apparent presence of ser133-phosphorylated cyclic amp res · apparent presence of...

Page 1

Apparen

tp

resence

of

Ser1

33

-ph

osp

ho

rylated

cy

clicA

MP

respon

seelem

ent

bin

din

gp

rotein

(pC

RE

B)

inb

rainm

itoch

ond

riais

due

tocro

ss-reactivity

of

pC

RE

Ban

tibo

dies

with

py

ruvate

deh

yd

rogen

ase

Jan

Pla

ten

ik,V

lad

imir

J.

Ba

lcar

,Y

ukio

Yo

ne

da

,B

arb

ara

Mio

dusze

wska

,R

ich

ard

Buch

al,

Ra

do

van

Hyne

k,

Lu

kasz

Kilia

ne

k,N

obu

yukiK

ura

moto

,G

rze

go

rzW

ilczynski,

Kiy

oka

zu

Ogita

,Y

oic

hiN

akam

ura

,a

nd

Le

szek

Kaczm

are

k

1,2

,32

23

14

52

36

23

99

.12

12

3

45

6

Inst.M

ed.B

ioch

em

.,1

st

Fac.

Me

d.,

Cha

rles

Un

iv.,P

rah

a2,

CZ

EC

HR

EP

UB

LIC

.La

b.

Mol.

Pha

rma

co

l.,D

iv.P

ha

rm.

Sci.,

Ka

nazaw

aU

niv.,

Kan

azaw

a,JA

PA

N.

De

pt.

Mo

l.C

ell.

Neu

robio

l.,N

en

ckiIn

st.,

Wa

rsaw

,P

OL

AN

D.

Dep

t.B

ioche

m.&

Mic

rob

iol.,

Inst.

Ch

em

.Te

ch

no

l.,P

raha

,C

ZE

CH

RE

PU

BL

IC.

La

b.C

on

foca

lM

icro

scop

y,N

enckiIn

st.,

Wars

aw

,P

OL

AN

D.

De

pt.

Ph

arm

aco

l.,S

ets

una

nU

niv.,

Osa

ka

,JA

PA

N.

Th

ecy

clic

AM

Pre

spon

se

ele

me

nt

bin

din

gp

rote

in(C

RE

B)

isan

ubiq

uito

usly

and

cons

titutiv

ely

expre

ssed

transcrip

tion

facto

rof

the

ba

sic

/leucin

ezip

pe

rfa

mily

tha

tre

cog

niz

es

aD

NA

consen

sus

sequ

ence

calle

dC

RE

(cyclic

AM

P/C

a2

+re

sponse

ele

men

t).Its

ability

toactiv

ate

tran

scrip

tion

of

the

regu

late

dg

en

ecritic

ally

dep

end

so

nph

ospho

rylatio

no

fa

serin

ere

sid

ue

num

ber

13

3in

itstra

nsa

ctivatio

ndo

main

,w

hich

isd

on

eby

varie

tyo

fp

rote

inkin

ases

inre

spon

seto

ele

vatio

nofin

trace

llula

rca

lcium,

cAM

Por

neu

rotro

ph

insig

nalin

g.

With

over

500

0re

ports

rela

ted

toC

RE

Bin

Me

dlin

eto

date

this

pro

tein

ha

sa

lso

be

com

eo

ne

of

the

mo

ststu

die

dtra

nsc

riptio

nfa

ctors

.In

neuro

scie

nce

the

CR

EB

has

bee

nw

ide

lyim

plica

ted

inthe

synap

ticpla

stic

ityund

erlyin

glo

ng

-term

me

mory

con

solid

ation

,an

dals

oin

the

neu

rona

lpro

-surv

iva

lsig

nalin

gpa

thw

ay

s.

Th

epre

vailin

gvie

wis

thatC

RE

Bis

perm

an

ently

and

exclu

sive

lyp

resent

inth

ecell

nucle

us.

Ho

wev

er,

there

have

be

ensevera

lmutu

ally

contro

versia

lre

po

rtsd

escrib

ing

an

extra

nu

clear

loca

liza

tion

of

CR

EB

.S

pecific

ally,

syna

ptic

,cy

toso

lic,

and

inp

artic

ula

rm

itoch

on

dria

llo

ca

liza

tion

wa

sre

po

rted.

He

rew

epre

sentre

sults

ofo

ur

ow

nstu

dy

on

sub

cellu

lar

loca

liza

tion

ofC

RE

B.

Intro

du

ctio

n

Me

tho

ds

Th

esu

bce

llula

rlo

ca

lizatio

nof

CR

EB

wa

sexam

ine

dby

mea

ns

of:

-S

ubc

ellu

lar

fractio

nation

ofa

du

ltra

tb

rain

corte

xfo

llowe

dby

We

ste

rnim

uno

blo

tting

with

CR

EB

/pC

RE

Ba

ntib

odie

sand

ge

lshift

with

CR

Ep

rob

e.S

uita

ble

nucle

ar,

mito

chon

dria

lan

dsyn

aptic

ma

rkers

als

oe

mplo

yed

.

-Im

mu

no

fluo

rescen

cesta

inin

gand

con

foca

lmicro

scop

yof

cultu

red

ratb

rain

ne

uro

nalan

dastro

glia

lce

lls

-Im

mu

no

pre

cip

itatio

nofC

RE

Bor

pC

RE

BIR

from

nucle

ar

or

mito

ch

on

drial

frac

tions,

follo

wed

by

We

stern

blottin

gan

alysis

and

identific

ation

ofp

recip

itate

dprote

ins

by

MA

LD

I-TO

Fm

as

sspectro

me

try

The

follow

ing

comm

ercialC

RE

B/pC

RE

Ba

ntibo

die

s(A

b's)

we

reu

sed

:

Pho

sph

o-C

RE

B(S

er13

3)A

b,

Ce

llSig

nallin

gT

ech

no

log

y(C

ST,

#9

191

)P

ho

sph

o-C

RE

BA

bfro

mU

psta

teB

iotech

no

log

y(#

06-5

19)

CR

EB

an

tibod

y(a

gain

sta

nep

itope

at

the

CR

EB

C-term

inus

),CS

T(#

919

2)

C-2

1C

RE

BA

b(a

gain

stC

RE

BD

NA

-bin

din

gd

om

ain

),S

anta

Cru

zB

iotech

nolo

gy

(sc-18

6).

Hm

CR

EB

antib

od

y(fro

m"h

om

e-m

ad

e")

was

raise

din

Y.

Yon

eda

'sla

bo

rato

rya

gain

stp

ep

tide

KR

RE

ILS

RR

PS

YR

KC

(resid

ues

123

-136

of

mou

se

or

rat

CR

EB

,en

comp

assing

unp

ho

sph

oryla

tedS

er13

3),a

nd

affin

ity-p

urifie

dus

ing

the

sam

ep

eptid

e.

(pCR

EB

...C

RE

Bp

hosp

hory

lated

at

Ser1

33,

IR...

imm

un

ore

activity,

MA

LD

I-TOF...

matrix-as

siste

dlase

rde

sorp

tion/io

nisa

tiontim

e-o

f-flight)

1)

Su

bce

llula

rfra

ctio

natio

np

roc

ed

ure

Fra

ctio

ns

ob

tain

ed:

HP1

P2

SP3

ABCDE

(orig

inalh

om

og

ena

te)

(nucle

ar

fractio

n,

but

als

oco

nta

inin

ga

lot

of

mito

cho

nd

riafro

mce

llde

bris)

(cru

de

mito

ch

ond

ria/sy

na

pto

so

me

s)

(cyto

sol)

(mic

roso

mal)

(pro

ba

bly

cell

me

mbra

nes,

Go

lgi)

(syn

apto

so

mes)

(no

n-sy

na

ptic

mito

ch

ond

ria)

(syn

aptic

mem

bra

ne

s,b

ut

als

ocon

tainin

ga

lot

of

syna

ptic

mito

cho

nd

ria)

(syn

aptic

mito

ch

ond

ria)

Pro

tein

con

ce

ntra

tion

and

yie

ldW

este

rnb

lotting

for

CR

EB

,p

CR

EB

,N

MD

Are

cep

tor

2B

sub

un

itC

yto

ch

rom

eo

xida

se

activity

Ge

lsh

iftfo

rC

RE

DN

Ab

ind

ing

activ

ity

Ineac

hfra

ctio

nm

ea

su

red

:

2)

pC

RE

BIR

isp

rom

ine

nt

inm

itoch

on

dria

lfra

ctio

ns

,b

ut

CR

EB

IRis

no

t

A),

C),

D):

repre

senta

tiveW

este

rnblo

tsofth

esubcellula

rfra

ctions

pro

bed

with

vario

us

pC

RE

Band

CR

EB

antibo

die

sas

indic

ate

d.B

):D

ensito

me

trice

valu

atio

no

fth

epC

RE

BAb

CS

TIR

(inse

t)in

com

pariso

nto

dis

tributio

no

fN

MD

Are

cep

tor

2B

subunit

IR(s

ynaptic

marke

r)an

da

zide

-inhib

itable

cytoch

rom

eoxid

ase

en

zym

ea

ctivity(m

itoch

ondria

lm

ark

er).

Me

an

±S

D,

N=

4in

dep

ende

ntexperim

en

ts,only

for

cyto

chro

me

oxid

ase

N=2

ind

epen

dente

xperim

ents.

*…p<0

.05

,**…

p<0.0

1vs.

orig

inalhom

ogen

ate

),o

ne-w

ay

AN

OVA

with

Dunn

ett's

pos

tte

st.

Th

es

ign

al

with

two

diffe

ren

tp

CR

EB

Ab

'sw

as

stro

ng

inm

itoc

ho

nd

ria-en

rich

ed

frac

tion

s(C

,D

,E

inp

artic

ula

r),a

nd

qu

an

titativ

ed

istrib

utio

no

fp

CR

EB

IRc

orre

late

dw

ithth

em

itoc

ho

nd

rial

ma

rker

ra

the

r

tha

ns

yn

ap

tico

ne

.In

co

ntra

st,

the

ba

nd

dete

cte

dw

ithtw

oC

RE

Ba

ntib

od

ies

ag

ain

st

diffe

ren

te

pito

pe

sw

as

se

en

mo

stly

inn

uc

lea

rfra

ctio

n,

so

me

inS

an

dP

3,

bu

ta

lmo

st

ab

se

nt

from

mito

ch

on

dria

lfra

ctio

ns

.

3)

Ge

lsh

ift:m

itoc

ho

nd

rial/s

yn

ap

ticC

RE

DN

Ab

ind

ing

activ

ityd

iffere

nt

from

nu

cle

ar

CR

EB

Gelsh

iftw

ith32P

-labelle

dC

RE

pro

be

perfo

rmed

with

allth

esu

bce

llula

rfra

ctio

ns,

for

nu

clear

and

mito

chond

rial

fractio

ns

also

nclu

din

gsu

persh

iftw

ithva

rious

CR

EB

/pC

RE

BA

b's

as

indic

ate

d).

The

dom

ina

ntlo

wer

band

appearin

gin

the

nucle

ar

fractio

nw

as

su

pers

hifte

db

ytw

oC

RE

BA

b's

which

qua

lifies

this

band

as

auth

entic

CR

EB

.In

con

trast,

the

CR

E-

bin

din

gactiv

ityin

mito

chon

dria

l/sy

nap

ticfra

ctio

ns

diffe

redin

ele

ctro

pho

retic

mobility

an

dw

as

una

ffecte

dby

CR

EB

Ab's.

4)

Nu

cle

ar

co

nta

min

atio

nof

P2

fractio

nw

as

low

bu

td

ete

cta

ble

DA

PI

sta

ino

ffre

sh

lyp

rep

are

dP

1an

dP

2fra

ctio

nre

veale

da

ple

nty

of

nu

cle

ia

nd

blo

od

ves

selfra

gm

en

tsin

the

P1,b

ut

only

very

few

nu

cle

iin

the

P2.

Lik

ew

ise,p

rese

nce

of

his

ton

eH

3d

ete

cte

dw

ithW

este

rnim

mu

no

blo

tting

was

res

tricte

dto

the

P1

fractio

n.S

eve

ral

dilu

tion

so

fP

1fra

ctio

nw

ere

inclu

ded

tosh

ow

that

the

fain

th

isto

ne

ban

din

P2

actu

ally

did

no

tre

pre

se

nt

mo

reth

an

ab

ou

t1%

of

his

ton

eco

nce

ntra

tion

inP

1.

5)

Ele

ctro

ph

ore

ticm

ob

ilityo

fm

itoc

ho

nd

rial

pC

RE

BIR

ma

rked

lyd

iffere

dfro

mn

uc

lea

rp

CR

EB

/CR

EB

IR

A):

The

CR

EB

IRo

nW

este

rnblo

tsde

tecte

dw

ithth

eC

RE

BA

bC

ST

inth

enuc

lear

fractio

nconsiste

ntly

app

eare

da

sa

double

ban

d,

likely

rep

resentin

gtw

oC

RE

Bsp

licin

gvaria

nts,

alp

ha

(the

upp

er

ban

d)

and

delta

(the

low

er

band

).Allo

the

rfra

ctions

app

are

ntly

con

tain

ed

only

delta

CR

EB

.T

heblo

tsdete

cte

dw

ithpC

RE

BA

bU

psta

teals

odis

pla

yed

ano

n-consta

ntdou

ble

band

inth

en

ucle

ar

P1

fractio

n,w

hile

inth

em

itochond

rialP

2fra

ctio

nth

ere

wa

salw

ays

asin

gle

band

with

positio

ncorre

spondin

gto

the

P1

low

erban

d.

