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Overexpression of the Shb SH2 Domain-Protein in Insulin-Producing Cells Leads to Altered Signaling Through the IRS-1 and IRS-2 Proteins

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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.

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References

  1. Weir GC, Bonner-Weir S, Leahy JL. (1990) Islet mass and function in diabetes and transplantation. Diabetes 39: 401–405.

    Article  CAS  PubMed  Google Scholar 

  2. Brüning JC, Winnay J, Bonner-Weir S, Taylor SI, Accili D, Kahn CR. (1997) Development of a novel polygenic model of NIDDM in mice heterozygous for IR and IRS-1 null alleles. Cell 88: 561–572.

    Article  PubMed  Google Scholar 

  3. Withers DJ, Gutierrez JS, Towery H, et al. (1998) Disruption of IRS-2 causes type 2 diabetes in mice Nature 391: 900–904.

    Article  CAS  PubMed  Google Scholar 

  4. Withers DJ, Burks DJ, Towery HH, Altamuro SL, Flint CL, White MF. (1999) Irs-2 coordinates Igf-1 receptor-mediated beta-cell development and peripheral insulin signalling. Nature Gen. 23: 32–40.

    Article  CAS  Google Scholar 

  5. Leibiger IB, Leibiger B Moede T, Berggren PO. (1998) Exocytosis of insulin promotes insulin gene transcription via the insulin receptor/PI-3 kinase/p70 s6 kinase and CaM kinase pathways. Mol. Cell 1: 933–938.

    Article  CAS  PubMed  Google Scholar 

  6. Kulkarni RN, Brüning JC, Winnay JN, Postic C, Magnuson MA, Kahn CR. (1999) Tissue-specific knockout of the insulin receptor in pancreatic beta cells creates an insulin secretory defect similar to that in type 2 diabetes. Cell 96: 329–339.

    Article  CAS  PubMed  Google Scholar 

  7. Whitman M, Downes CP, Keeler M, Keller T, Cantley L. (1988) Type I phosphatidylinositol kinase makes a novel inositol phospholipid, phosphatidylinositol-3-phosphate. Nature 332: 644–646.

    Article  CAS  PubMed  Google Scholar 

  8. Franke TF, Kaplan DR, Cantley LC, Toker A. (1997) Direct regulation of the Akt proto-oncogene product by phosphatidylinositol-3,4-bisphosphate. Science 275: 665–668.

    Article  CAS  PubMed  Google Scholar 

  9. Stokoe D, Stephens LR, Copeland T, et al. (1997) Dual role of phosphatidylinositol-3,4,5-trisphosphate in the activation of protein kinase B. Science 277: 567–570.

    Article  CAS  PubMed  Google Scholar 

  10. Franke TF, Kaplan D, Cantley L. (1997) PI3K: downstream AKTion blocks apoptosis. Cell 88: 435–437.

    Article  CAS  PubMed  Google Scholar 

  11. Datta SR, Dudek H, Tao X, et al. (1997) Akt phosphorylation of BAD couples survival signals to the cell-intrinsic death machinery. Cell 91: 231–241.

    Article  CAS  PubMed  Google Scholar 

  12. Cardone MH, Roy N, Stennicke HR, et al. (1998) Regulation of cell death protease caspase-9 by phosphorylation. Science 282: 1318–1321.

    Article  CAS  PubMed  Google Scholar 

  13. Brunet A, Bonni A, Zigmond MJ, et al. (1999) Akt promotes cell survival by phosphorylating and inhibiting a Forkhead transcription factor. Cell 96: 857–868.

    Article  CAS  PubMed  Google Scholar 

  14. Maehama T, Dixon JE. (1998) The tumor suppressor, PTEN/MMAC1, dephosphorylates the lipid second messenger, phosphatidylinositol 3,4,5-trisphosphate. J. Biol. Chem. 273: 13375–13378.

    Article  CAS  PubMed  Google Scholar 

  15. Cantely LC, Neel BG. (1999) New insights into tumor suppression: PTEN suppresses tumor formation by restraining the phosphoinositide 3-kinase/AKT pathway. Proc. Natl. Acad. Sci. U. S. A. 96: 4240–4245.

    Article  Google Scholar 

  16. Welsh M, Mares J, Karlsson T, Lavergne C, Bréant B, Claesson-Welsh L. (1994) Shb is a ubiquitously expressed Src homology 2 protein. Oncogene 9: 19–27.

    PubMed  CAS  Google Scholar 

  17. Welsh M, Songyang Z, Frantz D, et al. (1998) Stimulation through the T cell receptor leads to interactions between SHB and several signaling proteins Oncogene 16: 891–902.

    Article  CAS  PubMed  Google Scholar 

  18. Karlsson T, Songyang Z, Landgren E, et al. (1995) Molecular interactions of the Src homology 2 domain protein Shb with phosphotyrosine residues, tyrosine kinase receptors and Src homology 3 domain proteins Oncogene 10: 1475–1483.

    PubMed  CAS  Google Scholar 

  19. Lindholm CK, Gylfe E, Zhang W, Samelson LE, Welsh M. (1999) Requirement of the Src homology 2 domain protein Shb for T cell receptor-dependent activation of the interleukin-2 gene nuclear factor for activation of T cells element in Jurkat T cells. J. Biol. Chem. 274: 28050–28057.

    Article  CAS  PubMed  Google Scholar 

  20. Karlsson T, Welsh M. (1996) Apoptosis of NIH3T3 cells overexpressing the Src homology 2 domain protein Shb. Oncogene 13: 955–961.

    PubMed  CAS  Google Scholar 

  21. Welsh M, Christmansson L, Karlsson T, Sandler S, Welsh N. (1999) Transgenic mice expressing Shb adaptor protein under the control of rat insulin promoter exhibit altered viability of pancreatic islet cells. Mol. Med. 5: 169–180.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Andersson A. (1978) Isolated mouse pancreatic islets in culture: effects of serum and different culture media on the insulin production of the islets. Diabetologia 14: 397–404.

    Article  CAS  PubMed  Google Scholar 

  23. Saldeen J, Curiel DT, Eizirik DL, et al. (1996) Efficient gene transfer to dispersed human pancreatic islet cells in vitro using adenovirus-polylysine/DNA complexes or polycationic liposomes. Diabetes 45: 1197–1203.

    Article  CAS  PubMed  Google Scholar 

  24. Saldeen J, Lee JC, Welsh N. (2001) Role of p38 mitogenactivated protein kinase (p38 MAPK) in cytokine-induced rat islet cell apoptosis. Biochem. Pharmacol. 61:1561–1569.

    Article  CAS  PubMed  Google Scholar 

  25. Welsh N. (1996) Interleukin-1 beta-induced ceramide and diacylglycerol generation may lead to activation of the c-Jun NH2-terminal kinase and the transcription factor ATF2 in the insulin-producing cell line RINm5F J. Biol. Chem. 271: 8307–8312.

    Article  CAS  PubMed  Google Scholar 

  26. Traynor-Kaplan AE, Thompson BL, Harris AL, Taylor P, Omann GM, Sklar LA. (1989) Transient increase in phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol trisphosphate during activation of human neutrophils. J. Biol. Chem. 264:15668–15673.

    PubMed  CAS  Google Scholar 

  27. Homqvist K, Cross M, Riley D, Welsh M. (in press) The Shb adaptor protein causes Sic-dependent cell spreading and activation of focal adhesion kinase in murine brain endothelial cells. Cell. Signal.

  28. Sonoda Y, Watanabe S, Matsumoto Y, Aizu-Yokota E, Kasahara T. (1999) FAK is the upstream signal protein of the phosphatidylinositol 3-kinase-Akt survival pathway in hydrogen peroxide-induced apoptosis of a human glioblastoma cell line. J. Biol. Chem. 274: 10566–10570.

