Insulin and phorbol ester stimulate phosphorylation of a 15,000 dalton membrane protein in rat diaphragm in a similar manner.
The effects of insulin on the phosphorylation of a 15 kilodalton (kDa) membrane protein in rat diaphragm in situ have been investigated. Incubation of the diaphragm with insulin or tumor-promoting phorbol ester increased the 32P-labelling of the 15 kDa protein at serine residues by 50 +/- 8% and 64 +/- 11%, (mean +/- S.E.), respectively. Thermolytic peptide mapping of the 15 kDa protein after insulin treatment of the diaphragm yielded two major phosphopeptides, one of which was absent from digests from control diaphragms. The same two phosphopeptides were identified after incubation of the diaphragm with phorbol ester and after phosphorylation of sarcolemma in vitro with [gamma-32P]ATP and protein kinase C. Additional experiments indicated that pretreatment of diaphragms with insulin or phorbol ester both increased the state of phosphorylation of the 15 kDa sarcolemma protein on phosphorylation sites regulated by protein kinase C. The stimulatory effect of insulin was decreased by staurosporine or by preincubation of the diaphragms with phorbol esters. These results indicate that the insulin-induced increases in protein kinase C activity previously found in rat diaphragm (Walaas et al. (1987) FEBS Lett. 220, 311-318) may be involved in insulin-mediated regulation of phosphorylation of the 15 kDa protein in situ.
Walaas
O
O
Institute of Medical Biochemistry, University of Oslo, Norway.
Horn
R S
RS
Walaas
S I
SI
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D003964:Diaphragm / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D007328:Insulin / Q000494:pharmacology*; D008565:Membrane Proteins / Q000378:metabolism*; D008970:Molecular Weight; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism; D051381:Rats; D011919:Rats, Inbred Strains; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*
D007328:Insulin; D008565:Membrane Proteins; D010761:Phosphorus Radioisotopes; D000255:Adenosine Triphosphate; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate