Identification of multiple epidermal growth factor-stimulated protein serine/threonine kinases from Swiss 3T3 cells.
Growth factor activation of serine/threonine protein kinases was studied by treating quiescent Swiss 3T3 cells with epidermal growth factor (EGF) and examining cytosolic extracts for protein kinase activity under conditions inhibitory to calcium- and cyclic nucleotide-dependent kinases. Cytosolic extracts of cells stimulated for 5 min were fractionated by Mono Q fast protein liquid chromatography. Eight peaks of kinase activity were resolved, of which five were stimulated by EGF treatment of cells. These peaks were revealed using the synthetic peptide Arg-Arg-Leu-Ser-Ser-Leu-Arg-Ala (S6 peptide), 40 S ribosomal S6 protein, glycogen synthase, microtubule-associated protein 2, and myelin basic protein as substrates. The peaks varied in the kinetics of their activation by EGF and in their response to insulin. Selected peaks were resolved further by sizing gel chromatography. The results together indicate that at least seven distinct fractions of cytosolic kinase activities are stimulated in Swiss 3T3 cells by EGF. One of these, which phosphorylates both S6 protein and S6 peptide, is similar to the S6 kinase characterized previously in this cell line by others. Four additional activities that also phosphorylate the S6 protein and S6 peptide appear unrelated to this enzyme. Finally, two kinase activities that phosphorylate both myelin basic protein and microtubule associated protein 2 are EGF stimulated. One is similar to an insulin-stimulated microtubule-associated protein 2 kinase described in other cell lines whereas the other seems to represent a novel activity. Several of these EGF-stimulated activities were inactivated by protein phosphatases, suggesting that they might be regulated by phosphorylation.
Ahn
N G
NG
Department of Pharmacology, University of Washington, Seattle 98195.
Weiel
J E
JE
Chan
C P
CP
Krebs
E G
EG
D000595:Amino Acid Sequence; D000818:Animals; D002478:Cells, Cultured; D002850:Chromatography, Gel; D003600:Cytosol / Q000201:enzymology; D004815:Epidermal Growth Factor / Q000494:pharmacology*; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D010455:Peptides / Q000138:chemical synthesis; D011494:Protein Kinases / Q000302:isolation & purification / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases; D013379:Substrate Specificity
D010455:Peptides; D004815:Epidermal Growth Factor; D011494:Protein Kinases; D017346:Protein Serine-Threonine Kinases