Overexpression of the Shb SH2 domain-protein in insulin-producing cells leads to altered signaling through the IRS-1 and IRS-2 proteins

Abstract

Background: Overexpression of the Src homology 2 domain protein Shb in β-cells of transgenic mice has been shown to promote an increased β-cell mass. To investigate the mechanisms by which Shb controls the β-cell mass, we have presently studied the effects of Shb overexpression on the IRS-1-induced signaling pathway in mouse islet β-cells and in insulin-producing RINm5F cells and correlated these effects to growth and death patterns. Materials and Methods: Shb overexpression was achieved in RINm5F cells by selection of stable clones or by FACS purification of transiently transfected cells. For Shb overexpression in primary mouse islet cells, a Shb-transgene mouse was used. Cell proliferation and death rates were determined using flow cytometry. Serum-, insulin-, and IGF-1-stimulated signaling events were studied by immunoblot, immunoprecipitation, and in vitro kinase procedures. Results: Transient Shb overexpression in RINm5F cells resulted in increased proliferation. Both Shb-overexpressing RINm5F cells and islet cells from transgenic mice (islet Shb) exhibited increased basal tyrosine phosphorylation of IRS-1. Shb overexpression resulted also in the assembly and activation of a multiunit complex consisting of at least Shb, IRS-1, IRS-2, FAK, and PI3K. Consequently, the phosphorylation of Akt was enhanced under basal conditions in Shb overexpressing cells. Finally, Shb overexpression did not affect insulin-induced phosphorylation of the PI3K-antagonist PTEN. Conclusion: It is concluded that the Shb-induced alterations in the IRS-1/PI3K/Akt pathway may be relevant to the understanding of growth and death patterns of insulin-producing cells.