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-Llysine. •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

Figure adapted from Raman and Cobb 2003

Alternative 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 107 81 47 35 27 19 JNK2:c-Jun(1-87)GST (µL) JNK2 (1:1 1:3 3:3 3:1) c-Jun JNK2 λ-PPT

His-JNK1 WT ApF DpE trunc
183

TpY185

IX-X C-terminal

Catalytic domain

kDa 107 81 47 35 27 19

JNK2 c-Jun(1-87)GST JNK2 c-Jun(1-87)GST

Autophosphorylation of JNK2(WT) + phospho-mimic mutants
Western Blot
JNK2-His WT T243A T183D Y185E DpE

Autophosphorylation Rxn 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) - + Pro-Q Diamond

  Mass spectrometry
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 hJNK2 hJNK3 VINGSQ386HPSSSSSVNDVSSMSTDP AVSSNA386TPSQSSSINDISSMSTEQ 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
INACTIVE pT183
JNK2 JNK2

JNK1

pY185
JNK2

MKK4/7 MKPs pY185
JNK1

pT386 MKK4/7 MKK4/7

pT386

pT386

MKK4/7 MKPs pY185 pT183
JNK1

Functional cross-talk

MKPs

MKPs pY185
JNK2

pT183 pY185 ACTIVE
JNK2

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?

[MAPKs]

[Phosphatases]

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
183

TPY185

IX-X C-terminal pospho-T386

Catalytic domain

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.