Insulin-activated protein kinase C beta 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), in sulin induced protein kinase Ca (PKC alpha) and beta activities. The expres sion of kinase-deficient IR mutants abolished insulin stimulation of these PKC isoforms, indicating that receptor kinase is necessary for PKC activati on by insulin. In L6hIR cells, inhibition of insulin receptor substrate 1 ( IRS-1) expression caused a 90% decrease in insulin-induced PKC alpha and -b eta activation and blocked insulin stimulation of mitogen activated protein kinase (MAPK) and DNA synthesis. Blocking PKC beta with either antisense o ligonucleotide or the specific inhibitor LY379196 decreased the effects of insulin on MAPK activity and DNA synthesis by > 80% but did not affect epid ermal growth factor (EGF)- and serum-stimulated mitogenesis. In contrast, b locking 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, PKC beta block did not affect Ras activity but almost completely inhibited insulin-induced Ra f kinase activation and coprecipitation with PKC beta. Finally, blocking PK C alpha expression by antisense oligonucleotide constitutively increased MA PK activity and DNA synthesis, with little effect on their insulin sensitiv ity. We make the following conclusions. (i) The tyrosine kinase activity of the IR is necessary for insulin activation of PKC alpha and -beta. (ii) IR S-1 phosphorylation is necessary for insulin activation of these PKCs in th e L6 cells. (iii) In these cells, PKC beta plays a unique Ras-independent r ole in mediating insulin but not EGF or other growth factor mitogenic signa ls.