Identification of a 42-kilodalton phosphotyrosyl protein as a serine(threonine) protein kinase by renaturation.
We have surveyed fibroblast lysates for protein kinases that might be involved in mitogenesis. The assay we have used exploits the ability of blotted, sodium dodecyl sulfate-denatured proteins to regain enzymatic activity after guanidine treatment. About 20 electrophoretically distinct protein kinases could be detected by this method in lysates from NIH 3T3 cells. One of the kinases, a 42-kilodalton serine(threonine) kinase (PK42), was found to possess two- to fourfold-higher in vitro activity when isolated from serum-stimulated cells than when isolated from serum-starved cells. This kinase comigrated on sodium dodecyl sulfate-gels with a protein (p42) whose phosphotyrosine content increased in response to serum stimulation. The time courses of p42 tyrosine phosphorylation and PK42 activation were similar, reaching maximal levels within 10 min and returning to basal levels within 5 h. Both p42 tyrosine phosphorylation and PK42 activation were stimulated by low concentrations of phorbol esters, and the responses of p42 and PK42 to TPA were abolished by chronic 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment. Chronic TPA treatment had less effect on serum-induced p42 tyrosine phosphorylation and PK42 activation. PK42 and p42 bound to DEAE-cellulose, and both eluted at a salt concentration of 250 mM. Thus, PK42 and p42 comigrate and cochromatograph, and the kinase activity of PK42 correlates with the tyrosine phosphorylation of p42. These findings suggest that PK42 and p42 are related or identical, that PK42 is activated by tyrosine phosphorylation, and that this tyrosine phosphorylation can be regulated by protein kinase C.
Department of Molecular and Cell Biology, University of California, Berkeley 94720.
J E
JE
Ferrell
G S
GS
Martin
D000818:Animals; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D002478:Cells, Cultured; D002848:Chromatography, DEAE-Cellulose; D003470:Culture Media; D004591:Electrophoresis, Polyacrylamide Gel; D005347:Fibroblasts / Q000201:enzymology; D015151:Immunoblotting; D007700:Kinetics; D051379:Mice; D008970:Molecular Weight; D010750:Phosphoproteins / Q000302:isolation & purification / Q000378:metabolism*; D019000:Phosphotyrosine; D011489:Protein Denaturation; D011494:Protein Kinases / Q000302:isolation & purification / Q000378:metabolism*; D014443:Tyrosine / Q000031:analogs & derivatives*
D003470:Culture Media; D010750:Phosphoproteins; D019000:Phosphotyrosine; D014443:Tyrosine; D011494:Protein Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases