Evidence that pp42, a major tyrosine kinase target protein, is a mitogen-activated serine/threonine protein kinase.

Abstract

ABSTRACTpp42, a low-abundance 42-kDa protein, becomes transiently phosphorylated on tyrosine after stimulation of fibroblasts by a variety of mitogens, including epidermal growth factor, platelet-derived growth factor, phorbol 12-myristate 13-acetate, thrombin, and insulin-like growth factor II. The induction of pp42 phosphorylation on tyrosine by such diverse mitogenic agents suggests an important role for pp42 in the cascade of events necessary for cell transition from Go into the cell cycle. However, as with most proteins identified on the basis of their tyrosine phosphorylation, the function of pp42 in cellular regulation is unknown. In this manuscript we report evidence that suggests that pp42 is a serine/threonine-specific protein kinase. Stimulation of 3T3-L1 cells with insulin has been shown to activate a cytosolic serine/threonine kinase capable of phosphorylating microtubule-associated protein 2 (MAP-2) and ribosomal protein S6 kinase H. This cytosolic serine/threonine protein kinase, which itself is phosphorylated on tyrosine, has been termed "MAP kinase." We now report that pp42 phosphorylation and MAP kinase activation occur in fibroblasts in response to similar mitogens, that the two proteins comigrate on one-and two-dimensional polyacrylamide gels, and that the two proteins copurify chromatographically. The major peptides generated from purified MAP kinase by V8 protease digestion are present as a subset of the peptides in digests of pp42 excised from two-dimensional gels. Thus, the results suggest that MAP kinase is tyrosine-phosphorylated pp42.Many growth-factor receptors and oncogene products are tyrosine protein kinases. To understand the mechanisms of intracellular signaling used by these kinases, it will be important to identify and characterize their cellular substrate proteins. Although numerous tyrosine-phosphorylated proteins have been identified by gel electrophoresis in oncogenically transformed or growth factor-stimulated cells, in most cases the biological significance of these phosphorylations has not been determined. pp42 is among the most widely studied of these tyrosine-phosphorylated proteins; it was identified by gel electrophoresis in cells stimulated by any of a number of diverse mitogens [epidermal growth factor (EGF), plateletderived growth factor, insulin-like growth factor II, thrombin, or phorbol 12-myristate 13-acetate (PMA; also called TPA)](1-7) and also has been found in at least some oncogenically transformed cells (8-10). Because ofthe wide variety ofagents that stimulate this phosphorylation, pp42 is believed to be involved in some unknown step in intracellular signaling that is shared by all of these mitogens.Recently a novel serine/threonine protein kinase was identified in insulin-stimulated 3T3-L1 cells (11). This protein kinase has been shown to phosphorylate microtubuleassociated protein 2 (MAP-2) as well as ribosomal protein S6 kinase II in vitro (12). The kinase was also found to migrate on sodium dodecyl sulfate (SDS)/polyacrylamide gels with a molecular ...

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