Insulin-activated protein kinase Cbeta bypasses Ras and stimulates mitogen-activated protein kinase activity and cell proliferation in muscle cells.
In L6 muscle cells expressing wild-type human insulin receptors (L6hIR), insulin induced protein kinase Calpha (PKCalpha) and beta activities. The expression of kinase-deficient IR mutants abolished insulin stimulation of these PKC isoforms, indicating that receptor kinase is necessary for PKC activation by insulin. In L6hIR cells, inhibition of insulin receptor substrate 1 (IRS-1) expression caused a 90% decrease in insulin-induced PKCalpha and -beta activation and blocked insulin stimulation of mitogen-activated protein kinase (MAPK) and DNA synthesis. Blocking PKCbeta with either antisense oligonucleotide or the specific inhibitor LY379196 decreased the effects of insulin on MAPK activity and DNA synthesis by >80% but did not affect epidermal growth factor (EGF)- and serum-stimulated mitogenesis. In contrast, blocking c-Ras with lovastatin or the use of the L61,S186 dominant negative Ras mutant inhibited insulin-stimulated MAPK activity and DNA synthesis by only about 30% but completely blocked the effect of EGF. PKCbeta block did not affect Ras activity but almost completely inhibited insulin-induced Raf kinase activation and coprecipitation with PKCbeta. Finally, blocking PKCalpha expression by antisense oligonucleotide constitutively increased MAPK activity and DNA synthesis, with little effect on their insulin sensitivity. We make the following conclusions. (i) The tyrosine kinase activity of the IR is necessary for insulin activation of PKCalpha and -beta. (ii) IRS-1 phosphorylation is necessary for insulin activation of these PKCs in the L6 cells. (iii) In these cells, PKCbeta plays a unique Ras-independent role in mediating insulin but not EGF or other growth factor mitogenic signals.
Dipartimento di Biologia e Patologia Cellulare e Molecolare and Centro di Endocrinologia ed Oncologia Sperimentale del CNR, Università di Napoli Federico II, Naples 80131, Italy.
P
P
Formisano
F
F
Oriente
F
F
Fiory
M
M
Caruso
C
C
Miele
M A
MA
Maitan
F
F
Andreozzi
G
G
Vigliotta
G
G
Condorelli
F
F
Beguinot
D015153:Blotting, Western; D002455:Cell Division; D002460:Cell Line; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004815:Epidermal Growth Factor / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D006801:Humans; D019161:Hydroxymethylglutaryl-CoA Reductase Inhibitors / Q000494:pharmacology; D007328:Insulin / Q000378:metabolism*; D055504:Insulin Receptor Substrate Proteins; D007527:Isoenzymes / Q000378:metabolism*; D008148:Lovastatin / Q000494:pharmacology; D020935:MAP Kinase Signaling System / Q000187:drug effects*; D009132:Muscles / Q000378:metabolism; D016376:Oligonucleotides, Antisense; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010750:Phosphoproteins / Q000037:antagonists & inhibitors; D010766:Phosphorylation; D011233:Precipitin Tests; D020033:Protein Isoforms; D011493:Protein Kinase C / Q000378:metabolism*; D064546:Protein Kinase C beta; D051571:Protein Kinase C-alpha; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D019908:Proto-Oncogene Proteins c-raf / Q000378:metabolism; D016283:Proto-Oncogene Proteins p21(ras) / Q000378:metabolism; D011972:Receptor, Insulin / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D015398:Signal Transduction; D013997:Time Factors; D014162:Transfection; D018631:ras Proteins / Q000378:metabolism
D004791:Enzyme Inhibitors; D019161:Hydroxymethylglutaryl-CoA Reductase Inhibitors; C526292:IRS1 protein, human; D007328:Insulin; D055504:Insulin Receptor Substrate Proteins; D007527:Isoenzymes; D016376:Oligonucleotides, Antisense; D010750:Phosphoproteins; D020033:Protein Isoforms; D011994:Recombinant Proteins; D004815:Epidermal Growth Factor; D006160:Guanosine Triphosphate; D008148:Lovastatin; D019869:Phosphatidylinositol 3-Kinases; D011505:Protein-Tyrosine Kinases; D011972:Receptor, Insulin; D019908:Proto-Oncogene Proteins c-raf; C496628:PRKCA protein, human; D011493:Protein Kinase C; D064546:Protein Kinase C beta; D051571:Protein Kinase C-alpha; C501009:HRAS protein, human; D016283:Proto-Oncogene Proteins p21(ras); D018631:ras Proteins