resi
dues
bas
ed o
n t
he
thre
e to
six
amin
o a
cids
C-t
erm
inal
to t
he
phosp
hory
late
d s
ite.
C
onver
sely
, th
e bin
din
g o
f P
TB
dom
ains
(appro
xim
atel
y 150 am
ino ac
ids
in le
ngth
) is
det
erm
ined
by th
e re
sidues
18
pre
cedin
g th
e phosp
hory
lati
on si
te [
70].
T
hes
e S
H2-
and P
TB
-dom
ain co
nta
inin
g
pro
tein
s in
clude
adap
tor
pro
tein
s su
ch a
s, G
rb2,
Grb
7,
Crk
, an
d G
ab1,
pro
tein
and
lipid
kin
ases
, S
rc a
nd
phosp
hat
idyli
nosi
tol
3-k
inase
(P
I3K
) re
spec
tivel
y,
and p
rote
in
phosp
hat
ases
such
as
SH
P1 a
nd S
HP
2.
Man
y o
f th
e c
yto
pla
smic
tar
get
s of
Erb
B r
ecep
tors
sig
nal
to
the
MA
P k
inase
pat
hw
ays
via
R
as an
d S
hc,
re
sult
ing in
ce
ll pro
life
rati
on.
In
ad
dit
ion,
the
PI3
K-
acti
vat
ed A
KT
pat
hw
ay a
nd t
he
p70S
6K
/p85S
6K
pat
hw
ays
that
are
involv
ed i
n c
ell
surv
ival
ar
e dow
nst
ream
of
most
E
rbB
-rec
epto
r si
gnal
ing.
W
hil
e a
gre
at dea
l of
over
lap e
xis
ts a
mong t
he
cyto
pla
smic
tar
get
s of
Erb
B r
ecep
tors
, a
num
ber
of
spec
ific
targ
ets
do e
xis
t.
Thes
e in
clu
de
Cbl
and p
ho
sphat
idyli
nosi
tol
3-k
inas
e (P
I3K
), w
hic
h
dem
onst
rate
spec
ific
ity f
or
the
EG
F r
ecep
tor
and E
rbB
-3,
resp
ecti
vel
y [
71,
72].
T
he
bin
din
g s
pec
ific
ity o
f si
gnal
ing p
rote
ins
dow
nst
ream
of
the
Erb
B r
ecep
tors
lar
gel
y
acco
unts
for
the
abil
ity o
f th
ese
recep
tors
to s
tim
ula
te s
ignal
ing p
athw
ays
resu
ltin
g i
n
so m
any v
arie
d b
iolo
gic
al a
ctiv
itie
s.
Bec
ause
Erb
B r
ecep
tors
init
iate
mit
ogen
ic s
ignal
ing p
athw
ays,
over
expre
ssio
n
or
muta
tions
that
al
ter
thei
r in
trin
sic
kin
ase
acti
vit
y ar
e oft
en su
ffic
ient
to ca
use
mal
ignan
t tr
ansf
orm
atio
n.
T
ransf
orm
ing ca
pab
ilit
ies
hav
e bee
n at
trib
ute
d to
E
rbB
rece
pto
rs a
s ea
rly a
s th
e 1
980s,
when
th
e av
ian e
ryth
robla
stosi
s tu
mor
vir
us
was
found
to e
nco
de
a tr
unca
ted f
orm
of
the
hum
an E
GF
rec
epto
r [7
3].
E
rbB
-2 w
as l
ater
iso
late
d
in r
oden
t S
chw
annom
as a
s an
onco
gen
e (o
rigin
ally
cal
led N
eu)
wit
h a
n a
ctiv
atin
g
poin
t m
uta
tion i
n i
ts t
ransm
embra
ne
segm
ent
[74].
M
ore
rec
entl
y,
the
EG
F r
ecep
tor
and E
rbB
-2 h
ave
bee
n i
mpli
cate
d i
n s
ever
al h
um
an n
eopla
sms.
F
or
exam
ple
, th
e E
GF
rece
pto
r is
over
expre
ssed
in b
ladder
, bre
ast,
hea
d a
nd n
eck,
kid
ney
, non
-sm
all
cell
lung,
and p
rost
ate
cance
rs [
75-7
7],
whil
e E
rbB
-2/H
ER
2/N
eu o
ver
expre
ssio
n i
s fo
und
in s
tom
ach a
nd m
amm
ary
car
cinom
as [
78].
O
ver
expre
ssio
n o
f th
e kin
ase-
def
ecti
ve,
Erb
B-3
, has
als
o b
een l
inked
to h
um
an c
ance
rs i
ncl
udin
g b
reas
t, c
olo
n,
and g
astr
ic
19
carc
inom
as,
wher
eas
Erb
B-4
upre
gula
tion h
as b
een f
ound i
n b
oth
bre
ast
cance
r an
d
gra
nulo
se c
ell
tum
ors
of
the
ovar
y [
75].
Can
cer
cell
s ar
e ab
le to
bypass
th
e nece
ssar
y ch
eckpoin
ts in
volv
ed in
th
e
regula
tion
of
Erb
B
rece
pto
r si
gnal
ing
as
desc
ribed
bel
ow
.
Gli
obla
stom
as
and
sarc
om
as a
cquir
e th
e ab
ilit
y t
o p
roduce
gro
wth
fac
tors
to w
hic
h t
hey
are
res
pon
sive,
ther
eby c
reat
ing a
posi
tive
feed
-bac
k l
oop r
efer
red t
o a
s au
tocr
ine
stim
ula
tion [
79].
Oth
er c
ance
r ce
lls
induce
the
over
expre
ssio
n o
f E
rbB
rec
epto
rs a
t th
e ce
ll s
urf
ace,
resu
ltin
g in
hyper
sensi
tivit
y to
oth
erw
ise am
bie
nt
level
s of
gro
wth
fa
ctor.
G
ross
over
expre
ssio
n o
f E
rbB
rec
epto
rs c
an e
lici
t li
gan
d-i
ndep
enden
t si
gnal
ing [
80],
as
can
muta
tions
in t
he
cell
surf
ace
rece
pto
r.
Oft
en t
hes
e m
uta
tions
involv
e tr
unca
tions
of
the
extr
acel
lula
r dom
ain,
resu
ltin
g
in
const
ituti
ve
acti
vat
ion
of
the
rece
pto
r.
Det
erm
inin
g th
e m
echan
ism
by w
hic
h tr
ansf
orm
ed ce
lls
hav
e ex
plo
ited
th
e E
rbB
signal
ing
pat
hw
ays
can
be
extr
emel
y
val
uab
le
in
dev
elopin
g
pro
per
tr
eatm
ent
stra
tegie
s fo
r th
e dis
ease
.
M
ost
rec
ent
ther
apeu
tic
effo
rts
hav
e bee
n d
irec
ted t
ow
ards
the
EG
F r
ecep
tor
and E
rbB
-2,
giv
en t
he
link b
etw
een t
he
over
expre
ssio
n o
f th
ese
pro
tein
s an
d t
um
or
cell
form
atio
n.
The
gre
ates
t ch
alle
nge
in i
den
tify
ing t
her
apeu
tic
agen
ts a
gai
nst
pro
tein
kin
ases
li
es
in th
e fa
ct th
at th
e kin
ase
dom
ain is
hig
hly
co
nse
rved
th
roughout
all
seri
ne/
thre
onin
e an
d
tyro
sine
kin
ases
.
The
maj
ori
ty
of
smal
l m
ole
cule
kin
ase
inhib
itors
ar
e A
TP
-com
pet
itiv
e co
mpounds,
th
ereb
y ru
nnin
g th
e ri
sk o
f in
hib
itin
g
mult
iple
pro
tein
kin
ases
, an
d m
ult
iple
si
gnal
ing
pat
hw
ays.
D
esp
ite
this
ch
alle
nge,
AT
P-c
om
pet
itiv
e in
hib
itors
hav
e bee
n d
evel
op
ed a
gai
nst
the
EG
F r
ecep
tor
and E
rbB
-
2,
and a
re s
urp
risi
ngly
sel
ecti
ve
du
e to
the
rela
tiv
ely d
eep n
ucl
eoti
de-
bin
din
g p
ock
et
of
the
Erb
B p
rote
ins.
T
he
most
sel
ect
ive
inhib
itors
of
the
Erb
B r
ecep
tors
shar
e th
e
bas
ic q
uin
azoli
ne
stru
cture
[8
1,
82].
T
hes
e in
clude
com
pounds
like
Ires
sa (
ZD
-1839),
PK
I-66 a
nd T
arce
va
(OS
I-774).
Ir
essa
was
init
iall
y a
ppro
ved
in t
he
trea
tmen
t of
non-
20
smal
l-lu
ng c
ell
cance
r (N
SC
LC
), w
her
e th
e re
spo
nse
to I
ress
a w
as l
inked
to p
atie
nts
conta
inin
g sp
ecif
ic m
uta
tions
at cr
itic
al si
tes
wit
hin
th
e E
GF
re
cepto
r ac
tive
site
,
resu
ltin
g i
n a
n i
nduce
d f
it o
f th
e dru
g [
83].
C
urr
ent
monocl
onal
anti
body t
reat
men
t of
cance
r in
cludes
tre
atm
ent
of
colo
rect
ora
l ca
nce
r w
ith
an a
nti
-EG
F r
ecep
tor
anti
body,
Erb
itux
TM
, an
d
bre
ast
cance
r tr
eatm
ent
wit
h
the
Erb
B-2
m
onocl
onal
an
tibody,
Her
cepti
nT
M.
Erb
itux
TM
bin
ds
to t
he
EG
F r
ecep
tor
wit
h h
igh a
ffin
ity,
pre
ven
ting t
he
bin
din
g o
f E
GF
or
TG
F-!
and t
her
eby b
lock
ing r
ecep
tor
acti
vat
ion.
The
mec
han
ism
of
inhib
itio
n o
f E
rbB
-2 b
y H
erce
pti
nT
M i
s sl
ightl
y m
ore
com
pli
cate
d d
ue
to t
he
lack
of
an E
rbB
-2 l
igan
d. C
onse
quen
tly,
a gre
at d
eal
of
tim
e has
bee
n i
nves
ted i
n d
eter
min
ing
the
mec
han
ism
of
Her
cepti
nT
M i
nhib
itio
n.
It
is n
ow
thought
that
Her
cepti
nT
M n
ot
only
blo
cks
rece
pto
r bin
din
g
in
het
erodim
ers,
but
als
o
incr
ease
s th
e
rate
of
rece
pto
r
inte
rnal
izat
ion, re
sult
ing i
n t
he
reduct
ion o
f tu
mor
gro
wth
[84].
Des
pit
e th
ese
sig
nif
ican
t gai
ns,
sev
eral
fac
tors
con
tinue
to l
imit
the
effi
cacy
of
Erb
B-t
arget
ed t
her
apeu
tics
. F
or
exam
ple
, kin
ase-
inac
tive
Erb
B p
rote
ins
can r
etai
n
thei
r ab
ilit
y t
o s
ignal
when
com
ple
xed
to o
ther
Erb
B r
ecep
tors
. I
n a
ddit
ion,
gro
wth
fact
ors
can
act
ivat
e E
rbB
sig
nal
ing b
y a
ctiv
atin
g c
yto
pla
smic
tyro
sine
kin
ase
par
tner
s,
such
as
Src
. F
inal
ly,
man
y re
gula
tory
si
gnal
ing pat
hw
ays
involv
ed in
th
e dow
n-
regula
tion o
f E
rbB
rec
epto
r si
gnal
ing,
such
as
Cbl-
cata
lyze
d u
biq
uit
inat
ion,
requir
e
the
kin
ase
acti
vit
y o
f th
e re
cepto
r to
be
inta
ct.
Dow
n-r
egula
tion o
f E
rbB
rec
epto
r
signal
ing h
as,
and w
ill
conti
nue
to b
e a
sought
afte
r m
eans
of
inte
rven
ing a
gai
nst
unco
ntr
oll
ed c
ell
gro
wth
.
Cla
thri
n-m
edia
ted
En
docy
tosi
s
Cla
thri
n-m
edia
ted
endocy
tosi
s is
a
wel
l-st
udie
d
and
hig
hly
ch
arac
teri
zed
pro
cess
in w
hic
h c
ell
surf
ace
rece
pto
rs b
ecom
e in
tern
aliz
ed i
nto
the
cyto
pla
sm.
Not
only
is
cl
athri
n-m
edia
ted
endocy
tosi
s re
spon
sib
le
for
the
upta
ke
of
cell
su
rfac
e
21
rece
pto
rs,
but
it i
s al
so u
tili
zed b
y v
irtu
ally
all
eukar
yoti
c ce
lls
to i
nte
rnal
ize
nutr
ients
,
anti
gen
s, gro
wth
fa
ctors
, an
d pat
hogen
s fr
om
th
e pla
sma
mem
ber
to
in
trac
ellu
lar
com
par
tmen
ts [
85].
C
lath
rin-m
edia
ted e
ndocy
tosi
s ca
n e
ither
occ
ur
const
ituti
vel
y o
r
in r
esponse
to e
xte
rnal
sti
mu
li.
Cla
thri
n c
oat
ed v
esi
cles
(C
CV
s) a
re e
ncap
sula
ted i
nto
a ca
ge
form
ed b
y t
he
inte
ract
ion o
f cl
athri
n t
risk
elio
ns.
T
he
clat
hri
n t
risk
elio
n e
xis
ts
as a
thre
e-le
gged
str
uct
ure
com
pose
d o
f th
ree
hea
vy c
hai
ns
and t
hre
e li
ght
chai
ns.
T
he
carb
oxyl-
term
inal
en
ds
of
the
clat
hri
n hea
vy ch
ains
are
join
ed at
th
e hub re
gio
n,
mak
ing u
p t
he
tris
kel
ion s
truct
ure
[86].
T
hes
e hea
vy c
hai
ns
are
bound t
o o
ne
of
two
light
chai
ns,
LC
a an
d L
Cb,
wher
e th
e li
ght
chai
ns
hav
e bee
n s
ugges
ted
to r
egula
te t
he
asse
mbly
of
the
clat
hri
n tr
iskel
ions
[87].
P
roje
ctin
g aw
ay fr
om
th
e hub,
the
N-
term
inal
glo
bula
r dom
ain
ex
ists
as
a
bet
a-pro
pel
ler
and co
nta
ins
a bin
din
g m
oti
f
resp
onsi
ble
for
clat
hri
n’s
inte
ract
ions
wit
h v
ario
us
endocy
tic
pro
tein
s, i
ncl
udin
g t
he
adap
tor
pro
tein
, A
P-2
.
The
maj
ori
ty
of
clat
hri
n-b
indin
g
pro
tein
s as
soci
ate
wit
h
clat
hri
n t
hro
ugh c
onse
rved
moti
fs,
gen
eral
ly k
now
n a
s cl
athri
n b
ox m
oti
fs,
incl
udin
g
the
LL
NL
D (
Leu
-Leu
-Asn
-Leu
-Asp
) se
quen
ce o
f th
e "
2 s
ubunit
of
AP
-2.
E
ndocy
tosi
s of
cell
surf
ace
recep
tors
is
a ti
ghtl
y r
egula
ted,
sequen
tial
pro
cess
.
It i
nvolv
es s
ever
al c
yto
soli
c pro
tein
s, r
efer
red t
o a
s ac
cess
ory
pro
tein
s, a
s w
ell
as
the
mec
han
ical
m
eans
to
def
orm
th
e m
embra
ne
into
a
ves
icula
r bud
that
ult
imat
ely
pin
ches
off
to f
orm
a f
ree C
CV
(F
igure
1.5
).
This
pro
cess
beg
ins
wit
h t
he r
ecru
itm
ent
of
the
adap
tor
pro
tein
, A
P-2
, to
th
e pla
sma
mem
bra
ne.
A
P-2
is
a het
erote
tram
er
com
pri
sed o
f tw
o l
arge 1
00
-kD
a su
bunit
s, !
and "
, an
d t
wo s
mal
ler
chai
ns µ
2 a
nd
#2,
50 a
nd 2
0 k
Da,
res
pec
tivel
y.
AP
-2 s
erves
to
co
nce
ntr
ate
the
carg
o i
n t
he
emer
gin
g
bud,
as w
ell
as
to l
ink c
lath
rin t
o t
he p
lasm
a m
em
bra
ne
and c
oord
inat
e th
e st
ruct
ura
l
asse
mbly
of
the c
lath
rin c
oat
[88-9
1].
T
he "
sub
unit
of
AP
-2 i
nte
ract
s w
ith c
lath
rin
[92]
whil
e th
e µ
2
subunit
re
cogniz
es
tyro
sine-
bas
ed
endocy
tic
sort
ing
moti
fs
of
mult
iple
pro
tein
s, t
her
eby s
elec
ting t
he
appro
pri
ate
inte
gra
l pro
tein
s fo
r en
docy
tosi
s
22
Fig
ure
1.5
A
sch
emati
c d
iagra
m o
f cl
ath
rin
-med
iate
d e
nd
ocy
tosi
s of
cell
su
rface
rece
pto
rs.
A
dap
ted
fr
om
W
ate
rman
an
d
Yard
en
[152].
Cla
thri
n-m
edia
ted
endocy
tosi
s is
init
iate
d w
ith t
he
recr
uit
men
t of
the
adap
tor
pro
tein
, A
P-2
, to
the
cell
surf
ace.
A
P-2
ser
ves
to c
once
ntr
ate
the
car
go i
nto
the
emer
gin
g b
ud,
as w
ell
as r
ecru
it
clat
hri
n
to
the
pla
sma
mem
bra
ne.
Ass
embly
of
the
clat
hri
n
latt
ice
lead
s to
th
e
invag
inat
ion o
f th
e m
embra
ne
form
ing a
cla
thri
n-c
oat
ed p
it.
The
GT
Pas
e, d
ynam
in,
is
then
ass
emble
d a
t th
e nec
k o
f th
e cl
athri
n-c
oat
ed p
it,
wher
e G
TP
hydro
lysi
s re
sult
s in
fiss
ion t
o f
orm
a f
ree
clat
hri
n-c
oat
ed v
esic
le.
23
24
[93,
94].
O
nce
AP
-2 h
as b
een
rec
ruit
ed t
o t
he
cel
l m
embra
ne
and t
he
ass
embly
of
clat
hri
n
has
bee
n
init
iate
d,
the
mem
bra
ne
acquir
es
incr
easi
ng
curv
ature
unti
l an
invag
inat
ed pit
fo
rms.
A
s th
e cl
athri
n pit
m
ature
s, th
e fi
nal
st
ep in
volv
es fi
ssio
n
thro
ugh t
he
acti
on o
f th
e G
TP
ase,
dynam
in [
95].
D
ynam
in,
a 100-k
Da
pro
tein
, is
a
rath
er uniq
ue
mem
ber
of
the
GT
Pas
e su
per
fam
ily giv
en it
s lo
w af
finit
y fo
r G
TP
(~30 µ
M)
and hig
h in
trin
sic
and st
imula
ted ra
tes
of
GT
P hydro
lysi
s. It
is
now
bel
ieved
that
dynam
in i
s re
cruit
ed t
o t
he
cell
surf
ace
in t
he
GD
P-b
ound o
r nucl
eoti
de-
free
fo
rm an
d is
dis
trib
ute
d th
roughout
the
clath
rin la
ttic
e. G
DP
-GT
P ex
chan
ge
trig
ger
s dynam
in a
ssem
bly
at
the
nec
k o
f th
e cl
athri
n-c
oat
ed p
it,
form
ing a
hel
ical
coll
ar.
The
exac
t m
ole
cula
r m
echan
ism
of
fiss
ion i
s st
ill
uncl
ear,
though i
t se
ems
to
requir
e th
e hydro
lysi
s of
GT
P,
whic
h m
ay d
irec
tly o
r in
dir
ectl
y t
ighte
n t
he
asse
mble
d
dynam
in c
oll
ar r
esult
ing i
n a
fre
e cl
athri
n-c
oat
ed v
esic
le.
Once
the
carg
o h
as
bee
n
inte
rnal
ized
, en
ergy
-dep
enden
t unco
atin
g
rest
ore
s a
ves
icle
w
hic
h
can
even
tual
ly
mat
ure
into
an i
ntr
acel
lula
r co
mpar
tmen
t.
C
lath
rin-c
oat
ed v
esic
les
wer
e in
itia
lly i
den
tifi
ed b
y t
hei
r dis
tinct
ive
mole
cula
r
appea
rance
in e
lect
ron m
icro
gra
phs,
att
ribute
d t
o t
he
clat
hri
n l
atti
ces
that
mak
e u
p t
he
clat
hri
n c
oat
. T
his
ord
ered
str
uct
ure
tak
es
on t
he a
ppea
rance
of
a bask
et m
ade u
p o
f
pen
tagons
and
hex
agon
s [9
6,
97].
The
pen
tagons
appea
r to
fu
lfil
l th
e re
quir
ed
geo
met
ry o
f a
close
d p
oly
gonal
bas
ket
, tw
elve b
eing t
he
requir
ed n
um
ber
, but
the
num
ber
of
hex
agons
var
y [
97]
and a
ppea
r to
contr
ibute
to t
he
size
of
the
ves
icle
. F
or
exam
ple
, ves
icle
s in
the b
rain
are
~76
nm
and
co
nta
in 2
0 h
exag
on
s, w
her
eas
those
in
fibro
bla
sts
are
~120 n
m a
nd c
onta
in 6
0 h
exag
ons
[98].
C
oat
ed
pit
s co
ver
ap
pro
xim
atel
y
0.5
-2%
of
the
cell
su
rfac
e [9
9].
The
form
atio
n of
coat
ed pit
s ap
pea
rs to
st
art
at sp
eci
fic
asse
mbly
si
tes
on
th
e pla
sma
mem
bra
ne
call
ed ‘
pit
zones
’ [1
00].
T
he
fact
ors
that
def
ine
the
asse
mbly
sit
es
hav
e not
bee
n
full
y
iden
tifi
ed,
how
ever
, phosp
hat
idyli
nosi
tides
em
bed
ded
in
to
the
pla
sma
25
mem
bra
ne
hav
e been
found
to r
ecr
uit
AP
-2 t
o t
he
cell
surf
ace.
In
par
ticu
lar,
AP
-2 h
as
a hig
h b
indin
g a
ffin
ity f
or
phosp
hat
idyli
nosi
tol
(4,5
)-bis
phosp
hat
e (
PI(
4,5
)P2)
and
phosp
hat
idyli
nosi
tol
(4,5
,6)-
tris
pho
sphat
e (P
I(3,4
,5)P
3)
[101].
The
bin
din
g
of
PI(
4,5
)P2 r
equir
es p
osi
tivel
y c
har
ged
lysi
ne
resi
dues
on t
he !
-adap
tin s
ubunit
(am
ino
acid
s 21
-80),
wher
e m
uta
tion o
f th
ese
lysi
nes
red
uce
s th
e af
finit
y o
f A
P-2
for
the
pla
sma
mem
bra
ne
[102].
In
addit
ion,
mas
kin
g
PI(
4,5
)P2
wit
h
neo
myci
n,
a hig
h
affi
nit
y l
igan
d f
or
PI(
4,5
)P2,
or
the
PH
dom
ain o
f phosp
holi
pas
e C"
(!"PL
C")
, in
hib
its
the
recr
uit
men
t of
AP
-2 to
th
e en
doso
mes
an
d th
e pla
sma
mem
bra
ne
[103,
104].
Dynam
in-1
an
d
dynam
in-2
hav
e al
so
bee
n
report
ed
to
inte
ract
w
ith
phosp
hat
idyli
nosi
tides
PI(
4,5
)P2
and P
I(4)P
, al
bei
t w
ith l
ow
aff
init
y t
hro
ugh t
hei
r P
H
dom
ains
[105
, 106].
In
th
is ca
se,
pho
sphat
idyli
nosi
tides
ar
e not
likel
y to
re
cruit
dynam
in to
th
e ce
ll su
rfac
e, but
once
dynam
in is
co
nce
ntr
ated
in
th
e co
ated
pit
s,
subse
quen
t in
tera
ctio
ns
wit
h p
hosp
hat
idyli
nosi
tides
appea
r to
pla
y a
sig
nif
ican
t ro
le i
n
fiss
ion f
rom
the
pla
sma
mem
bra
ne.
T
his
model
has
been
support
ed b
y i
n v
itro s
tudie
s
wher
e in
tera
ctio
ns
bet
wee
n d
ynam
in a
nd P
I(4,5
)P2 r
esult
in t
he
dra
mat
ic e
nhan
cem
ent
of
dynam
in G
TP
ase
acti
vit
y [
107].
O
nce
th
e cl
athri
n-c
oat
ed
pit
has
pin
ched
off
fr
om
th
e pla
sma
mem
bra
ne
form
ing t
he
free
cla
thri
n-c
oat
ed v
esic
le,
the i
nte
rnal
ized
ves
icle
even
tual
ly u
nder
goes
unco
atin
g t
o f
orm
the
earl
y e
ndoso
me.
T
his
intr
acel
lula
r co
mpar
tmen
t dev
elops
into
a
rela
tivel
y l
arge
per
inucl
ear
ves
icle
mad
e up o
f m
ult
iple
inte
rnal
ves
icle
s [1
08],
and i
s
refe
rred
to a
s th
e m
ult
ives
icula
r body
(M
VB
s) o
r th
e la
te e
ndo
som
e.
Lat
e en
do
som
es
are
char
acte
rize
d b
y a
n a
ccum
ula
tion o
f hydro
lyti
c en
zym
es,
and a
low
inte
rnal
pH
,
suff
icie
ntl
y
acid
ic
to
dis
soci
ate
som
e
ligan
ds.
Stu
die
s per
form
ed
wit
h
recy
clin
g
rece
pto
rs
and
kin
ase
-def
ecti
ve
muta
nts
of
the
EG
F
rece
pto
r im
ply
th
at
the
late
endoso
me
is t
he
maj
or
site
of
sort
ing f
or
lyso
som
al d
egra
dat
ion.
In t
hes
e st
udie
s,
tran
sfer
rin a
nd k
inas
e-def
ecti
ve
EG
F r
ecep
tors
are
confi
ned
to t
he
ves
icula
r port
ion o
f
26
the
late
endoso
me,
whil
e ac
tive
EG
F r
ecep
tors
acc
um
ula
te i
n t
he
inte
rior
[109,
110].
It i
s th
ought
that
this
tra
nsl
oca
tion o
r co
mpar
tmen
tili
zati
on i
s cr
itic
al f
or
the
rem
oval
of
the
acti
ve
rece
pto
rs
from
th
e re
cycl
ing
pat
hw
ay
to
the
lyso
som
al
deg
radat
ion
pat
hw
ay,
thus
mai
nta
inin
g t
he
appro
pri
ate
rece
pto
r le
vel
s at
th
e ce
ll s
urf
ace.
Rec
epto
r en
docy
tosi
s an
d
deg
radat
ion
are
key
co
mponen
ts
in
the
dow
n-
regula
tion
of
gro
wth
fa
ctor-
med
iate
d
signal
ing
pat
hw
ays.
Giv
en
the
succ
ess
of
ther
apeu
tics
li
ke
Her
cepti
nT
M
in
slow
ing
tu
mor
gro
wth
by
incr
easi
ng
Erb
B-2
deg
radat
ion,
alte
ring th
e ra
tes
for
rece
pto
r en
docy
tosi
s has
bee
n sh
ow
n to
b
e an
effi
cien
t m
eans
of
inhib
itin
g t
um
ori
gen
esis
. O
ur
labora
tory
has
iden
tifi
ed t
wo o
ther
pote
nti
al p
arti
cipan
ts i
n E
GF
rec
epto
r deg
radat
ion,
nam
ely t
he
non-r
ecep
tor
tyro
sine
kin
ase,
A
CK
2,
and
its
subst
rate
, th
e so
rtin
g
nex
in
pro
tein
, S
H3P
X1.
G
iven
th
e
com
ple
xit
y
of
the
endocy
tic
pro
cess
an
d
its
pote
nti
al
import
ance
in
th
erap
euti
cs,
det
erm
inin
g t
he
role
of
AC
K2 a
nd S
H3P
X1 i
n r
ecep
tor
endocy
tosi
s an
d d
egra
dat
ion i
s
of
par
ticu
lar
inte
rest
.
Act
ivate
d C
dc4
2-a
ssoci
ate
d K
inase
-2
Act
ivat
ed
Cdc4
2-a
ssoci
ated
kin
ase-
2
(AC
K2)
is
an
83-k
Da
non-r
ecep
tor
tyro
sine
kin
ase
ori
gin
ally
clo
ned
fro
m a
bovin
e bra
in c
DN
A l
ibra
ry a
s a
targ
et o
f C
dc4
2 [
111].
AC
K2 is
th
e sm
alle
r is
ofo
rm of
AC
K1,
a 120-k
Da
pro
tein
cl
oned
fr
om
a
hu
man
hip
poca
mpal
cD
NA
lib
rary
[112].
A
CK
pro
tein
s ar
e ac
tivat
ed b
y C
dc4
2 i
n i
ts G
TP
-
bound s
tate
, an
d r
ecen
t fi
ndin
gs
confi
rm i
mport
ant
role
s fo
r both
iso
form
s in
sig
nal
tran
sduct
ion p
athw
ays.
T
he
pri
mar
y s
equen
ce of
AC
K2 i
ndic
ates
the
pre
sen
ce o
f
mult
iple
sig
nal
ing d
om
ains,
incl
udin
g a
tyro
sine k
inas
e d
om
ain,
an S
H3 d
om
ain,
a
CR
IB
(Cdc4
2/R
ac
inte
ract
ive-
bin
din
g)
moti
f,
two
pro
line-
rich
dom
ains,
an
d
a
clat
hri
n-b
indin
g d
om
ain [
111,
113]
(Fig
ure
1.6
).
A B
LA
ST
sea
rch o
f th
e N
atio
nal
Cen
ter
of
Bio
tech
nolo
gy I
nfo
rmat
ion d
atab
ase
wit
h t
he
AC
K2 s
equen
ce i
den
tifi
es t
he
27
regio
n
bet
wee
n
resi
dues
13
2
and
378
as
the
tyro
sine
kin
ase
dom
ain,
shar
ing
sim
ilar
itie
s w
ith a
num
ber
of
tyro
sine
kin
ases
, in
cludin
g T
nk1,
the
EG
F r
ecep
tor,
Hck
,
FA
K,
and P
YK
-2 [
114].
A
CK
2 t
yro
sine
phosp
hory
lati
on i
s st
imula
ted b
y a
num
ber
of
extr
acel
lula
r li
gan
ds
incl
udin
g e
pid
erm
al g
row
th f
acto
r (E
GF
), b
radykin
in,
and c
ell
adhes
ion m
ole
cule
s, d
emonst
rati
ng i
ts r
ole
in m
ult
iple
sig
nal
ing p
athw
ays
incl
udin
g
those
in
itia
ted
by
rece
pto
r ty
rosi
ne
kin
ases
an
d
those
in
itia
ted
by
hep
tahel
ical
/G
pro
tein
-couple
d r
ecep
tors
[111].
AC
K2 w
as
init
iall
y i
den
tifi
ed a
s a
hig
hly
spec
ific
tar
get
of
Cdc4
2,
bin
din
g
only
to t
he
acti
vat
ed f
orm
s of
the
G p
rote
in [
111].
A
CK
2 i
s a
spec
ific
Cdc4
2 e
ffec
tor
as i
t does
not
bin
d t
o t
he
act
ivat
ed f
orm
s of
Rac
, R
hoA
, or
Ras
. T
he
bin
din
g b
etw
een
AC
K2 a
nd C
dc4
2 o
ccurs
thro
ugh t
he
CR
IB m
oti
f of
AC
K2 (
resi
dues
454-4
77)
and a
regio
n b
etw
een s
wit
ch I
and s
wit
ch I
I of
the
G p
rote
in (
resi
dues
36-6
8),
des
ignat
ed t
he
AC
K-b
indin
g (
AB
) dom
ain [
115,
116].
B
indin
g t
o C
dc4
2 i
s not
suff
icie
nt
to a
ctiv
ate
AC
K2 i
n vit
ro,
how
ever
, co
-expre
ssio
n o
f A
CK
2 w
ith w
ild
-type
Cdc4
2 o
r C
dc4
2-
Q61L
en
han
ces
AC
K2
auto
phosp
hory
lati
on
in
CO
S-7
ce
lls.
Thes
e re
sult
s,
in
com
bin
atio
n w
ith t
he
inhib
itio
n o
f A
CK
2 a
uto
pho
sphory
lati
on w
ith o
ver
expre
ssio
n o
f
the
dom
inan
t-neg
ativ
e m
uta
nt,
Cdc4
2-D
57Y
[111],
confi
rm t
hat
the
tyro
sine
kin
ase
acti
vit
y o
f A
CK
2 i
s st
imula
ted i
n c
ells
by a
ctiv
ated
form
s of
Cdc4
2.
Cel
l ad
hes
ion h
as a
lso b
een
show
n t
o a
ctiv
ate
AC
K2 [
117].
T
his
act
ivat
ion
likel
y o
ccurs
via
inte
gri
ns
whic
h t
ransm
it s
ignal
s fr
om
extr
acel
lula
r m
atri
x p
rote
ins
and i
nit
iate
a n
um
ber
of
com
ple
x s
ignal
ing p
athw
ays
resp
onsi
ble
for
cell
moti
lity
,
gro
wth
, an
d
dif
fere
nti
atio
n
[118-1
22].
Furt
her
ev
iden
ce
of
inte
gri
n
involv
emen
t
incl
udes
co-i
mm
unopre
cipit
atio
n o
f A
CK
2 w
ith i
nte
gri
n #
1,
and a
dec
reas
e in
AC
K2
phosp
hory
lati
on u
pon t
reat
men
t w
ith a
n a
nti
-inte
gri
n #
1 a
nti
body a
nd R
GD
pep
tides
,
reag
ents
that
blo
ck t
he
inte
ract
ion b
etw
een f
ibro
nec
tin a
nd i
nte
gri
ns.
A
CK
2
28
Fig
ure
1.6
A
dep
icti
on
of
the
dom
ain
str
uct
ure
s o
f ty
rosi
ne
kin
ase
, A
CK
2 a
nd
its
sub
stra
te,
SH
3P
X1.
29
30
acti
vat
ion,
unli
ke
that
of
the
foca
l ad
hes
ion
kin
ase
(FA
K),
does
not
requir
e ce
ll
spre
adin
g,
as no dif
fere
nce
w
as obse
rved
in
A
CK
2 phosp
hory
lati
on bet
wee
n ce
lls
atta
ched
to
poly
lysi
ne-
an
d
fibro
nec
tin-c
oat
ed
pla
tes
[117].
Inte
rest
ingly
, A
CK
2
acti
vat
ion h
as b
een s
ugges
ted t
o d
ecr
ease
the
rate
of
cell
spre
adin
g o
f H
eLa
cell
s onto
fibro
nec
tin [
123].
T
hes
e ef
fect
s ap
pea
r to
be
mod
ula
ted b
y C
rkII
, p130C
as,
Rac
1 a
nd
Rap
1,
wher
e over
expre
ssio
n o
f C
rkII
, P
130C
as a
nd R
ac1 r
ever
se t
he
effe
cts
of
AC
K2
on c
ell
spre
adin
g,
and o
ver
expre
ssio
n o
f a
dom
inan
t-neg
ativ
e fo
rm o
f R
ap1 r
esto
res
the
abil
ity o
f A
CK
2 t
o s
low
cel
l sp
read
ing.
Giv
en t
he
inver
se r
elat
ionsh
ip b
etw
een
cell
spre
adin
g a
nd
cel
l m
oti
lity
, th
e in
crea
sed
moti
lity
exhib
ited
by A
CK
2-e
xpre
ssin
g
cell
s w
as c
onfi
rmed
[123].
Chan
ges
in c
ell
shap
e, c
ell
gro
wth
, an
d t
he f
orm
atio
n o
f fo
cal
com
ple
xes
hav
e
bee
n
attr
ibute
d
to
the
expre
ssio
n
of
AC
K2
in
induci
ble
ce
ll
lines
[1
24].
T
he
morp
holo
gy c
han
ges
rel
ated
to t
he e
xpre
ssio
n o
f A
CK
2 i
n N
IH3T
3 c
ells
incl
ude
a
signif
ican
t deg
ree
of
bra
nch
ing,
wit
h s
om
e of
the
bra
nch
es e
xte
ndin
g t
o a
len
gth
of
200 µ
m,
acco
mpan
ied b
y l
ong s
pik
es e
xte
ndin
g f
rom
the
cell
body.
Alo
ng w
ith
this
morp
holo
gic
al c
han
ge,
induci
ble
expre
ssio
n o
f A
CK
2 i
nhib
ited
cel
l gro
wth
in s
ever
al
cell
li
nes
. T
hes
e ch
anges
in
m
orp
holo
gy
and
cell
pro
life
rati
on
appea
r to
be
indep
enden
t of
AC
K2
kin
ase
act
ivit
y
as
the
induci
ble
ex
pre
ssio
n
of
kin
ase
-dea
d
AC
K2-K
158R
sh
ow
s si
mil
ar
bra
nch
ing
and
inhib
itio
n
of
gro
wth
, th
ough
less
pro
nounce
d w
hen
com
par
ed t
o w
ild-t
ype
AC
K2.
The
kin
ase
acti
vit
y o
f A
CK
2 d
oes,
how
ever
, ap
pea
r to
infl
uen
ce t
he
acti
n c
yto
skel
eton,
resu
ltin
g i
n t
he
dis
ass
embly
of
stre
ss f
iber
s an
d f
oca
l ad
hes
ion
com
ple
xes,
agai
n a
ssoci
atin
g A
CK
2 w
ith c
ell
moti
lity
and c
han
ges
in c
ell
morp
holo
gy.
Of
thes
e obse
rvat
ions,
per
hap
s th
e in
hib
itio
n o
f ce
ll
gro
wth
in A
CK
2 e
xpre
ssin
g c
ells
is
the
mo
st i
ntr
iguin
g,
bec
ause
as
desc
ribed
bel
ow
, it
is c
onsi
sten
t w
ith a
role
for
AC
K2 i
n E
GF
rec
epto
r dow
n-r
egula
tion.
31
Furt
her
evid
ence
for
this
role
surf
aced
wit
h t
he
iden
tifi
cati
on o
f cl
athri
n a
s a
bin
din
g p
artn
er f
or
AC
K2.
Cla
thri
n w
as i
den
tifi
ed i
n G
ST
pull
-dow
n e
xper
imen
ts
usi
ng
GS
T
fusi
on
pro
tein
s th
at
conta
ined
th
e tw
o
carb
oxyl-
term
inal
pro
line-
rich
dom
ains
of
AC
K2 [
113].
C
lath
rin w
as f
ound t
o i
nte
ract
wit
h t
he
over
lappin
g r
egio
ns
bet
wee
n t
he
two c
onst
ruct
s an
d a
seq
uen
ce
alig
nm
ent
confi
rmed
that
AC
K2 c
onta
ined
a co
nse
rved
cla
thri
n-b
indin
g m
oti
f, L
IDF
, sh
ared
by a
ll e
ndocy
tic
adap
tor
pro
tein
s.
Pre
dic
tably
, over
expre
ssio
n of
AC
K2 blo
cks
tran
sfer
rin en
docy
tosi
s by co
mpet
ing
wit
h t
he
endocy
tic
adap
tor
AP
-2,
for
clat
hri
n b
indin
g.
Inte
rest
ingly
, C
dc4
2 n
egat
ivel
y
regula
tes
the
bin
din
g o
f A
CK
2 t
o c
lath
rin a
nd c
on
sequen
tly r
ever
ses
the i
nhib
itio
n o
f
tran
sfer
rin r
ecep
tor
endocy
tosi
s by A
CK
2.
The
iden
tifi
cati
on o
f th
e so
rtin
g n
exin
, S
H3P
X1/s
ort
ing n
exin
9 (
SN
X9),
as
a
bin
din
g p
artn
er f
or
AC
K2 e
xpan
ded
the
role
of
the
non-r
ecep
tor
tyro
sine
kin
ase
to
incl
ude
rece
pto
r so
rtin
g a
nd
deg
radat
ion [
8].
T
he
AC
K2-S
H3P
X1 i
nte
ract
ion o
ccurs
bet
wee
n t
he
pro
line-
rich
dom
ain (
PR
D1)
of
AC
K2 a
nd t
he
SH
3 d
om
ain o
f S
H3P
X1.
SH
3P
X1 i
s not
only
an A
CK
2-b
indin
g p
artn
er,
but
also
a s
ubst
rate
, under
goin
g E
GF
-
dep
enden
t phosp
hory
lati
on
m
edia
ted via
th
e C
dc4
2-p
rom
ote
d ac
tivat
ion of
AC
K2,
wher
e th
e phosp
hory
lati
on
of
SH
3P
X1 is
ab
rogat
ed by th
e over
expre
ssio
n of
the
kin
ase-
def
icie
nt
muta
nt,
AC
K2-K
158R
.
In a
ddit
ion,
AC
K2,
SH
3P
X1,
and c
lath
rin
hav
e bee
n
show
n
to
form
a
com
ple
x
in
cell
s w
her
e th
e A
CK
2-d
epen
den
t
phosp
hory
lati
on o
f S
H3P
X1 r
esult
s in
EG
F r
ecep
tor
deg
radat
ion.
Thes
e fi
ndin
gs
are
of
par
ticu
lar
inte
rest
as
man
y c
ance
rs a
re l
inked
to t
he
over
expre
ssio
n o
f th
e E
GF
rece
pto
r or
EG
F re
cepto
r fa
mil
y m
ember
s an
d re
cepto
r dow
n-r
egula
tion is
a
key
mec
han
ism
in c
ontr
oll
ing r
ecep
tor
level
s at
the
cell
surf
ace.
32
SH
3P
X1
S
H3P
X1,
also
know
n a
s so
rtin
g n
exin
9 (
SN
X9),
was
iden
tifi
ed a
s a
bin
din
g
par
tner
for
AC
K2 i
n m
amm
alia
n c
ells
thro
ugh a
ser
ies
of
GS
T p
ull
-dow
n a
ssay
s [8
].
The
77
-kD
a S
H3P
X1 i
s a
mem
ber
of
the
sort
ing n
exin
fam
ily,
a gro
win
g f
amil
y o
f
cyto
soli
c an
d
mem
bra
ne-
asso
ciat
ed
pro
tein
s in
volv
ed
in
var
ious
asp
ects
of
endocy
tosi
s an
d s
ort
ing o
f ce
ll s
urf
ace
rece
pto
rs.
The
hal
lmar
k o
f th
is f
amil
y i
s th
e
pre
sence
of
the
PX
dom
ain,
a se
quen
ce o
f ap
pro
xim
atel
y 1
00-1
30 a
min
o a
cids
that
has
rece
ntl
y
bee
n
show
n
to
bin
d
var
ious
phosp
hat
idyli
nosi
tol
phosp
hat
es
(Ptd
InsP
s),
ther
eby s
pec
ific
ally
tar
get
ing t
hes
e pro
tein
s to
cel
lula
r m
embra
nes
enri
ched
in t
hes
e
phosp
holi
pid
s [1
25,
126].
Sev
eral
m
ember
s of
the
sort
ing
nex
in
fam
ily
conta
in
addit
ional
sig
nal
ing d
om
ains,
contr
ibuti
ng t
o t
he
loca
liza
tion o
f th
ese
pro
tein
s an
d
thei
r ab
ilit
y t
o f
orm
lar
ge
com
ple
xes
wit
hin
the
cell
. C
urr
entl
y,
twen
ty-f
ive
hu
man
sort
ing n
exin
s hav
e bee
n i
den
tifi
ed a
nd s
tudie
s ar
e u
nder
way
to d
eter
min
e th
e ro
les
of
thes
e pro
tein
s in
the r
egula
tion o
f m
embra
ne
traf
fick
ing.
Mem
ber
s of
the
sort
ing
nex
in f
amil
y c
onta
in l
ittl
e pri
mar
y s
equen
ce h
om
olo
gy,
wit
h t
he
exce
pti
on o
f th
e P
X
dom
ain.
This
dom
ain w
as o
rigin
ally
iden
tifi
ed a
s a
conse
rved
moti
f in
the p
40
ph
ox a
nd
p47
ph
ox
subunit
s of
the
neu
trophil
N
AD
PH
oxid
ase
(phox)
super
oxid
e-gen
erat
ing
com
ple
x [
127].
P
X d
om
ains
hav
e li
mit
ed s
equen
ce h
om
olo
gy a
side
from
a p
roli
ne-
rich
m
oti
f,
PxxP
, en
abli
ng
the
pro
tein
to
in
tera
ct
wit
h
SH
3-d
om
ain
conta
inin
g
pro
tein
s.
Upst
ream
of
the
pro
line-
rich
dom
ain i
s a
phosp
holi
pid
-inte
ract
ion m
oti
f,
whic
h c
an i
nte
ract
wit
h a
wid
e ra
nge
of
phophosp
holi
pid
s.
In a
ddit
ion t
o t
he
sort
ing
nex
ins,
PX
dom
ains
are
also
found i
n c
ell
signal
ing p
rote
ins
such
as
pho
spholi
pas
e D
(PL
D),
phosp
hat
idyli
nosi
tol
3-k
inas
e (P
I3K
), a
s w
ell
as y
east
pro
tein
s in
volv
ed i
n b
ud
emer
gen
ce a
nd
cel
l pola
rity
, B
em1 a
nd B
em3 [
128
, 129].
T
he
stru
cture
of
the
PX
dom
ain o
f p40
ph
ox w
as r
ecen
tly s
olv
ed a
nd h
as b
een s
how
n t
o a
dopt
a novel
fold
that
33
consi
sts
of
thre
e !
-sheet
s pack
ed a
gai
nst
a h
elic
al s
ubdom
ain,
mad
e up o
f fo
ur
alpha
hel
ices
("
2, "
3, "
4 a
nd "
4’)
, a
31
0 h
elix
and a
type
II p
oly
pro
line
hel
ix (
PP
II)
[130].
The
sort
ing nex
in fa
mil
y has
bee
n div
ided
in
to th
ree
sub
-cla
sses
base
d on
conse
rved
dom
ain s
truct
ure
. S
NX
1-2
, S
NX
4-8
, S
NX
15,
and S
NX
16 c
om
pri
se t
he
firs
t cl
ass.
They
al
l hav
e lo
ng
carb
oxyl-
term
inal
ex
tensi
ons
conta
inin
g
1-3
char
acte
rist
ic c
oil
ed-c
oil
mo
tifs
all
ow
ing
for
hom
o-
or
het
ero-o
ligom
eriz
atio
n w
ith
oth
er s
ort
ing n
exin
s, o
r pro
tein
-pro
tein
inte
ract
ions
wit
h n
on
-SN
X p
rote
ins
[131].
T
he
seco
nd su
b-c
lass
, m
ade u
p o
f S
NX
3,
SN
X10,
SN
X11,
SN
X12,
and S
NX
22
-24,
is
char
acte
rize
d
by
the
abse
nce
of
addit
ional
si
gn
alin
g
dom
ains
asid
e fr
om
th
e P
X
dom
ain (
ie.
SH
2,
SH
3,
pro
line-
rich
dom
ains)
. T
he
final
sub-c
lass
of
sort
ing n
exin
s is
mad
e up
of
the
rem
ainin
g
fam
ily
mem
ber
s co
nta
inin
g
var
ious
pro
tein
-pro
tein
inte
ract
ion m
oti
fs su
ch as
S
H3 dom
ains
[132,
133],
m
embra
ne-
targ
etin
g dom
ains
incl
udin
g h
ydro
phobic
seq
uen
ces
[134, 135],
and G
pro
tein
reg
ula
tory
seq
uen
ces
[136,
137].
SH
3P
X1 f
alls
into
this
las
t su
b-c
lass
of
sort
ing n
exin
s.
It c
onta
ins
an a
min
o-
term
inal
SH
3 d
om
ain,
whic
h h
as b
een i
mpli
cate
d i
n i
nte
ract
ions
wit
h A
CK
2 a
nd t
he
Wis
kott
-Ald
rich
sy
ndro
me
pro
tein
(W
AS
P),
th
e la
tter
b
ein
g
involv
ed
in
acti
n
poly
mer
izat
ion a
nd c
yto
skel
etal
org
aniz
atio
n [
138],
and m
ore
rec
entl
y,
the
endocy
tic
pro
tein
, dynam
in-2
[139].
S
H3P
X1 h
as a
lso
bee
n s
how
n t
o b
ind
to c
lath
rin a
nd
AP
-2,
thus
linkin
g t
he
sort
ing n
exin
to c
lath
rin
-med
iate
d e
ndocy
tosi
s an
d c
ell
traf
fick
ing
[139].
T
he
bin
din
g o
f S
H3P
X1 t
o c
lath
rin a
nd A
CK
2 w
as f
irst
dem
onst
rate
d i
n C
OS
-
7 c
ells
[8].
In
this
syst
em,
the
SH
3P
X1-c
lath
rin i
nte
ract
ion a
ppea
rs t
o b
e m
edia
ted
thro
ugh A
CK
2 a
s th
e cl
athri
n-b
indin
g d
efec
tive
muta
nt,
AC
K2
-LI2
A,
and t
he
SH
3
dom
ain-d
efec
tive
mu
tant,
A
CK
2-2
W2A
, dim
inis
hed
S
H3P
X1
bin
din
g
[8].
More
rece
ntl
y,
SH
3P
X1 d
elet
ion m
uta
nts
hav
e bee
n s
how
n t
o b
ind t
o c
lath
rin a
nd t
he
AP
-2
34
"-a
ppen
dag
e in
vit
ro [
139].
T
his
bin
din
g o
ccurs
thro
ugh t
he
regio
n b
etw
een t
he
SH
3
and P
X d
om
ains
of
SH
3P
X1 [
139].
H
ow
ever
, th
is d
irec
t bin
din
g i
nte
ract
ion i
s gre
atly
dim
inis
hed
when
full
-len
gth
SH
3P
X1 i
s use
d a
s th
e bai
t.
Thes
e ob
serv
atio
ns
sugg
est
that
the
bin
din
g s
ites
for
clat
hri
n a
nd A
P-2
may
be
obst
ruct
ed i
n f
ull
-len
gth
SH
3P
X1,
only
to b
e ex
pose
d i
n t
he d
elet
ion m
uta
nts
, per
haps
acco
unti
ng
for
the
dif
fere
nce
s in
bin
din
g b
etw
een i
n v
itro
and i
n v
ivo s
yst
ems.
Dow
nst
ream
of
the
SH
3
dom
ain
in S
H3P
X1
lies
a
Phox
hom
olo
gy (P
X)
dom
ain.
PX
dom
ains
are
hig
hly
conse
rved
in t
he
sort
ing n
exin
fam
ily a
nd a
re t
hought
to m
edia
te t
he
bin
din
g o
f phosp
hoin
osi
tide
seco
nd m
esse
nger
s, c
onse
quen
tly t
arget
ing
thes
e pro
tein
s to
th
e pla
sma
mem
bra
ne.
A
seri
es
of
lipid
-bin
din
g
exper
imen
ts
det
erm
ined
S
H3P
X1
to
be
pro
mis
cuous
in
the
bin
din
g
of
var
ious
phosp
hat
idyli
nosi
tides
[1
39].
SH
3P
X1
has
been
sh
ow
n
to
bin
d
PE
, P
S,
PI3
P,
PI(
3,4
)P2,
PI(
4,5
)P2,
and P
I(3,4
,5)P
3.
More
over
, th
e P
X d
om
ain o
f S
H3P
X1 s
eem
s
to r
equir
e at
lea
st o
ne
expose
d p
hosp
horo
us
gro
up a
s th
ere
was
no o
bse
rved
bin
din
g o
f
PI
[139].
F
inal
ly,
the
last
tw
enty
-eig
ht
amin
o a
cids
are
pre
dic
ted to
hav
e a
hig
h
pro
bab
ilit
y fo
r a
coil
ed-c
oil
fo
rmat
ion,
whic
h is
im
pli
cate
d in
fo
rmin
g fu
nct
ional
mult
imer
ic
com
ple
xes
to
th
is
C-t
erm
inal
re
gio
n.
Rec
ent
studie
s su
gges
t th
at
the
dim
eriz
atio
n o
f S
H3P
X1 m
ay b
e c
riti
cal
for
ph
osp
hory
lati
on o
f th
e so
rtin
g n
exin
,
show
n b
y a
loss
of
AC
K2
-bin
din
g a
ctiv
ity a
nd p
hosp
hory
lati
on o
f tr
unca
tion m
uta
nts
of
as l
ittl
e as
13 a
min
o a
cids
[140].
T
he
Dro
sophil
a
ort
holo
gue
of
SH
3P
X1,
DS
H3P
X1,
is
a 63-k
Da
pro
tein
involv
ed i
n a
xon g
uid
ance
[141].
T
his
pro
tein
was
ori
gin
ally
iden
tifi
ed a
s th
e sm
alle
st
mem
ber
of
a pro
tein
com
ple
x p
uri
fied
fro
m S
2 i
nse
ct c
ells
by t
he S
H2 d
om
ain o
f th
e
adap
tor
pro
tein
, D
ock
. D
ock
con
sist
s of
thre
e ta
ndem
SH
3 d
om
ains
foll
ow
ed b
y a
n
SH
2 d
om
ain a
nd w
as o
rigin
ally
iden
tifi
ed a
s an
ess
enti
al p
rote
in i
n t
he g
uid
ance
of
photo
rece
pto
r ax
ons
in
thir
d
inst
ar
larv
a [1
42].
DS
H3P
X1
has
bee
n
show
n
to
35
asso
ciat
e w
ith
the
Dro
sophil
a
ort
holo
gue
of
the
Dow
ns
Syndro
me
cell
ad
hes
ion
mole
cule
(D
scam
) re
cepto
r, a
s w
ell
as t
he
Dro
sophil
a o
rtholo
gue
of
WA
SP
and t
he
clat
hri
n-c
oat
adap
tor
pro
tein
, A
P-5
0 [
141].
A
ddit
ional
stu
die
s id
enti
fied
DS
H3P
X1 a
s
the
subst
rate
for
Dro
sophil
a A
CK
(D
Ack
) an
d d
efin
ed a
maj
or
site
of
phosp
hory
lati
on
as Y
56 [
143].
T
his
phosp
ho
ryla
tion e
ven
t ap
pea
rs t
o m
edia
te t
he
bin
din
g b
etw
een
com
pet
ing p
artn
ers
WA
SP
and D
ock
in D
roso
phil
a a
xonal
guid
ance
syst
ems.
W
hil
e th
e pre
cise
role
of
SH
3P
X1 p
ho
sphory
lati
on r
emai
ns
to b
e e
stab
lish
ed,
var
ious
lines
of
evid
ence
poin
t to
a s
ort
ing f
unct
ion i
nvolv
ing t
he
EG
F r
ecep
tor
and
rela
ted f
amil
y m
ember
s.
Sort
ing n
exin
1 (
SN
X1)
was
the
firs
t m
amm
alia
n s
ort
ing
nex
in t
o b
e ch
arac
teri
zed [
144].
It
was
iso
late
d i
n a
yea
st-t
wo-h
ybri
d s
cree
n u
sing t
he
kin
ase
dom
ain a
nd t
he
lyso
som
al t
arget
ing s
equen
ce (
tyro
sine
or
di-
leuci
ne
moti
fs)
of
the
EG
F r
ecep
tor
as b
ait.
T
he
port
ion o
f S
NX
1 t
hat
was
iso
late
d i
n t
his
scr
een,
the
carb
oxyl-
term
inal
co
iled
-coil
se
quen
ces,
in
tera
cted
sp
ecif
ical
ly w
ith th
e ly
soso
mal
targ
etin
g m
oti
f, T
yr-
Leu
-Val
-Ile
. I
mport
antl
y,
over
expre
ssio
n o
f fu
ll-l
ength
SN
X1 i
n
CV
1 (
Afr
ican
gre
en m
onkey
kid
ney
) ce
lls
mar
ked
ly d
ow
n-r
egula
ted E
GF
rec
epto
rs.
Oth
er
sort
ing
nex
ins,
in
cludin
g
SN
X2
and
SN
X4,
hav
e bee
n
show
n
to
co-
imm
unopre
cipit
ate
wit
h
rece
pto
rs
that
bin
d
EG
F,
insu
lin,
pla
tele
t-der
ived
gro
wth
fact
or
(PD
GF
) an
d t
he
long f
orm
of
the
lepti
n r
ece
pto
r [1
45],
whil
e S
NX
6 h
as b
een
show
n t
o a
ssoci
ate
wit
h t
he
tran
sform
ing g
row
th f
acto
r !! (
TG
F-!
) re
cepto
r [1
46].
Rec
ent
work
su
gges
ts th
at S
H3P
X1 pla
ys
a ro
le in
m
edia
ting th
e ex
pre
ssio
n an
d
acti
vit
y o
f th
e in
suli
n r
ecep
tor
[147].
S
ubce
llula
r fr
acti
onat
ion s
tudie
s, c
onfi
rmed
by
imm
unofl
uore
scen
ce
exper
imen
ts,
show
th
at
insu
lin
trea
tmen
t st
imula
tes
the
movem
ent
of
SH
3P
X1 fr
om
th
e cy
toso
l to
th
e m
embra
ne.
In
ad
dit
ion,
SH
3P
X1
asso
ciat
es
wit
h t
he
insu
lin r
ecep
tor
in v
itro
and o
ver
expre
ssio
n l
eads
to a
red
uct
ion i
n
cell
surf
ace
insu
lin r
ecep
tors
. S
imil
arly
, re
cent
dat
a fr
om
our
labora
tory
sugges
t th
at
the
AC
K2-c
atal
yze
d p
hosp
hory
lati
on o
f S
H3P
X1 s
tim
ula
tes
EG
F r
ecep
tor
deg
radat
ion
36
in
CO
S-7
ce
lls
[8].
The
phosp
hory
lati
on
of
SH
3P
X1
appea
rs
to
be
crit
ical
in
dec
reas
ing E
GF
re
cepto
r le
vel
s as
th
e kin
ase
-defi
cien
t m
uta
nt,
A
CK
2-K
158R
, has
litt
le
effe
ct.
M
ore
re
cent
work
has
im
pli
cate
d
SH
3P
X1
in
the
endocy
tosi
s of
tran
sfer
rin
rece
pto
r.
A
lth
ou
gh
full
-len
gth
S
H3P
X1
has
no
det
ecta
ble
ef
fect
, th
e
carb
oxyl-
term
inal
tr
unca
tion
muta
nts
, #
C141
and #
C412,
aboli
sh
the
upta
ke
of
tran
sfer
rin
in
HeL
a ce
lls
[139].
Q
uan
tify
ing
tran
sfer
rin
upta
ke
thro
ugh
imm
unofl
uore
scen
ce a
nal
ysi
s of
K562
cel
ls c
onfi
rmed
this
res
ult
[139].
T
he
obse
rved
effe
cts
of
SH
3P
X1 o
n r
ecep
tor
endocy
tosi
s hav
e bee
n a
ttri
bute
d t
o t
he
inte
ract
ion
bet
wee
n th
e S
H3 an
d pro
line-
rich
dom
ains
of
SH
3P
X1 an
d dynam
in,
resp
ecti
vel
y
[148].
R
econst
ituti
on a
ssay
s det
aili
ng t
he
liposo
me
bin
din
g o
f dynam
in a
nd S
H3P
X1
show
that
thes
e tw
o p
rote
ins
hav
e a
pre
fere
nce
for
phosp
hat
idyli
nosi
tols
. I
n t
hes
e
exper
imen
ts,
dynam
in is
un
able
to
bin
d phosp
hat
idyli
nosi
tols
in
ce
lls
dep
lete
d of
endogen
ous
SH
3P
X1,
how
ever
bin
din
g i
s re
store
d u
pon t
he a
ddit
ion o
f re
com
bin
ant
SH
3P
X1.
Support
ing e
vid
ence
show
s dep
leti
on o
f S
H3P
X1 i
n H
eLa
cell
s by R
NA
inte
rfer
ence
(R
NA
i) r
esult
s in
mis
loca
liza
tion o
f dynam
in-2
(D
yn2)
from
the
pla
sma
mem
bra
ne.
A
ddit
ional
ly,
SH
3P
X1 has
bee
n sh
ow
n to
st
imula
te dynam
in’s
bas
al
GT
Pas
e ac
tivit
y b
y s
even
fold
[149].
G
iven
thes
e new
rep
ort
s, w
e ar
e in
tere
sted
in
dev
elopin
g
a sy
stem
in
w
hic
h
we
can
furt
her
as
sess
th
e ro
le
of
SH
3P
X1
phosp
hory
lati
on
in
med
iati
ng
the
com
ple
x
pro
ces
ses
of
rece
pto
r en
docy
tosi
s an
d
deg
radat
ion.
Over
vie
w o
f d
isse
rtati
on
stu
die
s
Init
iall
y,
the
obje
ctiv
es o
f th
is d
isse
rtat
ion i
ncl
uded
iden
tify
ing t
he
tyro
sine
phosp
hory
lati
on s
ites
on S
H3P
X1,
dev
elopin
g i
n v
itro
kin
ase
assa
ys
thro
ugh w
hic
h
smal
l m
ole
cule
in
hib
itors
of
AC
K-c
atal
yze
d
SH
3P
X1
phosp
hory
lati
on
could
b
e
37
iden
tifi
ed,
and d
evel
opin
g a
syst
em i
n w
hic
h t
hese
dom
inan
t-neg
ativ
e re
agen
ts c
ould
be
use
d i
n s
tudyin
g t
he
effe
cts
of
SH
3P
X1 p
hosp
hory
lati
on o
n r
ecep
tor
pro
cess
ing.
To
do
so,
I des
crib
e th
e sy
nth
esis
an
d
use
of
SH
3P
X1
carb
oxyl-
term
inal
del
etio
n m
uta
nts
an
d ty
rosi
ne-
to-p
hen
yla
lanin
e p
oin
t m
uta
nts
in
ef
fort
s to
id
enti
fy
AC
K2-c
atal
yze
d
pho
sphory
lati
on
site
s in
ch
apte
r tw
o.
In
ad
dit
ion,
I det
ail
the
gen
erat
ion an
d puri
fica
tion of
reco
mbin
ant
pro
tein
s, H
is-A
CK
2 an
d H
is-S
H3P
X1,
from
in
sect
ce
ll an
d bac
teri
al ce
ll ex
pre
ssio
n sy
stem
s, re
spec
tivel
y.
I
incl
ude
the
pre
par
atio
n
of
the
mas
s sp
ectr
om
etry
sa
mple
th
rough
whic
h
tyro
sine
287
was
iden
tifi
ed a
s th
e m
ajor
AC
K2
-cat
alyze
d s
ite
on S
H3P
X1,
and t
he
condit
ions
for
the
in
vitr
o k
inas
e ass
ay u
sed i
n t
he
hig
h-t
hro
ughput
scre
ens
at M
erck
& C
o.
As
a re
sult
of
thes
e sc
reen
s, P
arke-
Dav
is c
om
pound,
PD
158780,
was
show
n t
o i
nhib
it A
CK
2 k
inas
e
acti
vit
y w
ith a
n I
C5
0 o
f ~
80 p
M.
In c
hap
ter
thre
e, I
go o
n t
o d
escr
ibe
the
iden
tifi
cati
on
of
two
pre
vio
usl
y
unre
port
ed
tyro
sine
kin
ases
fo
r S
H3P
X1,
FA
K
and
Src
.
I
char
acte
rize
thei
r bin
din
g i
nte
ract
ions
and m
odes
of
SH
3P
X1 p
hosp
hory
lati
on,
and
show
that
they
var
y c
on
sider
ably
fro
m r
esult
s se
en w
ith A
CK
2.
More
over
, I
des
crib
e
tech
niq
ues
use
d t
o i
den
tify
tyro
sine
resi
du
es 1
77,
239,
269,
294,
and 5
61 f
rom
Src
-
cata
lyze
d S
H3P
X1 p
hosp
hory
lati
on i
n c
ells
.
38
Ref
eren
ces
1.
Ols
on,
M.F
., A
. A
shw
ort
h,
and A
. H
all,
An e
ssenti
al
role
for
Rho,
Rac,
and
Cdc4
2
GT
Pase
s in
ce
ll
cycl
e p
rogre
ssio
n
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54
CH
AP
TE
R T
WO
RE
GU
LA
TIO
N O
F A
CK
2-C
AT
AL
YZ
ED
SH
3P
X1 P
HO
SP
HO
RY
LA
TIO
N
Ab
stra
ct
Act
ivat
ed C
dc4
2-a
sso
ciat
ed k
inas
e-2 (
AC
K2)
is a
non-r
ecep
tor
tyro
sine
kin
ase
that
funct
ions
as a
dow
nst
ream
tar
get
of
the
Rho-f
amil
y G
pro
tein
, C
dc4
2.
This
83
-
kD
a is
ofo
rm o
f A
CK
was
ori
gin
ally
clo
ned
fro
m a
bovin
e bra
in l
ibra
ry a
nd b
ased
on
pri
mar
y s
equen
ce a
nal
ysi
s, i
s co
mpri
sed
of
sever
al
signal
ing d
om
ains,
in
cludin
g S
H3,
pro
line-
rich
, an
d
clat
hri
n-b
indin
g
dom
ains.
N
ot
surp
risi
ngly
, A
CK
2
has
bee
n
impli
cate
d i
n m
edia
ting t
he
acti
vat
ion o
f num
erous
cell
sig
nal
ing p
athw
ays
that
res
ult
in
dif
fere
nt
bio
logic
al
acti
vit
ies.
Of
thes
e,
an
emer
gin
g
role
fo
r A
CK
2
in
the
endocy
tosi
s an
d t
raff
ickin
g o
f gro
wth
fac
tor
rece
pto
rs f
or
deg
radat
ion,
hold
s par
ticu
lar
inte
rest
. T
his
role
for
AC
K2 l
ikel
y i
nvolv
es i
ts a
bil
ity t
o p
hosp
hory
late
the
sort
ing
nex
in,
SH
3P
X1.
Thus
far,
SH
3P
X1 i
s th
e only
know
n c
ellu
lar
phosp
ho-s
ubst
rate
for
AC
K2,
and p
hosp
hory
lati
on o
f S
H3P
X1 h
as b
een l
inked
to t
he
deg
radat
ion o
f E
GF
rece
pto
rs i
n C
OS
-7 c
ells
. I
n o
rder
to f
urt
her
elu
cidat
e th
e m
ole
cula
r m
echan
ism
s
under
lyin
g g
row
th f
acto
r re
cepto
r en
docy
tosi
s an
d d
egra
dat
ion,
and m
ore
pre
cise
ly
def
ine
the
role
of
AC
K2 a
nd S
H3P
X1 i
n t
his
com
ple
x c
ellu
lar
pro
cess
, w
e hav
e se
t
out
to g
ener
ate
reag
ents
, nam
ely,
phosp
hory
lati
on-d
efec
tive
muta
nts
of
SH
3P
X1 a
nd
smal
l m
ole
cule
inhib
itors
agai
nst
the
tyro
sine
kin
ase
acti
vit
y o
f A
CK
2.
To t
his
end,
we
hav
e gen
erat
ed a
pan
el o
f ca
rboxyl-
term
inal
del
etio
n c
onst
ruct
s of
SH
3P
X1 t
o h
elp
def
ine
the
regio
n o
f S
H3P
X1 p
hosp
hory
late
d b
y A
CK
2.
Our
findin
gs
dem
onst
rate
that
AC
K2 l
ose
s it
s ab
ilit
y t
o p
hosp
hory
late
SH
3P
X1
in c
onst
ruct
1-5
11
wher
e th
e C
-
term
inal
84 a
min
o a
cids
hav
e bee
n r
emoved
. M
ass
spect
rom
etry
anal
ysi
s of
AC
K2-
cata
lyze
d
pho
sphory
lati
on
of
SH
3P
X1
revea
ls
tyro
sine
287
as
the
maj
or
site
of
phosp
hory
lati
on.
T
hes
e dat
a,
toget
her
w
ith
oth
er
report
s,
argue
for
a re
gula
tory
55
mec
han
ism
that
involv
es t
he
dim
eriz
atio
n o
f S
H3P
X1 t
hro
ugh t
he
C-t
erm
inal
BA
R
dom
ain.
In a
ddit
ion,
we
hav
e i
den
tifi
ed a
com
pound,
PD
158780,
whic
h a
cts
as a
pote
nt
inhib
itor
of
AC
K2 k
inas
e ac
tivit
y i
n v
itro
(IC
50 ~
80 p
M).
T
he
gen
erat
ion o
f
phosp
hory
lati
on-d
efec
tive
SH
3P
X1 m
uta
nts
and
the
iden
tifi
cati
on o
f A
CK
2 i
nhib
itors
should
hel
p p
rovid
e a
bet
ter
under
stan
din
g o
f th
e m
echan
ism
s by w
hic
h g
row
th f
acto
r
rece
pto
r le
vel
s in
the
pla
sma
mem
bra
ne
are
tightl
y r
egula
ted.
Intr
od
uct
ion
Reg
ula
tion o
f gro
wth
fac
tor
rece
pto
r ex
pre
ssio
n a
nd k
inas
e a
ctiv
ity l
evel
s is
esse
nti
al fo
r th
e pro
pag
atio
n of
sever
al
signal
tr
ansd
uct
ion pat
hw
ays,
par
ticu
larl
y
those
that
med
iate
mit
ogen
ic r
esp
onse
s in
cel
ls.
Norm
al c
ell
gro
wth
is
achie
ved
by
mai
nta
inin
g t
he
pro
per
bal
ance
bet
wee
n g
row
th f
acto
r re
cepto
r ac
tivat
ion,
signal
ing,
endocy
tosi
s, d
egra
dat
ion,
and r
ecycl
ing.
As
a r
esult
, th
e over
expre
ssio
n o
f gro
wth
fact
or
rece
pto
rs o
r m
uta
tions
that
lea
d t
o i
ncr
ease
s in
thei
r ty
rosi
ne
kin
ase
act
ivit
y
dis
rupt
this
del
icat
e bal
ance
, an
d i
s su
ffic
ient
to c
ause
mal
ignan
t tr
ansf
orm
atio
n.
Of
the
var
ious
know
n
gro
wth
fa
ctor
rece
pto
rs,
the
epid
erm
al
gro
wth
fa
ctor
(EG
F)
rece
pto
r an
d
oth
er
Erb
B
fam
ily
mem
ber
s ar
e th
e bes
t st
udie
d
and
most
hig
hly
char
acte
rize
d.
O
ver
expre
ssio
n of
the
Erb
B fa
mil
y of
rece
pto
rs ap
pea
rs to
pla
y a
causa
tive
role
in
v
ario
us
form
s of
cance
r. A
w
ell-
docu
men
ted ex
ample
in
volv
es
Erb
B-2
/HE
R2/N
eu,
as t
he
over
expre
ssio
n o
f th
is E
rbB
fam
ily m
ember
has
bee
n f
ound
in 2
5-3
0%
of
all
hum
an b
reas
t ca
nce
r ca
ses
and
appea
rs t
o b
e pre
dic
tive
of
a poor
pro
gnosi
s fo
r th
is d
isea
se [
1].
C
urr
entl
y,
the
use
of
monocl
onal
anti
bodie
s to
blo
ck
the
Erb
B-2
/HE
R2/N
eu r
ecep
tor,
pai
red w
ith t
radit
ional
chem
oth
erap
y,
has
bee
n s
how
n
to b
e ef
fica
cious
in t
he
trea
tmen
t of
bre
ast
cancer
pat
ients
. C
on
sequen
tly,
ther
e is
much
in
tere
st in
el
uci
dat
ing
th
e m
echan
ism
under
lyin
g th
is im
munoth
erap
y.
It
is
curr
entl
y b
elie
ved
that
Her
cepti
nT
M n
ot
only
blo
cks
the
form
atio
n o
f het
erodim
ers
56
bet
wee
n E
rbB
-2 a
nd E
GF
rec
epto
rs,
a st
ep t
hat
is
esse
nti
al f
or
rece
pto
r ac
tivit
y,
but
also
incr
ease
s th
e ra
te o
f E
rbB
-2 i
nte
rnal
izat
ion a
nd s
ubse
quen
t deg
radat
ion o
f th
e
rece
pto
r, t
hus
resu
ltin
g i
n t
he
reduct
ion o
f tu
mor
gro
wth
[2].
H
erce
pti
nT
M s
tudie
s hav
e
pro
vid
ed s
ubst
anti
al i
nsi
ght
into
the
abil
ity o
f re
cepto
r en
docy
tosi
s an
d d
egra
dat
ion t
o
counte
r th
e ab
erra
nt
gro
wth
of
tran
sform
ed c
ells
.
As
a re
sult
, m
uch
focu
s has
bee
n o
n n
ot
only
ch
arac
teri
zing t
he
mec
han
ism
s
beh
ind r
ecep
tor
endocy
tosi
s an
d d
egra
dat
ion b
ut
also
in i
den
tify
ing a
nd c
har
acte
rizi
ng
key
pro
tein
s in
volv
ed i
n t
hes
e ev
ents
.
Rec
entl
y,
two n
ew p
arti
cipan
ts h
ave
bee
n
impli
cate
d in
E
GF
re
cepto
r deg
radat
ion,
nam
ely th
e non-r
ecep
tor
tyro
sine
kin
ase,
AC
K2,
and i
ts p
hosp
ho-s
ubst
rate
, th
e so
rtin
g n
exin
pro
tein
, S
H3P
X1.
Pre
vio
usl
y,
AC
K2 h
as
bee
n a
ssoci
ated
wit
h s
uch
cel
lula
r pro
cess
es
as i
nte
gri
n s
ignal
ing [
3],
cel
l
gro
wth
re
gula
tion
[4
], an
d ax
on guid
ance
sy
stem
s in
D
roso
phil
a [5
]. A
CK
2 an
d
SH
3P
X1 h
ave
since
bee
n l
inked
to e
ndocy
tosi
s th
rough t
hei
r ab
ilit
y t
o a
ssoci
ate
wit
h
var
ious
pro
tein
s in
volv
ed i
n t
his
pro
cess
, an
d a
lter
cel
l su
rfac
e re
cepto
r num
ber
s w
hen
over
expre
ssed
. S
pec
ific
ally
, A
CK
2 a
nd S
H3P
X1 h
ave
bee
n s
how
n t
o f
orm
com
ple
xes
wit
h e
ndocy
tic
pro
tein
s, i
ncl
udin
g c
lath
rin,
dynam
in,
and A
P-2
[6-9
].
Not
only
do
thes
e pro
tein
s as
soci
ate
wit
h k
ey e
ndocy
tic
par
tner
s, b
ut
over
expre
ssio
n o
f A
CK
2,
eith
er w
ith w
ild-t
ype
SH
3P
X1 o
r var
ious
SH
3P
X1 d
elet
ion m
uta
nts
, has
bee
n s
how
n
to a
ffec
t th
e pro
cess
ing a
nd t
raff
ickin
g o
f both
EG
F a
nd t
ransf
erri
n r
ecep
tors
[7,
10].
Spec
ific
ally
, dat
a fr
om
our
labora
tory
su
gges
t th
at
the
AC
K2-m
edia
ted
phosp
hory
lati
on o
f S
H3P
X1 s
tim
ula
tes
EG
F r
ecep
tor
deg
radat
ion i
n C
OS
-7 c
ells
[10].
Her
e,
the
phosp
hory
lati
on
of
SH
3P
X1
appea
rs
to
be
crit
ical
in
dec
reas
ing
EG
F
rece
pto
r le
vel
s as
the
kin
ase-
def
icie
nt
muta
nt,
AC
K2-K
158R
, has
lit
tle
effe
ct.
More
rece
nt
work
has
im
pli
cate
d S
H3P
X1 i
n t
he
endocyto
sis
of
tran
sfer
rin r
ecep
tors
, w
her
e
the
carb
oxyl-
term
inal
tru
nca
tion m
uta
nts
, #
C141 a
nd #
C412,
aboli
sh t
he
upta
ke o
f
tran
sfer
rin i
n H
eLa
cell
s [7
].
57
Whil
e th
e pre
cise
ro
le
of
AC
K2
-dep
enden
t p
hosp
hory
lati
on
of
SH
3P
X1
rem
ains
to
be
esta
bli
shed
, var
ious
lines
of
evid
ence
poin
t to
a
sort
ing
funct
ion
involv
ing t
he
EG
F r
ecep
tor
and r
elat
ed f
amil
y m
ember
s.
SH
3P
X1 i
s a
mem
ber
of
the
gro
win
g f
amil
y o
f so
rtin
g n
exin
s, c
yto
soli
c pro
tein
s in
volv
ed i
n t
he
traf
fick
ing a
nd
deg
radat
ion o
f ce
ll s
urf
ace
rece
pto
rs.
Wit
hin
this
fam
ily,
sort
ing n
exin
1 (
SN
X1)
serv
es a
s a
pro
toty
pe
show
n t
o b
ind t
o t
he
cyto
soli
c dom
ain o
f th
e E
GF
rec
epto
r in
yea
st-t
wo-h
ybri
d s
cree
ns
and t
o d
ow
n-r
egula
te E
GF
rec
epto
rs i
n C
V1 (
Afr
ican
gre
en
monkey
kid
ney
) ce
lls
[11].
O
ther
sort
ing n
exin
mem
ber
s hav
e b
een a
ssoci
ated
wit
h
the
pro
cess
ing o
f G
pro
tein
-couple
d r
ecep
tors
, pro
teas
e-ac
tivat
ed r
ecep
tor-
1 (
PA
R1)
[12,
13],
low
-den
sity
lip
opro
tein
rec
epto
r (L
DL
R)
[14],
pla
tele
t-der
ived
gro
wth
fac
tor
(PD
GF
) re
cepto
r [1
5],
and l
epti
n r
ecep
tor
[15].
R
ecen
t w
ork
suggest
s th
at S
H3P
X1
pla
ys
a ro
le
in
med
iati
ng
the
expre
ssio
n
and
ac
tivit
y
of
the
insu
lin
rece
pto
r,
dem
onst
rate
d i
n s
ub
cell
ula
r fr
acti
onat
ion a
nd i
mm
unofl
uore
scen
ce s
tudie
s [1
6].
T
his
,
toget
her
wit
h i
ts e
ffec
ts o
n t
he
traf
fick
ing o
f E
GF
and t
ransf
erri
n r
ecep
tors
, ar
gues
for
a gen
eral
role
for
SH
3P
X1
in p
roce
ssin
g t
ransm
embra
ne
rece
pto
rs.
In o
rder
to d
efin
itiv
ely l
ink t
he
phosp
hory
lati
on o
f S
H3P
X1 t
o g
row
th f
acto
r
rece
pto
r dow
n-r
egula
tion
and
deg
radat
ion,
it
wil
l ult
imat
ely
be
nec
essa
ry
to
man
ipula
te it
s phosp
hory
lati
on st
atus
thro
ugh th
e use
of
phosp
hory
lati
on-d
efec
tive
muta
nts
, an
d i
dea
lly,
smal
l m
ole
cule
inhib
itors
. A
s a
firs
t st
ep t
ow
ard a
chie
vin
g t
hes
e
goal
s, w
e hav
e u
sed a co
mbin
atio
n of
bio
chem
ical
ap
pro
aches
, in
cludin
g del
etio
n
anal
ysi
s, s
ite-
dir
ecte
d m
uta
gen
esi
s, a
nd m
ass
spec
trom
etry
, to
more
full
y c
har
acte
rize
the
inte
ract
ions
bet
wee
n
AC
K2
and
SH
3P
X1,
and
the
site
s of
AC
K2
-cat
alyze
d
phosp
hory
lati
on.
We
hav
e a
lso i
nit
iate
d a
sea
rch f
or
smal
l m
ole
cule
s th
at a
re c
apab
le
of
inhib
itin
g t
hes
e phosp
hory
lati
on e
ven
ts.
58
Mate
rials
an
d M
eth
od
s
Mate
rials
—A
nti
-HA
an
d
anti
-Myc
anti
bodie
s w
ere
from
C
ovan
ce.
A
nti
-
phosp
hoty
rosi
ne
(4G
10)
anti
body w
as f
rom
Upst
ate
Bio
tech
nolo
gy,
Inc.
T
4 D
NA
ligas
e, L
ipofe
ctam
ine
Tra
nsf
ecti
on R
eagen
t, P
rote
in G
agar
ose
bea
ds,
and t
he
Bac
-to-
Bac
Bacu
lovir
us
Expre
ssio
n S
yst
em w
ere
from
Invit
rogen
. T
he
Quik
Chan
ge
Sit
e-
Dir
ecte
d M
uta
gen
esis
kit
was
fro
m S
trat
agen
e an
d t
he
Qia
quic
k G
el E
xtr
acti
on k
it
was
fro
m Q
iagen
. [
32P
]-A
TP
was
fro
m P
erkin
Elm
er.
Con
stru
ctio
n
of
SH
3P
X1
Del
etio
n
Mu
tan
ts—
The
pcD
NA
3-S
H3P
X1
wil
d-t
ype
pla
smid
was
use
d a
s a t
empla
te t
o c
onst
ruct
the c
arboxyl-
term
inal
del
etio
n m
uta
nts
.
Bri
efly
, D
NA
pri
mer
s to
gen
erat
e ea
ch d
elet
ion
const
ruct
(S
H3P
X1
-#C
84, #
C197,
#C
340, #
C395, #
C447)
wer
e en
gin
eere
d w
ith B
amH
1 a
nd E
coR
1 r
estr
icti
on s
ites
,
and t
hen
thes
e pri
mer
s w
ere
use
d i
n a
PC
R r
eact
ion (
PC
R S
pri
nt,
Hybai
d).
T
he
PC
R
pro
duct
s w
ere
ligat
ed in
to th
e pC
R2-T
OP
O vec
tor,
foll
ow
ed by r
estr
icti
on d
iges
ts
wit
h B
amH
1 a
nd E
coR
1.
These
DN
A p
roduct
s w
ere
reso
lved
on a
1%
agar
ose
gel
conta
inin
g 8
0 n
g/m
L e
thid
ium
bro
mid
e, a
nd t
hen
the
inse
rts
wer
e ex
cise
d f
rom
the
gel
and p
uri
fied
wit
h t
he
Qia
quic
k G
el E
xtr
acti
on k
it.
The
resu
ltin
g D
NA
inse
rts
wer
e
then
lig
ated
in
to t
he
HA
-tag
ged
pcD
NA
3 e
xpre
ssio
n v
ecto
r usi
ng T
4 D
NA
lig
ase.
Con
stru
ctio
n o
f S
H3P
X1 P
oin
t M
uta
nts
—S
H3P
X1 t
yro
sine-
to-p
hen
yla
lanin
e poin
t
muta
nts
wer
e al
so g
ener
ated
in
a p
cDN
A3-S
H3P
X1 w
ild
-type
bac
kgro
und.
Conse
rved
muta
nts
of
Dro
sophil
a a
nd h
um
an o
rtholo
gs:
Y9F
, Y
56F
, Y
287F
, Y
496F
, Y
546F
,
Y578F
, as
w
ell
as m
ult
iple
m
uta
nts
: Y
546F
/Y578F
, Y
546F
/Y561F
/Y563F
/Y578F
,
and
Y546F
/Y561F
/Y563
F/Y
570F
/Y578F
w
ere
gen
erat
ed
by
PC
R
usi
ng
the
Quik
Chan
ge
Sit
e-D
irec
ted M
uta
gen
esis
kit
fro
m S
trat
agen
e.
SH
3P
X1 w
ild-t
ype
and p
oin
t m
uta
nts
wer
e su
bcl
oned
into
the
V5-p
cDN
A 3
.1
vec
tor
usi
ng t
he
Dir
ecti
onal
TO
PO
Expre
ssio
n kit
fro
m I
nvit
rogen
. P
rim
ers
wer
e
des
igned
bas
ed
on
the
man
ufa
cture
r’s
inst
ruct
ions
and
SH
3P
X1
wil
d-t
ype
and
59
tyro
sine-
to-p
hen
yla
lanin
e m
uta
nt
DN
A i
nse
rts
were
gen
erat
ed b
y P
CR
usi
ng t
he H
A-
tagged
pcD
NA
3-S
H3P
X1 co
nst
ruct
s as
bac
kgro
und.
The
blu
nt-
end P
CR
pro
duct
s
wer
e re
solv
ed o
n a
1%
agar
ose
gel
conta
inin
g 8
0 n
g/m
L e
thid
ium
bro
mid
e, a
nd t
hen
the
DN
A
inse
rts
wer
e ex
cise
d
from
th
e gel
an
d
puri
fied
w
ith
the
Qia
quic
k
Gel
Extr
acti
on k
it.
The
resu
ltin
g D
NA
inse
rts
wer
e th
en l
igat
ed i
nto
the
pcD
NA
3.1
/V5-
His
-TO
PO
pla
smid
.
Cel
l C
ult
ure
, T
ran
sfec
tion
, an
d P
rep
ara
tion
of
Lysa
tes—
CO
S-7
an
d H
EK
293
cell
s w
ere c
ult
ure
d i
n D
ulb
ecco
’s m
odif
ied E
agle
’s m
ediu
m (
DM
EM
) co
nta
inin
g 1
0%
feta
l bo
vin
e se
rum
(F
BS
).
The
cel
l li
nes
wer
e m
ainta
ined
in 5
% C
O2 a
t 37°C
. T
o
expre
ss t
he
var
ious
form
s of
AC
K2,
SH
3P
X1,
and d
ynam
in-2
in c
ells
, M
yc-
, H
A-,
or
V5-t
agged
co
nst
ruct
s en
codin
g
the
pro
tein
s w
ere
tran
sfec
ted
into
ce
lls
usi
ng
Lip
ofe
ctam
ine
Tra
nsf
ecti
on
Rea
gen
t acc
ord
ing
to
the
man
ufa
cture
r’s
inst
ruct
ions
(Invit
rogen
).
Tw
enty
-four
ho
urs
foll
ow
ing t
he
tran
sfec
tion,
the
cell
s w
ere
rinse
d w
ith
phosp
hat
e-buff
ered
sal
ine
(PB
S)
and l
yse
d w
ith c
old
mam
mal
ian c
ell
lysi
s buff
er (
10
mM
Tri
s (p
H 7
.4),
5 m
M M
gC
l 2,
150 m
M N
aC
l, 1
% T
rito
n X
-100,
1 m
M s
odiu
m
ort
hovan
adat
e,
5
mM
bet
a-gly
cero
l phosp
hat
e,
10 µ
g/m
L
apro
tinin
, 10 µ
g/m
L
leupep
tin)
foll
ow
ed
by
cell
sc
rapin
g.
T
he
cell
ly
sate
s w
ere
then
cl
eare
d
by
centr
ifugat
ion a
t 16,1
00 r
pm
in a
mic
rofu
ge
for
12 m
inute
s.
Pro
tein
conce
ntr
atio
ns
wer
e det
erm
ined
by d
iluti
ng t
he
lysa
tes
1:2
50 w
ith 1
x B
radfo
rd r
eagen
t an
d m
easu
ring
the
abso
rban
ce b
y v
isib
le l
amp o
n t
he
spec
trophoto
met
er.
Imm
un
op
reci
pit
ati
on
—C
ell
lysa
tes
wer
e in
cubat
ed w
ith M
yc,
HA
or
V5 a
nti
bodie
s
for
2-2
0 h
ours
, w
ith r
ota
tion a
t 4°C
. P
rote
in G
agar
ose
bea
ds
wer
e ad
ded
and r
ota
ted
for
an a
ddit
ional
hour.
T
he
bea
ds
wer
e th
en p
reci
pit
ated
in a
mic
rofu
ge
and w
ashed
3-
4 t
imes
wit
h c
old
mam
mal
ian
cel
l ly
sis
buff
er.
The
bea
ds
wer
e re
susp
ended
in 5
x
SD
S l
oad
ing b
uff
er a
nd t
he
asso
ciat
ed p
rote
ins
were
res
olv
ed b
y S
DS
-PA
GE
. T
he
gel
was
then
tra
nsf
erre
d t
o a
PV
DF
mem
bra
ne
and s
ubje
cted
to W
este
rn b
lott
ing a
nal
ysi
s.
60
Inse
ct
Cel
l E
xp
ress
ion
—S
f21
cell
s w
ere
main
tain
ed
in
Gra
ces
Inse
ct
Med
ia
(Invit
rogen
) su
pple
men
ted w
ith 1
0%
fet
al b
ovin
e se
rum
. V
iruse
s en
codin
g H
is-t
agged
and unta
gged
ver
sions
of
AC
K2,
AC
K2-K
158R
, an
d S
H3P
X1 w
ere
pre
par
ed fo
r
inse
ct ce
ll ex
pre
ssio
n usi
ng th
e In
vit
rogen
B
ac-t
o-B
ac B
aculo
vir
us
Expre
ssio
n kit
.
Sf2
1
cell
s w
ere
infe
cted
w
ith
the
vir
use
s an
d
expre
ssio
n
of
each
co
nst
ruct
w
as
det
erm
ined
by W
este
rn b
lott
ing a
nal
ysi
s fo
llow
ing 2
4-,
48-,
and 7
2-h
our
infe
ctio
n
per
iods.
A
CK
2 k
inas
e ac
tivit
y w
as
assa
yed
by c
o-i
nfe
ctin
g S
f21 c
ells
wit
h A
CK
2 a
nd
SH
3P
X1 vir
use
s, re
solv
ing ce
ll ly
sate
s by S
DS
-PA
GE
, tr
ansf
erri
ng to
P
VD
F,
and
imm
unoblo
ttin
g f
or
tyro
sine-
phosp
hory
late
d S
H3P
X1.
Rec
om
bin
an
t P
rote
in E
xp
ress
ion
an
d P
uri
fica
tion
—E
xpre
ssio
n of
His
-SH
3P
X1
was
car
ried
out
in t
he
BL
21 E
. co
li s
trai
n.
Wil
d-t
ype
SH
3P
X1 w
as c
loned
into
the
pE
T28A
vec
tor
for
reco
mbin
ant
expre
ssio
n.
O
ver
nig
ht
cult
ure
s fr
om
co
lonie
s of
BL
21 c
ells
(N
ovag
en)
tran
sform
ed w
ith t
he
expre
ssio
n v
ecto
r w
ere
gro
wn a
t 37°C
.
Thes
e w
ere
use
d t
o i
nocu
late
one-
lite
r cu
lture
s of
super
bro
th,
an e
nri
ched
bac
teri
al
gro
wth
med
ia.
Bac
teri
al c
ult
ure
s w
ere
gro
wn t
o a
n O
D6
00 o
f 0.8
at
37°C
and i
nduce
d
wit
h
200 µ
M
IPT
G
over
nig
ht
at
room
te
mpera
ture
.
Cel
ls
wer
e har
ves
ted
by
centr
ifugat
ion at
4000 rp
m fo
r 10 m
inute
s an
d fr
oze
n at
"
80°C
. A
ll su
bse
quen
t
puri
fica
tion s
teps
wer
e c
arri
ed o
ut
at 4
°C.
Bac
teri
al p
elle
ts w
ere
resu
spen
ded
in
Ni2
+
bin
din
g
buff
er
(20
mM
T
ris
(pH
7.9
),
500
mM
N
aCl,
20
mM
im
idaz
ole
, 10%
gly
cero
l) su
pple
men
ted w
ith pro
teas
e in
hib
itors
(1 m
M P
MS
F,
10 µ
g/m
L ea
ch of
apro
tinin
and l
eupep
tin,
10
µM
ben
zam
idin
e) a
nd l
yse
d b
y t
hre
e pas
sag
es t
hro
ugh a
Fre
nch
Pre
ssu
re C
ell.
T
he
extr
acts
wer
e th
en s
onic
ated
for
5 m
inute
s an
d t
he
lysa
tes
wer
e cl
arif
ied by ult
race
ntr
ifugat
ion at
40,0
00 rp
m fo
r 45 m
inute
s. T
he
clar
ifie
d
lysa
tes
wer
e in
cubat
ed f
or
30
min
ute
s w
ith N
i2+ b
ead
s (A
mer
sham
) at
4°C
. T
he
bea
ds
wer
e th
en w
ash
ed w
ith
bin
din
g b
uff
er a
nd t
he p
rote
in r
ecover
ed w
ith e
luti
on b
uff
er
(20 m
M T
ris
(pH
7.9
), 5
00 m
M N
aCl,
200 m
M i
mid
azole
, 10%
gly
cero
l).
Sam
ple
s
61
from
th
e pro
tein
puri
fica
tion
w
ere
then
re
solv
ed by S
DS
-PA
GE
an
d st
ained
w
ith
Coom
assi
e blu
e.
In
V
itro K
inase
Rea
ctio
ns—
Pre
lim
inar
y k
inas
e re
acti
ons
wer
e per
form
ed w
ith H
is-
AC
K2 a
nd H
is-S
H3P
X1 i
n a
kin
ase
reac
tion b
uff
er (
10 m
M H
epes
(pH
7.4
), 5
mM
MgC
l 2,
150 m
M N
aCl)
in t
he p
rese
nce
of
1 m
M N
a 3V
O4 a
nd 1
mM
AT
P f
or
0.5
-18
hours
at
30°C
. T
he kin
ase re
acti
ons
wer
e quen
ched
w
ith th
e ad
dit
ion of
5x
S
DS
load
ing buff
er.
T
he
reac
tio
n sa
mple
s w
ere
boil
ed,
reso
lved
by S
DS
-PA
GE
, an
d
tran
sfer
red t
o a
PV
DF
mem
bra
ne
wher
e su
bst
rate
phosp
hory
lati
on w
as m
easu
red b
y
imm
unoblo
ttin
g w
ith a
nti
-phosp
hoty
rosi
ne
anti
body.
AC
K2
Hig
h-t
hro
ugh
pu
t S
cree
n—
The
LA
NC
ET
M
tim
e-re
solv
ed
fluore
scen
t
reso
nan
ce e
ner
gy t
ransf
er s
yst
em f
rom
Per
kin
Elm
er w
as i
nit
iall
y u
sed f
or
scre
enin
g
for
smal
l m
ole
cule
inhib
itors
of
AC
K2 a
ctiv
ity.
LA
NC
ET
M i
s base
d o
n t
he
ener
gy
tran
sfer
bet
wee
n
donor
(euro
piu
m
chel
ate)
an
d
acce
pto
r (a
llophyco
cyan
in—
AP
C)
reag
ents
that
are
bro
ught
wit
hin
pro
xim
ity b
y a
spec
ific
bin
din
g e
ven
t.
In t
he
case
of
the
kin
ase
reac
tion,
euro
piu
m i
s co
nju
gat
ed t
o a
n a
nti
-phosp
hoty
rosi
ne
anti
body a
nd
AP
C i
s co
nju
gat
ed t
o s
trep
tavid
in b
eads.
E
ner
gy t
ransf
er o
ccurs
in t
he
pre
sence
of
a
tyro
sine
phosp
hory
late
d,
bio
tinyla
ted s
ubst
rate
. U
sing t
his
syst
em,
kin
ase
reac
tions
wer
e ca
rrie
d o
ut
wit
h 4
.15 µ
g/m
L A
CK
2,
200 n
M S
H3P
X1,
2 m
M A
TP
in 5
0 m
M
Hep
es,
10 m
M M
gC
l 2,
and 0
.1%
Tri
ton X
-100 f
or
0-2
hours
at
room
tem
per
ature
. T
he
reac
tion
sam
ple
s w
ere
then
quen
ched
w
ith
the
addit
ion
of
AP
C-c
onju
gat
ed
stre
pta
vid
in an
d E
uro
piu
m-c
hel
ated
an
ti-p
hosp
hoty
rosi
ne
anti
body.
S
ample
s w
ere
then
rea
d b
y a
flu
ore
scen
t pla
te r
eader
.
In s
ubse
quen
t sc
reen
s, a
[3
3P
]-A
TP
fil
ter
assa
y w
as u
sed t
o s
impli
fy d
etec
tion.
The
condit
ions
wer
e sl
ightl
y a
lter
ed b
y u
sing 8
.3 µ
g/m
L A
CK
2,
200 n
M S
H3P
X1,
50 µ
M A
TP
, an
d 0
.2 µ
Ci/
wel
l fo
r 0-1
8 h
ours
at
room
tem
per
ature
. F
oll
ow
ing t
he
kin
ase
reac
tions,
pro
tein
s w
ere
pre
cipit
ated
wit
h 3
0%
tri
cholo
roac
etic
aci
d (
TC
A)
and
62
left
on i
ce f
or
1 h
our.
T
he
reac
tion m
ixtu
re w
as
then
tra
nsf
erre
d t
o a
pre
-wet
ted 9
6-
wel
l fi
lter
pla
te,
foll
ow
ed b
y 5
was
hes
wit
h a
sodiu
m p
yro
phosp
hat
e/15%
TC
A w
ash
buff
er.
The
filt
er
was
dri
ed
and
scin
till
atio
n
fluid
w
as
added
, al
low
ing
for
the
mea
sure
men
t of
[33P
]O4
3-
inco
rpora
tion
into
ad
her
ent
pro
tein
s.
L
ater
, 1:3
se
rial
dil
uti
ons
wer
e ca
rrie
d o
ut
wit
h t
yrp
host
in,
AG
1478,
an E
GF
rec
epto
r in
hib
itor,
as
wel
l
as w
ith t
he
Par
ke-
Dav
is p
yri
do-p
yri
mid
ine,
PD
158780,
and t
he
SU
Gen
com
pound,
SU
6656,
in r
epli
cate
s of
four.
Res
ult
s
Much
inte
rest
has
bee
n d
irec
ted a
t dis
sect
ing t
he
mec
han
ism
beh
ind A
CK
2-d
epen
den
t
phosp
hory
lati
on o
f S
H3P
X1 a
nd i
ts e
ffec
ts o
n E
GF
rec
epto
r deg
radat
ion i
n c
ells
. A
s a
firs
t st
ep t
ow
ard g
ener
atin
g r
eagen
ts t
o s
tudy t
his
com
ple
x p
roce
ss,
we
wer
e in
tere
sted
in i
den
tify
ing t
he
tyro
sine
pho
sphory
lati
on s
ite(
s) o
n t
he
sort
ing n
exin
.
Carb
oxyl-
term
inal
Tru
nca
tion
M
uta
nts
of
SH
3P
X1
are
D
efec
tive
for
AC
K2-
dep
end
ent
Ph
osp
hory
lati
on
To d
elin
eate
the
regio
n o
n S
H3P
X1 t
hat
conta
ins
the
phosp
hory
lati
on s
ite(
s)
for
AC
K2,
a se
ries
of
C-t
erm
inal
tru
nca
tion m
uta
nts
was
const
ruct
ed.
Eac
h o
f th
ese
const
ruct
s w
as d
esig
ned
to
co
nta
in t
he
N-t
erm
inal
SH
3 d
om
ain,
as w
e hav
e sh
ow
n t
hat
this
reg
ion i
s ess
enti
al f
or
AC
K2-S
H3P
X1 b
indin
g (
Fig
ure
2.1
).
Phosp
hory
lati
on o
f
the
HA
-epit
ope
tagged
muta
nts
#C
84, #
C197, #
C340, #
C395, #
C447 w
as a
sses
sed
by
co-e
xpre
ssio
n
wit
h
the
Myc-
AC
K2
const
ruct
in
C
OS
-7
cell
s,
foll
ow
ed
by
imm
unopre
cipit
atio
n w
ith a
nti
-HA
anti
body,
SD
S-P
AG
E,
and W
este
rn b
lott
ing w
ith
anti
-phosp
hoty
rosi
ne
anti
body.
Whil
e A
CK
2-c
ataly
zed t
yro
sine
pho
sphory
lati
on w
as
det
ecte
d in
w
ild-t
ype
SH
3P
X1 (F
igure
2.2
A,
lane
3),
it
w
as not
obse
rved
in
th
e
carb
oxyl-
term
inal
tru
nca
tion m
uta
nts
#C
84, #
C197 (
Fig
ure
2.2
A,
lanes
1 a
nd 2
) an
d
63
Fig
ure
2.1
C
arb
oxyl-
term
inal
Del
etio
n M
uta
nts
of
SH
3P
X1.
Car
boxyl-
term
inal
del
etio
n m
uta
nts
of
SH
3P
X1 w
ere
des
igned
so t
hat
they
mai
nta
ined
the
amin
o-t
erm
inal
SH
3 d
om
ain t
hat
has
bee
n s
ho
wn t
o b
e cr
itic
al f
or
bin
din
g A
CK
2.
Muta
nts
#
C84, #
C197, #
C340, #
C395, #
447 w
ere
engin
eere
d
wit
h
Bam
H1
and
Eco
R1 r
estr
icti
on s
ites
, w
ith
the
resu
ltin
g P
CR
pro
duct
s li
gat
ed i
nto
the
pC
R2-T
OP
O
vec
tor.
T
he
ligat
ion p
roduct
s w
ere
then
dig
este
d w
ith B
amH
1 a
nd E
coR
1 r
est
rict
ion
enzy
mes
.
Thes
e D
NA
pro
duct
s w
ere
reso
lved
on
a 1%
ag
arose
gel
co
nta
inin
g
80 n
g/m
L e
thid
ium
bro
mid
e, a
nd t
he
inse
rts
wer
e ex
cise
d f
rom
the
gel
and p
uri
fied
wit
h t
he
Qia
quic
k G
el E
xtr
acti
on k
it.
The
resu
ltin
g D
NA
inse
rts
wer
e th
en l
igat
ed
into
the
HA
-tag
ged
pcD
NA
3 e
xpre
ssio
n v
ecto
r usi
ng T
4 D
NA
lig
ase.
64
65
Fig
ure
2.2
Th
e tr
un
cati
on
m
uta
nt !
C84
is
def
ecti
ve
for
AC
K2-d
epen
den
t
ph
osp
hory
lati
on
.
Myc-
AC
K2
and
HA
-SH
3P
X1
trunca
tion
muta
nts
w
ere
co-
tran
sfec
ted i
n C
OS
-7 c
ells
. T
wen
ty-f
our
to s
eventy
-tw
o h
ours
aft
er t
ransf
ecti
on,
the
cell
s w
ere
lyse
d
and
the
whole
ce
ll
lysa
tes
wer
e re
solv
ed
by
SD
S-P
AG
E
and
tran
sfer
red t
o P
VD
F m
embra
ne.
T
he
sam
ple
s w
ere
then
subje
cted
to
West
ern b
lott
ing
anal
ysi
s w
ith a
nti
-phosp
hoty
rosi
ne
(A)
and a
nti
-HA
anti
bodie
s (B
).
66
67
!C
339 (
dat
a not
show
n).
T
hes
e dat
a in
dic
ate
that
the
del
etio
n o
f th
e la
st 8
4 a
min
o
acid
s fr
om
th
e C
-ter
min
al en
d of
SH
3P
X1 re
sult
s in
a
com
ple
te lo
ss of
AC
K2-
cata
lyze
d t
yro
sine
phosp
hory
lati
on.
Giv
en t
his
res
ult
, w
e th
en s
et o
ut
to m
uta
te e
ach
of
the
tyro
sine
resi
dues
in t
he
C-t
erm
inal
84
amin
o
acid
re
gio
n
of
SH
3P
X1
to
phen
yla
lanin
e.
S
H3P
X1
poin
t
muta
nts
Y546F
, Y
561F
, Y
56
3F
, Y
570F
and Y
578F
wer
e gen
erat
ed u
sing s
ite-
dir
ecte
d
muta
gen
esis
, an
d m
ult
iple
rounds
of
muta
gen
esis
wer
e per
form
ed t
o y
ield
mult
iple
poin
t m
uta
nts
Y546F
/Y578
F a
nd Y
546F
/Y561F
/Y563F
/Y570F
/Y578F
. T
he
resu
ltin
g
muta
nts
w
ere
co-e
xpre
ssed
w
ith A
CK
2 in
C
OS
-7 ce
lls.
F
oll
ow
ing ce
ll ly
sis,
th
e
SH
3P
X1 m
uta
nts
wer
e pre
cipit
ated
fro
m w
hole
-cel
l ly
sate
s w
ith a
nti
-HA
anti
body,
and t
hen
im
munoblo
tted
wit
h a
nti
-phosp
hoty
rosi
ne
anti
body.
Unex
pec
tedly
, A
CK
2-
dep
enden
t phosp
hory
lati
on w
as s
till
obse
rved
wit
h e
ach o
f th
ese m
uta
nts
, in
cludin
g
the
const
ruct
that
conta
ins
subst
ituti
ons
for
each
of
the
C-t
erm
inal
tyro
sine
resi
dues
(SH
3P
X1-F
546/F
561/F
563/F
570/F
578),
co
mpar
ed
to
the
neg
ativ
e co
ntr
ol,
A
CK
2
kin
ase-
def
icie
nt
muta
nt
AC
K2-K
158R
wit
h S
H3P
X1 (
Fig
ure
2.3
, co
mpar
e la
ne
2 w
ith
lanes
3-8
).
Ther
e ap
pea
r to
be
two p
oss
ible
expla
nat
ions
for
this
obse
rvat
ion:
eit
her
AC
K2 i
s unab
le t
o b
ind t
o t
hes
e C
-ter
min
al d
elet
ion m
uta
nts
, th
us
acco
unti
ng f
or
the
loss
of
phosp
hory
lati
on o
f th
e !
C84 c
on
stru
ct,
or
AC
K2 i
s cap
able
of
phosp
hory
lati
ng
mult
iple
tyro
sine
resi
dues
, in
cludin
g t
ho
se t
hat
lie
at
posi
tion
s upst
ream
fro
m t
he
C-
term
inal
tyro
sine
resi
dues
.
To a
ddre
ss t
he
firs
t poss
ibil
ity,
we
car
ried
out
in viv
o
exper
imen
ts t
o a
ssay
the
bin
din
g b
etw
een A
CK
2 a
nd t
he
var
iou
s S
H3P
X1 d
elet
ion
muta
nts
. M
yc-
AC
K2 a
nd t
he
HA
-tag
ged
tru
nca
tion m
uta
nts
wer
e co
-expre
ssed
in
CO
S-7
cel
ls,
foll
ow
ed b
y i
mm
unopre
cipit
atio
n o
f w
hole
cel
l ly
sate
s w
ith a
nti
-Myc
anti
body a
nd i
mm
unoblo
ttin
g w
ith a
nti
-HA
anti
body
. H
ow
ever
, th
ese
exper
imen
ts
gav
e m
ixed
res
ult
s an
d d
id n
ot
per
mit
a d
efin
itiv
e in
terp
reta
tion.
We
then
subcl
oned
AC
K2 i
nto
the
V5-e
pit
ope
tagged
pcD
NA
3.1
vec
tor,
giv
en t
hat
this
pla
smid
appea
rs
68
Fig
ure
2.3
A
CK
2 p
hosp
hory
late
s C
-ter
min
al
tyro
sin
e-to
-ph
enyla
lan
ine
mu
tan
ts.
Myc-
AC
K2 w
as c
o-t
ransf
ecte
d w
ith t
he
indic
ated
HA
-SH
3P
X1 p
oin
t m
uta
nts
in C
OS
-
7 ce
lls.
T
wen
ty-f
our
to se
ven
ty-t
wo hours
af
ter
tran
sfec
tion,
the
SH
3P
X1 m
uta
nts
wer
e im
munopre
cipit
ated
wit
h a
nti
-HA
anti
body.
The
resu
ltin
g p
reci
pit
ated
sam
ple
s
wer
e th
en
reso
lved
by
SD
S-P
AG
E,
tran
ferr
ed
to
PV
DF
m
embra
ne,
an
d
Wes
tern
blo
tted
wit
h a
nti
-phosp
hoty
rosi
ne
and a
nti
-HA
anti
bodie
s.
69
70
to g
ive
rise
to a
more
robust
expre
ssio
n o
f A
CK
2.
The
resu
ltin
g V
5-A
CK
2 c
onst
ruct
was
co
-expre
ssed
in
C
OS
-7
cell
s w
ith
th
e S
H3P
X1
del
etio
n
muta
nts
, w
her
e th
e
AC
K2-S
H3P
X1 i
nte
ract
ions
wer
e as
sess
ed b
y p
reci
pit
atin
g A
CK
2 f
rom
cel
l ly
sate
s
wit
h an
ti-V
5 an
tibody,
and im
munoblo
ttin
g fo
r as
soci
ated
H
A-S
H3P
X1 co
nst
ruct
s
usi
ng a
n a
nti
-HA
anti
body.
As
show
n i
n F
igure
2.4
, under
condit
ions
wher
e fu
ll-
length
HA
-SH
3P
X1 w
as c
lear
ly a
ble
to b
e co
-im
munopre
cipit
ated
wit
h V
5-A
CK
2,
all
of
the
C-t
erm
inal
SH
3P
X1 d
elet
ion m
uta
nts
wer
e in
effe
ctiv
e in
bin
din
g t
o A
CK
2.
Thus,
thes
e fi
ndin
gs
indic
ate
that
the
inab
ilit
y o
f th
e C
-ter
min
al t
runca
tion m
uta
nts
of
SH
3P
X1 t
o b
e phosp
hory
late
d b
y A
CK
2 i
s a
dir
ect
conse
quen
ce
of
thei
r in
abil
ity t
o
bin
d t
o t
he
tyro
sine
kin
ase.
All
Con
serv
ed T
yro
sin
e R
esid
ues
of
SH
3P
X1 A
re S
usc
epti
ble
to A
CK
2-c
ata
lyze
d
Ph
osp
hory
lati
on
Giv
en t
he
resu
lts
des
crib
ed i
n t
he
pre
cedin
g s
ecti
on w
hic
h i
ndic
ated
that
th
e C
-
term
inal
dom
ain o
f S
H3P
X1 d
id n
ot
conta
in t
he
phosp
hory
lati
on s
ite(
s) f
or
AC
K2,
we
then
exam
ined
those
tyro
sine
resi
dues
of
SH
3P
X1 k
now
n t
o b
e co
nse
rved
bet
wee
n
dif
fere
nt
spec
ies.
Speci
fica
lly,
we
gen
erat
ed
tyro
sine-
to-p
hen
yla
lanin
e poin
t
muta
tions
of
conse
rved
res
idues
bet
wee
n h
um
an,
mouse
and Drosophila o
rtholo
gues
.
The
foll
ow
ing S
H3P
X1 m
uta
nts
, Y
9F
, Y
56F
, Y
287F
, Y
496F
, Y
546F
, an
d Y
578F
wer
e gen
erat
ed t
hro
ugh s
ite-
dir
ecte
d m
uta
gen
esis
. T
he
abil
ity o
f th
ese
muta
nts
to b
e
phosp
hory
late
d
in
an
AC
K2-d
epen
den
t m
ann
er
was
ev
alu
ated
upon
thei
r co
-
expre
ssio
n
wit
h
AC
K2
in
CO
S-7
ce
lls.
The
cell
ly
sate
s w
ere
subje
ct
to
imm
unopre
cipit
atio
n w
ith a
nti
-HA
anti
body,
foll
ow
ed b
y i
mm
unoblo
ttin
g w
ith a
nti
-
phosp
hoty
rosi
ne
anti
body.
We
mea
sure
d t
he
AC
K2
-cat
alyze
d p
hosp
hory
lati
on o
f th
e
poin
t m
uta
nts
agai
nst
wil
d-t
ype
SH
3P
X1,
usi
ng c
ells
co-e
xpre
ssin
g k
inas
e-def
ecti
ve
AC
K2 (
AC
K2
-K158R
) w
ith S
H3P
X1 a
s a
neg
ativ
e co
ntr
ol
for
thes
e ex
per
imen
ts.
71
Fig
ure
2.4
AC
K2
is
un
ab
le
to
bin
d
to
SH
3P
X1
del
etio
n
mu
tan
ts.
A s
chem
atic
rep
rese
nta
tion o
f th
e S
H3P
X1 d
elet
ion c
onst
ruct
s (A
).
CO
S-7
cel
ls w
ere
tran
sfec
ted
wit
h
V5-A
CK
2
and
HA
-SH
3P
X1
carb
oxyl-
term
inal
del
etio
n
muta
nts
.
Tw
enty
-four
to s
even
ty-t
wo h
ours
aft
er t
ransf
ecti
on,
AC
K2 w
as i
mm
unopre
cipit
ated
wit
h a
nti
-V5 a
nti
body a
nd t
he s
ample
s w
ere r
esolv
ed b
y S
DS
-PA
GE
. T
he
gel
was
then
tra
nsf
erre
d t
o P
VD
F m
embra
ne
and s
ubje
cted
to W
este
rn b
lott
ing w
ith a
nti
-HA
anti
body (
B).
72
73
Tyro
sine
56
has
bee
n
report
ed
to
be
the
maj
or
phosp
hory
lati
on
site
in
Dro
sophil
a [
5].
H
ow
ever
, w
e fo
und t
hat
eac
h o
f th
e si
ngle
poin
t m
uta
nts
ret
ained
the
abil
ity t
o b
e ty
rosi
ne
phosp
hory
late
d i
n a
n A
CK
2-d
epen
den
t m
anner
(F
igure
2.5
, la
nes
4-1
0).
In
fac
t, t
her
e w
as n
o d
etec
table
dec
reas
e in
phosp
hory
lati
on s
ignal
of
any o
f th
e
poin
t m
uta
nts
com
par
ed t
o w
ild-t
ype.
T
his
sugges
ted t
hat
ther
e m
ay b
e m
ult
iple
sit
es
on S
H3P
X1 f
or
AC
K2-c
atal
yze
d p
hosp
hory
lati
on, as
we
hav
e not
bee
n a
ble
to d
etec
t a
signif
ican
t dec
reas
e in
phosp
hory
lati
on f
or
any o
f th
e su
bst
ituti
ons
thus
far
exam
ined
.
Ther
e ar
e 21 t
yro
sine
amin
o a
cids
in t
he h
um
an o
rtholo
gue
of
SH
3P
X1.
This
,
couple
d
wit
h
the
fact
th
at
ther
e ar
e no re
port
ed
conse
nsu
s se
quen
ces
fo
r A
CK
2
tyro
sine
phosp
hory
lati
on,
contr
ibute
s to
th
e
chal
lenge
of
usi
ng
site
-dir
ecte
d
muta
gen
esis
to i
den
tify
the
maj
or
tyro
sine
phosp
hory
lati
on s
ites
on S
H3P
X1.
Thus,
as
an a
lter
nat
ive
appro
ach,
we
hav
e tu
rned
to m
ass
spec
trom
etry
. T
his
req
uir
es t
hat
we
dev
elop
reco
mbin
ant
sourc
es
of
AC
K2
and
S
H3P
X1
in
ord
er
to
per
form
th
e
phosp
hory
lati
on
assa
ys
and
pho
spho-p
epti
de
map
pin
g
anal
ysi
s in
a
wel
l-def
ined
,
puri
fied
syst
em.
Tyro
sin
e re
sid
ue
287 w
as
Iden
tifi
ed a
s a P
hosp
ho-s
ite
by M
ass
Sp
ectr
om
etry
We
init
iall
y s
et o
ut
to i
den
tify
the
AC
K2-c
atal
yzed
phosp
hory
lati
on s
ites
on
SH
3P
X1,
usi
ng
our
inse
ct c
ell
pre
par
atio
n o
f re
com
bin
ant
AC
K2 a
nd
the
E.
coli
-
expre
ssed
His
-SH
3P
X1.
How
ever
, w
e so
on f
ound t
hat
we
wer
e unab
le t
o g
ener
ate
suff
icie
nt
level
s of
(AC
K2-c
atal
yze
d)
phosp
hory
late
d
SH
3P
X1
from
our
in
vitr
o
assa
ys
to p
erm
it a
def
init
ive
iden
tifi
cati
on o
f th
e phosp
hory
lati
on s
ite(
s).
As
a r
esult
,
we
looked
for
condit
ions
whic
h w
ould
giv
e an
enhan
ced p
hosp
hory
lati
on o
f S
H3P
X1,
hopin
g
that
w
e co
uld
w
ork
bac
kw
ards
tow
ard
iden
tify
ing
the
AC
K2-c
atal
yze
d
phosp
hory
lati
on s
ites
. T
his
led
us
to e
xam
ine
FA
K a
nd S
rc a
s poss
ible
kin
ases
for
SH
3P
X1 g
iven
the
sim
ilar
itie
s bet
wee
n t
hei
r re
spec
tive
kin
ase
dom
ains
and t
hat
of
74
Fig
ure
2.5
T
he
AC
K2-d
epen
den
t p
hosp
hory
lati
on
of
SH
3P
X1 c
on
stru
cts
bea
rin
g
sub
stit
uti
on
s at
the
con
serv
ed t
yro
sin
e re
sid
ues
is
com
para
ble
to
that
of
wil
d-
typ
e S
H3P
X1.
C
OS
-7 ce
lls
wer
e tr
ansf
ecte
d w
ith M
yc-
AC
K2 an
d H
A-S
H3P
X1
tyro
sine-
to-p
hen
yla
lanin
e poin
t m
uta
nts
.
Tw
enty
-four
to
seven
ty-t
wo
hours
af
ter
tran
sfec
tion,
SH
3P
X1 a
nd t
he
indic
ated
poin
t m
uta
nts
wer
e im
munopre
cipit
ated
wit
h
anti
-HA
anti
body a
nd r
esolv
ed b
y S
DS
-PA
GE
. T
he
gel
was
then
tra
nsf
erre
d t
o P
VD
F
mem
bra
ne
and s
ubje
cted
to i
mm
unoblo
ttin
g w
ith a
nti
-phosp
hoty
rosi
ne
anti
body.
75
76
AC
K2.
Spec
ific
ally
, an
im
munopre
cipit
ated
sam
ple
fro
m H
EK
293
cel
ls e
xpre
ssin
g
Src
an
d S
H3P
X1 w
as su
bm
itte
d to
th
e C
orn
ell
Mas
s S
pec
trom
etry
C
ore
F
acil
ity.
Liq
uid
chro
mat
ogra
phy M
S/M
S w
as u
sed t
o i
denti
fy t
yro
sine
resi
dues
Y177,
Y239,
Y269,
Y294, an
d Y
561 a
s S
rc-c
atal
yze
d p
hosp
hory
lati
on s
ites
(F
igure
2.6
).
We
then
tes
ted t
he
abil
ity o
f A
CK
2 t
o p
ho
sphory
late
the
resu
ltin
g t
yro
sine-
to-
phen
yla
lanin
e m
uta
nts
. A
s sh
ow
n i
n F
igure
2.7
, ea
ch o
f th
e p
oin
t m
uta
nts
, in
cludin
g
muta
nt
Y17
7F
/Y239F
/Y269F
/Y294F
/Y561
(Fig
ure
2.7
. la
nes
4-9
),
wer
e
phosp
hory
late
d b
y A
CK
2,
indic
atin
g t
hat
AC
K2
and S
rc p
hosp
hory
late
dis
tinct
sit
es
on S
H3P
X1.
Conse
quen
tly
, w
e use
d s
imil
ar c
ondit
ions
to s
ubm
it a
sam
ple
fro
m H
EK
293 c
ells
tra
nsf
ecte
d w
ith A
CK
2 a
nd S
H3P
X1.
The
cell
s w
ere
lyse
d 2
4 h
ours
aft
er
tran
sfec
tion a
nd V
5-S
H3P
X1 w
as p
reci
pit
ated
fro
m t
he
pre
par
ed l
ysa
tes
(~10 m
g t
ota
l
pro
tein
) w
ith a
nti
-V5 a
nti
bod
y.
The
pre
cipit
ated
sam
ple
was
res
olv
ed b
y S
DS
-PA
GE
,
stai
ned
wit
h C
oom
assi
e, a
nd e
xci
sed (
Fig
ure
2.8
A).
A
liquots
fro
m t
his
sam
ple
wer
e
set
asid
e an
d t
yro
sine
pho
sphory
lati
on w
as c
onfi
rmed
by W
este
rn B
lott
ing (
Fig
ure
2.8
B).
T
his
sam
ple
was
then
sub
mit
ted t
o t
he
Corn
ell
Bio
tech
nolo
gy R
eso
urc
e C
ente
r
Pro
teom
ics
and M
ass
Spec
tro
met
ry C
ore
Fac
ilit
y.
An a
ppro
ach u
sing
mas
s re
acti
on
monit
ori
ng
(MR
M-I
DA
) te
chniq
ues
id
enti
fied
ty
rosi
ne
287
as
a si
te
of
AC
K2-
cata
lyze
d p
hosp
hory
lati
on.
Tyro
sine
287 i
s a
conse
rved
tyro
sine
resi
due
loca
ted i
n t
he
PX
dom
ain
of
SH
3P
X1.
W
e
curr
entl
y
bel
iev
e th
at
ther
e ar
e
addit
ional
A
CK
2-
cata
lyze
d
phosp
hory
lati
on
site
s as
earl
ier
muta
gen
esis
an
aly
ses
indic
ated
th
at
the
Y287F
muta
nt
was
sti
ll p
hosp
hory
late
d b
y A
CK
2.
As
a r
esult
, ad
dit
ional
sam
ple
s ar
e
bei
ng p
repar
ed t
o c
onfi
rm a
nd
pote
nti
ally
iden
tify
new
sit
es.
77
Fig
ure
2.6
S
equ
ence
ali
gn
men
t of
hu
man
, m
ou
se a
nd
Drosophila o
rth
olo
gu
es o
f
SH
3P
X1.
78
------------------+-------------------+-------------------+-------------------+-
10 20 30 40
M A T K A R V M Y D F A A E P G N N E L T V N E G E I I T I T N P D V G G G W L SH3PX1 homo
M A T K A R V M Y D F A A E P G N N E L T V T E G E I I T V T N P N V G G G W L SH3PX1 mouse
M T S Y V R A M Y D F T G E P G S S E L S I A T G D V L S V T R S D V G E G W W SH3PX1 drosophila
------------------+-------------------+-------------------+-------------------+-
50 60 70 80
E G R N I K G E R G L V P T D Y V E I L P S D G K D Q F S C G N S V A D Q A F L SH3PX1 homo
E G K N N K G E Q G L V P T D Y V E I L P N D G K D P F S C G N S V A D Q A F L SH3PX1 mouse
E G K N A R G Q I G L F P A A Y V E V M S A A E A Q K L S A S G A T S V P Q V P SH3PX1 drosophila
------------------+-------------------+-------------------+-------------------+-
90 100 110 120
D S L S A S T A H A S S S A A S N N H Q V G S G N D P W S A W S A S K S G N W E SH3PX1 homo
D S L T A S T A Q T N S S S A N S N N Q V G G G N D P W T A W N A P K P G N W D SH3PX1 mouse
D P F A S P P L P R Y D Q T A D D D - - - - - - - - - - - G - - S - - - - N W D SH3PX1 drosophila
------------------+-------------------+-------------------+-------------------+-
130 140 150 160
S S E G W G A Q P E G A G A Q R N T N T P N N W D T A F G H P Q A Y Q G P A T G SH3PX1 homo
S S D A W G S R T D G T S A Q R N S - S A N N W D T G F G H P Q A Y Q G P A T G SH3PX1 mouse
D E D D W S D D N D T Y S E I G P G - - - - - - - - - - G Q K S A G Q S A R A G SH3PX1 drosophila
------------------+-------------------+-------------------+-------------------+-
170 180 190 200
D D D D W D E D W D G P K S S S - Y F K D S E S A D A G G A Q R G N S R A S S S SH3PX1 homo
D D D E W D E D W D D P K S S S P Y F K D S E P A E A G G I Q R G N S R A G A S SH3PX1 mouse
G L S H S T S D Y D N K H L P I A P N D D T Q S L A S V A G G T G A A G T V K K SH3PX1 drosophila
------------------+-------------------+-------------------+-------------------+-
210 220 230 240
S M K I P L N K F P G F A K P G T E Q Y L L A K Q L A K P K E K I P I I V G D Y SH3PX1 homo
S M K L P L N K F P G F A K P G M E Q Y L L A K Q L A K P K E K I A I I V G D Y SH3PX1 mouse
G M F A K S S D S Y I L G L S S T K E K I P E C E M A Y I T Q - - - - - V E D S SH3PX1 drosophila
------------------+-------------------+-------------------+-------------------+-
250 260 270 280
G P M W V Y P T S T F D C V V A D P R K G S K M Y G L K S Y I E Y Q L T P T N T SH3PX1 homo
G P M W V Y P T S T F D C V V A D P R K G S K M Y G L K S Y I E Y Q L T P T N T SH3PX1 mouse
I Y Q W T Q N H S P Y S V V V A S P K K E S K F K G M K T F I A Y Q L T P S F N SH3PX1 drosophila
------------------+-------------------+-------------------+-------------------+-
290 300 310 320
N R S V N H R Y K H F D W L Y E R L L V K F G S A I P I P S L P D K Q V T G R F SH3PX1 homo
N R S V N H R Y K H F D W L Y E R L L V K F G S A I P I P S L P D K Q V T G R F SH3PX1 mouse
N I S V S R R Y K H F D W L H E R L V D K F - C L I P V P P L P D K Q I S G R Y SH3PX1 drosophila
------------------+-------------------+-------------------+-------------------+-
330 340 350 360
E E E F I K M R M E R L Q A W M T R M C R H P V I S E S E V F Q Q F L N F R D E SH3PX1 homo
E E E F I K M R M E R L Q A W M T R M C R H P V V S E S E V F Q Q F L N F R D E SH3PX1 mouse
E E Q F V E H R R V Q L Q E F V D W V C R H P V I S K C E V W Y H F L T C R D E SH3PX1 drosophila
------------------+-------------------+-------------------+-------------------+-
370 380 390 400
K E W K T G K R K A E R D E L A G V M I F S T M E P E A P D L D L V E I E Q K C SH3PX1 homo
K E W K T G K R K A E K D E L V G V M I F S T M E P E A P D L D L I E I E Q K C SH3PX1 mouse
K I W K S G K R K A E R D P Y M G V N Y C L A I S P P D K N L L H S K V D A Q V SH3PX1 drosophila
------------------+-------------------+-------------------+-------------------+-
410 420 430 440
E A V G K F T K A M D D G V K E L L T V G Q E H W K R C T G P L P K E Y Q K I G SH3PX1 homo
D A V G K F T K A M D D G V K E L L T V G Q E H W K R C T G P L P K E Y Q K I G SH3PX1 mouse
E L G T Q F I H S M D V A V R N L N N I S N D M A K R S M S Q S K K E F Q R I G SH3PX1 drosophila
------------------+-------------------+-------------------+-------------------+-
450 460 470 480
K A L Q S L A T V F S S S G Y Q G - - - - E T D L N D A I T E A G K T Y E E I A SH3PX1 homo
K A L Q S L A A V F S S S G Y Q G - - - - E T D L N D A I T E A G K T Y E E I A SH3PX1 mouse
D G L S D L A K A L A I D E R R A P T R N A V P L S E S V G R I G G I F I G I G SH3PX1 drosophila
------------------+-------------------+-------------------+-------------------+-
490 500 510 520
S L V A E Q P K K D L H F L M E C N H E Y K G F L G C F P D I I G T H K G A I E SH3PX1 homo
S L V A E Q P K K D L H F L M E C N H E Y K G F L G C F P D I I G A H K G A I E SH3PX1 mouse
Q A F G D Q P K H D W I P L S D R L H I Y R G V L N C F P D I F S T H K G A I Q SH3PX1 drosophila
79
Fig
ure
2.6
(C
on
tin
ued
)
------------------+-------------------+-------------------+-------------------+-
530 540 550 560
K V K E S D K L V A T S K I T L Q D K Q N M V K R V S I M S Y A L Q A E M N H F SH3PX1 homo
K V K E S D K L V A T S K I T P Q D K Q T M V K R V G T M S Y A L Q A E M N H F SH3PX1 mouse
K R K D C E K L A G E G R M N N P Q L H D V N R R T D V V S Y T V L A E L T H F SH3PX1 drosophila
------------------+-------------------+-------------------+-------------------+-
570 580 590 600
H S N R I Y D Y N S V I R L Y L E Q Q V Q F Y E T I A E K L R Q A L S R F P V M SH3PX1 homo
H S N R I Y D Y N S V I R L Y L E Q Q V Q F Y E T I A E K L R Q A L S R F P V M SH3PX1 mouse
K S E R D T H L K H T L K N F I A E Q I K F Y Q G V V A R L Q E A S R Q I E SH3PX1 drosophila
Y.....Conserved tyrosine residues between human, mouse and Drosophila
Y.....Src-catalyzed phosphorylation sites identified by mass spectrometry
Y.....ACK2-catalyzed phosphorylation site identified by mass spectrometry
80
Fig
ure
2.7
A
CK
2-c
ata
lyze
d p
hosp
hory
lati
on
sit
es o
n S
H3P
X1 a
re d
isti
nct
fro
m
those
of
Src
. M
yc-
AC
K2 w
as
co-t
ransf
ecte
d w
ith t
he
indic
ated
HA
-SH
3P
X1 p
oin
t
muta
nts
in
C
OS
-7 ce
lls.
T
wen
ty-f
our
to se
ven
ty-t
wo hours
af
ter
tran
sfec
tion,
the
SH
3P
X1 m
uta
nts
w
ere im
munopre
cipit
ated
w
ith
an
ti-H
A an
tibody.
T
he
resu
ltin
g
pre
cipit
ated
sa
mple
s w
ere
then
re
solv
ed
by
SD
S-P
AG
E,
tran
ferr
ed
to
PV
DF
mem
bra
ne,
and W
este
rn b
lott
ed w
ith a
nti
-phosp
hoty
rosi
ne
and a
nti
-HA
anti
bodie
s.
81
82
Fig
ure
2.8
P
rep
ara
tion
of
the
AC
K2-c
ata
lyze
d S
H3P
X1 p
hosp
hory
lati
on
sam
ple
for
mass
sp
ectr
om
etry
an
aly
sis.
M
yc-
AC
K2 a
nd V
5-S
H3P
X1 w
ere
co-e
xpre
ssed
in
HE
K 2
93 c
ells
. V
5-S
H3P
X1 w
as i
mm
unopre
cipit
ated
fro
m t
he
whole
cel
l ly
sate
s
wit
h a
nti
-V5 a
nti
body,
reso
lved
by S
DS
-PA
GE
, an
d s
tain
ed w
ith C
oom
ass
ie (
A).
T
he
ban
d
conta
inin
g
SH
3P
X1
was
ex
cise
d
and
su
bm
itte
d
to
the
Corn
ell
Mass
Spec
trom
etry
Core
Fac
ilit
y.
Ali
quots
fro
m t
his
sam
ple
was
als
o r
esolv
ed b
y S
DS
-
PA
GE
, tr
ansf
erre
d t
o P
VD
F,
and s
ubje
cted
to W
este
rn b
lott
ing a
nal
ysi
s w
ith a
nti
-
phosp
hoty
rosi
ne
and a
nti
-V5 a
nti
bodie
s (B
).
83
84
Rec
om
bin
an
t P
rote
in G
ener
ati
on
In o
rder
to d
evel
op t
he
assa
y t
o s
cree
n f
or
smal
l m
ole
cule
inhib
itors
of
AC
K2
kin
ase
acti
vit
y,
it w
as fi
rst
nec
essa
ry to
ex
pre
ss an
d puri
fy re
com
bin
ant
form
s of
AC
K2 a
nd
SH
3P
X1.
Due t
o t
he s
ize o
f A
CK
2 a
nd S
H3P
X1 (
83 k
Da a
nd 7
7 k
Da,
resp
ecti
vel
y),
we
init
iall
y s
et o
ut
to e
xpre
ss t
hes
e pro
tein
s in
inse
ct c
ells
. V
iruse
s
enco
din
g
His
-tag
ged
as
wel
l as
unta
gged
ver
sions
of
AC
K2
and
SH
3P
X1
wer
e
dev
eloped
for
expre
ssio
n o
f th
ese
pro
tein
s in
Sf2
1 c
ells
usi
ng t
he
Invit
rogen
Bac
-to-
Bac
kit
. W
e fo
und t
hat
SH
3P
X1,
when
expre
ssed
as
a H
is-t
agged
pro
tein
in i
nse
ct
cell
s,
reta
ined
a
basa
l le
vel
of
tyro
sine
pho
sphory
lati
on.
G
iven
re
port
s th
at
Dro
sophil
a S
H3P
X1 i
s pho
sphory
late
d b
y D
roso
phil
a A
CK
2 [
5],
we
thought
that
the
bas
al
phosp
hory
lati
on
of
SH
3P
X1
could
poss
ibly
be
due
to
an
isofo
rm
of
AC
K
expre
ssed
endogen
ou
sly i
n t
he
Sf2
1 c
ells
. C
onse
quen
tly,
we
sought
to e
lim
inat
e th
is
phosp
hory
lati
on
signal
by
dev
elopin
g
a kin
ase
-def
ecti
ve
AC
K2-K
158R
vir
us.
How
ever
, co
-infe
ctin
g S
f21 c
ells
wit
h A
CK
2-K
158R
and S
H3P
X1 d
id n
ot
rever
se t
his
effe
ct.
Sti
ll,
by c
o-e
xpre
ssin
g A
CK
2 k
inas
e to
get
her
wit
h S
H3P
X1 i
n S
f21 c
ells
, w
e
wer
e ab
le t
o o
bse
rve
an i
ncr
ease
in t
he p
hosp
hory
lati
on o
f S
H3P
X1 r
ealt
ive
to t
hat
for
SH
3P
X1 a
lone
(Fig
ure
2.9
B,
com
par
e la
nes
1 a
nd 2
), t
hus
confi
rmin
g t
he
acti
vit
y o
f
AC
K2 f
rom
inse
ct c
ells
.
To t
ry t
o m
inim
ize
any a
mbig
uit
y a
risi
ng f
rom
the
bas
al p
hosp
hory
lati
on o
f
SH
3P
X1,
we
then
expre
ssed
it
as
a G
ST
fusi
on p
rote
in i
n E
. co
li.
Full
-len
gth
SH
3P
X1
was
clo
ned
into
the
pG
EX
-KG
vec
tor
for
reco
mbin
ant
expre
ssio
n.
Expre
ssio
n o
f th
e
GS
T-S
H3P
X1 f
usi
on p
rote
in w
as c
arri
ed o
ut
in t
he
BL
21 E
. co
li s
trai
n,
wher
e one-
lite
r cu
lture
s w
ere
gro
wn t
o a
n O
D6
00 o
f 0.8
in s
uper
bro
th a
nd i
nduce
d w
ith 2
00
µM
IPT
G
over
nig
ht.
C
ells
w
ere
har
ves
ted
by
ce
ntr
ifugat
ion
and
GS
T-S
H3P
X1
was
puri
fied
on glu
tath
ione-
agar
ose
bea
ds
(Sig
ma)
. P
reli
min
ary kin
ase
reac
tions
wer
e
carr
ied o
ut
by i
ncu
bat
ing H
is-A
CK
2 (
puri
fied
fro
m i
nse
ct c
ells
) w
ith G
ST
-SH
3P
X1
85
Fig
ure
2.9
S
H3P
X1 c
an
be
ph
osp
hory
late
d b
y A
CK
2 u
pon
co
-exp
ress
ion
of
thes
e
pro
tein
s in
in
sect
ce
lls.
Vir
use
s en
codin
g
His
-AC
K2
and
His
-SH
3P
X1
wer
e
gen
erat
ed u
sing t
he
Bac
-to-B
ac B
aculo
vir
us
expre
ssio
n s
yst
em.
The
resu
ltin
g v
iruse
s
wer
e use
d t
o i
nfe
ct S
f21 c
ells
for
24
-72 h
ours
pri
or
to c
ell
lysi
s.
The
whole
cel
l
lysa
tes
wer
e re
solv
ed b
y S
DS
-PA
GE
and t
ransf
erre
d t
o P
VD
F m
embra
ne.
H
is-A
CK
2
expre
ssio
n
was
sh
ow
n
by
West
ern
blo
ttin
g
whole
-cel
l ly
sate
s w
ith
anti
-AC
K2
anti
body (
A).
T
he
kin
ase
acti
vit
y o
f re
com
bin
ant
AC
K2 w
as a
ssess
ed b
y c
o-i
nfe
ctin
g
AC
K2 a
nd S
H3P
X1 i
n S
f21 c
ells
. T
wen
ty-f
our
to s
even
ty-t
wo h
ours
aft
er i
nfe
ctio
n
the
cell
s w
ere
lyse
d
and
SH
3P
X1
was
im
munopre
cipit
ated
w
ith
anti
-SH
3P
X1
anti
body,
and s
ample
s w
ere
reso
lved
by S
DS
-PA
GE
. T
he
gel
was
then
tra
nsf
erre
d t
o
PV
DF
mem
bra
ne
and i
mm
unoblo
tted
wit
h a
nti
-phosp
hoty
rosi
ne
anti
body (
B).
86
87
on g
luta
thio
ne-
agar
ose
bea
ds
in k
inas
e re
acti
on b
uff
er (
10 m
M H
epes
(pH
7.4
), 5
mM
MgC
l 2,
150 m
M N
aCl,
1 m
M N
a 3V
O4,
1 m
M A
TP
) fo
r 30 m
inute
s at
30°C
. K
inas
e
reac
tion s
ample
s w
ere
quen
ched
wit
h 5
x S
DS
loadin
g b
uff
er,
and p
hosp
hory
lati
on o
f
GS
T-S
H3P
X1 w
as d
etec
ted b
y i
mm
unoblo
ttin
g w
ith a
nti
-phosp
hoty
rosi
ne
anti
body
(dat
a not
show
n).
A
ltho
ugh th
e phosp
hory
lati
on of
GS
T-S
H3P
X1 co
nfi
rmed
th
e
acti
vit
y o
f th
e re
com
bin
ant
pro
tein
, w
e fo
und t
hat
GS
T-S
H3P
X1 e
xis
ts i
n m
onom
eric
and v
ario
us
oli
gom
eric
com
ple
xes
as
det
erm
ined
by p
uri
fica
tion o
n a
siz
e ex
clusi
on
colu
mn (
wit
h t
he
oli
gom
eric
com
ple
xes
elu
ting a
s a
bro
ad p
eak j
ust
aft
er t
he
void
volu
me)
.
To d
eter
min
e if
the
oli
gom
eric
com
ple
xes
of
SH
3P
X1 r
efle
cted
an i
nher
ent
abil
ity o
f th
e pro
tein
to u
nder
go s
elf-
asso
ciat
ion (
e.g.
dim
eriz
atio
n o
f th
e C
-ter
min
al
BA
R m
oti
f),
we
expre
ssed
S
H3P
X1
as
a
His
-tag
ged
pro
tein
. S
H3P
X1 w
as
firs
t
cloned
into
the
pE
T28A
vec
tor
and t
ransf
orm
ed i
nto
BL
21 c
ells
. O
ne-
lite
r cu
lture
s
wer
e gro
wn t
o a
n O
D6
00 o
f 0.8
in s
up
er b
roth
at
37°C
and i
nduce
d w
ith 2
00 µ
M I
PT
G
over
nig
ht
at r
oom
tem
per
ature
. C
ells
wer
e har
ves
ted a
nd t
he
pro
tein
was
puri
fied
on a
nic
kel
colu
mn.
Furt
her
puri
fica
tion b
y s
ize
excl
usi
on r
evea
led t
he
monom
eric
nat
ure
of
the
His
-tag
ged
S
H3P
X1.
A
s sh
ow
n in
F
igu
re 2.1
0,
His
-SH
3P
X1 re
com
bin
ant
pro
tein
is
able
to b
e phosp
hory
late
d b
y H
is-A
CK
2 f
rom
inse
ct c
ells
(F
igure
2.1
0B
,
lane
1).
G
iven
the
pro
pen
sity
of
GS
T-S
H3P
X1 t
o a
ggre
gat
e, w
e dec
ided
that
the
His
-
tagged
SH
3P
X1 w
ould
be
use
d i
n f
utu
re s
tudie
s.
Park
e-D
avis
co
mp
ou
nd
P
D158780 w
as
iden
tifi
ed in
a H
igh
-th
rou
gh
pu
t A
CK
2
Kin
ase
Scr
een
As
an a
lter
nat
ive
mea
ns
for
exam
inin
g t
he
cell
ula
r an
d b
iolo
gic
al c
onse
quen
ces
of
the
AC
K2-c
atal
yze
d p
hosp
hory
lati
on o
f S
H3P
X1,
we
wer
e in
tere
sted
in i
den
tify
ing s
mal
l
mole
cule
inhib
itors
of
AC
K2 k
inas
e ac
tivit
y. F
or
this
purp
ose
, w
e use
d t
he
hig
h-
88
Fig
ure
2.1
0
E
xp
ress
ion
an
d
act
ivit
y
of
reco
mb
inan
t S
H3P
X1
in
E.
coli
.
Over
nig
ht
cult
ure
s fr
om
co
lonie
s of
BL
21
cell
s (N
ovag
en)
tran
sform
ed
wit
h
the
expre
ssio
n v
ecto
r w
ere
gro
wn a
t 37°C
. T
hes
e w
ere
use
d t
o i
nocu
late
one-
lite
r cu
lture
s
of
super
bro
th.
Bac
teri
al c
ult
ure
s w
ere
gro
wn t
o a
n O
D6
00 o
f 0.8
at
37°C
and i
nduce
d
wit
h
200 µ
M
IPT
G
over
nig
ht
at
room
te
mpera
ture
.
Cel
ls
wer
e har
ves
ted
by
centr
ifugat
ion at
4000 rp
m fo
r 10 m
inute
s an
d fr
oze
n at
!
80°C
. A
ll su
bse
quen
t
puri
fica
tion s
teps
wer
e c
arri
ed o
ut
at 4
°C.
Bac
teri
al p
elle
ts w
ere
resu
spen
ded
in
Ni2
+
bin
din
g
buff
er
(20
mM
T
ris
(pH
7.9
),
500
mM
N
aCl,
20
mM
im
idaz
ole
, 10%
gly
cero
l) su
pple
men
ted w
ith pro
teas
e in
hib
itors
(1 m
M P
MS
F,
10 µ
g/m
L ea
ch of
apro
tinin
and l
eupep
tin,
10
µM
ben
zam
idin
e) a
nd l
yse
d b
y t
hre
e pas
sag
es t
hro
ugh a
Fre
nch
Pre
ssu
re C
ell.
T
he
extr
acts
wer
e th
en s
onic
ated
for
5 m
inute
s an
d t
he
lysa
tes
wer
e cl
arif
ied by ult
race
ntr
ifugat
ion at
40,0
00 rp
m fo
r 45 m
inute
s. T
he
clar
ifie
d
lysa
tes
wer
e in
cubat
ed f
or
30
min
ute
s w
ith N
i2+ b
ead
s (A
mer
sham
) at
4°C
. T
he
bea
ds
wer
e th
en w
ash
ed w
ith
bin
din
g b
uff
er a
nd t
he p
rote
in r
ecover
ed w
ith e
luti
on b
uff
er
(20 m
M T
ris
(pH
7.9
), 5
00 m
M N
aCl,
200 m
M i
mid
azole
, 10%
gly
cero
l).
Sam
ple
s
from
th
e pro
tein
puri
fica
tion
w
ere
then
re
solv
ed by S
DS
-PA
GE
an
d st
ained
w
ith
Coom
assi
e b
lue
(A).
T
he
abil
ity o
f S
H3P
X1 t
o b
e phosp
hory
late
d b
y A
CK
2 w
as
exam
ined
by i
ncu
bat
ing
the
puri
fied
pro
tein
wit
h r
ecom
bin
ant
His
-AC
K2 f
rom
inse
ct
cell
s fo
r 30 m
inute
s at
30°C
in H
EP
ES
/MgC
l 2/N
aCl 2
buff
er.
The
kin
ase
react
ion w
as
quen
ched
w
ith 5x S
DS
-PA
GE
sa
mple
buff
er an
d sa
mple
s w
ere
reso
lved
by S
DS
-
PA
GE
and l
ater
subje
cted
to W
este
rn b
lott
ing w
ith a
nti
-phosp
hoty
rosi
ne
anti
body (
B).
89
90
thro
ughput
smal
l m
ole
cule
sc
reen
ing
faci
lity
in
th
e A
uto
mat
ed
Bio
tech
nolo
gy
dep
artm
ent
of
Mer
ck
&
Co.
In
itia
lly,
we
set
out
to
use
th
e w
ell-
char
acte
rize
d
det
ecti
on s
yst
em,
LA
NC
ET
M,
a ti
me-
reso
lved
flu
ore
scen
t re
son
ance
ener
gy t
ransf
er
syst
em,
from
Per
kin
Elm
er.
LA
NC
ET
M i
s bas
ed o
n t
he
ener
gy t
ransf
er b
etw
een d
onor
(euro
piu
m c
hel
ate)
and a
ccep
tor
(all
ophyco
cyan
in—
AP
C)
reag
ents
that
are
bro
ught
wit
hin
pro
xim
ity by a
spec
ific
bin
din
g ev
ent.
I
n th
e ca
se of
the
kin
ase
reac
tion,
euro
piu
m i
s co
nju
gat
ed t
o a
n a
nti
-phosp
hoty
rosi
ne
anti
body a
nd A
PC
is
conju
gat
ed t
o
stre
pta
vid
in
bea
ds.
Ener
gy
tran
sfer
occ
urs
in
th
e pre
sence
of
a ty
rosi
ne
phosp
hory
late
d,
bio
tinyla
ted s
ubst
rate
(F
igure
2.1
1).
Opti
mal
kin
ase
reac
tion co
ndit
ions
wer
e det
erm
ined
by per
form
ing a
tim
e
cours
e fo
r th
e A
CK
2-c
atal
yze
d p
hosp
hory
lati
on r
eact
ion.
The
gre
ates
t ch
alle
nge
was
gen
erat
ing
enough
SH
3P
X1
phosp
hory
lati
on
to
be
read
out
by
th
e fl
uore
scen
t-
det
ecti
on
syst
em.
W
e ad
dre
ssed
th
is
lim
itat
ion
by
alte
ring
reac
tion
condit
ions
incl
udin
g,
incr
easi
ng t
he
kin
ase c
once
ntr
atio
n a
nd
chan
gin
g t
he
buff
er s
alin
ity,
whil
e
at th
e sa
me ti
me,
per
form
ing posi
tive
contr
ols
w
ith re
com
bin
ant
insu
lin re
cepto
r.
Thro
ugh t
hes
e ad
just
men
ts,
we
ob
serv
ed t
hat
the o
pti
mal
AC
K2 k
inas
e ac
tivit
y w
as
obse
rved
in 5
0 m
M H
epes
/10 m
M M
gC
l 2/0
.1%
Tri
ton X
-100 b
uff
er,
wher
e so
diu
m
chlo
ride
was
fo
und
to
sever
ely
inhib
it
sub
stra
te
phosp
hory
lati
on.
U
nder
th
ese
condit
ions,
the
reac
tion r
each
ed c
om
ple
tion
aft
er 5
-10 m
inute
s at
room
tem
per
ature
(Fig
ure
2.1
2).
T
o i
nhib
it p
ote
nti
al n
onsp
ecif
ic i
nte
ract
ions
bet
wee
n A
CK
2 a
nd t
he
reac
tion p
late
, w
e in
cluded
det
ergen
ts a
nd B
SA
in s
ubse
quen
t en
zym
e ti
trat
ions.
When
the
addit
ion
of
det
ergen
t an
d
BS
A
did
not
alte
r our
resu
lts,
w
e per
form
ed
a
stau
rosp
ori
ne
dose
re
sponse
to
co
nfi
rm th
at th
e si
gnal
w
as due
to kin
ase
acti
vit
y
(Fig
ure
2.1
3).
91
Fig
ure
2.1
1
L
AN
CE
TM
ti
me-
reso
lved
fl
uore
scen
t re
son
an
ce
ener
gy
tr
an
sfer
syst
em.
E
xci
tati
on
of
a E
uro
piu
m-c
onju
gat
ed
anti
-phosp
hoty
rosi
ne
anti
body
in
pro
xim
ity
of
the
fluore
scen
t pro
be
allo
phyco
cyan
in
(AP
C),
re
sult
s in
an
en
ergy
tran
sfer
that
can
be
read
at
the
emis
sion w
avel
ength
of
AP
C b
y a
flu
ore
scen
t pla
te
read
er.
92
93
Fig
ure
2.1
2 A
CK
2 a
ctiv
ity i
n t
he
LA
NC
ET
M d
etec
tion
syst
em.
Rec
om
bin
ant
His
-AC
K2 (
4.1
5 µ
g/m
L)
and H
is-S
H3P
X1 (
200 n
M)
wer
e ad
ded
to a
kin
ase
reac
tion m
ixtu
re c
onta
inin
g 2
mM
AT
P,
50 m
M H
EP
ES
, 10 m
M M
gC
l 2,
and
0.1
% T
rito
n X
-100.
The
kin
ase
reac
tion w
as a
llow
ed t
o p
roce
ed a
t ro
om
tem
per
ature
for
the
indic
ated
tim
e in
terv
als
afte
r w
hic
h t
he
reac
tion s
ample
s w
ere
then
quen
ched
wit
h
the
addit
ion
of
AP
C-c
onju
gat
ed
stre
pta
vid
in
and
Euro
piu
m-c
hel
ated
an
ti-
phosp
hoty
rosi
ne
anti
body. S
ample
s w
ere
then
rea
d b
y a
flu
ore
scen
t pla
te r
eader
.
94
95
Fig
ure
2.1
3
T
he
kin
ase
act
ivit
y
of
AC
K2
is
inh
ibit
ed
by
stau
rosp
ori
ne.
The
kin
ase
reac
tions
wer
e ca
rrie
d o
ut
wit
h 4
.15 µ
g/m
L A
CK
2 a
nd 2
00 n
M S
H3P
X1 i
n
kin
ase
buff
er (
2 m
M A
TP
, 50 m
M H
EP
ES
, 10 m
M M
gC
l 2,
and 0
.1%
Tri
ton X
-100)
in
the
pre
sence
of
20 µ
M-8
0
nM
st
auro
spori
ne.
Rea
ctio
ns
wer
e quen
ched
af
ter
5
min
ute
s at
room
tem
per
ature
wit
h t
he
addit
ion o
f A
PC
-conju
gat
ed s
trep
tavid
in a
nd
Euro
piu
m-c
hel
ated
an
ti-p
hosp
hoty
rosi
ne
anti
body.
S
ample
s w
ere
then
re
ad
by
a
fluore
scen
t pla
te r
eader
.
96
97
Ult
imat
ely,
we
moved
to a
[3
3P
]-A
TP
fil
ter
assa
y t
o s
impli
fy d
etec
tion (
Fig
ure
2.1
4).
Foll
ow
ing
the
kin
ase
reac
tion,
the
pro
tein
s w
ere
pre
cipit
ated
w
ith
tric
hlo
roac
etic
aci
d. T
he
reac
tion m
ixtu
re w
as t
hen
tra
nsf
erre
d t
o a
fil
ter
pla
te w
her
e
[3
3P
] in
corp
ora
tion o
f ad
her
ent
pro
tein
s w
as m
easu
red.
A c
om
par
ativ
e ti
me
cours
e
was
ca
rrie
d
out
under
th
e sa
me
condit
ions
as
the
LA
NC
ET
M
syst
em.
H
ow
ever
,
inco
rpora
tion of
[33P
]-A
TP
w
as ver
y lo
w,
attr
ibute
d to
th
e re
lati
vel
y poor
kin
ase
acti
vit
y o
f A
CK
2.
We
then
exte
nded
the
reac
tion t
ime
to o
ver
nig
ht
condit
ions
to
max
imiz
e th
e si
gnal
-to-b
ack
gro
und
rati
o
to
2.5
:1,
wit
hin
th
e ra
nge
of
scre
enin
g
(Fig
ure
2.1
5).
T
he
nex
t st
ep w
as
to a
uto
mat
e t
he
assa
y,
wit
h e
ach r
eact
ion c
om
ponen
t
dis
trib
ute
d
by
an
auto
mat
ed
inst
rum
ent.
Unfo
rtunat
ely,
the
DM
SO
co
ntr
ol
pla
te
show
ed s
ever
al s
pik
es i
n s
ignal
, m
ost
lik
ely
due
to t
he
low
sig
nal
-to
-bac
kgro
und r
atio
as w
ell
as to
a
man
ual
st
ep nec
essa
ry in
th
e pro
cess
. W
e did
not
pro
ceed
to
an
auto
mat
ed
scre
en
bec
ause
w
e fe
lt
that
th
e ar
tifa
cts
would
sk
ew
any
true
hit
s.
How
ever
, th
e si
gnal
-to-b
ackg
round r
atio
was
hig
h e
nough t
o s
cree
n c
om
pound
s in
repli
cate
s.
Dose
-res
ponse
pro
file
s w
ere
per
form
ed u
sing t
yrp
host
in A
G1478 (
a know
n
EG
F r
ecep
tor
kin
ase
inhib
itor)
, P
arke-
Dav
is p
yri
do-p
yro
mid
ine,
PD
158780,
and t
he
SU
Gen
com
pound,
SU
6656.
We
found t
hat
the
Par
k-D
avis
com
pound w
as a
hig
hly
pote
nt
inhib
itor
of
AC
K2 k
inas
e ac
tivit
y i
n v
itro w
ith a
n I
C50 o
f ~
80 p
M (
Fig
ure
2.1
6).
Dis
cuss
ion
Due
to t
he
role
of
Erb
B r
ecep
tors
in i
nit
iati
ng m
itog
enic
sig
nal
ing p
athw
ays,
over
expre
ssio
n o
r m
uta
tions
that
alt
er t
he
intr
insi
c kin
ase
acti
vit
y o
f th
ese
rece
pto
rs
are
oft
en s
uff
icie
nt
to c
ause
mal
ignan
t tr
ansf
orm
atio
n.
Can
cer
cell
s ar
e ab
le t
o b
ypas
s
the
nec
essa
ry c
heck
poin
ts i
nvolv
ed i
n t
he
regula
tion o
f E
rbB
rec
epto
r si
gnal
ing i
n
sever
al w
ays,
in
cludin
g th
e over
expre
ssio
n of
gro
wth
fa
ctor
rece
pto
rs at
th
e ce
ll
surf
ace,
res
ult
ing i
n h
yper
sensi
tivit
y t
o o
ther
wis
e am
bie
nt
level
s of
gro
wth
fac
tor.
98
Fig
ure
2.1
4 F
ilte
r ass
ay f
or
ph
osp
hory
lati
on
rea
ctio
ns.
Foll
ow
ing
in
vit
ro
kin
ase
reac
tions,
th
e pro
tein
s ar
e pre
cipit
ated
w
ith
chil
led
tric
hlo
roac
etic
aci
d (
TC
A)
and t
ransf
erre
d t
o 9
6-w
ell
filt
er p
late
s.
The
sam
ple
s ar
e
was
hed
w
ith
TC
A
in
the
filt
er
pla
tes
and
inco
rpora
ted
radio
acti
ve
phosp
hat
e is
mea
sure
d i
n t
he
pre
sence
of
scin
till
atio
n f
luid
.
99
100
Fig
ure
2.1
5
AC
K2-c
ata
lyze
d k
inase
rea
ctio
ns
ass
ayed
by f
iltr
ati
on
.
Kin
ase
reac
tion
s w
ere
carr
ied o
ut
wit
h 8
.3 µ
g/m
L A
CK
2,
200 n
M S
H3P
X1,
50 µ
M
AT
P,
0.2
µC
i/w
ell
for
18-h
our
reac
tion a
t ro
om
tem
per
ature
. F
oll
ow
ing i
n v
itro k
inas
e
reac
tions,
the
pro
tein
s w
ere
pre
cipit
ated
wit
h c
hil
led
tri
chlo
roac
etic
aci
d (
TC
A)
and
tran
sfer
red t
o 9
6-w
ell
filt
er p
late
s.
The
sam
ple
s w
ere
was
hed
wit
h T
CA
in t
he
filt
er
pla
tes
and
inco
rpora
ted
radio
acti
ve
phosp
hat
e w
as
mea
sure
d
in
the
pre
sence
of
scin
till
atio
n f
luid
.
101
102
Fig
ure
2.1
6
Park
e-D
avis
pyri
dop
yri
mid
ine
inh
ibit
s A
CK
2 k
inase
act
ivit
y w
ith
an
IC5
0 ~
80 p
M.
Str
uct
ure
of
Par
ke-
Dav
is c
om
pound P
D158780 (
A).
K
inas
e re
acti
ons
wer
e ca
rrie
d o
ut
wit
h 8
.3 µ
g/m
L A
CK
2,
200 n
M S
H3P
X1,
50 µ
M A
TP
+ 0
.2 µ
Ci/
wel
l
for
18 h
ours
at
room
tem
per
ature
. F
oll
ow
ing i
n vit
ro k
inas
e re
acti
on
s, t
he
pro
tein
s
wer
e pre
cipit
ated
w
ith ch
ille
d tr
ichlo
roac
etic
ac
id an
d tr
ansf
erre
d to
96
-wel
l fi
lter
pla
tes.
T
he
sam
ple
s w
ere
was
hed
w
ith T
CA
in
th
e fi
lter
pla
tes
and in
corp
ora
ted
radio
acti
ve
ph
osp
hat
e w
as m
easu
red i
n t
he
pre
sence
of
scin
till
atio
n f
luid
(B
).
103
104
Gro
ss
over
expre
ssio
n
of
Erb
B
rece
pto
rs
has
ev
en
bee
n
show
n
to
induce
li
gan
d-
indep
enden
t si
gnal
ing [
17],
thus
enhan
cing c
ell
pro
life
rati
on i
n t
he
abse
nce
of
exte
rnal
signal
s. A
noth
er
pote
nti
al
cause
of
can
cer
is
the
loss
of
gro
wth
fa
ctor
rece
pto
r
deg
radat
ion.
In c
ells
wit
h e
levat
ed l
evel
s of
gro
wth
fac
tor
rece
pto
rs a
t th
e ce
ll s
urf
ace,
one
mec
han
ism
fo
r re
stori
ng norm
al pro
life
rati
ve
rate
s in
volv
es th
e upta
ke
of
the
rece
pto
rs f
rom
the
pla
sma
mem
bra
ne.
A
wel
l-st
ud
ied m
echan
ism
for
dow
n-r
egula
ting
gro
wth
fa
ctor
rece
pto
rs
is
clat
hri
n-m
edia
ted
endocy
tosi
s,
a hig
hly
ch
arac
teri
zed
pro
cess
in
w
hic
h
cel
l su
rfac
e re
cepto
rs
beco
me
inte
rnal
ized
in
to
the
cyto
pla
sm.
Endocy
tosi
s of
cell
su
rfac
e re
cepto
rs is
a
tightl
y re
gula
ted,
sequen
tial
pro
cess
. It
involv
es s
ever
al c
yto
soli
c pro
tein
s, r
efer
red t
o a
s ac
cess
ory
pro
tein
s, a
s w
ell
as t
he
mec
han
ical
m
eans
to
def
orm
th
e m
embra
ne
into
a
ves
icula
r bud
that
ult
imat
ely
pin
ches
off
fro
m t
he
mem
bra
ne
to f
orm
the
free
cla
thri
n-c
oat
ed v
esic
le.
The
pri
nci
pal
pro
tein
s in
cl
athri
n-m
edia
ted en
docy
tosi
s in
clude,
cl
athri
n,
a
tris
kel
ion p
rote
in m
akin
g u
p t
he
cage o
f th
e cl
athri
n-c
oat
ed v
esic
le,
AP
-2,
resp
onsi
ble
for
conce
ntr
atin
g t
he
carg
o a
nd r
ecru
itin
g c
lath
rin t
o t
he
pla
sma
mem
bra
ne,
and t
he
GT
Pas
e,
dynam
in,
involv
ed
in
pro
vid
ing
the
mec
han
ical
fo
rce
from
w
hic
h
the
clat
hri
n-c
oat
ed v
esic
le p
ropel
s it
self
fro
m t
he
mem
bra
ne.
In
tere
stin
gly
, A
CK
2 a
nd
SH
3P
X1 c
onta
in s
ignal
ing d
om
ains
that
all
ow
them
to i
nte
ract
wit
h k
ey a
ccess
ory
pro
tein
s in
the
endocy
tic
pro
cess
. A
CK
2 h
as b
een s
how
n t
o b
ind t
o c
lath
rin i
n G
ST
pull
-dow
n
exper
imen
ts
wher
e th
e tw
o
carb
oxy
l-te
rmin
al
pro
line-
rich
dom
ains
of
AC
K2 w
ere
show
n t
o m
edia
te t
his
act
ion [
6].
A
cla
thri
n-b
indin
g m
oti
f, L
IDF
, has
since
bee
n
iden
tifi
ed
bet
wee
n
the
two
pro
line-
rich
m
oti
fs.
S
H3P
X1
conta
ins
an
amin
o-t
erm
inal
SH
3 d
om
ain,
whic
h h
as b
een i
mpli
cate
d i
n i
nte
ract
ions
wit
h A
CK
2,
the
Wis
kott
-Ald
rich
sy
ndro
me
pro
tein
(W
AS
P),
an
d m
ore
re
centl
y,
the
endocy
tic
105
GT
Pas
e, d
ynam
in-2
[7].
T
his
sort
ing n
exin
has
als
o b
een s
how
n t
o b
ind t
o c
lath
rin
and A
P-2
, th
us
stre
ngth
enin
g t
he
link t
o c
lath
rin-m
edia
ted e
nd
ocy
tosi
s an
d s
ort
ing [
7].
Whil
e th
e ex
act
funct
ion o
f S
H3P
X1 i
n r
ecep
tor
endocy
tosi
s an
d d
egra
dat
ion
is s
till
not
under
stood,
one
can m
ake
a re
asonab
le a
ssum
pti
on b
ased
on w
hat
has
bee
n
det
erm
ined
for
oth
er m
ember
s of
the
sort
ing n
exin
fam
ily.
Sort
ing n
exin
1 (
SN
X1),
the
firs
t m
amm
alia
n s
ort
ing n
exin
to b
e ch
arac
teri
zed,
has
bee
n s
how
n t
o i
nte
ract
wit
h
EG
F r
ecep
tors
and r
egula
te a
ctiv
ated
rec
epto
r le
vel
s in
CV
1 (
Afr
ican
gre
en m
onkey
kid
ney
) ce
lls
[11].
O
ther
sort
ing n
exin
s, S
NX
2 a
nd S
NX
4,
co-i
mm
unopre
cipit
ate
wit
h
rece
pto
rs t
hat
bin
d E
GF
, in
suli
n,
pla
tele
t-der
ived
gro
wth
fac
tor
(PD
GF
) an
d t
he
long
form
of
the
lepti
n r
ecep
tor
[15].
M
ore
over
, over
expre
ssio
n o
f S
H3P
X1 t
oget
her
wit
h
AC
K2 in
C
OS
-7 ce
lls
was
sh
ow
n to
al
ter
the
pro
cess
ing an
d tr
affi
ckin
g of
EG
F,
insu
lin,
and t
ransf
erri
n r
ecep
tors
[10]
[7,
16].
In o
rder
to u
ltim
atel
y u
nder
stan
d t
he r
ole
s of
AC
K2 a
nd S
H3P
X1 i
n g
row
th
fact
or
rece
pto
r deg
radat
ion,
it w
ill
be
import
ant
to c
har
acte
rize
the
AC
K2-S
H3P
X1
inte
ract
ion,
iden
tify
the
site
(s)
on t
he
sort
ing n
exin
that
are
phosp
hory
late
d b
y A
CK
2,
and d
eter
min
e th
e si
gnif
ican
ce o
f th
e phosp
hory
lati
on e
ven
t to
cel
lula
r pro
cess
ing.
To
dat
e, w
e hav
e dem
onst
rate
d t
he l
oss
of
AC
K2-c
atal
yze
d p
hosp
hory
lati
on i
n t
he !
C84
muta
nt
of
SH
3P
X1 b
y d
elet
ion a
nal
ysi
s.
Our
init
ial
hypoth
esis
was
th
at t
his
car
boxyl-
term
inal
reg
ion o
f S
H3P
X1 c
onta
ined
the
AC
K2 p
hosp
hory
lati
on s
ites
. I
nst
ead,
we
det
erm
ined
that
the
loss
of
pho
sphory
lati
on w
as a
res
ult
of
the
inab
ilit
y o
f A
CK
2 t
o
bin
d t
o t
his
car
boxyl-
term
inal
tru
nca
tion m
uta
nt
of
SH
3P
X1.
Rec
ent
report
s cl
aim
ing
that
the
carb
oxyl-
term
inal
BA
R m
oti
f is
cri
tica
l fo
r dim
eriz
atio
n o
f th
e so
rtin
g n
exin
[18]
may
expla
in t
his
obse
rvat
ion,
as d
imer
izat
ion m
ay b
e cr
itic
al f
or
the
bin
din
g o
f
AC
K2 a
nd s
ubse
quen
t phosp
hory
lati
on o
f th
e so
rtin
g n
exin
.
Pre
vio
usl
y,
the
tyro
sine
phosp
hory
lati
on
site
of
the
Dro
sophil
a
SH
3P
X1
ort
holo
gue
has
bee
n re
port
ed as
Y
56
[5].
H
ow
ever
, m
uta
ting th
e co
rres
pondin
g
106
resi
dues
in
th
e hum
an
isofo
rm
of
SH
3P
X1
did
n
ot
blo
ck
the
AC
K2-c
atal
yze
d
phosp
hory
lati
on o
f S
H3P
X1.
This
and o
ther
exper
imen
ts l
ed u
s to
the
real
izat
ion t
hat
AC
K2 li
kel
y phosp
hory
late
s S
H3P
X1 on m
ult
iple
ty
rosi
ne
resi
dues
. A
ttem
pts
to
iden
tify
the
AC
K2-c
atal
yze
d s
ites
by m
ass
spec
trom
etry
hav
e le
d t
o t
he
iden
tifi
cati
on
of
tyro
sine
287 a
s a
site
of
pho
sphory
lati
on.
This
tyro
sine
resi
due
is l
oca
ted
in t
he
PX
dom
ain o
f S
H3P
X1,
resp
onsi
ble
for
targ
etin
g t
he
pro
tein
to t
he
pla
sma
mem
bra
ne
thro
ugh
inte
ract
ions
wit
h
phosp
hat
idyli
nosi
tols
.
One
could
im
agin
e th
e
phosp
hory
lati
on o
f Y
287 a
s a
regula
ting f
acto
r in
dic
tati
ng t
he
loca
liza
tion o
f th
e
sort
ing n
exin
in t
he
cell
. D
espit
e th
is p
osi
tive
iden
tifi
cati
on,
our
dat
a sh
ow
that
the
SH
3P
X1 m
uta
nt
Y287F
ret
ains
som
e l
evel
of
ph
osp
hory
lati
on w
hen
co-e
xpre
ssed
in
cell
s.
As
a re
sult
, w
e bel
ieve
ther
e ar
e ad
dit
ional
sit
es o
f m
odif
icat
ion a
nd a
re t
akin
g
mea
sure
s to
id
enti
fy th
ese
site
s. In
th
e fu
ture
, w
e bel
ieve
that
th
e gen
erat
ion of
SH
3P
X1 m
uta
nts
def
ecti
ve
for
AC
K2-c
atal
yze
d p
hosp
hory
lati
on s
hould
be
inval
uab
le
in e
stab
lish
ing t
he
role
for
SH
3P
X1 p
hosp
hory
lati
on i
n r
ecep
tor
endocy
tosi
s.
As
a
com
ple
men
tary
ap
pro
ach
to
war
d
pro
bin
g
the
import
ance
of
AC
K2
acti
vit
y a
nd i
ts p
hosp
hory
lati
on o
f S
H3P
X1,
we
set
out
to i
den
tify
sm
all
mole
cule
inhib
itors
of
AC
K2.
Thro
ugh a
ver
y g
ener
ous
rela
tionsh
ip w
ith M
erck
, w
e w
ere
able
to
uti
lize
th
e hig
h-t
hro
ughput
scre
enin
g
faci
lity
of
the
Auto
mat
ed
Bio
tech
nolo
gy
dep
artm
ent.
H
ere
we
wer
e ab
le t
o i
den
tify
the
Park
e-D
avis
com
pound,
PD
158780 a
s
an i
n v
itro
inhib
itor
of
AC
K2 k
inas
e ac
tivit
y.
PD
158780 i
s a
pyri
dopyri
mid
ine,
a
der
ivia
tive
of
the
quin
azoli
ne
fam
ily o
f co
mpounds.
T
hes
e a
rom
atic
sm
all
mole
cule
s
hav
e bee
n
show
n
to
effe
ctiv
ely
inhib
it
EG
F
rece
pto
r fa
mil
y
pro
tein
s.
In
dee
d,
PD
158780 i
nhib
its
EG
F r
ecep
tor
kin
ase
acti
vit
y w
ith a
n I
C50 ~
8 p
M [
19].
T
hus,
it
wil
l
be
dif
ficu
lt t
o u
se t
his
com
pound t
o s
elec
tivel
y e
xam
ine
the
acti
ons
of
AC
K2 (
IC5
0
~80 p
M),
unle
ss e
xper
imen
ts w
ere
per
form
ed u
sing o
ther
inhib
itors
that
blo
ck o
nly
EG
F r
ecep
tor
acti
vit
y,
as a
mea
ns
of
dis
tinguis
hin
g t
he
conse
quen
ces
of
inhib
itin
g
107
EG
F r
ecep
tors
fro
m t
hose
res
ult
ing f
rom
the
inhib
itio
n o
f A
CK
2.
Futu
re s
tudie
s ar
e
bei
ng d
irec
ted t
ow
ard i
den
tify
ing m
ore
sp
ecif
ic i
nhib
itors
of
AC
K2 t
hat
do n
ot
affe
ct
the
EG
F r
ecep
tor.
108
Refe
ren
ces
1.
Sla
mon,
D.J
., e
t al
., H
um
an b
reast
cance
r: c
orr
elati
on o
f re
lapse
and s
urv
ival
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ssoci
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yrosi
ne k
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g,
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he
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cepto
r ty
rosi
ne
kina
se A
CK
2,
a s
pec
ific
targ
et f
or
Cdc4
2 a
nd a
neg
ati
ve r
egula
tor
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wth
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oca
l adhes
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hem
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Worb
y,
C.A
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t al
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roso
phil
a A
ck t
arg
ets
its
subst
rate
, th
e so
rtin
g n
exin
DSH
3P
X1,
to a
pro
tein
co
mple
x in
volv
ed i
n a
xonal
guid
ance
. J
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l C
hem
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Yan
g,
W.,
et
al., T
he
Cdc4
2 t
arg
et A
CK
2 d
irec
tly
inte
ract
s w
ith c
lath
rin a
nd
infl
uen
ces
clath
rin a
ssem
bly
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l C
hem
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7.
Lundm
ark,
R.
and S
.R.
Car
lsso
n,
Sort
ing nexi
n 9 part
icip
ate
s in
cl
ath
rin-
med
iate
d e
ndocy
tosi
s th
rough i
nte
ract
ion
s w
ith
the
core
com
ponen
ts.
J B
iol
Chem
, 2003. 278(4
7):
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1.
8.
Lundm
ark,
R.
and S
.R.
Car
lsso
n,
The
bet
a-a
ppen
dages
of
the
fou
r adapto
r-
pro
tein
(A
P)
com
ple
xes:
str
uct
ure
and b
indin
g p
roper
ties
, and i
den
tifi
cati
on o
f
sort
ing n
exin
9 a
s an
acc
ess
ory
pro
tein
to
AP
-2.
Bio
chem
J,
2002.
362(P
t 3):
p.
597-6
07.
109
9.
Soule
t,
F.,
et
al
.,
SN
X9
regula
tes
dyn
am
in
ass
embly
and
is
requir
ed
for
effi
cien
t cl
ath
rin
-med
iate
d e
ndocy
tosi
s. M
ol
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l C
ell,
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58-
67.
10.
Lin
, Q
.,
et
al.,
The
Cdc4
2
targ
et
AC
K2
inte
ract
s w
ith
sort
ing
nex
in
9
(SH
3P
X1)
to r
egula
te e
pid
erm
al
gro
wth
fact
or
rece
pto
r deg
radati
on.
J B
iol
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, 2002. 277(1
2):
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11.
Kurt
en,
R.C
., D
.L.
Cad
ena,
an
d G
.N.
Gil
l, E
nhance
d deg
radati
on of
EG
F
rece
pto
rs b
y a s
ort
ing n
exin
, SN
X1.
Sci
ence
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264):
p. 1008-1
0.
12.
Wan
g,
Y.,
et
al
.,
Dow
n-r
egula
tion
of
pro
tease
-act
ivate
d
rece
pto
r-1
is
regula
ted b
y so
rtin
g n
exin
1.
Mol
Bio
l C
ell,
2002. 13(6
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6.
13.
Gull
apal
li,
A.,
et
al
.,
An
esse
nti
al
role
fo
r SN
X1
in
lyso
som
al
sort
ing
of
pro
tease
-act
ivate
d
recep
tor-
1:
evid
ence
fo
r re
trom
er-,
H
rs-,
and
Tsg
101-
indep
enden
t fu
nct
ions
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ing n
exi
ns.
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Bio
l C
ell,
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38.
14.
Burd
en,
J.J.
, et
al.
, So
rtin
g m
oti
fs i
n t
he
intr
ace
llula
r dom
ain
of
the
low
den
sity
lipopro
tein
rec
epto
r in
tera
ct w
ith a
nove
l do
main
of
sort
ing n
exin
-17.
J B
iol
Chem
, 2004. 279(1
6):
p. 16237-4
5.
15.
Haf
t, C
.R.,
et
al.,
Iden
tifi
cati
on o
f a f
am
ily
of
sort
ing n
exin
mole
cule
s and
chara
cter
izati
on
of
thei
r ass
oci
ati
on
wit
h
rece
pto
rs.
Mol
Cel
l B
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16.
MaC
aula
y,
S.L
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t al
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nsu
lin s
tim
ula
tes
move
ment
of
sort
ing n
exin
9 b
etw
een
cell
ula
r co
mpart
men
ts:
a
puta
tive
ro
le
med
iati
ng
cell
su
rface
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cepto
r
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ssio
n a
nd i
nsu
lin
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ioch
em J
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t 1):
p.
123-3
4.
17.
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iore
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al
.,
Erb
B-2
is
a pote
nt
onco
gen
e w
hen
ove
rexp
ress
ed in
NIH
/3T
3 c
ells
. S
cien
ce, 1987. 237:
p.
178-1
82.
110
18.
Chil
dre
ss,
C.,
Q.
Lin
, an
d W
. Y
ang,
Dim
eri
zati
on i
s re
quir
ed f
or
SH
3P
X1
tyro
sine
phosp
ho
ryla
tion in
re
sponse
to
ep
ider
mal
gro
wth
fa
ctor
signall
ing
and i
nte
ract
ion w
ith A
CK
2.
Bio
chem
J,
2006.
394(P
t 3):
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.
19.
Rew
cast
le,
G.W
.,
et
al.,
T
yrosi
ne
kina
se
inhib
itors
. 14.
Str
uct
ure
-act
ivit
y
rela
tionsh
ips
for
met
hyl
am
ino-s
ub
stit
ute
d
der
ivati
ves
of
4-[
(3-
bro
mophen
yl)a
min
o]-
6-(
met
hyl
am
ino)-
pyr
ido[3
,4-d
]pyr
imid
ine
(PD
158780),
a p
ote
nt
and s
pec
ific
inhib
itor
of
the
tyro
sine
kin
ase
act
ivit
y of
rece
pto
rs f
or
the
EG
F f
am
ily
of
gro
wth
fact
ors
. J
Med
Chem
, 1998.
41(5
): p
. 742-5
1.
111
CH
AP
TE
R T
HR
EE
SH
3P
X1 I
S A
PH
OS
PH
O-S
UB
ST
RA
TE
FO
R B
OT
H T
HE
FO
CA
L A
DH
ES
ION
KIN
AS
E A
ND
SR
C
Ab
stra
ct
A
ro
le fo
r th
e so
rtin
g nex
in,
SH
3P
X1,
in th
e co
mple
x pro
cess
of
clat
hri
n-
med
iate
d e
ndocy
tosi
s h
as b
een
incr
easi
ngly
sugg
este
d i
n r
ecen
t yea
rs.
For
exam
ple
,
SH
3P
X1 b
een s
how
n t
o b
ind t
o k
ey e
ndocy
tic
pro
tein
s in
cludin
g a
dap
tor
pro
tein
-2
(AP
-2),
cl
athri
n,
and
dynam
in.
M
ore
over
, knock
ing
dow
n
endogen
ous
SH
3P
X1
expre
ssio
n i
n c
ells
usi
ng S
H3P
X1-R
NA
i, w
as
found t
o b
lock
dynam
in t
raff
ickin
g t
o
the
pla
sma
mem
bra
ne.
A
s a
resu
lts
of
thes
e an
d s
imil
ar f
indin
gs,
ther
e is
a g
row
ing
inte
rest
in e
luci
dat
ing e
xact
ly h
ow
SH
3P
X1 i
s re
gula
ted a
nd f
unct
ions
in e
ndocy
tic
pro
cess
es.
Tyro
sine
phosp
hory
lati
on a
ppea
rs t
o b
e one
mec
han
ism
by w
hic
h S
H3P
X1 i
s
regula
ted d
uri
ng t
he
endocy
tic
pro
cess
. O
ur
lab
ora
tory
and o
ther
s hav
e pre
vio
usl
y
dem
onst
rate
d t
hat
tyro
sine
ph
osp
hory
lati
on o
f S
H3P
X1 e
nhan
ces
dynam
in t
raff
ickin
g
to t
he
pla
sma
mem
bra
ne,
an
d s
ubse
quen
t en
docy
tosi
s an
d d
egra
dat
ion o
f th
e E
GF
rece
pto
r in
cel
ls.
Giv
en t
he
appar
ent
import
ant
of
SH
3P
X1 p
hosp
hory
lati
on,
we
wer
e
inte
rest
ed
in
iden
tify
ing
pro
tein
kin
ases
resp
onsi
ble
an
d
the
loca
tion
of
the
phosp
hory
lati
on si
tes.
O
ur
resu
lts
des
crib
ed in
ch
apte
r 2 id
enti
fy A
CK
2 as
on
e
pro
tein
ty
rosi
ne
kin
ase
that
ca
n
phosp
hory
late
S
H3P
X1,
wit
h
the
maj
or
site
of
phosp
hory
lati
on b
eing Y
287.
Her
e, w
e hav
e ex
amin
ed w
het
her
addit
ional
kin
ases
mig
ht
phosp
hory
late
SH
3P
X1,
nam
ely F
AK
and S
rc.
We
consi
der
ed f
oca
l ad
hes
ion
kin
ase
(FA
K)
to b
e a p
ote
nti
al c
andid
ate b
ased
on
the
fact
that
the
kin
ase d
om
ains
of
FA
K
and
AC
K2
shar
e a
hig
h
deg
ree
of
hom
olo
gy,
and
seco
ndly
, th
at
FA
K
phosp
hory
late
s en
dophil
in,
a pro
tein
wit
h s
imil
ar d
om
ain f
eatu
res
as
SH
3P
X1.
Src
112
was
ch
ose
n
as
an
addit
ional
ca
ndid
ate
giv
en
its
know
n
asso
ciat
ion
and
tandem
signal
ing w
ith F
AK
in c
ells
. H
ere,
we s
how
th
at F
AK
and S
rc c
an p
hosp
hory
late
SH
3P
X1 b
oth
in v
ivo a
nd i
n v
itro
. W
e d
emonst
rate
that
the
FA
K-
and
Src
-med
iate
d
phosp
hory
lati
on of
SH
3P
X1 is
m
uch
m
ore
pro
nounce
d th
an th
e A
CK
2-c
atal
yze
d
phosp
hory
lati
on.
M
ore
over
, w
e go
on to
dem
onst
rate
th
at
C-t
erm
inal
tr
unca
tion
muta
nts
of
SH
3P
X1 t
hat
are
def
ecti
ve
in t
hei
r ab
ilit
y t
o b
e phosp
hory
late
d b
y A
CK
2,
are
stil
l ca
pab
le
of
bei
ng
phosp
hory
late
d
by
FA
K
and
Src
.
Mas
s sp
ectr
om
etry
anal
ysi
s of
Src
-cat
alyze
d
pho
sphory
lati
on
of
SH
3P
X1
iden
tifi
ed
5
site
s of
phosp
hory
lati
on i
ncl
udin
g Y
177,
Y239,
Y269,
Y294,
and Y
561.
Y239 w
as s
how
n t
o
be
the
maj
or
phosp
hory
lati
on s
ite
by F
AK
and S
rc,
and m
uta
tion o
f al
l 5 t
yro
sine
resi
dues
resu
lted
in a
pho
sphory
lati
on-d
efec
tive
SH
3P
X1 m
uta
nt.
T
aken
toget
her
, our
resu
lts
suggest
that
SH
3P
X1 m
ay r
ecei
ve
spec
ific
sig
nal
s an
d/o
r par
tici
pat
e in
dis
tinct
signal
ing
pat
hw
ays
resu
ltin
g
from
A
CK
2-c
atal
yze
d,
and
FA
K-
and
Src
-cat
alyze
d
SH
3P
X1 p
hosp
hory
lati
on.
Intr
od
uct
ion
Sort
ing
nex
ins
are
a fa
mil
y
of
pro
tein
s th
at
hav
e bee
n
impli
cate
d
in
the
traf
fick
ing an
d pro
cess
ing of
cell
su
rfac
e re
cepto
rs.
T
he
hal
lmar
k of
this
div
erse
fam
ily o
f pro
tein
s is
the
PX
dom
ain,
iden
tifi
ed a
s a
conse
rved
moti
f in
the
p40
ph
ox a
nd
p47
ph
ox
subunit
s of
the
neu
trophil
N
AD
PH
oxid
ase
(phox)
super
oxid
e-gen
erat
ing
com
ple
x [
1].
C
om
par
ing t
he
PX
dom
ains
from
dif
fere
nt
pro
tein
s sh
ow
s th
at t
hey
hav
e
lim
ited
seq
uen
ce h
om
olo
gy a
side
from
a c
on
serv
ed p
roli
ne-
rich
moti
f, P
xxP
, w
hic
h
enab
les
PX
-dom
ain-c
onta
inin
g
pro
tein
s to
in
tera
ct
wit
h
SH
3
dom
ain-c
onta
inin
g
pro
tein
s.
Upst
ream
of
the
pro
line-
rich
dom
ain i
s a
phosp
holi
pid
-inte
ract
ion m
oti
f,
(R/K
)(R
/K)(
Y/F
)xxF
xxL
xxxL
and R
(R/K
)xxL
xx(Y
/F),
whic
h c
an i
nte
ract
wit
h a
wid
e
range
of
phophosp
holi
pid
s, th
ereb
y sp
ecif
ical
ly ta
rget
ing th
ese
pro
tein
s to
ce
llula
r
113
mem
bra
nes
enri
ched
in
phosp
hat
idyli
nosi
tols
[2,
3].
T
he
pho
spholi
pid
-inte
ract
ing
moti
fs ar
e oft
en co
nse
rved
am
ong P
X dom
ain-c
onta
inin
g pro
tein
s. W
ithin
th
ese
conse
nsu
s se
quen
ces,
th
e ly
sine
and ar
gin
ine
resi
dues
se
rve
to cr
eate
a
posi
tivel
y
char
ged
su
rfac
e on
the
pro
tein
, w
hic
h
is
attr
acte
d
to
neg
ativ
ely
char
ged
phosp
hat
idyli
nosi
tols
em
bed
ded
in t
he
pla
sma
mem
bra
ne.
In a
ddit
ion t
o t
he
sort
ing n
exin
s, P
X d
om
ains
are
also
found i
n s
ever
al o
ther
signal
ing
pro
tein
s su
ch
as
phosp
holi
pase
D
(P
LD
),
phosp
hat
idyli
nosi
tol
3-k
inas
e
(PI3
K),
and B
em1 a
nd B
em3,
two y
east
pro
tein
s in
volv
ed i
n b
ud e
mer
gen
ce a
nd c
ell
pola
rity
[4
, 5].
It
has
bee
n sh
ow
n th
at th
e P
X d
om
ains
of
the ab
ove m
enti
oned
pro
tein
s ar
e al
l in
volv
ed i
n b
indin
g t
o p
hosp
hat
idyli
nosi
tols
, as
wel
l as
med
iati
ng a
num
ber
of
pro
tein
-pro
tein
inte
ract
ions
thro
ugh t
hei
r pro
line-
rich
moti
fs {
Coll
ey,
1997
#179[6
, 7].
A
uniq
ue
role
for
the
PX
dom
ain
of
PL
D h
as a
lso b
een
rec
entl
y r
eport
ed.
It w
as d
emonst
rate
d t
hat
this
par
ticu
lar
PX
dom
ain
funct
ions
as a
GT
Pas
e-ac
tivat
ing
pro
tein
(G
AP
),
cata
lyzi
ng
the
hydro
lysi
s of
GT
P-b
ound
dynam
in
[8].
Arg
inin
e
resi
dues
128
an
d 197 of
PL
D hav
e bee
n im
pli
cate
d in
ca
taly
zing dynam
in G
TP
hydro
lysi
s, t
hus
funct
ionin
g a
s th
e ar
gin
ine
finger
of
this
puta
tive
GA
P.
The
sort
ing n
exin
s hav
e b
een c
lass
ifie
d i
nto
subfa
mil
ies
bas
ed o
n t
he
pre
sence
or
abse
nce
of
cert
ain s
ignal
ing
or
stru
ctura
l do
mai
ns
conta
ined
wit
hin
eac
h f
amil
y
mem
ber
. O
ne
subcl
ass,
com
pri
sed o
f S
NX
1-2
, S
NX
4-8
, S
NX
15,
and S
NX
16,
all
hav
e
long
carb
oxyl-
term
inal
ex
tensi
ons
conta
inin
g
1-3
ch
arac
teri
stic
co
iled
-coil
m
oti
fs
whic
h a
llow
for
hom
o-
or
het
ero-o
ligom
eriz
atio
n w
ith o
ther
sort
ing n
exin
pro
tein
s [9
].
The
seco
nd s
ub
-cla
ss,
mad
e u
p o
f S
NX
3,
SN
X10,
SN
X11,
SN
X12,
and S
NX
22-2
4,
is
char
acte
rize
d b
y t
he
abse
nce
of
addit
ional
sig
nal
ing d
om
ains
(ie.
SH
2,
SH
3,
pro
line-
rich
) outs
ide
of
the
PX
dom
ain.
The
final
sub
-cla
ss o
f so
rtin
g n
exin
s is
char
acte
rize
d
by t
he
pre
sence
of
var
ious
pro
tein
-pro
tein
inte
ract
ion m
oti
fs s
uch
as
SH
3 d
om
ains
[10,
11],
mem
bra
ne-
targ
etin
g d
om
ains
incl
udin
g h
ydro
phobic
seq
uen
ces
[12,
13],
and G
114
pro
tein
reg
ula
tory
seq
uen
ces
[14,
15].
S
H3P
X1 f
alls
into
this
last
sub
-cla
ss o
f so
rtin
g
nex
ins.
SH
3P
X1,
also
ref
erre
d t
o a
s so
rtin
g n
exin
9 (
SN
X9),
is
a 77-k
Da
pro
tein
, w
ith
an
amin
o-t
erm
inal
S
H3
dom
ain,
foll
ow
ed
by
its
PX
dom
ain,
and
a
Bin
/am
phip
hysi
n/R
vs
(BA
R)
moti
f at
the c
arboxy
l-te
rmin
us.
B
AR
moti
fs h
ave b
een
iden
tifi
ed i
n a
num
ber
of
scaf
fold
pro
tein
s an
d c
onsi
st o
f a
curv
ed d
imer
wit
h p
osi
tive
char
ges
dis
trib
ute
d al
ong th
e m
embra
ne-
inte
ract
ing co
nca
ve fa
ce.
T
hese
dom
ain
s
com
monly
show
up i
n t
andem
wit
h P
X o
r P
H d
om
ains,
and b
ecau
se t
hey
exis
t as
curv
ed,
alpha-
hel
ical
bundle
s, t
hey
are
sen
siti
ve
to m
embra
ne
curv
ature
, pre
ferr
ing
hig
hly
curv
ed m
embra
nes
[16,
17].
O
ne
exam
ple
of
this
pre
fere
nce
is
seen
in t
he
abil
ity o
f th
e G
TP
ase
acti
vat
ing p
rote
ins
(GA
Ps)
, oli
gophre
nin
and c
enta
uri
n"
2,
to
bin
d t
o l
iposo
mes
wit
h s
pec
ific
mem
bra
ne
curv
ature
thro
ugh t
hei
r re
spec
tive
BA
R
dom
ains
[16].
In
SH
3P
X1,
the
BA
R m
oti
f has
no
t only
bee
n s
ugges
ted t
o t
raff
ic t
his
pro
tein
to t
he
pla
sma
mem
bra
ne,
but
is a
lso t
hought
to p
rom
ote
the
dim
eriz
atio
n o
f
this
so
rtin
g nex
in w
ith it
self
an
d pote
nti
ally
oth
er m
ember
s of
the
sort
ing nex
in
fam
ily.
The
sort
ing a
nd t
raff
ickin
g o
f ce
ll s
urf
ace
rece
pto
rs f
rom
the
pla
sma
mem
bra
ne
is
a ro
le
that
m
ember
s of
the
sort
ing
nex
in
fam
ily
hav
e bee
n
impli
cate
d
in.
Tra
dit
ional
ly,
the
com
ple
x p
roce
ss o
f re
cepto
r en
docy
tosi
s in
volv
es
sever
al p
rote
ins,
whic
h w
ork
in c
on
cert
to d
eform
the m
embra
ne
into
a v
esi
cula
r bu
d t
hat
ult
imat
ely
pin
ches
off
to f
orm
a f
ree
clat
hri
n-c
oat
ed v
esi
cle.
T
he
core
pro
tein
s in
volv
ed i
n t
his
endocy
tic
pro
cess
in
clude
the
adap
tor
pro
tein
, A
P-2
, cl
athri
n,
and
the
GT
Pas
e,
dynam
in.
Upon r
ecru
itm
ent
to t
he
pla
sma
mem
bra
ne,
AP
-2 s
erves
to c
once
ntr
ate t
he
carg
o i
n t
he
emer
gin
g b
ud,
as w
ell
as t
o l
ink c
lath
rin t
o t
he
pla
sma
mem
bra
ne
[18-2
1].
Once
the
asse
mbly
of
clat
hri
n h
as b
een
init
iate
d a
t th
e em
ergin
g b
ud,
the
mem
bra
ne
115
acquir
es in
crea
sing
curv
ature
unti
l an
in
vag
inat
ed pit
fo
rms.
A
s th
e cl
athri
n pit
mat
ure
s, t
he
final
ste
p, fi
ssio
n, re
quir
es t
he
acti
on o
f th
e G
TP
ase,
dynam
in [
22].
Dynam
in,
a 100-k
Da
pro
tein
, is
a
rath
er
uniq
ue
mem
ber
of
the
GT
Pas
e
super
fam
ily d
ue
to i
ts l
ow
aff
init
y f
or
GT
P (
~30 µ
M)
and h
igh i
ntr
insi
c ra
te o
f G
TP
hydro
lysi
s.
Dynam
in i
s re
cruit
ed t
o t
he
cell
surf
ace
wher
e G
DP
-GT
P e
xch
ange
occ
urs
and s
om
ehow
tri
gger
s dynam
in a
ssem
bly
into
a h
elic
al c
oll
ar a
round t
he
nec
k o
f th
e
clat
hri
n-c
oat
ed p
it.
The
hydro
lysi
s of
GT
P-b
ound d
ynam
in t
ighte
ns
the
dynam
in-
asse
mb
led h
elic
al c
oll
ar u
nti
l a
free
cla
thri
n-c
oat
ed v
esic
le i
s gen
erat
ed.
Curr
entl
y,
the
mec
han
ism
for
dynam
in r
ecru
itm
ent
to t
he
nec
k o
f cl
athri
n-c
oat
ed p
its
is n
ot
wel
l
under
stood.
What
is
clea
r is
that
the
inte
ract
ion b
etw
een t
he
PH
dom
ain o
f dynam
in
and phosp
hat
idyli
nosi
tides
in
th
e pla
sma
mem
bra
ne
is not
suff
icie
nt
to re
cruit
th
e
GT
Pas
e to
the
cell
surf
ace
[23
].
Rat
her
, it
is
thought
that
oth
er c
yto
soli
c pro
tein
s m
ust
be
resp
onsi
ble
for
the
recr
uit
men
t of
dynam
in t
o t
he
site
s of
emer
gin
g b
uds.
SH
3P
X1 w
as r
ecen
tly i
den
tifi
ed a
s a
bin
din
g p
artn
er f
or
dynam
in-2
in K
562
hum
an er
yth
role
ukem
ia ce
lls
[24].
T
he
bin
din
g bet
wee
n dynam
in-2
an
d S
H3P
X1
occ
urs
thro
ugh t
he
SH
3 d
om
ain o
f th
e so
rtin
g n
exin
and t
he
pro
line-
rich
reg
ion o
f
dynam
in,
since
a
poin
t m
uta
tion
in
the
SH
3
dom
ain
of
SH
3P
X1
aboli
shes
the
inte
ract
ion
wit
h
dynam
in-2
.
Not
only
does
S
H3P
X1
bin
d
to
dynam
in-2
, but
incr
easi
ng
evid
ence
al
so
sugges
ts
that
dynam
in
may
be
traf
fick
ed
to
the
pla
sma
mem
bra
ne
by S
H3P
X1.
This
was
dem
onst
rate
d u
sing a
rec
onst
ituti
on a
ssay
in w
hic
h
liposo
mes
mim
ickin
g t
he
pla
sma
mem
bra
ne,
wer
e in
cubat
ed w
ith l
ysa
tes
from
cel
ls
that
wer
e dep
lete
d o
f S
H3P
X1 u
sing R
NA
i.
The
resu
lts
show
ed t
hat
dynam
in-2
co
uld
asso
ciat
e w
ith
the
liposo
mes
only
w
hen
S
H3
PX
1
was
pre
sent
in
the
cyto
sol.
How
ever
, if
rec
om
bin
ant
SH
3P
X1 w
as a
dded
back
to t
he
SH
3P
X1
-dep
lete
d c
yto
sol,
the
dynam
in-l
iposo
me
inte
ract
ion c
ould
be
rest
ore
d [
25].
A
noth
er l
ine
of
evid
ence
support
ing
a ro
le
for
SH
3P
X1
in
traf
fick
ing
dynam
in-2
co
mes
fr
om
116
imm
unofl
uore
scen
ce s
tudie
s th
at s
ho
wed
that
dy
nam
in-2
was
mis
loca
lized
fro
m t
he
pla
sma
mem
bra
ne
in H
eLa
cell
s ex
pre
ssin
g S
H3
PX
1-R
NA
i.
Tak
en t
oget
her
, th
ese
dat
a ar
gue
for
a cr
itic
al r
ole
for
SH
3P
X1 i
n m
edia
ting e
ndocy
tosi
s.
Tyro
sine
phosp
hory
lati
on of
SH
3P
X1 m
ay cr
uci
al in
th
is tr
affi
ckin
g ev
ent,
wher
e S
H3P
X1 an
d dynam
in-2
dem
onst
rate
th
e gre
ates
t bin
din
g to
pre
par
ed
cell
mem
bra
nes
in t
he
pre
sence
of
AT
P,
GT
P#S
, ort
hovan
adat
e at
37°C
. T
he
nec
essi
ty o
f
AT
P an
d ort
hovan
adat
e ar
gues
fo
r th
e si
gnif
ican
ce of
tyro
sine
phosp
hory
lati
on of
SH
3P
X1 i
n t
arget
ing d
ynam
in-2
to t
he
pla
sma
mem
bra
ne.
In
dee
d,
a pre
ceden
t has
alre
ady
bee
n
esta
bli
shed
fo
r th
e im
port
ance
of
SH
3P
X1
phosp
hory
lati
on
in
Dro
sophil
a,
resu
ltin
g i
n d
iver
gin
g s
ignal
ing p
ath
way
s.
Tyro
sine
phosp
hory
lati
on o
f
Dro
sophil
a S
H3P
X1 (
DS
H3
PX
1)
has
bee
n s
how
n t
o p
rom
ote
bin
din
g t
o t
he
adap
tor
pro
tein
, D
ock
, over
that
of
Wis
cott
-Ald
rich
syndro
me
pro
tein
(W
AS
P),
the
pre
ferr
ed
bin
din
g p
artn
er o
f unphosp
hory
late
d S
H3P
X1.
Whil
e, t
he
role
of
SH
3P
X1 i
n d
ynam
in t
raff
ickin
g i
s sl
ow
ly c
om
ing t
o l
ight,
ther
e re
mai
n
a num
ber
of
ques
tion
s to
ad
dre
ss.
N
amel
y,
iden
tify
ing
the
phosp
hory
lati
on s
ites
on t
he
sort
ing n
exin
res
ponsi
ble
for
the
regula
tion o
f dynam
in
and d
evel
opin
g a
syst
em i
n w
hic
h t
hes
e si
tes
can b
e m
uta
ted a
nd u
sed i
n e
xper
imen
ts
to
furt
her
el
uci
dat
e how
th
e phosp
hory
lati
on
infl
uen
ces
the
pro
cess
of
rece
pto
r
endocy
tosi
s.
As
a fi
rst
step
in d
evel
opin
g t
his
exper
imen
tal
syst
em,
we
set
out
to
iden
tify
kin
ases
that
are
resp
onsi
ble
for
the
pho
sphory
lati
on o
f S
H3P
X1.
To t
his
end,
we
hav
e id
enti
fied
tw
o n
ovel
kin
ases,
FA
K a
nd S
rc,
that
can
robust
ly p
hosp
hory
late
SH
3P
X1.
Conse
quen
tly,
we
focu
sed o
n c
har
acte
rizi
ng t
he
inte
rpla
y b
etw
een F
AK
,
Src
and S
H3P
X1,
map
pin
g t
he
tyro
sine
phosp
hory
lati
on s
ites
on t
he
sort
ing n
exin
, an
d
com
par
ing t
he
pat
tern
s of
ph
osp
hory
lati
on b
etw
een F
AK
and S
rc, an
d A
CK
2.
117
Mate
rials
an
d M
eth
od
s
Mate
rials
—A
nti
-HA
an
d
anti
-c-M
yc
anti
bodie
s w
ere
from
C
ovan
ce,
anti
-
phosp
hoty
rosi
ne
(4G
10)
anti
body
and
reco
mbin
ant
His
-Src
w
ere
from
U
pst
ate
Bio
tech
nolo
gy,
Inc.
, an
ti-H
is
poly
clonal
an
tibody
was
fr
om
C
ell
Sig
nal
ing
Tec
hnolo
gy,
and a
nti
-dynam
in a
nti
body w
as f
rom
BD
Tra
nsd
uct
ion L
abora
tori
esT
M.
T4 D
NA
lig
ase,
Lip
ofe
ctam
ine
Tra
nsf
ecti
on R
eagen
t, P
rote
in-G
agar
ose
bea
ds,
anti
-
V5
anti
body,
and
the
Bac
-to-B
ac
Bac
ulo
vir
us
Expre
ssio
n
Syst
em
wer
e fr
om
Invit
rogen
. T
he
Quik
Chan
ge
Sit
e-D
irec
ted M
uta
gen
esis
kit
was
fro
m S
trat
agen
e an
d
the
Qia
quic
k G
el E
xtr
acti
on k
it w
as f
rom
Qia
gen
. R
ecom
bin
ant
His
-FA
K w
as f
rom
Act
ive
Moti
f an
d [
32P
]-A
TP
was
fro
m P
erkin
Elm
er.
Con
stru
ctio
n
of
SH
3P
X1
Del
etio
n
Mu
tan
ts—
The
pcD
NA
3-S
H3P
X1
wil
d-t
ype
pla
smid
was
use
d a
s a t
empla
te t
o c
onst
ruct
the c
arboxyl-
term
inal
del
etio
n m
uta
nts
.
Bri
efly
, D
NA
pri
mer
s to
gen
erat
e ea
ch d
elet
ion
const
ruct
(S
H3P
X1
-!C
84, !
C197,
!C
339, !
C395, !
C547)
wer
e en
gin
eere
d w
ith B
amH
1 a
nd E
coR
1 r
estr
icti
on s
ites
,
and t
hen
thes
e D
NA
pri
mer
s w
ere
use
d i
n a
PC
R r
eact
ion (
PC
R S
pri
nt,
Hybai
d).
The
PC
R
pro
duct
s w
ere
ligat
ed
into
th
e pC
R2
-TO
PO
vec
tor,
fo
llow
ed
by
rest
rict
ion
dig
ests
wit
h B
amH
1 a
nd E
coR
1.
Thes
e D
NA
pro
duct
s w
ere
reso
lved
on a
1%
agar
ose
gel
conta
inin
g 8
0 n
g/m
L e
thid
ium
bro
mid
e, a
nd t
hen
the
DN
A i
nse
rts
wer
e ex
cise
d
from
the
gel
and p
uri
fied
wit
h t
he
Qia
quic
k G
el E
xtr
acti
on k
it.
The
resu
ltin
g D
NA
inse
rts
wer
e li
gat
ed i
nto
the
HA
-tag
ged
pcD
NA
3 e
xpre
ssio
n v
ecto
r usi
ng T
4 D
NA
ligas
e. C
onst
ruct
ion o
f S
H3P
X1 P
oin
t M
uta
nts
—S
H3P
X1 t
yro
sine-
to-p
hen
yla
lanin
e
poin
t m
uta
nts
w
ere
also
gen
erat
ed on a
pcD
NA
3-S
H3P
X1 w
ild-t
ype
bac
kgro
und.
Muta
nts
Y
177F
, Y
239F
, Y
269F
, Y
294F
, Y
561F
, as
w
ell
as
quin
tuple
m
uta
nt,
F177/F
239/F
269/F
294/F
561,
wer
e gen
erat
ed
by
PC
R
usi
ng
the
Quik
Chan
ge
Sit
e-
Dir
ecte
d M
uta
gen
esis
kit
fro
m S
trat
agen
e.
118
SH
3P
X1 w
ild-t
ype
and p
oin
t m
uta
nts
wer
e su
bcl
oned
into
the
V5-p
cDN
A 3
.1
vec
tor
usi
ng t
he
Dir
ecti
onal
TO
PO
Expre
ssio
n kit
fro
m I
nvit
rogen
. P
rim
ers
wer
e
des
igned
bas
ed
on
the
man
ufa
cture
r’s
inst
ruct
ions
and
SH
3P
X1
wil
d-t
ype
and
tyro
sine-
to-p
hen
yla
lanin
e m
uta
nt
DN
A i
nse
rts
were
gen
erat
ed b
y P
CR
usi
ng t
he H
A-
tagged
pcD
NA
3 S
H3P
X1
co
nst
ruct
s as
bac
kgro
und.
The
blu
nt-
end P
CR
pro
duct
s
wer
e re
solv
ed o
n a
1%
agar
ose
gel
conta
inin
g 8
0 n
g/m
L e
thid
ium
bro
mid
e, a
nd t
hen
the
DN
A
inse
rts
wer
e ex
cise
d
from
th
e gel
an
d
puri
fied
w
ith
the
Qia
quic
k
Gel
Extr
acti
on k
it.
The
resu
ltin
g D
NA
inse
rts
wer
e li
gat
ed i
nto
the
pcD
NA
3.1
/V5-H
is-
TO
PO
pla
smid
.
Cel
l C
ult
ure
, T
ran
sfec
tion
an
d P
rep
ara
tion
of
Lysa
tes—
CO
S-7
and H
EK
29
3 c
ells
wer
e cu
lture
d i
n D
ulb
ecco
’s m
odif
ied E
agle
’s m
ediu
m (
DM
EM
) co
nta
inin
g 1
0%
fet
al
bovin
e se
rum
. T
he
cel
l li
nes
wer
e m
ainta
ined
in
5%
CO
2 a
t 37°C
. T
o e
xpre
ss t
he
var
ious
form
s of
SH
3P
X1,
FA
K,
and S
rc i
n c
ells
, M
yc-
, H
A-,
or
V5-t
agged
const
ruct
s
enco
din
g t
he
pro
tein
s w
ere
tran
sfec
ted i
nto
cel
ls u
sing L
ipofe
ctam
ine
Tra
nsf
ecti
on
Rea
gen
t ac
cord
ing t
o t
he
man
ufa
cture
r’s
inst
ruct
ions
(Invit
rogen
).
Tw
enty
-four
hours
foll
ow
ing t
ransf
ecti
on,
the
cell
s w
ere
rinse
d w
ith p
hosp
hat
e-buff
ered
sal
ine
(PB
S)
and
lyse
d w
ith c
old
mam
mal
ian c
ell
lysi
s buff
er (
10 m
M T
ris
(pH
7.4
), 5
mM
MgC
l 2,
150
mM
N
aCl,
1%
T
rito
n
X-1
00,
1
mM
so
diu
m
ort
hovan
adat
e,
5
mM
bet
a-gly
cero
l
phosp
hat
e, 1
0 µ
g/m
L a
pro
tin
in,
10 µ
g/m
L l
eupep
tin)
foll
ow
ed b
y c
ell
scra
pin
g.
The
cell
lysa
tes
wer
e t
hen
cle
ared
by
cen
trif
ugat
ion a
t 16,1
00 r
pm
in a
mic
rofu
ge
for
12
min
ute
s.
Pro
tein
conce
ntr
atio
ns
wer
e det
erm
ined
by d
iluti
ng t
he
lysa
tes
1:2
50 w
ith 1
x
Bra
dfo
rd r
eagen
t.
Imm
un
op
reci
pit
ati
on
—C
ell
lysa
tes
wer
e in
cubate
d w
ith M
yc,
HA
, or
V5
anti
body
for
2-2
0 h
ours
, w
ith r
ota
tion a
t 4°C
. P
rote
in-G
agar
ose
bea
ds
wer
e ad
ded
, ro
tati
ng
for
an a
ddit
ional
hour.
T
he
bea
ds
wer
e th
en p
reci
pit
ated
in a
mic
rofu
ge
and w
ashed
3-4
119
tim
es w
ith c
old
mam
mal
ian c
ell
lysi
s buff
er.
They
wer
e th
en r
esusp
ended
in 5
x S
DS
load
ing b
uff
er a
nd t
he
asso
ciat
ed p
rote
ins
wer
e re
solv
ed b
y S
DS
-PA
GE
.
Mass
Sp
ectr
om
etry
An
aly
sis—
HE
K 2
93 c
ells
wer
e tr
ansf
ecte
d w
ith H
A-S
rc a
nd V
5-
SH
3P
X1.
The
cell
s w
ere
lyse
d a
fter
24 h
ours
in c
old
mam
mal
ian c
ell
lysi
s buff
er (
10
mM
Tri
s (p
H 7
.4),
5 m
M M
gC
l 2,
150 m
M N
aC
l, 1
% T
rito
n X
-100,
1 m
M s
odiu
m
ort
hovan
adat
e,
5
mM
bet
a-gly
cero
l phosp
hat
e,
10 µ
g/m
L
apro
tinin
, 10 µ
g/m
L
leupep
tin)
and V
5-S
H3P
X1 w
as
imm
unopre
cipit
ated
fro
m t
he
pre
par
ed l
ysa
tes
(~10
mg t
ota
l pro
tein
) w
ith a
nti
-V5
anti
body.
The
pre
cip
itat
ed s
ample
s w
ere
boil
ed,
run o
n
an S
DS
-PA
GE
gel
, st
ained
wit
h C
oom
assi
e, a
nd e
xci
sed.
Ali
quots
fro
m t
he
sam
ple
s
wer
e se
t as
ide a
nd t
yro
sine
phosp
hory
lati
on w
as
confi
rmed
by W
este
rn b
lott
ing.
The
sam
ple
s w
ere
then
su
bm
itte
d
to
the
Corn
ell
Bio
tech
nolo
gy
Reso
urc
e C
ente
r
Pro
teom
ics
and M
ass
Spec
trom
etry
Core
Fac
ilit
y.
Rec
om
bin
an
t P
rote
in E
xp
ress
ion
an
d P
uri
fica
tion
—E
xpre
ssio
n of
His
-SH
3P
X1
was
car
ried
out
in t
he
BL
21 E
. co
li s
trai
n.
The
cDN
A e
nco
din
g w
ild
-type
SH
3P
X1
was
clo
ned
in
to t
he
pE
T28A
vec
tor
for
reco
mbin
ant
expre
ssio
n.
Over
nig
ht
cult
ure
s
from
colo
nie
s of
BL
21 c
ells
(N
ovag
en)
tran
sform
ed w
ith t
he
expre
ssio
n v
ect
or
wer
e
gro
wn
at
37°C
. T
hes
e w
ere
use
d
to
inocu
late
one-
lite
r cu
lture
s of
super
bro
th,
enri
ched
bac
teri
al m
edia
. B
acte
rial
cult
ure
s w
ere
gro
wn t
o a
n O
D6
00 o
f 0.8
at
37°C
and i
nduce
d w
ith 2
00 µ
M I
PT
G o
ver
nig
ht
at r
oom
tem
per
ature
. C
ells
wer
e har
vest
ed
by c
entr
ifugat
ion a
t 4000 r
pm
for
10 m
inute
s an
d f
roze
n a
t !
