Abstract
c-Abl is a cytoplasmic tyrosine kinase involved in several signal transduction pathways. Here we report that c-Abl is involved also in insulin receptor signaling. Indeed, c-Abl tyrosine kinase is activated upon insulin stimulation. Inhibition of c-Abl tyrosine kinase by STI571 attenuates the effect of insulin on Akt/GSK-3beta phosphorylation and glycogen synthesis, and at the same time, it enhances the effect of insulin on ERK activation, cell proliferation, and migration. This effect of STI571 is specific to c-Abl inhibition, because it does not occur in Abl-null cells and is restored in c-Abl-reconstituted cells. Numerous evidences suggest that focal adhesion kinase (FAK) is involved in mediating this c-Abl effect. First, anti-phosphotyrosine blots indicate that c-Abl tyrosine kinase activation is concomitant with FAK dephosphorylation in response to insulin, whereas c-Abl inhibition is accompanied by FAK phosphorylation in response to insulin, a response similar to that observed with IGF-I. Second, the c-Abl effects on insulin signaling are not observed in cells devoid of FAK (FAK(-/-) cells). Taken together these results suggest that c-Abl activation by insulin, via a modification of FAK response, may play an important role in directing mitogenic versus metabolic insulin receptor signaling.
Highlights
Despite the high similarity between IR and IGF-IR, these two receptors display different effects: IR mainly elicits metabolic effects, including glucose uptake and glycogen synthesis, and IGF-IR mainly stimulates cell proliferation, survival, and differentiation
Insulin Biological Effects Are Influenced by the Inhibition of c-Abl Tyrosine Kinase Activity—To explore whether c-Abl tyrosine kinase is involved in insulin biological effects, we incubated HepG2 (Fig. 1, left panel) and MCF-7 cells (Fig. 1, right panel) in the presence or the absence of the Abl inhibitor STI571 and evaluated both metabolic and nonmetabolic effects in response to insulin
Evaluation of cell proliferation by methyl thiazolyl tetrazolium (MTT) assay indicated that exposure to STI571 significantly enhanced the effect of tion and migration in response to insulin in ablWTargϪ/Ϫ cells (Fig. 2, B and C, compare bars 8 with bars 11) in a manner similar to that observed in HepG2 (Fig. 1B)
Summary
Reagents and Compounds—STI571 was by Novartis (Basel, Switzerland). The dimerizer AP20187 was provided by Ariad Pharmaceuticals Inc. (Cambridge, MA). Silencing of c-Abl Expression by siRNA—The cells were plated onto six-well plates (105/well), maintained in antibioticfree medium for 24 h, and transfected with a mixture containing Opti-MEM, 8 l/well Lipofectamine 2000 (Invitrogen, San Diego, CA), and either 0.5 g/well scramble small interfering RNA (siRNA) or a mixture of four c-Abl siRNA (Dharmacon Research, Inc., Lafayette, CO) for 5 h. The sequences of these siRNAs are available from the manufacturer, and their specificity has been tested by microarray analysis.
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