jnk2 retreat

8/14/2019 JNK2 retreat

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Proteomic Investigationof

Inactive and Active JNK2

Pimienta G, Ficarro SB, Gutierrez GJ, Bhoumik A,Peters EC, Ronai Z and Pascual JCell Cycle Journal (2007) 6:1751-60


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Outline

Introduction

Results

Conclusions/Perspectives


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Background

Kyriakis JM and Avruch J (J Biol Chem. 1990)pp54 microtubule-associated protein 2 kinase. A novel serine/threonineprotein 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 areexpressed in primary glial tumors.Cui J et al.,Wong AJ (J Biol Chem. 2005)Identification of a specific domain responsible for JNK2alpha2autophosphorylation.

Cui J et al.,Wong AJ (Cancer Res. 2006)c-Jun NH(2)-terminal kinase 2alpha2 promotes the tumorigenicity ofhuman glioblastoma cells.


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The MAPK signaling cascade

Figure adapted from Raman and Cobb 2003

MAPKSignalosome

AlternativeMAPK activation


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JNK1 is activated by MKK4/SEK1 and/or MKK7via a two-step phosphorylation mechanism:

First Tyr185, then Thr183

Figure adapted from Kishimoto et al.,Nishina 2003


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?

?

Figure adapted from Liu et al., Lin 2004

Active JNK1 is the main in vivokinase componentfor most JNK signaling outputs


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Outline

Introduction

Results



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JNK2 WT, but not JNK1 is autophosphorylated.Recombinant JNK2 WTphosphorylates c-Jun in vitro

His-JNK1 WT ApF DpE trunc

His-JNK2

kDa

10781

47

35

27

19

kDa

10781

47

35

27

19JNK2:c-Jun(1-87)GST(L) JNK2

(1:1 1:3 3:3 3:1) c-Jun JNK2 -PPT

c-Jun(1-87)GST

JNK2

c-Jun(1-87)GST

JNK2

Catalytic domain

IX-X

C-terminal183TpY185


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Autophosphorylation of JNK2(WT) + phospho-mimic mutants

-T386A is unstable, suggesting a structural role

-Thr183 (activation loop) is the main phospho-site-Phospho-site Thr243 is not important for JNKs autoactivation

-The double mutant DpE fails to auto-phosphorylate

Total JNK

pTyr

pThr-Pro

JNK2-His WT T243A T183D Y185E DpE

JNK2-His WT T243A T183D Y185E DpE

Autophosphorylation RxnWestern Blot32P-ATP


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UV(45J) - +

Pro-Q Diamond

Sypro Ruby

MS/MS analysis of JNK2 phosphosites

purified from 293Tcells

large scale transfection: 10 plates ; tag: Flag-JNK2

Mass spectrometryBasal Flag-JNK2

TACTNFMMpT183PY185VVTRTACTNFMMT183PpY185VVTRSSNApT386PSQSSI

UV-treated Flag-JNK2

TACTNFMMpT183PY185VVTRTACTNFMMT183PpY185VVTRTACTNFMMpT183PpY185VVTRSSNApT386PSQSSI


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JNK1 crystal structure Yong-Seok et al., 2004

Structural localization of the new phospho-site

C-terminal extension

hJNK1 VINGSQ386HPSSSSSVNDVSSMSTDP

hJNK2 AVSSNA386TPSQSSSINDISSMSTEQ

hJNK3 AVNSSE386SLPPSSSVNDISSMSTDQ

COO-


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293T cellspost-UV irradiation purified JNK2 sample followed bya time course/MS relative quantificationof pJNK2 peptides

pY185pT183

pT386 pT183/pY185

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


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Outline

Introduction

Results



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JNK1

pY185

pT183

JNK1

pY185

JNK1

WORKING MODEL

pT386

Functional cross-talk

INACTIVE

ACTIVE

MKPs

MKPs

MKK4/7

MKK4/7

JNK2

JNK2

pY185

pT386

JNK2

pY185

pT386

MKK4/7

pT386

JNK2

pT183

JNK2

pY185pT183

pT386

MKPs MKPs

MKK4/7

Active JNK2 appears first but is diluted


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Figure adapted from Liu et al., Lin 2004

The JNK1 JNK2 functional compensationmay underpinthe opposed signaling outputs:

JNK2 could have a role other thandestabilizing c-Jun in basal conditions

Active JNK2 appears first but is diluted;therefore it may contribute to

the Dose-Response activation curve shape

Short term JNK2 activation causes cell proliferation whereas long term JNK1, apoptosis


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Future experiments

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

Is any of these phosphositesup/down-regulated over the cell cycle?a protein-protein interaction surface?

How do the phosphosites we find here

shape the JNK activation curvein vivo

?

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

[Phosphatases][MAPKs]


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Outline

Introduction

Results



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CONCLUDING REMARKS

The pY185 autophosphorylation of JNK2 and its multisite

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

-the emerging notion of in vivocompensatory crosstalk among JNKs.

-saturation by JNK2(pY185) of MKK4/7 and VHR makesthe JNK cascade ultrasensitive & bistable

-the establishment of a positive feedback loop (hysteresis)

JNK i i d i bi bl d h h i


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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


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Figure adapted from Oliver et al.,Pearl 2007

It has been proposed thatdimerization-dependent autophosphorylation

is pervasive among protein kinases

Catalytic domain

IX-X

C-terminal183TPY185

In agreement, JNK2 (a long MAPK)autophosphorylates

pT386 on the unique C-terminal tail stabilizes the protein

pospho-T386


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Acknowledgements

Genaro Pimienta

Eric Peters (GNF)

Ronai lab.

jnk2 retreat

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