Insulin and phorbol ester induce distinct phosphorylations of pp60c-src in the BC3H-1 murine myocyte cell line.
Insulin and phorbol esters have been shown to produce similar, non-additive metabolic effects in BC3H-1 murine myocytes. Recently, it has been demonstrated that insulin stimulation of these cells increases production of diacylglycerol, a known activator of protein kinase C (PK-C). To determine if insulin stimulation results in the activation of PK-C, we have examined the effects of insulin and the tumor promoting phorbol ester, 12-0-tetradecanoyl-phorbol-13-acetate (TPA), on the phosphorylation of a known PK-C substrate in vivo, the cellular proto-oncogene product, pp60c-src. Differentiated BC3H-1 monocytes showed an approximate twofold elevation in the [32P] content of pp60c-src following stimulation with insulin or TPA for 20 min, with no detectable change in the level of immunoprecipitable c-src protein. The enhanced phosphorylation in response to each agent localized to serine residues in the amino terminal 16 kD staphylococcal V8 proteolytic fragment. Tryptic phosphopeptide analysis revealed that TPA stimulation resulted in an approximate 18-fold increase in phosphorylation of the serine 12-containing tryptic fragment. Insulin stimulation, however, resulted in an approximate 10-fold increase in phosphorylation of the serine 17-containing tryptic fragment with little or no accompanying increase in serine 12 phosphorylation. In cells exposed to high concentrations of TPA for 16 h to deplete PK-C activity, insulin, but not TPA, stimulated phosphorylation of pp60c-src. These data suggest that insulin and phorbol ester induce phosphorylation of pp60c-src by distinct protein kinases.
Luttrell
L M
LM
Department of Pharmacology, University of Virginia School of Medicine, Charlottesville 22908.
Luttrell
D K
DK
Parsons
S J
SJ
Rogol
A D
AD
D000818:Animals; D002460:Cell Line; D000242:Cyclic AMP / Q000494:pharmacology; D007328:Insulin / Q000494:pharmacology*; D051379:Mice; D009132:Muscles / Q000166:cytology / Q000378:metabolism; D010449:Peptide Mapping; D010766:Phosphorylation; D010768:Phosphoserine / Q000378:metabolism; D011493:Protein Kinase C / Q000502:physiology; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D016392:Proto-Oncogene Proteins pp60(c-src); D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*
D007328:Insulin; D011518:Proto-Oncogene Proteins; D010768:Phosphoserine; D000242:Cyclic AMP; D016392:Proto-Oncogene Proteins pp60(c-src); D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate