Evidence that pp42, a major tyrosine kinase target protein, is a mitogen-activated serine/threonine protein kinase.
pp42, 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 G0 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 II. 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.
Rossomando
A J
AJ
Department of Microbiology, University of Virginia School of Medicine, Charlottesville 22908.
Payne
D M
DM
Weber
M J
MJ
Sturgill
T W
TW
D000818:Animals; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D002478:Cells, Cultured; D004789:Enzyme Activation; D004815:Epidermal Growth Factor / Q000494:pharmacology*; D051379:Mice; D008970:Molecular Weight; D010449:Peptide Mapping; D010748:Phosphopeptides / Q000302:isolation & purification; D010750:Phosphoproteins / Q000302:isolation & purification; D010766:Phosphorylation; D011494:Protein Kinases / Q000302:isolation & purification / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D012697:Serine Endopeptidases; D013379:Substrate Specificity
D010748:Phosphopeptides; D010750:Phosphoproteins; D004815:Epidermal Growth Factor; D011494:Protein Kinases; D011505:Protein-Tyrosine Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase