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Insulin-activated protein kinase Cbeta bypasses Ras and stimulates mitogen-activated protein kinase activity and cell proliferation in muscle cells.

ABSTRACT: 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.

SUBMITTER: Formisano P

PROVIDER: S-EPMC86107 | biostudies-literature | 2000 Sep

REPOSITORIES: biostudies-literature

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Insulin-activated protein kinase Cbeta bypasses Ras and stimulates mitogen-activated protein kinase activity and cell proliferation in muscle cells.

Formisano P P Oriente F F Fiory F F Caruso M M Miele C C Maitan M A MA Andreozzi F F Vigliotta G G Condorelli G G Beguinot F F

Molecular and cellular biology 20000901 17

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 o ...[more]

PMID: 10938109

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Project description:Signalling by the epidermal growth factor (EGF) receptor (EGFR) has been studied intensively, but for most cell types the analysis is complicated by the fact that EGFR not only homodimerizes but can also form heterodimers with other EGFR family members. Heterodimerization is a particular problem in the study of EGFR mutants, where the true phenotype of the mutants is confounded by the contribution of the heterodimer partner to signal transduction. We have made use of the murine hemopoietic cell line BaF/3, which does not express EGFR family members, to express wild-type (WT) EGFR, three kinase-defective EGFR mutants (V741G, Y740F, and K721R), or a C-terminally truncated EGFR (CT957) and have measured their responses to EGF. We found that under the appropriate conditions EGF can stimulate cell proliferation of BaF/3 cells expressing WT or CT957 EGFRs but not that of cells expressing the kinase-defective mutants. However, EGF promotes the survival of BaF/3 cells expressing either of the kinase-defective receptors (V741G and Y740F), indicating that these receptors can still transmit a survival signal. Analysis of the early signalling events by the WT, V741G, and Y740F mutant EGF receptors indicated that EGF stimulates comparable levels of Shc phosphorylation, Shc-GRB-2 association, and activation of Ras, B-Raf, and Erk-1. Blocking the mitogen-activated protein kinase (MAPK) signalling pathway with the specific inhibitor PD98059 abrogates completely the EGF-dependent survival of cells expressing the kinase-defective EGFR mutants but has no effect on the EGF-dependent proliferation mediated by WT and CT957 EGFRs. Similarly, the Src family kinase inhibitor PP1 abrogates EGF-dependent survival without affecting proliferation. However blocking phosphatidylinositol-3-kinase or JAK-2 kinase with specific inhibitors does arrest growth factor-dependent cell proliferation. Thus, EGFR-mediated mitogenic signalling in BaF/3 cells requires an intact EGFR tyrosine kinase activity and appears to depend on the activation of both the JAK-2 and PI-3 kinase pathways. Activation of the Src family of kinases or of the Ras/MAPK pathway can, however, be initiated by a kinase-impaired EGFR and is linked to survival.

Dataset Information

Insulin-activated protein kinase Cbeta bypasses Ras and stimulates mitogen-activated protein kinase activity and cell proliferation in muscle cells.

Publications

Insulin-activated protein kinase Cbeta bypasses Ras and stimulates mitogen-activated protein kinase activity and cell proliferation in muscle cells.

Similar Datasets

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