80°C
. A
ll s
ubse
quen
t
puri
fica
tion s
teps
wer
e c
arri
ed o
ut
at 4
°C.
Bac
teri
al p
elle
ts w
ere
resu
spen
ded
in
Ni2
+
bin
din
g
buff
er
(20
mM
T
ris
(pH
7.9
),
500
mM
N
aCl,
20
mM
im
idaz
ole
, 10%
gly
cero
l) su
pple
men
ted w
ith pro
teas
e in
hib
itors
(1 m
M P
MS
F,
10 µ
g/m
L ea
ch of
apro
tinin
and l
eupep
tin,
10
µM
ben
zam
idin
e) a
nd l
yse
d b
y t
hre
e pas
sag
es t
hro
ugh a
Fre
nch
Pre
ssu
re C
ell.
T
he
extr
acts
wer
e th
en s
onic
ated
for
5 m
inute
s an
d t
he
lysa
tes
wer
e cl
arif
ied by ult
race
ntr
ifugat
ion at
40,0
00 rp
m fo
r 45 m
inute
s. T
he
clar
ifie
d
120
lysa
tes
wer
e in
cubat
ed f
or
30
min
ute
s w
ith N
i2+ b
ead
s (A
mer
sham
) at
4°C
. T
he
bea
ds
wer
e th
en w
ash
ed w
ith
bin
din
g b
uff
er a
nd t
he p
rote
in r
ecover
ed w
ith e
luti
on b
uff
er
(20 m
M T
ris
(pH
7.9
), 5
00 m
M N
aCl,
200 m
M i
mid
azole
, 10%
gly
cero
l).
Sam
ple
s
from
th
e pro
tein
puri
fica
tion
w
ere
then
re
solv
ed by S
DS
-PA
GE
an
d st
ained
w
ith
Coom
assi
e blu
e.
In
Vit
ro
Tyro
sin
e K
inase
Ass
ay—
In v
itro
tyro
sine
phosp
hory
lati
on o
f S
H3P
X1 w
as
det
erm
ined
by
incu
bat
ing
50
ng
reco
mbin
ant
His
-Src
or
His
-FA
K
wit
h
500
ng
bac
teri
al p
uri
fied
His
-SH
3P
X1 i
n k
inas
e re
acti
on b
uff
er (
50 m
M T
ris-
HC
l (p
H 7
.4),
2
mM
MgC
l 2,
10 m
M M
nC
l 2"#,
1 #µ
M A
TP
, 10 µ
Ci
[32P
]-A
TP
).
Rea
ctio
ns
wer
e ca
rrie
d
out
at
room
te
mper
ature
fo
r var
ious
leng
ths
of
tim
e (5
-120
min
ute
s),
and
then
quen
ched
wit
h t
he
addit
ion o
f 5x S
DS
load
ing b
uff
er.
The
reac
tion s
ample
s w
ere
boil
ed,
reso
lved
by S
DS
-PA
GE
, an
d th
e gel
st
ain
ed w
ith C
oom
ass
ie.
T
he
ban
ds
corr
espondin
g t
o S
H3P
X1 w
ere
exci
sed a
nd t
he
exte
nt
of
phosp
hory
lati
on w
as r
ead
out
by
det
erm
inin
g
the
amo
unt
of
[32P
]-A
TP
in
corp
ora
ted
into
S
H3P
X1
usi
ng
a
scin
till
atio
n c
ounte
r.
Alt
ernat
ivel
y,
som
e of
the k
inas
e re
act
ions
wer
e r
esolv
ed b
y
SD
S-P
AG
E g
el a
nd t
ransf
erre
d t
o P
VD
F m
embra
nes
. T
he
exte
nt
of
phosp
hory
lati
on
was
det
erm
ined
by e
xposi
ng t
he
mem
bra
nes
to x
-ray
fil
m.
Res
ult
s Tyro
sine
phosp
hory
lati
on e
ven
ts a
re a
hal
lmar
k o
f ce
llula
r si
gnal
ing a
ctiv
itie
s
and p
lay a
n i
nte
gra
l ro
le i
n m
edia
ting n
earl
y a
ll c
ellu
lar
pro
cess
es.
SH
3P
X1 i
s a
pro
tein
that
has
bee
n l
inked
to t
he
endocy
tosi
s and s
ort
ing o
f ce
ll s
urf
ace
rece
pto
rs.
Stu
die
s hav
e dem
on
stra
ted t
hat
SH
3P
X1 i
s ty
rosi
ne
phosp
hory
late
d b
y A
CK
2 a
nd t
hat
this
phophory
lati
on
even
t is
im
port
ant
for
SH
3P
X1
to
med
iate
E
GF
re
cepto
r
endocy
tosi
s [2
9].
H
ere,
w
e hav
e se
t out
to det
erm
ine
whet
her
ad
dit
ional
pro
tein
121
kin
ases
ca
taly
ze
the
phosp
hory
lati
on
of
SH
3P
X1,
and
if
so,
do
thes
e kin
ases
phosp
hory
late
the
sam
e ty
rosi
ne
resi
dues
on S
H3P
X1 a
s A
CK
2 d
oes
.
We
chose
to
ex
amin
e w
het
her
F
AK
m
ight
phosp
hory
late
S
H3P
X1 fo
r tw
o
reas
ons.
F
irst
, F
AK
is
close
ly r
elat
ed t
o t
he
tyro
sin
e fa
mil
y o
f A
CK
s.
The
seco
nd
reas
on
is
base
d
on
pre
vio
us
findin
gs
whic
h
show
th
at
FA
K
can
pho
sphory
late
endophil
in,
a pro
tein
that
shar
es f
unct
ional
dom
ains
wit
h S
H3P
X1 a
nd p
lays
a ro
le i
n
endocy
tosi
s an
d t
raff
ickin
g o
f ce
ll s
urf
ace
rece
pto
rs.
Giv
en t
hat
Src
bin
ds
to F
AK
and
oft
en si
gnal
s in
ta
ndem
w
ith it
, w
e al
so ex
amin
ed th
e poss
ibil
ity th
at S
rc co
uld
phosp
hory
late
S
H3P
X1.
In
th
is
chap
ter,
w
e pro
vid
e ev
iden
ce
that
F
AK
an
d
its
signal
ing
par
tner
, S
rc,
can
phosp
hory
late
S
H3P
X1
on
tyro
sine
resi
dues
th
at
are
dis
tinct
fro
m t
hose
phosp
hory
late
d b
y A
CK
2.
FA
K P
hosp
hory
late
s S
H3P
X1 I
n v
ivo
In o
ur
pre
vio
us
work
, w
e hav
e sh
ow
n t
hat
AC
K2
can
phosp
hory
late
SH
3P
X1
both
in
vi
vo an
d in
vi
tro.
U
sing a
sim
ilar
ap
pro
ach,
we
exam
ined
w
het
her
F
AK
phosp
hory
late
s S
H3P
X1.
W
e fi
rst
co-t
ransf
ecte
d C
OS
-7 ce
lls
wit
h S
H3P
X1 an
d
eith
er w
ild-t
ype
FA
K,
the
kin
ase-
def
icie
nt
FA
K-Y
397F
muta
nt
or
wil
d-t
ype
AC
K2.
The
foll
ow
ing d
ay,
the
cell
s w
ere
pla
ced i
n m
ediu
m w
ithout
seru
m f
or
24 h
ours
, an
d
the
des
ignat
ed s
ample
s w
ere
stim
ula
ted w
ith c
om
ple
te m
edia
(co
nta
inin
g 1
0%
FB
S)
for
20
min
ute
s.
A
ll
of
the
cell
cu
lture
s w
ere
then
ly
sed
and
SH
3P
X1
was
imm
unopre
cipit
ated
fr
om
th
e w
hole
ce
ll
extr
acts
w
ith
anti
-V5
anti
body.
T
he
imm
unopre
cipit
ated
sa
mple
s w
ere
subje
cted
to
W
este
rn
blo
t an
alysi
s w
ith
an
ti-
phosp
hoty
rosi
ne
anti
body.
Fig
ure
3.1
show
s th
at w
hen
SH
3P
X1 w
as e
xpre
ssed
alo
ne
in C
OS
-7 c
ells
, no p
hosp
hory
lati
on w
as d
etec
ted (
Fig
ure
3.1
, la
ne
3).
C
onsi
sten
t w
ith
pre
vio
us
findin
gs,
SH
3P
X1 s
how
ed a
sli
ght,
but
consi
sten
t pho
sphory
lati
on w
hen
co-
expre
ssed
wit
h A
CK
2 (
Fig
ure
3.1
, la
ne
4).
T
his
phosp
hory
lati
on w
as n
ot
det
ecte
d
122
Fig
ure
3.1
F
AK
ph
osp
hory
late
s S
H3P
X1 i
n c
ells
.
CO
S-7
cel
ls w
ere
tran
sfec
ted w
ith t
he
var
ious
expre
ssio
n p
lasm
ids
indic
ated
. T
wen
ty-
four
to f
ort
y-e
ight
hours
aft
er t
ransf
ecti
on,
the
cell
s w
ere
pla
ced
in m
ediu
m c
onta
inin
g
1%
FB
S f
or
an a
ddit
ional
day
. J
ust
pri
or
to c
ell
lysi
s, s
om
e of
the
cell
cult
ure
s w
ere
stim
ula
ted w
ith m
ediu
m c
onta
inin
g 1
0%
FB
S f
or
20 m
inute
s.
V5-t
agged
SH
3P
X1
was
im
munopre
cipit
ated
w
ith an
ti-V
5 an
tibody an
d th
e pre
cipit
ated
sa
mple
s w
ere
reso
lved
by
SD
S-P
AG
E
and
then
su
bje
cted
to
W
este
rn
blo
ttin
g
wit
h
anti
-
phosp
hoty
rosi
ne
anti
body a
nd a
nti
-V5 a
nti
body.
123
124
when
SH
3P
X1 w
as e
xpre
ssed
wit
h a
kin
ase-
def
ecti
ve
form
of
AC
K2,
AC
K2-K
158R
(Fig
ure
3.1
, la
ne
5).
H
ow
ever
, w
hen
FA
K w
as c
o-e
xpre
ssed
wit
h S
H3P
X1,
a ro
bust
tyro
sine
phosp
hory
lati
on o
f S
H3P
X1 w
as d
etec
ted (
Fig
ure
3.1
, co
mpar
e la
ne
4 t
o l
ane
6).
W
hen
quan
tita
ted,
the
exte
nt
of
SH
3P
X1 p
ho
sphory
lati
on b
y F
AK
was
more
than
10 f
old
gre
ater
than
that
of
AC
K2.
Expre
ssio
n o
f th
e kin
ase-
def
ecti
ve
FA
K-Y
397F
muta
nt
did
not
resu
lt
in
the
tyro
sine
phosp
hory
lati
on
of
SH
3P
X1,
even
under
condit
ions
wher
e th
e se
rum
-sta
rved
sam
ple
s w
ere
stim
ula
ted w
ith m
ediu
m c
onta
inin
g
10%
FB
S (
Fig
ure
3.1
, la
nes
8 a
nd 9
).
Tak
en t
oget
her
, th
ese
findin
gs
indic
ate
that
incr
ease
s in
FA
K a
ctiv
ity i
n c
ells
lea
ds
to a
n e
xte
nt
of
SH
3P
X1 p
hosp
hory
lati
on t
hat
is s
ignif
ican
tly g
reat
er t
han
that
cat
alyze
d b
y A
CK
2.
We
nex
t as
ked
whet
her
FA
K c
an d
irec
tly p
hosp
hory
late
SH
3P
X1.
In o
rder
to
addre
ss t
his
ques
tion,
we
turn
ed t
o a
n i
n v
itro
kin
ase
assa
y.
His
-tag
ged
SH
3P
X1 w
as
expre
ssed
and p
uri
fied
fro
m b
acte
rial
cel
ls w
her
eas
His
-tag
ged
FA
K w
as
expre
ssed
and p
uri
fied
fro
m i
nse
ct c
ells
. K
inas
e re
acti
ons
wer
e ca
rrie
d o
ut
at r
oom
tem
per
ature
for
up t
o 2
hours
. F
igure
3.2
A s
how
s a
repre
sen
tati
ve
auto
radio
gra
ph o
f th
e kin
ase
assa
y.
The
FA
K-c
atal
yze
d p
hosp
hory
lati
on o
f S
H3P
X1 w
as d
etec
ted a
s ear
ly a
s 15
min
ute
s, w
ith m
axim
al p
hosp
hory
lati
on o
ccurr
ing w
ithin
45 m
inute
s.
The
resu
ltin
g
subst
rate
phosp
hory
lati
on w
as
also
quan
tifi
ed b
y e
xci
sing t
he
SH
3P
X1 b
and
s fr
om
the
gel
s an
d t
hen
dir
ectl
y m
easu
ring [
32P
]-phosp
hat
e in
corp
ora
tion u
sing a
sci
nti
llat
ion
counte
r.
Fig
ure
3.2
B s
how
s th
at F
AK
sti
mula
ted t
he
inco
rpora
tion o
f [3
2P
]-A
TP
into
SH
3P
X1
by
nea
rly
25-f
old
over
co
ntr
ol
cell
s.
T
hes
e fi
ndin
gs
concl
usi
vel
y
dem
onst
rate
that
FA
K c
an d
irec
tly p
hosp
hory
late
SH
3P
X1.
Src
Als
o A
cts
as
a K
inase
for
SH
3P
X1
FA
K a
nd S
rc o
ften
cooper
ate
in c
ells
as
a si
gnal
ing c
om
ple
x.
The
mec
han
ism
for
this
cooper
atio
n h
as b
een w
ell
esta
bli
shed
and i
s in
itia
ted w
ith t
he
125
Fig
ure
3.2
F
AK
ph
osp
hory
late
s S
H3P
X1 i
n v
itro.
Rec
om
bin
ant
FA
K (
8.3
ng
) an
d S
H3P
X1 (
500 n
g)
wer
e ad
ded
to
a k
inas
e r
eact
ion
mix
ture
conta
inin
g 5
0 m
M T
ris-
HC
l (p
H 7
.4),
2 m
M M
gC
l 2,
10 m
M M
nC
l 2,
1 µ
M
AT
P,
10 µ
Ci
[32P
]-A
TP
. T
he
kin
ase
reac
tion w
as
allo
wed
to p
roce
ed f
or
the
indic
ated
tim
e in
terv
als
and t
hen
the
reac
tion
was
quen
ched b
y t
he
addit
ion o
f 5x
SD
S s
ample
buff
er.
SD
S s
ample
buff
er (
5x)
was
added
to t
he
t=0 s
ample
bef
ore
the
addit
ion o
f
kin
ase.
E
ach
kin
ase
reac
tion
was
re
solv
ed
by
SD
S-P
AG
E,
tran
sfer
red
to
PV
DF
mem
bra
ne,
and e
xpose
d t
o x
-ray
fil
m.
The
sam
e b
lot
was
then
pro
bed
wit
h a
nti
-His
anti
body t
o s
how
that
the
amount
of
reco
mbin
ant
SH
3P
X1 w
as p
rese
nt
in e
ach k
inase
reac
tion (
A).
A
lter
nat
ivel
y,
kin
ase
reac
tion m
ixtu
res
wer
e re
solv
ed b
y S
DS
-PA
GE
and s
tain
ed w
ith C
oom
ass
ie b
lue.
B
ands
of
SH
3P
X1 w
ere
exci
sed a
nd t
he e
xte
nt
of
phosp
hory
lati
on w
as r
ead o
ut
by
det
erm
inin
g t
he a
mount
of
[32P
]-A
TP
inco
rpora
ted
into
SH
3P
X1 u
sing a
sci
nti
llat
ion c
ounte
r (B
).
126
127
auto
phosp
hory
lati
on o
f F
AK
at
tyro
sine
397.
This
cre
ates
a b
indin
g s
ite
for
the
SH
2-
dom
ain o
f S
rc [
26].
T
he
bin
din
g o
f S
rc t
o F
AK
all
ow
s S
rc t
o f
urt
her
phosp
hory
late
FA
K a
t ad
dit
ional
sit
es i
ncl
udin
g r
esid
ues
407,
57
6,
577,
861,
and 9
25,
whic
h l
ead
s to
enhan
ced
FA
K
acti
vit
y
and
a
subse
quen
t in
crea
se
in
the
pho
sphory
lati
on
of
dow
nst
ream
eff
ecto
rs [
27]
[26,
28].
Thus,
w
e w
ere
inte
rest
ed
in
sein
g
whet
her
S
rc
mig
ht
also
ca
taly
ze
the
phosp
hory
lati
on o
f S
H3P
X1.
To t
est
this
poss
ibil
ity,
we
took t
he
sam
e ap
pro
ach a
s
we
did
to e
stab
lish
that
FA
K p
hosp
hory
late
s S
H3
PX
1.
HE
K 2
93 c
ells
expre
ssin
g S
rc
and S
H3P
X1 w
ere
lyse
d a
nd
SH
3P
X1 w
as i
mm
unopre
cipit
ated
fro
m t
he
whole
cel
l
lysa
tes
wit
h
anti
-V5
anti
body.
Im
munoblo
t an
alysi
s of
the
imm
unopre
cipit
ated
sam
ple
s w
ith a
nti
-phosp
hoty
rosi
ne
anti
body s
how
ed t
hat
Src
expre
ssio
n r
esult
ed i
n a
pote
nt
phosp
hory
lati
on
of
SH
3P
X1
(Fig
ure
3.3
).
T
he
phosp
hory
lati
on
signal
gen
erat
ed b
y S
rc w
as
signif
ican
tly a
nd
consi
sten
tly e
nhan
ced o
ver
that
obse
rved
wit
h
FA
K (F
igure
3.3
, co
mpar
e la
nes
2 an
d 4).
In
tere
stin
gly
, w
e did
not
obse
rve
an
incr
ease
in
S
H3P
X1
phosp
hory
lati
on
in
sam
ple
s w
her
e S
rc
and
FA
K
wer
e both
expre
ssed
wit
h S
H3P
X1 (
Fig
ure
3.3
, co
mpar
e la
nes
4 a
nd 5
), s
ugges
ting t
hat
Src
can
phosp
hory
late
eac
h o
f th
e si
tes
use
d b
y F
AK
. F
igure
3.3
(la
ne
6)
show
s th
at t
he
kin
ase-
def
icie
nt
FA
K-F
397
muta
nt,
w
as
unab
le
to
blo
ck
Src
-cat
alyze
d
phosp
hory
lati
on o
f S
H3P
X1.
W
e th
en e
xam
ined
whet
her
Src
can
dir
ectl
y p
hosp
hory
late
SH
3P
X1.
Fig
ure
3.4
show
s th
at t
he S
rc-c
atal
yze
d p
ho
sphory
lati
on o
f S
H3P
X1 w
as s
een
as
earl
y a
s 5
min
ute
s, a
nd t
hat
the
phosp
hory
lati
on s
ignal
conti
nued
to i
ncr
ease
thro
ugh 2
hours
.
Th
e P
hosp
hory
lati
on
of
SH
3P
X1 b
y F
AK
an
d S
rc D
iffe
rs f
rom
th
at
of
AC
K2
We
hav
e sh
ow
n
that
A
CK
2-c
atal
yzed
phosp
hory
lati
on
of
SH
3P
X1
is
elim
inat
ed w
hen
84 a
min
o a
cids
from
the
carb
oxyl-
term
inal
end w
ere
rem
oved
. T
his
128
Fig
ure
3.3
S
rc i
s a m
ore
eff
ecti
ve
kin
ase
for
SH
3P
X1 i
n c
ells
.
V5-t
agged
SH
3P
X1 w
as t
ransf
ecte
d a
long w
ith w
ith e
ither
HA
-tag
ged
Src
or
HA
-
tagged
FA
K i
n H
EK
293 c
ells
. A
ppro
xim
atel
y 2
4 h
ours
foll
ow
ing t
ransf
ecti
on,
the
cell
s w
ere
lyse
d.
SH
3P
X1 w
as i
mm
unopre
cipit
ated
wit
h a
nti
-V5 a
nti
body f
rom
cel
l
lysa
tes,
an
d
then
re
solv
ed
by
SD
S-P
AG
E.
T
he
gel
s w
ere
tran
sfer
red
to
PV
DF
mem
bra
ne
and s
uje
cted
to W
este
rn b
lott
ing a
nal
ysi
s w
ith a
nti
-phosp
hoty
rosi
ne
and
anti
-V5 a
nti
bodie
s.
129
130
Fig
ure
3. 4 S
rc p
hosp
hory
late
s S
H3P
X1 i
n v
itro.
Rec
om
bin
ant
Src
(5
0 ng)
and S
H3P
X1 (2
00 ng)
wer
e ad
ded
to
a
kin
ase
reac
tion
mix
ture
conta
inin
g 5
0 m
M T
ris-
HC
l (p
H 7
.4),
2 m
M M
gC
l 2,
10 m
M M
nC
l 2,
1 µ
M
AT
P,
10 µ
Ci
[32P
]-A
TP
. T
he
kin
ase
reac
tion w
as
allo
wed
to p
roce
ed f
or
the
indic
ated
tim
e in
terv
als
and t
hen
the
reac
tion
was
quen
ched b
y t
he
addit
ion o
f 5x
SD
S s
ample
buff
er.
SD
S s
ample
buff
er (
5x)
was
added
to t
he
t=0 s
ample
bef
ore
the
addit
ion o
f
kin
ase.
E
ach
kin
ase
reac
tion
was
re
solv
ed
by
SD
S-P
AG
E,
tran
sfer
red
to
PV
DF
mem
bra
ne,
and e
xpose
d t
o x
-ray
fil
m.
131
132
is l
ikel
y d
ue
to t
he
fact
that
AC
K2 i
s unab
le t
o b
ind t
o t
hes
e m
uta
nts
. A
fter
we
iden
tifi
ed F
AK
and S
rc a
s p
rote
ins
capab
le o
f phosp
hory
lati
ng S
H3P
X1,
we
wer
e
inte
rest
ed i
n c
om
par
ing t
hei
r ab
ilit
y t
o b
ind t
o S
H3P
X1.
Com
bin
atio
ns
of
FA
K,
Src
,
and S
H3P
X1 w
ere
tran
sfec
ted
into
HE
K 2
93 c
ells
, an
d t
hen
the
cell
s w
ere
lyse
d a
nd
FA
K
or
Src
w
as
imm
unopre
cipit
ated
w
ith
anti
-HA
an
tibody.
T
he
imm
unopre
cipit
atio
n s
ample
s w
ere
subje
cted
to W
este
rn b
lott
ing a
nal
ysi
s w
ith a
nti
-
V5 a
nti
body t
o d
etec
t as
soci
ated
SH
3P
X1.
Fig
ure
3.5
show
s th
at d
espit
e a
robust
expre
ssio
n o
f both
FA
K a
nd
Src
, S
H3P
X1 d
id n
ot
co-i
mm
unopre
cipit
ate
wit
h e
ither
pro
tein
. S
ince
w
e hav
e dem
onst
rate
d th
at both
F
AK
an
d S
rc ca
n phosp
hory
late
SH
3P
X1,
but
we
do n
ot
det
ect
an a
ssoci
atio
n b
etw
een F
AK
or
Src
and
SH
3P
X1,
we
can i
nfe
r th
at t
he
kin
ase-
sub
stra
te i
nte
ract
ions
bet
wee
n t
hes
e pro
tein
s ar
e tr
ansi
ent,
whic
h a
llow
for
rapid
enzy
mat
ic t
urn
over
. O
ne
way
of
test
ing t
his
hypoth
esis
would
be
to a
ttem
pt
to t
rap t
he
kin
ase
-sub
stra
te i
nte
ract
ion b
y u
sing k
inase
-def
ecti
ve
muta
nts
of
FA
K a
nd S
rc.
We
then
ask
ed w
het
her
FA
K a
nd S
rc p
hosp
hory
late
d t
he
sam
e si
tes
as A
CK
2.
We
took
advan
tage
of
the
C-t
erm
inal
del
etio
n
const
ruct
s of
SH
3P
X1
that
w
ere
pre
vio
usl
y
use
d
to
inves
tigat
e A
CK
2-c
atal
yze
d
phosp
hory
lati
on
of
SH
3P
X1.
A
schem
atic
of
thes
e c
onst
ruct
s is
show
n i
n F
igure
3.6
A.
All
of
thes
e c
onst
ruct
s, w
hen
co-e
xpre
ssed
alo
ng w
ith A
CK
2 i
n c
ells
, w
ere
able
to c
om
ple
tely
blo
ck t
he
abil
ity o
f
AC
K2 t
o b
ind t
o a
nd p
hosp
hory
late
SH
3P
X1 (
see
Fig
ure
2.4
in C
hap
ter
2).
T
hes
e
resu
lts
sugges
t th
at del
etin
g th
e ca
rboxyl-
term
inal
84 am
ino ac
ids
of
SH
3P
X1 is
suff
icie
nt
to e
lim
inat
e A
CK
2’s
abil
ity t
o p
hosp
hory
late
SH
3P
X1.
Fig
ure
s 3.6
and 3
.7
show
the
resu
lts
of
exper
imen
ts w
her
e F
AK
and S
rc,
resp
ecti
vel
y,
wer
e ex
amin
ed f
or
thei
r ab
ilit
ies
to p
hosp
hory
late
the
var
ious
C-t
erm
inal
tru
nca
tion m
uta
nts
of
SH
3P
X1.
Bri
efly
, th
ese
exper
imen
ts w
ere
carr
ied o
ut
by c
oex
pre
ssin
g F
AK
or
Src
wit
h e
ach
SH
3P
X1 d
elet
ion m
uta
nt,
SH
3P
X1-!
C84, !
C197, an
d !
C340 i
n H
EK
293 c
ells
. T
he
133
Fig
ure
3.5
F
AK
an
d S
rc d
o n
ot
bin
d t
o S
H3P
X1.
Pla
smid
s en
codin
g e
ither
HA
-FA
K o
r H
A-S
rc w
ere
co-t
ransf
ecte
d w
ith V
5-S
H3P
X1
in C
OS
-7 c
ells
. A
ppro
xim
atel
y 4
8 h
ours
foll
ow
ing t
he
tran
sfec
tion,
the
cell
s w
ere
lyse
d.
HA
-tag
ged
FA
K (
A)
or
Src
(B
) w
ere
imm
unopre
cipit
ated
fro
m w
hole
cel
l
lysa
tes
wit
h a
nti
-HA
anti
body
and r
esolv
ed b
y S
DS
-PA
GE
. T
he
gel
s w
ere
tran
sfer
red
to P
VD
F a
nd t
hen
the
blo
t w
as s
ubje
cted
to W
est
ern b
lott
ing a
nal
ysi
s usi
ng a
nti
-V5
anti
body t
o d
etec
t S
H3P
X1 b
indin
g.
134
135
Fig
ure
3.6
F
AK
ph
osp
hory
late
s th
e ca
rboxyl-
term
inal
SH
3P
X1 d
elet
ion
mu
tan
ts
!C
84, !
C197,
an
d !
C340.
A s
chem
atic
rep
rese
nta
tion o
f th
e S
H3P
X1 C
-ter
min
al
del
etio
n c
onst
ruct
s th
at w
ere
use
d i
n t
hese
stu
die
s (A
). T
he
HA
-FA
K p
lasm
id w
as c
o-
tran
sfec
ted w
ith t
he
var
ious
C-t
erm
inal
del
etio
n c
onst
ruct
s of
SH
3P
X1 o
utl
ined
ab
ove.
About
24 h
ours
foll
ow
ing t
ransf
ecti
on,
the
cell
s w
ere
lyse
d a
nd t
he d
elet
ion c
onst
ruct
s
of
SH
3P
X1 w
ere
imm
unop
reci
pit
ated
fro
m t
he
cell
extr
acts
usi
ng a
nti
-Myc
anti
body.
The
sam
ple
s w
ere
reso
lved
by S
DS
-PA
GE
, tr
ansf
erre
d t
o P
VD
F,
and i
mm
unoblo
ting
anal
ysi
s w
ith a
nti
-phosp
hoty
rosi
ne
and a
nti
-Myc
anti
bodie
s w
as p
erfo
rmed
(B
).
136
137
Fig
ure
3.7
S
rc p
hosp
hory
late
s th
e ca
rboxyl-
term
inal
SH
3P
X1 d
elet
ion
mu
tan
ts
!C
84, !
C197,
an
d !
C340.
A s
chem
atic
rep
rese
nta
tion o
f th
e S
H3
PX
1 C
-ter
min
al
del
etio
n c
onst
ruct
s gen
erat
ed (
A).
The
HA
-Src
pla
smid
was
co-t
ransf
ecte
d w
ith t
he
var
ious
C-t
erm
inal
del
etio
n
const
ruct
s of
SH
3P
X1.
A
ppro
xim
atel
y
24
hours
foll
ow
ing t
ransf
ecti
on,
the
cell
s w
ere
lyse
d a
nd
the
del
etio
n c
on
stru
cts
of
SH
3P
X1
wer
e im
munopre
cipit
ated
fr
om
th
e ce
ll
extr
acts
usi
ng
anti
-Myc
anti
body.
T
he
sam
ple
s w
ere
reso
lved
b
y
SD
S-P
AG
E,
tran
sfer
red
to
PV
DF
an
d
imm
unoblo
ting
anal
ysi
s w
ith a
nti
-phosp
hoty
rosi
ne
and a
nti
-Myc
anti
bodie
s w
as p
erfo
rmed
(B
).
138
139
cell
s w
ere
then
lyse
d a
nd t
he
SH
3P
X1 m
uta
nts
wer
e im
munopre
cipit
ated
wit
h a
nti
-HA
anti
body
and
anal
yze
d
by
Wes
tern
blo
ttin
g
wit
h
anti
-phosp
hoty
rosi
ne
anti
body.
Contr
ary t
o w
hat
was
ob
serv
ed w
ith A
CK
2,
we
found t
hat
FA
K (
Fig
ure
3.6
B)
and S
rc
(Fig
ure
3.7
B)
not
only
phosp
hory
late
d t
he
SH
3P
X1-!
C84 c
onst
ruct
, but
they
ret
ained
the
abil
ity t
o p
hosp
hory
late
all
of
the S
H3P
X1 C
-ter
min
al d
elet
ion m
uta
nts
. T
hus,
the
dat
a su
ggest
th
at F
AK
an
d S
rc li
kel
y pho
sphory
late
dis
tinct
re
sidues
on
S
H3P
X1
rela
tive
to A
CK
2.
Tyro
sin
e R
esid
ues
177,
23
9,
269,
294,
an
d 5
61 a
re P
hosp
hory
late
d I
n v
ivo b
y S
rc
Our
labora
tory
an
d
oth
ers
hav
e sh
ow
n
that
ty
rosi
ne
phosp
hory
lati
on
of
SH
3P
X1
has
co
nse
quen
tial
ef
fect
s on
EG
F
rece
pto
r pro
cess
ing
and
dynam
in
traf
fick
ing [
25,
29].
In
par
ticu
lar,
AC
K2
-cat
alyze
d p
hosp
hory
lati
on o
f S
H3P
X1 i
s
crit
ical
fo
r th
e deg
radat
ion
of
EG
F
rece
pto
rs
in
cell
s [2
9],
an
d
SH
3P
X1
phosp
hory
lati
on i
s th
ought
to b
e im
port
ant
for
loca
lizi
ng d
ynam
in-2
to s
ites
on t
he
pla
sma
mem
bra
ne
wh
ere
endocy
tic
bud
s fo
rm.
[25].
G
iven
the
appar
ent
import
ance
of
SH
3P
X1 p
hosp
hory
lati
on o
n E
GF
rec
epto
r pro
cess
ing,
and t
he
fact
that
we
hav
e
iden
tifi
ed F
AK
and S
rc a
s novel
kin
ases
that
can
phosp
hory
late
SH
3P
X1,
we
wer
e
inte
rest
ed in
id
enti
fyin
g th
e in
div
idual
phosp
hory
lati
on si
tes
on S
H3P
X1 th
at ar
e
phosp
hory
late
d b
y F
AK
and S
rc.
We
turn
ed t
o m
ass
spec
trom
etry
as
a m
eans
to
iden
tify
the
site
s of
tyro
sine
phosp
hory
lati
on o
n S
H3P
X1.
In o
rder
to e
nsu
re a
via
ble
sam
ple
for
mas
s sp
ectr
om
etry
, w
e to
ok a
dvan
tage
of
Src
-cat
alyze
d p
hosp
hory
lati
on o
f
SH
3P
X1,
as i
t ex
hib
ited
the
most
act
ivit
y i
n c
ells
and i
n v
itro.
Bas
ed o
n o
ur
findin
gs
show
ing
that
co
-expre
ssio
n
of
SH
3P
X1
and
S
rc
in
cell
s re
sult
ed
in
robust
phosp
hory
lati
on of
SH
3P
X1 (s
ee F
igure
3.3
), w
e dec
ided
to
u
se th
is ap
pro
ach to
gen
erat
e a
sam
ple
fo
r m
ass
spec
trom
etry
an
alysi
s. In
bri
ef,
HE
K 293 ce
lls
wer
e
140
tran
sfec
ted
wit
h
HA
-Src
an
d
V5-S
H3P
X1.
T
wen
ty-f
our
hours
fo
llow
ing
the
tran
sfec
tion,
the
cell
s w
ere
lyse
d
and
V5-S
H3P
X1
was
im
munopre
cipit
ated
fr
om
appro
xim
atel
y
10
mg
of
tota
l cel
l ex
trac
t u
sing
an
anti
-V5
anti
body.
T
he
imm
unopre
cipit
atio
ns
wer
e div
ided
and
each
set
was
res
olv
ed o
n s
epar
ate S
DS
-PA
GE
gel
s.
One
gel
was
sta
ined
wit
h C
oom
ass
ie b
lue t
o v
isual
ize
all
pro
tein
s th
at c
ame
dow
n i
n t
he
imm
unopre
cipit
atio
n w
hil
e th
e se
cond g
el w
as t
ransf
erre
d t
o P
VD
F a
nd
then
subje
cted
to W
est
ern b
lot
anal
ysi
s w
ith a
n a
nti
-phosp
hoty
rosi
ne
anti
body (
Fig
ure
3.8
B).
The
imm
unoblo
t an
alysi
s co
nfi
rmed
th
at
the
SH
3P
X1
was
in
fact
phosp
hory
late
d b
y S
rc.
The
ban
d c
orr
espondin
g t
o p
hosp
hory
late
d S
H3P
X1 o
n t
he
Coom
assi
e-st
ained
gel
(F
igure
3.8
A)
was
then
exci
sed a
nd t
he
sam
ple
subm
itte
d t
o t
he
Corn
ell
Bio
tech
nolo
gy
Res
ourc
e C
ente
r P
rote
om
ics
and
Mas
s S
pec
trom
etry
C
ore
Fac
ilit
y.
