Inactivation of p42 MAP kinase by protein phosphatase 2A and a protein tyrosine phosphatase, but not CL100, in various cell lines.
BACKGROUND
Mitogen-activated protein (MAP) kinase is central to a signal transduction pathway that triggers cell proliferation or differentiation. Activation of the p42mapk isoform requires its phosphorylation at two residues, Thr 183 and Tyr 185, and this phosphorylation is catalysed by MAP kinase kinase (MAPKK). Relatively little is known, however, about the enzymes that dephosphorylate these residues, thereby inactivating the pathway. Recently, the CL100 phosphatase has been shown to inactivate p42mapk in vitro by dephosphorylating Thr 183 and Tyr 185 at similar rates. CL100, the product of an immediate early gene, is synthesized within one hour of stimulating cells with growth factors or exposure to oxidative stress or heat shock. Incubation of NIH 3T3 fibroblasts with cycloheximide prevents both synthesis of CL100 and inactivation of p42mapk after stimulation with serum.


RESULTS
Depleting cells of CL100 and preventing its induction using cycloheximide stopped the inactivation of p42mapk in Swiss 3T3 fibroblasts following stimulation with epidermal growth factor (EGF), but had no effect on the rapid inactivation of p42mapk in response to EGF in adipose (3T3-L1) or chromaffin (PC12) cells or in response to platelet-derived growth factor (PDGF) in endothelial (PAE) cells. Moreover, maximal induction of CL100 mRNA and a CL100-like activity did not trigger inactivation of p42mapk, which was sustained at a high level after stimulation of PC12 cells with nerve growth factor, PAE cells with serum, or Swiss 3T3 cells with PDGF. Dephosphorylation of Tyr 185 but not Thr 183 of p42mapk was suppressed by vanadate in EGF-stimulated PC12 cells; dephosphorylation of Thr 183, by contrast, was elicited by a vanadate-insensitive activity. Protein phosphatase-2A was the only vanadate-insensitive phosphatase acting on Thr 183 of p42mapk or on MAPKK to be detected in PC12 cell extracts. Phosphorylation of Thr 183 also inhibited the dephosphorylation of Tyr 185 in vitro by the major vanadate-sensitive Tyr 185-specific phosphatase, explaining why dephosphorylation of Thr 183 is rate-limiting for p42mapk inactivation in PC12 cells after stimulation with EGF.


CONCLUSIONS
The rapid inactivation of p42mapk initiated five minutes after stimulation of endothelial, adipose and chromaffin cells with growth factor is not catalysed by CL100, but rather by protein phosphatase 2A and by a protein tyrosine phosphatase distinct from CL100. Induction of CL100 is not accompanied by the inactivation of p42mapk in a number of situations.
Alessi
D R
DR
Department of Biochemistry, University of Dundee, UK.
Gomez
N
N
Moorhead
G
G
Lewis
T
T
Keyse
S M
SM
Cohen
P
P
D016475:3T3 Cells; D000818:Animals; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000037:antagonists & inhibitors*; D018797:Cell Cycle Proteins; D002460:Cell Line; D002478:Cells, Cultured; D003513:Cycloheximide / Q000494:pharmacology; D054638:Dual Specificity Phosphatase 1; D004730:Endothelium, Vascular / Q000187:drug effects / Q000378:metabolism; D015870:Gene Expression / Q000187:drug effects*; D006133:Growth Substances / Q000494:pharmacology*; D017874:Immediate-Early Proteins / Q000096:biosynthesis / Q000378:metabolism*; D007700:Kinetics; D051379:Mice; D020929:Mitogen-Activated Protein Kinase Kinases; D008970:Molecular Weight; D009414:Nerve Growth Factors / Q000494:pharmacology; D016716:PC12 Cells; D010749:Phosphoprotein Phosphatases / Q000096:biosynthesis / Q000378:metabolism*; D010982:Platelet-Derived Growth Factor / Q000494:pharmacology; D011494:Protein Kinases / Q000378:metabolism*; D054645:Protein Phosphatase 1; D054648:Protein Phosphatase 2; D017027:Protein Tyrosine Phosphatases / Q000096:biosynthesis / Q000378:metabolism*; D012333:RNA, Messenger / Q000032:analysis / Q000096:biosynthesis; D051381:Rats; D013997:Time Factors
D018797:Cell Cycle Proteins; D006133:Growth Substances; D017874:Immediate-Early Proteins; D009414:Nerve Growth Factors; D010982:Platelet-Derived Growth Factor; D012333:RNA, Messenger; D003513:Cycloheximide; D011494:Protein Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D020929:Mitogen-Activated Protein Kinase Kinases; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1; D054648:Protein Phosphatase 2; D054638:Dual Specificity Phosphatase 1; C517020:Dusp1 protein, mouse; C517021:Dusp1 protein, rat; D017027:Protein Tyrosine Phosphatases