B)

2-D

ele

ctroph

ore

sis

ofP

1(u

pper

pa

nel)

and

P2

(mid

dle

pan

el)

fractio

ns

follo

wed

by

We

stern

imm

uno

blo

tting

with

pC

RE

BA

bU

psta

te.

The

P1

blo

tth

en

subje

cted

tostrip

pin

gpro

cedure

,develo

pe

dw

ithsecondary

antib

od

yto

verify

com

ple

tere

mova

lof

the

pC

RE

BA

b,an

dre

pro

bed

with

the

CR

EB

Ab

CS

T(b

otto

mpan

el).

Mo

st

imp

orta

ntly,

the

po

sitio

ns

of

nu

cle

ar

do

ub

leb

an

ds

dete

cte

dw

ithC

RE

BA

bC

ST

an

dp

CR

EB

Ab

Up

sta

ted

idn

ot

co

rresp

on

dto

eac

ho

the

r.R

ath

er,

the

ban

dd

ete

cte

dw

ithC

RE

BA

bC

ST

co

rresp

on

ded

toth

eb

an

dd

ete

cte

dw

ithp

CR

EB

Ab

Up

sta

te.

2-D

ele

ctro

ph

ore

sis

reso

lve

dth

en

uclea

ran

dm

itoch

on

dria

lp

CR

EB

IR's

into

two

sp

ots

with

su

ch

ab

igd

iffere

nce

in

ap

pare

nt

isoe

lec

tricp

oin

ts,

that

the

ym

ust

be

co

mp

lete

lyd

iffere

nt

pro

tein

s.T

he

mito

cho

nd

rial

pC

RE

BIR

infa

ct

rep

resen

tsan

oth

er

mito

ch

ond

rial

pro

tein

,w

hic

hc

ross-

rea

cts

with

the

pC

RE

BA

b's

.

low

er

up

per

6)

pC

RE

BA

b’s

sta

ine

dm

itoch

on

dria

,in

ad

ditio

nto

nu

cle

i,o

fg

lialc

ells

inp

rima

ryc

ortic

alc

ultu

res

The

prim

ary

neu

rona

lcultu

res

were

made

ofnew

-born

ratcortice

s,cyto

sine

ara

bin

osid

ea

dded

atd

ay

2;a

nd

cultu

res

used

for

imuno

fluores

cence

stain

ing

at

day

3-6

.M

ostof

the

cells

were

neu

rone

s,w

hile

glia

le

lem

ents

acc

oun

ted

forle

ss

than

5%

.A

ntib

odie

sag

ain

stpC

RE

B/C

RE

B,

cyto

chro

me

oxid

ase

(CO

X)

or

astro

cytic

marke

rG

FA

Pw

ere

used

for

stainin

g,a

nd

the

pre

para

tions

were

exa

min

ed

by

confo

calm

icrosc

opy.

pC

RE

Bantib

od

yU

psta

testa

ined

nu

clei

ofa

llcells,

buta

lso

mito

chondria

ofglia

lelem

ents,

where

itco

-loca

lized

with

CO

X.T

he

mito

cho

ndria

lsig

nalw

as

evid

en

teve

nin

unstim

ula

ted

cultu

res

(A),

bu

tw

as

stron

ger

wh

en

the

cultu

res

were

challe

nge

dw

ithfo

rsko

lin(a

ctivato

ro

fpro

tein

kin

ase

A)

prior

tofix

atio

n(B

,C).

IfC

RE

BA

bw

as

used

for

stain

ing

,only

fain

tnu

clear

sign

alc

ould

be

ob

taine

din

this

experim

enta

lsy

stem

.

7)

Th

ree

pro

tein

sp

rec

ipita

ted

by

pC

RE

BA

bfro

mP

2fra

ctio

nw

ere

iden

tified

as

PD

H

Su

cc

es

sfu

lim

mu

no

pre

cip

itatio

no

fth

e'fa

lse'p

CR

EB

IRfr

om

P2

was

co

nsis

ten

tlya

sso

cia

ted

with

3C

oo

ma

ssie

-de

tec

tab

lep

rote

inb

an

ds

of

ap

pare

nt

MW

65

.5,

42

an

d3

1.5

kD

a.

Th

ese

3b

an

ds

we

rec

on

clu

sive

lyid

en

tified

by

MA

LD

I-TO

FM

Sas

the

E2

,E

1a

lpha

su

bu

nit,

an

dE

1b

eta

su

bu

nit

co

mp

on

en

ts,

res

pe

ctiv

ely

,o

fth

ew

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lar

Cell

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gy,

5th

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man

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20

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)