The
resu
lts
of
the
mas
s sp
ectr
om
etry
anal
ysi
s id
enti
fied
tyro
sine r
esid
ues
177,
239,
269,
294,
and 5
61 a
s S
rc-c
atal
yze
d p
hosp
hory
lati
on s
ites
(T
able
3.1
).
The
resi
due
and p
epti
de
sequen
ce
show
n i
n t
he
firs
t tw
o c
olu
mns
of
the
table
ref
er t
o t
he
indiv
idual
phosp
ho-p
epti
des
iden
tifi
ed b
y l
iquid
chro
mat
ogra
phy (
LC
) M
S-M
S,
whil
e
M/Z
rat
io r
efer
s to
the
mas
s-to
-char
ge
rati
o o
f th
e pep
tides
. T
he
mascot
score r
efer
s to
an al
go
rith
m pro
gra
m use
d to
det
erm
ine
the
likel
ihood of
phosp
hory
lati
on (3
0 is
consi
der
ed t
he
bas
elin
e),
and %
Pi
refe
rs t
o t
he
per
centa
ge
each
tyro
sine
resi
due
was
phosp
hory
late
d in
our
sam
ple
. F
igure
3.9
is
an
ex
ample
of
a M
S/M
S sp
ectr
um
show
ing t
he
smal
ler
ions
dev
eloped
fro
m t
he
seco
nd r
ound o
f m
ass
spec
trom
etry
. I
t is
wort
h m
enti
onin
g t
hat
anoth
er s
ample
of
imm
unopre
cipit
ated
SH
3P
X1 w
as s
ubm
itte
d
for
mas
s sp
ectr
om
etry
an
alysi
s fr
om
ce
lls
in
whic
h
SH
3P
X1,
alone,
w
as
over
expre
ssed
in
HE
K 2
93 c
ells
. T
he
mas
s sp
ect
rom
etry
res
ult
s id
enti
fied
no t
yro
sin
e
phosp
hory
late
d
resi
dues
, th
us,
fu
rther
co
nfi
rmin
g
that
S
rc-c
atal
yze
d
the
phosp
hory
lati
on s
ites
on S
H3P
X1 i
den
tifi
ed b
y m
ass
spec
trom
etry
anal
ysi
s.
141
Fig
ure
3.8
Pre
para
tion
of
the
ph
osp
hory
late
d
SH
3P
X1
sam
ple
fo
r m
ass
spec
trom
etry
an
aly
sis.
H
A-S
rc a
nd V
5-S
H3P
X1 w
ere
co-e
xpre
ssed
in H
EK
293
cell
s.
V5-S
H3P
X1 w
as
imm
unopre
cipit
ated
fro
m t
he
whole
cel
l ly
sate
s w
ith a
nti
-V5
anti
body,
reso
lved
by S
DS
-PA
GE
, an
d s
tain
ed w
ith C
oom
assi
e.
The
ban
d c
onta
inin
g
SH
3P
X1 w
as e
xci
sed a
nd s
ubm
itte
d t
o t
he
Corn
ell
Mas
s S
pec
trom
etry
Core
Fac
ilit
y
(A).
A
liquots
fr
om
th
is sa
mple
w
ere
also
re
solv
ed by S
DS
-PA
GE
, tr
ansf
erre
d to
PV
DF
, an
d s
ubje
cted
to W
est
ern b
lott
ing a
nal
ysi
s w
ith a
nti
-phosp
hoty
rosi
ne
and a
nti
-
V5 a
nti
bodie
s (B
).
142
143
Tab
le 3
.1
Tyro
sin
e re
sid
ues
177,
239,
269,
294 a
nd
561 w
ere
iden
tifi
ed a
s S
rc-
cata
lyze
d
ph
osp
hory
lati
on
si
tes
on
S
H3P
X1
by
mass
sp
ectr
om
etry
. T
he
phosp
hory
late
d p
epti
des
are
lis
ted i
n t
he
far-
left
colu
mns.
M/Z
ref
ers
to t
he
mas
s-to
-
char
ge
rati
o o
f ea
ch p
epti
de,
mas
cot
score
ref
ers
to a
n a
lgori
thm
pro
gra
m u
sed t
o
det
erm
ine
the
likel
ihood o
f phosp
hory
lati
on (
30 i
s co
nsi
der
ed t
o b
e th
e base
line)
, an
d
%
phosp
hory
lati
on
refe
rs
to
the
per
centa
ge
of
each
ty
rosi
ne
resi
due
that
is
phosp
hory
late
d.
144
145
Fig
ure
3.9
D
epic
tion
of
the
MS
/MS
sp
ectr
um
fro
m t
he
pep
tid
e in
clu
din
g Y
294.
146
147
Foll
ow
ing t
he
iden
tifi
cati
on o
f th
e S
rc-c
atal
yze
d t
yro
sine
phosp
ho
ryla
tion s
ites
by
mas
s sp
ect
rom
etry
, w
e nex
t w
ante
d
to
confi
rm
thes
e re
sult
s usi
ng
a gen
etic
appro
ach.
S
ingle
an
d
mult
iple
ty
rosi
ne-
to-p
hen
yla
lanin
e poin
t m
uta
nts
w
ere
engin
eere
d
usi
ng
site
-dir
ecte
d
muta
gen
esi
s.
T
he
SH
3P
X1
muta
nts
gen
erat
ed
incl
uded
: Y
177F
, Y
239F
, Y
269F
, Y
294F
, Y
561F
, as
wel
l as
a m
uta
nt
in w
hic
h a
ll o
f
the
tyro
sine
resi
des
wer
e m
uta
ted,
Y177F
/Y239F
/Y269F
/Y294F
/Y561F
. C
ells
wer
e
co-t
ransf
ecte
d w
ith S
rc a
nd
eac
h o
f th
e S
H3P
X1
poin
t m
uta
nts
, an
d a
fter
24
hours
of
expre
ssio
n,
the
cell
s w
ere
lyse
d
and
the
vari
ous
poin
t m
uta
nts
of
SH
3P
X1
imm
unopre
cipit
ated
w
ith
anti
-V5
anti
body.
Im
munoblo
t an
alysi
s of
the
imm
unopre
cipit
ated
sam
ple
s w
ith a
nti
-phosp
hoty
rosi
ne
anti
body w
as p
erfo
rmed
and
the
resu
lts
are
show
n i
n F
igure
3.1
0.
The
blo
t in
dic
ates
that
only
the
SH
3P
X1-Y
239F
and S
H3P
X1-Y
177F
/Y239F
/Y269F
/Y29
4F
/Y561F
co
nst
ruct
s sh
ow
ed a
dim
inis
hed
capac
ity
to
be
phosp
hory
late
d
by
Src
.
In
the
SH
3P
X1-
Y177F
/Y239F
/Y269F
/Y294F
/Y561F
m
uta
nt,
nea
rly
all
of
the
Src
-med
iate
d
phosp
hory
lati
on o
f S
H3P
X1 w
as l
ost
, w
hil
e th
e 2
39 m
uta
nt
show
ed a
n a
ppro
xim
atel
y
60%
dec
reas
e in
the
amount
of
SH
3P
X1 p
hosp
hory
lati
on.
More
over
, w
e w
ent
on t
o
dem
onst
rate
that
FA
K-c
atal
yze
d t
he
phosp
hory
lati
on o
f S
H3P
X1 a
t th
e sa
me
site
s as
Src
(F
igure
3.1
1).
T
hes
e fi
ndin
gs
furt
her
support
the
idea
that
FA
K a
nd S
rc w
ork
in
tandem
to
phosp
hory
late
S
H3P
X1,
and
that
th
e m
ajor
site
of
FA
K
and
Src
phosp
hory
lati
on o
n S
H3P
X1 i
s Y
239.
Dis
cuss
ion
Endocy
tosi
s of
cell
surf
ace
rece
pto
rs i
s a
tightl
y r
egula
ted,
sequen
tial
pro
cess
in w
hic
h ce
ll su
rfac
e re
cepto
rs bec
om
e in
tern
aliz
ed in
to th
e cy
topla
sm.
C
lath
rin-
med
iate
d e
ndocy
tosi
s ca
n e
ither
occ
ur
const
ituti
vely
or
in r
esponse
to e
xte
rnal
sti
muli
.
Whil
e m
any p
rote
ins
hav
e bee
n i
mpli
cate
d i
n r
egula
ting e
ndocy
tosi
s, A
P-2
, cl
athri
n,
148
Fig
ure
3.1
0
SH
3P
X1
mu
tan
t Y
177F
/Y239F
/Y269F
/Y294F
/Y561
is
ph
osp
hory
lati
on
-def
ecti
ve,
wh
ere
tyro
sin
e 239 i
s th
e m
ajo
r si
te p
hosp
hory
late
d
by
Src
.
Pla
smid
s en
codin
g
HA
-Src
an
d
V5-S
H3P
X1
poin
t m
uta
nts
w
ere
co-
tran
sfec
ted i
nto
HE
K 2
93 c
ells
. A
ppro
xim
atel
y 2
4 h
ours
aft
er t
ransf
ecti
on,
cell
s w
ere
lyse
d a
nd t
he S
H3P
X1 p
oin
t m
uta
nts
wer
e im
mun
opre
cipit
ated
wit
h a
nti
-V5 a
nti
body.
The
pre
cipit
ated
sa
mple
s w
ere
reso
lved
by
SD
S-P
AG
E,
afte
r w
hic
h
they
w
ere
tran
sfer
red t
o P
VD
F a
nd s
ubje
cted
to W
est
ern b
lott
ing w
ith a
nti
-phosp
hoty
rosi
ne
and
anti
-V5 a
nti
bodie
s.
149
150
Fig
ure
3.1
1
F
AK
is
u
nab
le
to
ph
osp
hory
late
th
e S
H3P
X1
mu
tan
t
Y177F
/Y239F
/Y269F
/Y294F
/Y561,
wh
ere
the
majo
r si
te
of
ph
osp
hory
lati
on
ap
pea
rs t
o b
e ty
rosi
ne
239.
Pla
smid
s en
codin
g H
A-F
AK
and V
5-S
H3P
X1 p
oin
t
muta
nts
w
ere
co-t
ransf
ecte
d
into
H
EK
293
ce
lls.
Appro
xim
atel
y
24
hours
af
ter
tran
sfec
tion,
cell
s w
ere
lyse
d a
nd t
he
SH
3P
X1 p
oin
t m
uta
nts
wer
e im
munopre
cipit
ated
wit
h a
nti
-V5 a
nti
body.
The
pre
cipit
ated
sam
ple
s w
ere
reso
lved
by S
DS
-PA
GE
, af
ter
whic
h t
hey
wer
e tr
ansf
erre
d t
o P
VD
F a
nd s
ubje
cted
to W
este
rn b
lott
ing w
ith a
nti
-
phosp
hoty
rosi
ne
and a
nti
-V5 a
nti
bodie
s.
151
152
and d
ynam
in h
ave
bee
n s
how
n t
o h
ave
esse
nti
al r
ole
s in
this
cel
lula
r pro
cess
. B
rief
ly,
the
acti
on o
f A
P-2
ser
ves
to i
nit
iate
the
endocy
tic
pro
cess
by c
once
ntr
atin
g t
he
carg
o
in t
he
emer
gin
g e
ndocy
tic b
ud a
s w
ell
as r
ecru
itin
g c
lath
rin t
o t
he
pla
sma
mem
bra
ne
[18-2
1].
The
tris
kel
ion
stru
cture
of
clat
hri
n
pro
vid
es
the
geo
met
ric
const
rain
ts
nec
essa
ry f
or
the
mem
bra
ne
to a
cquir
e as
incr
easi
ng c
urv
ature
form
ing a
n i
nvag
inat
ed
pit
. A
s th
e cl
athri
n p
it c
onti
nues
to m
ature
, th
e fi
nal
ste
p i
nvolv
es f
issi
on t
hro
ugh t
he
acti
on o
f th
e G
TP
ase,
dynam
in [
22].
SH
3P
X1 i
s a r
ecen
tly i
den
tifi
ed p
rote
in t
hought
to f
unct
ion i
n e
ndocy
tosi
s.
It
has
bee
n s
how
n t
o b
ind t
o d
ynam
in i
n K
562 h
um
an e
ryth
role
ukem
ia c
ells
[24]
and t
o
stim
ula
te d
ynam
in’s
bas
al G
TP
ase
acti
vit
y i
n v
itro
[30].
More
over
, a
convin
cing r
ole
for
SH
3P
X1 i
n m
edia
ting e
ndocy
tosi
s w
as d
emonst
rate
d b
y t
he
mis
loca
liza
tion o
f
dynam
in fr
om
th
e pla
sma
mem
bra
ne of
cell
s d
eple
ted of
SH
3P
X1 ex
pre
ssio
n by
SH
3P
X1-R
NA
i [2
5].
Our
labora
tory
an
d
oth
ers
hav
e re
port
ed
that
S
H3P
X1
is
tyro
sine
phosp
hory
late
d,
how
ever
the
effe
cts
of
this
tyro
sine
pho
sphory
lati
on e
ven
t on
the
funct
ion o
f S
H3P
X1 h
ave
yet
to b
e fu
lly c
har
acte
rize
d.
In th
is st
udy,
we
hav
e ch
ose
n to
fo
cus
on
es
tabli
shin
g w
het
her
ad
dit
ional
pro
tein
kin
ases
asi
de f
rom
AC
K2 c
an p
hosp
hory
late
SH
3P
X1.
To t
his
end,
we h
ave
iden
tifi
ed
two
pre
vio
usl
y
unre
port
ed
kin
ases
fo
r S
H3P
X1,
FA
K
and
Src
.
We
dem
onst
rate
d t
hat
over
expre
ssio
n o
f ei
ther
of
thes
e kin
ases
resu
lted
in
robust
SH
3P
X1
phosp
hory
lati
on.
F
AK
w
as
init
iall
y
iden
tifi
ed as
a
pote
nti
al
candid
ate
bas
ed
on
sim
ilar
itie
s bet
wee
n t
he
kin
ase
dom
ains
of
AC
K2 a
nd
FA
K,
note
d i
n a
BL
AS
T s
earc
h
of
the
Nat
ional
C
ente
r of
Bio
tech
nolo
gy
Info
rmat
ion
(NC
BI)
dat
abas
e [3
1].
Its
addit
ional
role
in e
ndophil
in p
hosp
hory
lati
on f
urt
her
hin
ted a
t it
s pote
nti
al r
ole
, bas
ed
on t
he
sim
ilar
itie
s in
dom
ain s
truct
ure
and f
unct
ion o
f S
H3P
X1 a
nd e
ndophil
in [
32].
Src
th
en bec
ame
an ad
dit
ional
ca
ndid
ate
giv
en it
s know
n as
soci
atio
n an
d ta
ndem
signal
ing w
ith F
AK
in c
ells
.
153
When
we
firs
t in
itia
ted t
hes
e ex
per
imen
ts,
we
exp
ecte
d F
AK
and S
rc t
o e
xhib
it
sim
ilar
bin
din
g a
nd p
ho
sphory
lati
on p
atte
rns
as t
hose
ob
serv
ed w
ith A
CK
2.
How
ever
,
we
hav
e fo
und
that
FA
K a
nd S
rc a
ppear
to s
ignal
in a
dis
tinct
man
ner
. T
he
FA
K-
and
Src
-cat
alyze
d S
H3P
X1 p
hosp
hory
lati
on i
s al
most
10 f
old
gre
ater
than
that
of
AC
K2 i
n
cell
s.
This
obse
rvat
ion i
s li
kel
y d
ue
in p
art
to t
he
incr
ease
d c
atal
yti
c ac
tivit
ies
of
FA
K
and S
rc.
Of
the
two
kin
ases,
Src
-cat
alyze
d p
ho
sphory
lati
on o
f S
H3P
X1 w
as t
he m
ost
inte
nse
, a
pro
per
ty
we
too
k
advan
tage
of
in
the
iden
tifi
cati
on
of
five
pri
mar
y
phosp
hory
lati
on s
ites
on S
H3P
X1
by m
ass
spec
trom
etry
, ty
rosi
ne
resi
dues
177,
239,
269,
294,
and 5
61.
A
side
from
tyro
sine
561,
four
of
the
tyro
sine
resi
dues
, Y
177,
Y239,
Y269 a
nd Y
294 a
re j
ust
N-t
erm
inal
of
the
PX
dom
ain.
This
is
intr
iguin
g g
iven
rece
nt
report
s th
at t
yro
sine
phosp
hory
lati
on o
f th
is r
egio
n m
ay b
e cr
itic
al i
n t
arget
ing
the
endocy
tic
pro
tein
, dynam
in,
to th
e ce
ll su
rfac
e [2
5].
N
one
of
the
FA
K/S
rc-
cata
lyze
d p
hosp
hory
lati
on s
ites
appea
r to
be
shar
ed w
ith t
hose
sit
es p
ho
sphory
late
d b
y
AC
K2.
This
sugges
ts t
hat
these
dif
fere
nt
phosp
hory
lati
on e
ven
ts a
re l
ikel
y t
o h
ave
dis
tinct
fu
nct
ional
co
nse
quen
ces.
The
gen
erat
ion
of
SH
3P
X1
muta
nts
th
at
are
def
ecti
ve
for
FA
K/S
rc-c
atal
yze
d p
hosp
hory
lati
on, ver
sus
muta
nts
def
ecti
ve
for
AC
K2-
pro
mote
d phosp
hory
lati
on,
should
be
inval
uab
le in
es
tabli
shin
g th
e ro
le fo
r th
ese
dif
fere
nt
phosp
hory
lati
on e
ven
ts i
n e
ndocy
tosi
s an
d E
GF
rec
epto
r deg
radat
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154
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Cdc4
2
targ
et
AC
K2
inte
ract
s w
ith
sort
ing
nex
in
9
(SH
3P
X1)
to r
egula
te e
pid
erm
al
gro
wth
fact
or
rece
pto
r deg
radati
on.
J B
iol
Chem
, 2002. 277(1
2):
p. 10134-8
.
30.
Soule
t,
F.,
et
al
.,
SN
X9
regula
tes
dyn
am
in
ass
embly
and
is
requir
ed
for
effi
cien
t cl
ath
rin
-med
iate
d e
ndocy
tosi
s. M
ol
Bio
l C
ell,
2005.
16(4
): p
. 20
58-
67.
31.
Yan
g,
W.
and R
.A.
Cer
ione,
Clo
nin
g a
nd c
hara
cter
izati
on o
f a n
ove
l C
dc4
2-
ass
oci
ate
d ty
rosi
ne
kinase
, A
CK
-2,
from
bovi
ne
bra
in.
J B
iol
Chem
, 1997.
272(4
0):
p.
24819-2
4.
157
32.
Wu,
X., e
t al
., F
AK
-med
iate
d s
rc p
hosp
hory
lati
on o
f en
dophil
in A
2 i
nhib
its
endocy
tosi
s of
MT
1-M
MP
and p
rom
ote
s E
CM
deg
radati
on.
Dev
Cel
l, 2
005.
9(2
): p
. 185-9
6.
158
CH
AP
TE
R F
OU
R
CO
NC
LU
SIO
N
The
regula
tion o
f gro
wth
fac
tor
rece
pto
r le
vel
s and k
inas
e a
ctiv
ity a
t th
e cel
l
surf
ace
is c
riti
cal
in m
edia
ting s
ignal
ing p
athw
ays
involv
ed i
n m
itogen
esis
. A
lter
ed
expre
ssio
n le
vel
s an
d m
uta
tions
resu
ltin
g in
unre
gula
ted kin
ase
acti
vit
y ar
e oft
en
suff
icie
nt
to c
ause
abnorm
al c
ell
gro
wth
and
pro
life
rati
on.
Of
the
sever
al m
echan
ism
s
uti
lize
d
by
the
cell
to
m
ainta
in
norm
al
pro
life
rati
on,
rece
pto
r en
docy
tosi
s an
d
deg
radat
ion p
lay k
ey r
ole
s by e
nsu
ring t
he
pro
per
hom
eost
asis
of
rece
pto
r ty
rosi
ne
kin
ases
. SH
3P
X1 h
as r
ecen
tly b
een i
mpli
cate
d i
n t
he
endo
cyti
c pro
cess
. A
mem
ber
of
the
sort
ing n
exin
fam
ily,
SH
3P
X1 h
as n
ot
only
been
show
n t
o b
ind
to A
P-2
, cl
athri
n,
and d
ynam
in,
but
when
tyro
sine-
phosp
hory
late
d,
it h
as a
lso b
een s
how
n t
o r
ecru
it
dynam
in t
o t
he
pla
sma
mem
bra
ne.
A
s a
resu
lt,
we
wer
e in
tere
sted
in c
har
acte
rizi
ng
this
phosp
hory
lati
on e
ven
t, a
nd d
evel
opin
g p
ote
nti
al d
om
inan
t-neg
ativ
e m
uta
nts
that
mig
ht
be
use
ful
for
furt
her
exam
inin
g t
he
role
of
SH
3P
X1 i
n e
ndocy
tosi
s.
In c
hap
ter
2,
I ch
arac
teri
ze t
he
inte
ract
ion
s bet
wee
n A
CK
2 a
nd S
H3P
X1 a
nd
the
non-r
ecep
tor
tyro
sine
kin
ase,
AC
K2,
a C
dc4
2-s
pec
ific
eff
ecto
r pro
tein
. A
ddit
ional
ly,
I des
crib
e in
vitr
o k
inas
e ass
ays
use
d t
o i
den
tify
pyri
dopyri
mid
ine,
PD
158780,
as a
pote
nt
inhib
itor
of
AC
K2 k
inas
e ac
tivit
y.
In c
hap
ter
3,
I fo
cus
on t
he
iden
tifi
cati
on o
f F
AK
and S
rc a
s
novel
kin
ases
for
SH
3P
X1.
I s
how
that
FA
K a
nd S
rc a
re m
uch
more
eff
ecti
ve
at
phosp
hory
lati
ng S
H3P
X1 c
om
par
ed t
o A
CK
2
It i
s in
tere
stin
g t
hat
wit
h t
he
exce
pti
on o
f Y
561,
all
of
the
phosp
hory
lati
on s
ites
are
loca
ted w
ithin
100 a
min
o a
cids
of
the P
X d
om
ain,
whic
h h
as b
een
im
pli
cate
d i
n
targ
etin
g p
rote
ins
to t
he
pla
sma
mem
bra
ne
[1].
O
ther
lab
ora
tori
es h
ave
report
ed t
hat
the
phosp
hory
lati
on o
f S
H3P
X1 w
ithin
this
reg
ion (
resi
dues
151
-185)
is c
riti
cal
for
159
med
iati
ng t
he
traf
fick
ing o
f dynam
in-2
[2].
In
thes
e st
udie
s, t
yro
sine
phosp
hory
lati
on
elim
inat
es b
indin
g o
f th
e gly
coly
tic e
nzy
me,
ald
ola
se.
In t
he
abse
nce
of
aldola
se
bin
din
g,
SH
3P
X1 re
gai
ns
its
abil
ity to
as
soci
ate
wit
h th
e pla
sma
mem
bra
ne,
th
us
traf
fick
ing d
ynam
in t
o i
ts r
egio
n o
f ac
tion a
t th
e nec
k o
f cl
athri
n-c
oat
ed p
its.
In
a
more
gen
eral
exam
ple
, phosp
hory
lati
on o
f se
rine
412 o
f !
-arr
esti
n h
as b
een s
how
n t
o
loca
lize
the
endocy
tic
pro
tein
to t
he
cell
mem
bra
ne
wher
e it
is
involv
ed i
n i
nit
iati
ng
endocy
tosi
s of
the !
-adre
ner
gic
re
cepto
r [3
],
thus
esta
bli
shin
g
a
pre
ceden
t fo
r
phosp
hory
lati
on a
s a
mea
ns
of
regula
ting p
rote
in t
raff
ickin
g.
Sev
eral
re
port
s hav
e su
gges
ted
that
S
H3P
X1
has
th
e ab
ilit
y
to
dim
eriz
e
thro
ugh i
ts C
-ter
min
al B
AR
dom
ain,
a co
iled
-coil
moti
f m
ade
up o
f th
e la
st 2
0 a
min
o
acid
s.
In c
hap
ter
2,
I des
crib
e th
e in
abil
ity o
f A
CK
2 t
o b
ind t
o a
nd p
hosp
hory
late
SH
3P
X1 m
ole
cule
s th
at l
ack t
his
C-t
erm
inal
dom
ain.
How
ever
, F
AK
and S
rc a
re a
ble
to p
hosp
hory
late
thes
e sa
me
const
ruct
s.
This
rai
ses
the
poss
ibil
ity t
hat
the
monom
er-
dim
er
equil
ibri
um
of
SH
3P
X1
det
erm
ines
w
hic
h
site
s bec
om
e
avai
lable
fo
r
phosp
hory
lati
on b
y t
hes
e d
iffe
rent
kin
ases
. F
or
exam
ple
, w
e bel
ieve
that
SH
3P
X1
dim
eriz
atio
n c
ould
pote
nti
ally
med
iate
its
phosp
hory
lati
on b
y m
ult
iple
pro
tein
kin
ases
.
Dim
eriz
atio
n o
f S
H3P
X1 m
ight
allo
w f
or
AC
K2
-cat
alyze
d p
hosp
hory
lati
on,
wher
eas,
FA
K a
nd S
rc m
ight
pre
fere
nti
ally
phosp
hory
late
th
e m
onom
eric
form
. T
his
could
pro
vid
e a
mec
han
ism
by w
hic
h S
H3P
X1 a
ccom
odat
es d
iver
se,
upst
ream
sig
nal
s su
ch
that
it
can p
arti
cipat
e in
FA
K/S
rc-p
rom
ote
d r
ecru
itm
ent
of
dynam
in t
o e
ndocy
tic
site
s
at
the
cell
su
rfac
e an
d
AC
K2-m
edia
ted
EG
F
rece
pto
r so
rtin
g
and
deg
radat
ion.
How
ever
, th
e sp
ecif
ic m
ech
anis
ms
by w
hic
h p
hosp
hory
late
d S
H3P
X1 m
edia
tes
the
recr
uit
men
t of
dynam
in o
r th
e so
rtin
g o
f E
GF
rec
epto
rs a
re l
ikel
y t
o b
e co
mple
x a
nd
wil
l be
the
focu
s of
futu
re s
tudie
s in
our
labora
tory
. O
ne
such
lin
e of
study w
ill
involv
e fu
rther
char
acte
riza
tion o
f th
e in
tera
ctio
ns
of
SH
3P
X1 w
ith d
ynam
in.
We
hav
e beg
un t
o e
xam
ine
this
an
d a
s sh
ow
n i
n F
igure
4.1
, w
e hav
e fo
und t
hat
muta
ting
160
Fig
ure
4.1
A
CK
2 a
nd
Dyn
am
in b
ind
to S
H3P
X1 t
hro
ugh
th
e S
H3 d
om
ain
of
SH
3P
X1.
C
OS
-7
cell
s w
ere
tran
sfec
ted
wit
h
the
var
ious
expre
ssio
n
pla
smid
s
indic
ated
. T
wen
ty-f
our
to s
even
ty-t
wo h
ours
aft
er
tran
sfec
tion,
cell
s w
ere
lyse
d a
nd
AC
K2 (
A)
and d
ynam
in-2
(B
) w
ere
imm
unopre
cipit
ated
wit
h a
nti
-Myc
and a
nti
-HA
anti
bodie
s,
resp
ecti
vel
y.
P
reci
pit
ated
sa
mple
s w
ere
reso
lved
by
SD
S-P
AG
E
and
tran
sfer
red t
o P
VD
F.
The
PV
DF
mem
bra
ne
was
then
pro
bed
wit
h a
nti
-Myc,
anti
-HA
,
and a
nti
-V5 a
nti
bodie
s.
161
162
the
conse
rved
try
pto
phan
res
idue
in t
he
SH
3 d
om
ain o
f S
H3P
X1,
whic
h i
s es
senti
al
for
bin
din
g
to
pro
line-
rich
dom
ains
on
oth
er
pro
tein
s,
elim
inat
es
the
abil
ity
of
SH
3P
X1 t
o b
ind t
o e
ither
AC
K2 o
r dynam
in-2
(F
igure
4.1
, la
ne
2 o
f A
and
B).
G
iven
that
the
bin
din
g p
artn
ers
report
ed f
or
SH
3P
X1 h
ave
each
bee
n s
how
n t
o b
ind t
hro
ugh
the
SH
3 d
om
ain o
f th
e so
rtin
g n
exin
, it
is
likel
y t
hat
ther
e ar
e co
mpet
ing i
nte
ract
ions.
Our
resu
lts
also
su
gges
t co
ndit
ions
that
al
low
A
CK
2-c
atal
yze
d phosp
hory
lati
on of
SH
3P
X1 m
ay c
om
pro
mis
e it
s ab
ilit
y t
o b
ind t
o d
ynam
in (
Fig
ure
4.2
B,
com
par
e la
nes
1 a
nd 2
), a
s en
han
ced
co
-im
munopre
cipit
atio
n o
f dynam
in w
ith S
H3P
X1 i
s obse
rved
in
cell
s w
her
e S
H3P
X1
has
been
co
-expre
ssed
w
ith
kin
ase
-def
ecti
ve
AC
K2.
How
ever
, w
e hope
to u
se p
hosp
hory
lati
on-d
efec
tive
muta
nts
of
SH
3P
X1 i
n o
rder
to
ver
ify t
hes
e pre
lim
inar
y o
bse
rvat
ions.
S
pec
ific
ally
, w
e ar
e in
tere
sted
in c
arry
ing o
ut
imm
unofl
uore
scen
ce s
tudie
s w
ith c
ells
expre
ssin
g p
hosp
hory
lati
on
-def
ecti
ve
SH
3P
X1
muta
nts
and e
xam
inin
g h
ow
this
infl
uen
ces
the
loca
liza
tion o
f en
dogen
ous
dyn
amin
.
It w
ill
be
inte
rest
ing t
o s
ee w
het
her
the
SH
3P
X1 m
uta
nt
that
is
def
ecti
ve
for
AC
K2
enhan
ces
the
inte
ract
ions
bet
ween
dynam
in a
nd S
H3P
X1.
We
would
als
o l
ike
to s
ee
whet
her
S
H3P
X1 m
uta
nts
th
at ar
e def
ecti
ve
for
phosp
hory
lati
on by F
AK
an
d S
rc
affe
ct d
ynam
in l
oca
liza
tion.
In p
arti
cula
r, w
e w
ould
lik
e to
know
whet
her
FA
K/S
rc-
cata
lyze
d
phosp
hory
lati
on
of
SH
3P
X1
repre
sen
ts
the
regula
tory
cu
e th
at
recr
uit
s
dynam
in t
o t
he
cell
surf
ace.
If
so,
it w
ould
sugges
t th
at t
he
dif
fere
nt
phosp
hory
lati
on
even
ts b
y A
CK
2 v
ersu
s F
AK
and S
rc h
ave o
pp
osi
ng e
ffec
ts o
n S
H3P
X1-d
ynam
in
inte
ract
ions.
L
ikew
ise,
it
w
ill
be
inte
rest
ing to
se
e if
th
e S
H3P
X1 m
uta
nt
that
is
def
ecti
ve
for
AC
K2-c
atal
yze
d
phosp
hory
lati
on
blo
cks
EG
F
rece
pto
r so
rtin
g
and
deg
radat
ion a
s w
ould
be
sug
ges
ted f
rom
our
earl
ier
work
show
ing a
lin
k b
etw
een
AC
K2-m
edia
ted p
hosp
hory
lati
on o
f S
H3P
X1 o
n E
GF
rec
epto
r deg
radat
ion [
4].
A l
egit
imat
e ro
le f
or
SH
3P
X1 i
n c
lath
rin
-med
iate
d e
ndocy
tosi
s an
d r
ecep
tor
pro
cess
ing h
as b
een i
ncr
easi
ngly
est
abli
shed
. T
o d
ate,
SH
3P
X1 h
as b
een i
mpli
cate
d
163
Fig
ure
4.2
S
H3P
X1 b
ind
ing t
o d
yn
am
in i
s n
ot
med
iate
d b
y A
CK
2.
In
ad
dit
ion
,
dyn
am
in-2
bin
ds
to u
np
ho
sph
ory
late
d S
H3P
X1.
HA
-dynam
in-2
was
tra
nsf
ecte
d
wit
h e
ither
Myc-
AC
K2 o
r V
5-S
H3P
X1 i
n C
OS
-7 c
ells
. T
wen
ty-f
our
to s
even
ty-t
wo
hours
af
ter
tran
sfec
tion,
AC
K2
and
SH
3P
X1
wer
e im
munopre
cipit
ated
fr
om
ce
ll
lysa
tes
wit
h a
nti
-Myc
and a
nti
-V5 a
nti
bodie
s, r
espec
tivel
y.
Pre
cipit
ated
sam
ple
s w
ere
reso
lved
by S
DS
-PA
GE
and
tra
nsf
erre
d t
o P
VD
F.
The
PV
DF
mem
bra
ne
was
then
subje
cted
to W
est
ern b
lott
ing a
nal
ysi
s w
ith a
nti
-HA
to m
easu
re d
ynam
in-2
bin
din
g
(A).
C
OS
-7 c
ells
wer
e tr
ansf
ecte
d w
ith V
5-S
H3P
X1,
HA
-dynam
in-2
, an
d e
ither
Myc-
AC
K2
or
Myc-
AC
K2
-K158R
.
Appro
xim
atel
y
24-7
2
hours
af
ter
tran
sfec
tion,
SH
3P
X1
was
im
munopre
cip
itat
ed
from
ce
ll
lysa
tes
wit
h
anti
-V5
anti
body.
T
he
sam
ple
s w
ere
reso
lved
by S
DS
-PA
GE
, an
d t
ransf
erre
d t
o P
VD
F m
embra
ne.
L
ater
, th
e
sam
ple
s w
ere
subje
cted
to
W
este
rn
blo
ttin
g
wit
h
anti
-HA
an
tibody
to
mea
sure
dynam
in b
indin
g i
n t
he
pre
sence
of
AC
K2 a
nd t
he
kin
ase-
def
ecti
ve
muta
nt
AC
K2-
K158R
(B
).
164
165
in t
he
traf
fick
ing o
f se
ver
al c
ell
surf
ace
rece
pto
rs i
ncl
udin
g t
he
insu
lin [
5],
EG
F [
4],
and
tran
sfer
rin
rece
pto
rs
[6].
Giv
en
the
import
ance
of
med
iati
ng
the
signal
ing
acti
vit
ies
and p
roper
dow
n-r
egula
tion o
f th
ese
rece
pto
rs,
we
bel
ieve
that
elu
cidat
ing
the
role
of
SH
3P
X1 a
nd i
ts p
hosp
hory
lati
on i
n r
ecep
tor
endocy
tosi
s w
ill
ult
imat
ely
lead
to
a
bet
ter
under
stan
din
g
of
the
bio
logic
al
pat
hw
ays
under
lyin
g
abnorm
al
pro
life
rati
ve
dis
ease
.
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