Overexpression of the Shb SH2 domain-protein in insulin-producing cells leads to altered signaling through the IRS-1 and IRS-2 proteins.
BACKGROUND
Overexpression of the Src homology 2 domain protein Shb in beta-cells of transgenic mice has been shown to promote an increased beta-cell mass. To investigate the mechanisms by which Shb controls the beta-cell mass, we have presently studied the effects of Shb overexpression on the IRS-1-induced signaling pathway in mouse islet beta-cells and in insulin-producing RINm5F cells and correlated these effects to growth and death patterns.


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.


CONCLUSIONS
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.
Welsh
Nils
N
Department of Medical Cell Biology, Uppsala University, Sweden. Nils.Welsh@medcellbiol.uu.se
Makeeva
Natalia
N
Welsh
Michael
M
D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D005434:Flow Cytometry; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; D049452:Green Fluorescent Proteins; D015151:Immunoblotting; D066298:In Vitro Techniques; D007328:Insulin / Q000378:metabolism* / Q000494:pharmacology; D055504:Insulin Receptor Substrate Proteins; D007334:Insulin-Like Growth Factor I / Q000494:pharmacology; D047908:Intracellular Signaling Peptides and Proteins; D007515:Islets of Langerhans / Q000378:metabolism*; D008164:Luminescent Proteins / Q000378:metabolism; D051379:Mice; D008822:Mice, Transgenic; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D051059:PTEN Phosphohydrolase; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism*; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism; D010766:Phosphorylation; D011233:Precipitin Tests; D017346:Protein Serine-Threonine Kinases; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000378:metabolism / Q000502:physiology*; D051057:Proto-Oncogene Proteins c-akt; D051381:Rats; D015398:Signal Transduction / Q000187:drug effects*; D014162:Transfection; D025521:Tumor Suppressor Proteins / Q000378:metabolism; D014443:Tyrosine; D018909:src Homology Domains / Q000502:physiology
D048868:Adaptor Proteins, Signal Transducing; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; D049452:Green Fluorescent Proteins; D007328:Insulin; D055504:Insulin Receptor Substrate Proteins; D007334:Insulin-Like Growth Factor I; D047908:Intracellular Signaling Peptides and Proteins; D008164:Luminescent Proteins; D020928:Mitogen-Activated Protein Kinases; D051059:PTEN Phosphohydrolase; D019869:Phosphatidylinositol 3-Kinases; D010750:Phosphoproteins; D010744:Phosphoric Monoester Hydrolases; D017346:Protein Serine-Threonine Kinases; D011505:Protein-Tyrosine Kinases; D011518:Proto-Oncogene Proteins; D051057:Proto-Oncogene Proteins c-akt; D025521:Tumor Suppressor Proteins; D014443:Tyrosine; C526295:Irs1 protein, mouse; C526296:Irs1 protein, rat; C526298:Irs2 protein, mouse; C526299:Irs2 protein, rat; C490081:SHB protein, human; C085634:Shb protein, mouse; C490082:Shb protein, rat; C496051:Ptk2 protein, mouse; C496052:Ptk2 protein, rat; C494920:Akt1 protein, rat