Nicotinic agonists, phorbol esters, and growth factors activate two extracellular signal-regulated kinases, ERK1 and ERK2, in bovine chromaffin cells.
Treatment of bovine chromaffin cells with nicotinic agonists, phorbol esters, and growth factors increases protein kinase activity toward microtubule-associated protein-2 and myelin basic protein (MBP) in vitro. To characterize the kinases that are activated by these agents, we separated chromaffin cell proteins by electrophoresis in sodium dodecyl sulfate-polyacrylamide gels into which MBP had been incorporated, allowed the proteins to renature, and then assayed MBP kinase activity by incubating the gels with [gamma-32P]ATP. Chromaffin cells contain a family of kinases that phosphorylate MBP in vitro. Two of these kinases, of M(r) 46,000 and 42,000 (PK46 and PK42), were activated by treatment of the cells with dimethylphenylpiperazinium (DMPP), phorbol 12,13-dibutyrate (PDBu), or insulin-like growth factor I (IGF-I). Activation of PK46 and PK42 by DMPP was dependent on extracellular Ca2+, whereas the effects of PDBu and IGF-I were Ca2+ independent. Down-regulation of protein kinase C by incubation of the cells with PDBu abolished the activation of PK46 and PK42 by DMPP, PDBu, and IGF-I. Staurosporine, a protein kinase C inhibitor, prevented the activation of PK46 and PK42 by DMPP and PDBu but did not block the activation of these kinases by IGF-I. Immunoblotting experiments with antiphosphotyrosine (anti-PTyr) antibodies demonstrated that agents that increased the kinase activities of PK46 and PK42 also increased the apparent PTyr content of M(r) 46,000 and 42,000 proteins. PK46 and PK42 comigrated with proteins that reacted with antibodies against extracellular signal-regulated kinases (ERKs). Thus, PK46 and PK42 appear to be the bovine homologues of ERK1 and ERK2.(ABSTRACT TRUNCATED AT 250 WORDS)
Pavlović-Surjancev
B
B
Department of Pediatrics, University of Chicago, IL 60637.
Cahill
A L
AL
Perlman
R L
RL
D000470:Alkaloids / Q000494:pharmacology; D000818:Animals; D002417:Cattle; D002478:Cells, Cultured; D002838:Chromaffin System / Q000166:cytology* / Q000201:enzymology*; D004246:Dimethylphenylpiperazinium Iodide / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000187:drug effects; D005731:Ganglionic Stimulants / Q000494:pharmacology*; D006133:Growth Substances / Q000494:pharmacology*; D015151:Immunoblotting; D007334:Insulin-Like Growth Factor I / Q000494:pharmacology; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D008970:Molecular Weight; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology; D010703:Phorbol Esters / Q000494:pharmacology*; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000502:physiology; D011494:Protein Kinases / Q000032:analysis / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000032:analysis / Q000378:metabolism*; D019311:Staurosporine
D000470:Alkaloids; D005731:Ganglionic Stimulants; D006133:Growth Substances; D010703:Phorbol Esters; D015240:Phorbol 12,13-Dibutyrate; D004246:Dimethylphenylpiperazinium Iodide; D007334:Insulin-Like Growth Factor I; D011494:Protein Kinases; D017346:Protein Serine-Threonine Kinases; D011493:Protein Kinase C; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D019311:Staurosporine