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Proteomic Investigation

of
Inactive and Active JNK2

Pimienta G, Ficarro SB, Gutierrez GJ, Bhoumik A,


Peters EC, Ronai Z and Pascual J
Cell Cycle Journal (2007) 6:1751-60
Outline
Introduction

Results

Conclusions/Perspectives
Background
•Kyriakis JM and Avruch J (J Biol Chem. 1990)
pp54 microtubule-associated protein 2 kinase. A novel serine/threonine
protein kinase regulated by phosphorylation and stimulated by poly-L-
lysine.
•Tsuiki et al.,…Wong AJ. (Cancer Res. 2003)
Constitutively active forms of c-Jun NH2-terminal kinase are
expressed in primary glial tumors.
• Cui J et al.,…Wong AJ (J Biol Chem. 2005)
Identification of a specific domain responsible for JNK2alpha2
autophosphorylation.
•Cui J et al.,…Wong AJ (Cancer Res. 2006)
c-Jun NH(2)-terminal kinase 2alpha2 promotes the tumorigenicity of
human glioblastoma cells.
The MAPK signaling cascade

MAPK
Signalosome

Alternative
Figure adapted from Raman and Cobb 2003 MAPK activation
JNK1 is activated by MKK4/SEK1 and/or MKK7
via a two-step phosphorylation mechanism:
First Tyr185, then Thr183

Figure adapted from Kishimoto et al.,…Nishina 2003


Active JNK1 is the main
in vivo kinase component
for most JNK signaling outputs

Figure adapted from Liu et al.,… Lin 2004


Outline
Introduction

Results

Conclusions/Perspectives
JNK2 WT, but not JNK1 is autophosphorylated.
Recombinant JNK2 WT phosphorylates c-Jun in vitro

His-JNK2

kDa His-JNK1 WT ApF DpE trunc


IX-X
107 TpY185
183
81 C-terminal
47 Catalytic domain
35

27

19
JNK2:c-Jun(1-87)GST (µL) JNK2
(1:1 1:3 3:3 3:1) c-Jun JNK2 λ-PPT

kDa
JNK2
107
81
c-Jun(1-87)GST
47

35 JNK2
27 c-Jun(1-87)GST
19
Autophosphorylation of JNK2(WT) + phospho-mimic mutants

Western Blot Autophosphorylation Rxn


JNK2-His WT T243A T183D Y185E DpE
32
P-γATP
JNK2-His WT T243A T183D Y185E DpE

Total JNK

pTyr

pThr-Pro

-T386A is unstable, suggesting a structural role

-Thr183 (activation loop) is the main phospho-site


-Phospho-site Thr243 is not important for JNK’s autoactivation
-The double mutant DpE fails to auto-phosphorylate
MS/MS analysis of JNK2 phosphosites
purified from 293T cells
large scale transfection: 10 plates ; tag: Flag-JNK2
UV(45J) - +
  Mass spectrometry
Pro-Q Diamond

Basal Flag-JNK2
TACTNFMMpT183PY185VVTR
TACTNFMMT183PpY185VVTR
SSNApT386PSQSSI

UV-treated Flag-JNK2
Sypro Ruby
TACTNFMMpT183PY185VVTR
TACTNFMMT183PpY185VVTR
TACTNFMMpT183PpY185VVTR
SSNApT386PSQSSI
Structural localization of the new phospho-site

­
COO
C-terminal extension
hJNK1 VINGSQ386HPSSSSSVNDVSSMSTDP

hJNK2 AVSSNA386TPSQSSSINDISSMSTEQ

hJNK3 AVNSSE386SLPPSSSVNDISSMSTDQ

JNK1 crystal structure Yong-Seok et al., 2004


293T cells post-UV irradiation purified JNK2 sample followed by
a time course/MS relative quantification of pJNK2 peptides

label-free quantification
(enough to look at the pattern of each phosphosite)

pT183
pY185

pT386
pT183/pY185
Outline
Introduction

Results

Conclusions/Perspectives
WORKING MODEL

pT183 pY185
JNK1
INACTIVE
JNK2 JNK2 JNK2
MKK4/7
pT386 pT386 pT386

MKPs
MKK4/7 MKK4/7
pY185

JNK1

MKK4/7 Functional cross-talk MKPs MKPs

MKPs
pT183 pY185 pY185
pY185 pT183
JNK2 JNK2
JNK1
ACTIVE pT386
pT386

Active JNK2 appears first but is diluted


The JNK1 JNK2 functional compensation
may underpin the opposed signaling outputs:
Short term JNK2 activation causes cell proliferation whereas long term JNK1, apoptosis

JNK2 could have a role other than


destabilizing c-Jun in basal conditions

Active JNK2 appears first but is diluted;


therefore it may contribute to
the Dose-Response activation curve shape

Figure adapted from Liu et al.,… Lin 2004


Future experiments
•To validate the phosphosites in vivo: MS/MS of endogenous JNKs

•Is any of these phosphosites


up/down-regulated over the cell cycle?
a protein-protein interaction surface?

•How do the phosphosites we find here


shape the JNK activation curve in vivo?

[Phosphatases]
[MAPKs]

Figure taken from Hornberg et al.,…Heinrich† and Westerhoff 2001


Outline
Introduction

Results

Conclusions/Perspectives
CONCLUDING REMARKS

The pY185 autophosphorylation of JNK2 and its multisite


phospho-pattern upon activation may account for JNKs
apparent redundancy resulting in:

-the emerging notion of in vivo compensatory crosstalk among JNKs.

-saturation by JNK2(pY185) of MKK4/7 and VHR makes


the JNK cascade ultrasensitive & bistable

-the establishment of a positive feedback loop (hysteresis)


JNK activation cascade is bistable and shows hysteresis
• converts graded stimulus into a switch-like (on/off) response
• has self-perpetuating properties

Switch-like response

Positive feedback loop

Figure taken from Cristoph et al.,…Ferrell 2001


It has been proposed that
dimerization-dependent autophosphorylation
is pervasive among protein kinases

IX-X
TPY185
183

C-terminal
Catalytic domain pospho-T386

In agreement, JNK2 (a long MAPK)


autophosphorylates

pT386 on the unique C-terminal tail


stabilizes the protein

Figure adapted from Oliver et al.,…Pearl 2007


Acknowledgements

Genaro Pimienta

Eric Peters (GNF)

Ronai lab.