a polybasic motif in erbb3-binding-protein 1 (ebp1) has key
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ACCEPTED MANUSCRIPT
Copyright 2016 The Author(s). This is an Accepted Manuscript; not the final Version of Record. Archiving permitted only in line with the archiving policy of Portland Press Limited (http://www.portlandpresspublishing.com/content/open-access-policy#Archiving Policy). All other rights reserved.
Cite as Biochemical Journal (2016) DOI: 10.1042/BCJ20160274
BIOCHEMICAL JOURNAL
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10.1042/BCJ20160274. Please cite using the DOI 10.1042/BCJ20160274http://dx.doi.org/up-to-date version is available at
encouraged to use the Version of Record that, when published, will replace this version. The most this is an Accepted Manuscript, not the final Version of Record. You are:Biochemical Journal
). http://www.portlandpresspublishing.com/content/open-access-policy#ArchivingArchiving Policy of Portland Press (which the article is published. Archiving of non-open access articles is permitted in accordance with the Use of open access articles is permitted based on the terms of the specific Creative Commons Licence under
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PA2G4
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E. coli
E.coli
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et al
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P P i.e.
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et alP
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in vitro
P Pin vitro
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P PP
P P
P
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P
P P
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PP
P
(A)(B)
(C)
(D)(E)
(D)
(A)
(B)(C)
(D)
(A)(B)
(C)
(D)(E)
P(A)
(B)(C)
P(D)
P (E)
P(A) P
(B)
(C)
P(A)
(B)
(C)P (D)
P (E)
P (F)P
(A) (B)
(C)
(D) C
Figure 1
C
S1P
PtdIns(3,4)P2
PtdIns(3,5)P2
PtdIns(4,5)P2
PtdIns(3,4,5)P3
PA
PS
LPA
LPC
PE
PC
PtdIns
PtdIns(3)P
PtdIns(4)P
PtdIns(5)P
Blank
E
GSTEBP1 PLC 1-PH GRP1-PH
1
2
3
9
8
7
6
5
4
14
13
12
11
10
15
4
5
6
11
14
11 11
4
6
15
A
EBP1
B
- + neomycin10
12 12 12
13
0
1
2
3
4
5
6
7
3P 4P 5P 34P2 35P2 45P2 PIP3 PA PS
0
0,2
0,4
0,6
0,8
1
1,2
1,4
1,6
3P 4P 5P 34P2 35P2 45P2 PIP3 PA PS
150 300
D
FmM NaCl 400
Den
sito
met
ry ra
tios
D
ensi
tom
etry
ratio
s (3
00/1
50)
N-term EBP1N-WT WTC-WT
C-term EBP1 Full Length EBP1
A
Figure 2
B
GST1 394
C-term motif:C-WT: 364RKTQKKKKKK373
C-K4A: 364RKTQKAAAAK373
N-term motif:N-WT: 65KKEKEMKK72
N-K3A: 65AAEAEMKK72
GST1 351
GST1 394352
K65K66K68
C Term 362
N-K3A C-K4A N-K3A/C-K4A
C
D
EBP1 355 KALLQSSAS-RKTQKKKKKKASKTSAP30L 77 HKNFIQSVR-N-KRKRK-TSPf1 103 C-------TVR---RKKR-EQKKEING2 256 CRGDNEKTMDKSTEKTKKD--RRSRBASP1 1 MGGKLS-KKKKGYNVN
FL
N-term
C-term
4
5
6
11 11
4
5
6
10
14
12
13
4
5
6
11
A EBP1 NPM Merge
putative NoLS: 357-85 LLQSSASRKTQKKKKKKASKTAENATSGEC-term PBR: 364-73 RKTQKKKKKK
B
C
EGFP
-tubulin
EGFP-EBP1
D
E
NPM
Mer
geEG
FP
N-K3AC-K4AWT N-K3A/C-K4A
Figure 3
- WT
0
20
40
60
80
100
120
pattern #1
pattern #2
pattern #3
WT
%
A
B C
D
Figure 4
E
PtdIns(3,4,5)P3
- -+ +
Nucleolin PtdIns(3,4,5)P3 Merge
NPM p110 Merge
%
pmol
PtdIns(4,5)P2
PtdIns(3,5)P2
PtdIns(3,4)P2
PtdIns(5)PPtdIns(4)PPtdIns(3)PPtdIns
EGFP
Nuc
leol
inM
erge
NLS-EGFP NLS-EGFP-GRP1-PH
- +
A
B C
Figure 5
- + anti-EBP1
PtdIns(3,4)P2
PtdIns(3,5)P2
PtdIns(4,5)P2
PtdIns(3,4,5)P3
PtdIns(5)PPtdIns(4)P
PtdIns(3)PEBP150
37
25
75
100150250
EBP1 PtdIns(3,4,5)P3 Merge
A
C
E
Figure 6
F
1
15
G393G350*
G384
T382
R349*
T377 S363
T366S375
T386
S360S351*
D394
A356
L357K355
S383
S361Q359
L346*
N380
E379E385
N390
A362A374 A378
L387A392
A381
V347*
L358
R364
K365K368
Q367K372
K373K376
E388E391E389
D345*
H [ppm] 8.5 8.0 7.5 7.0 6.5
N [p
pm]
130
1
25
120
1
15
1
SSP plot
intensity C16
intensity C8
B
D
115
G393G350*
G384
T382
R349*
T377 S363
T366S375
T386
S360S351*
D394
A356
L357K355
S383
S361Q359
L346*
N380
E379E385
N390
A362A374 A378
L387A392
A381
V347*
L358
R364
K365K368
Q367K372
K373K376
E388E391E389
D345*
1H [ppm] 8.5 8.0 7.5 7.0 6.5 N
[ppm
] 1
30
125
1
20
115
A
Figure 7
WT 364-RKTQKKKKKK-373K372N 364-RKTQKKKKNK-373
B
CK372N
EGFP NPM Merge
K37
2N
WT
PI(3,4,5)P3
D
-0,2
0
0,2
0,4
0,6
0,8
1
1,2
3P 4P 5P 34P2 35P2 45P2 345P3 PA PS
PI(3,4)P2
PA
PS
Den
sito
met
ry ra
tio K
372N
/WT
PI(4,5)P2
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