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 sig nal transduction pathway that triggers cell proliferation or different iation. Activation of the p42(mapk) isoform requires its phosphorylati on at two residues, Thr 183 and Tyr 185, and this phosphorylation is c atalysed by MAP kinase kinase (MAPKK). Relatively little is known, how ever, about the enzymes that dephosphorylate these residues, thereby i nactivating the pathway. Recently, the CL100 phosphatase has been show n to inactivate p42(mapk) in vitro by dephosphorylating Thr 183 and Ty r 185 at similar rates. CL100, the product of an immediate early gene, is synthesized within one hour of stimulating cells with growth facto rs or exposure to oxidative stress or heat shock. Incubation of NIH 3T 3 fibroblasts with cycloheximide prevents both synthesis of CL100 and inactivation of p42(mapk) after stimulation with serum. Results: Deple ting cells of CL100 and preventing its induction using cycloheximide s topped the inactivation of p42(mapk) in Swiss 3T3 fibroblasts followin g stimulation with epidermal growth factor (EGF), but had no effect on the rapid inactivation of p42(mapk) in response to EGF in adipose (3T 3-L1) or chromaffin (PC12) cells or in response to platelet-derived gr owth factor (PDGF) in endothelial (PAE) cells. Moreover, maximal induc tion of CL100 mRNA and a CL100-like activity did not trigger inactivat ion of p42(mapk), which was sustained at a high level after stimulatio n of PC12 cells with nerve growth factor, PAE cells with serum, or Swi ss 3T3 cells with PDGF. Dephosphorylation of Tyr 185 but not Thr 183 o f p42(mapk) was suppressed by vanadate in EGF-stimulated PC12 cells; d ephosphorylation of Thr 183, by contrast, was elicited by a vanadate-i nsensitive activity. Protein phosphatase-2A was the only vanadate-inse nsitive phosphatase acting on Thr 183 of p42(mapk) or on MAPKK to be d etected in PC12 cell extracts. Phosphorylation of Thr183 also inhibite d the dephosphorylation of Tyr 185 in vitro by the major vanadate-sens itive Tyr 185-specific phosphatase, explaining why dephosphorylation o f Thr 183 is rate-limiting for p42(mapk) inactivation in PC12 cells af ter stimulation with EGF. Conclusions: The rapid inactivation of p42(m apk) 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 phosphatas e distinct from CL100. Induction of CL100 is not accompanied by the in activation of p42(mapk) in a number of situations.