    Article  CAS  PubMed  Google Scholar 

  29. Lebrun P, Mothe-Satney I, Delahaye L, Obberghen EV, Baron V. (1998) Insulin receptor substrate-1 as a signaling molecule for focal adhesion kinase pp125(FAK) and pp60(src). J. Biol. Chem. 273: 32244–32253.

    Article  CAS  PubMed  Google Scholar 

  30. Chen HC, Appeddu PA, Isoda H, Guan JL. (1996) Phosphorylation of tyrosine 397 in focal adhesion kinase is required for binding phosphatidylinositol 3-kinase. J Biol. Chem. 271: 26329–26334.

    Article  CAS  PubMed  Google Scholar 

  31. Hungersford JE, Compton MT, Matter ML, Hoffstrom BG, Otey CA. (1996) Inhibition of pp125FAK in cultured fibroblasts results in apoptosis. J. Cell. Biol. 135: 1383–1390.

    Article  Google Scholar 

  32. Ueno H, Honda H, Nakamoto T, et al. (1997) The phosphatidylinositol 3′ kinase pathway is required for the survival signal of leukocyte tyrosine kinase. Oncogene 14: 3067–3072.

    Article  CAS  PubMed  Google Scholar 

  33. Zhou LF, Xu SQ, Dews M, Baserga R. (1997) Co-operation of simian virus 40 T antigen and insulin receptor substrate-1 in protection from apoptosis induced by interleukin-3 withdrawal. Oncogene 15: 961–970.

    Article  CAS  Google Scholar 

  34. Schneller M, Vuori K, Ruoslahti E. (1997) Alphavbeta3 integrin associates with activated insulin and PDGFbeta receptors and potentiates the biological activity of PDGF. EMBO J. 16: 5600–5607.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Anneren C. (2002) Dual role of the tyrosine kinase GTK and the adaptor protein SHB in beta-cell growth: enhanced beta-cell replication after 60% pancreatectomy and increased sensitivity to streptozotocin. J. Endocrinol. 172: 145–153.

    Article  CAS  PubMed  Google Scholar 

  36. Vazquez F, Ramaswamy S, Nakamura R, Sellers WR. (2000) Phosphorylation of the PTEN tail regulates protein stability and function. Mol. Cell. Biol. 20: 5010–5018.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Torres J, Pulido R. (2001) The tumor suppressor PTEN is phosphorylated by the protein kinase CK2 at its C terminus. Implications for PTEN stability to proteasome-mediated degradation. J. Biol. Chem. 276: 993–998.

    Article  CAS  PubMed  Google Scholar 

  38. Rothenberg PL, Willison LD, Simon J, Wolf BA. (1995) Glucose-induced insulin receptor tyrosine phosphorylation in insulin-secreting beta-cells. Diabetes 44: 802–809.

    Article  CAS  PubMed  Google Scholar 

  39. Kwon G, Xu G, Marshall CA, McDaniel ML. (1999) Tumor necrosis factor alpha-induced pancreatic beta-cell insulin resistance is mediated by nitric oxide and prevented by 15–de-oxy-Delta12,14-prostaglandin J2 and aminoguanidine. A role for peroxisome proliferator-activated receptor gamma activation and inos expression. J. Biol. Chem. 274: 18702–18708.

    Article  CAS  PubMed  Google Scholar 

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Acknowledgments

The excellent technical assistance of Ing-Marie Mörsare and Ing-Britt Hallgren is gratefully acknowledged. This work was supported by grants from the Swedish Medical Research Council (12X-109, 12X-11564, 31X-10822, 72P-12995), the Swedish Diabetes Association, the Nordic Insulin Fund, the Juvenile Diabetes Foundation International, the Wallenberg Foundation, and the Family Ernfors Fund.

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Authors and Affiliations

  1. Department of Medical Cell Biology, Uppsala University, Box 571, Biomedicum, S-75123, Uppsala, Sweden

    Nils Welsh, Natalia Makeeva & Michael Welsh

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  1. Nils Welsh
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  2. Natalia Makeeva
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  3. Michael Welsh
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Correspondence to Nils Welsh.

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Contributed by D. Steiner.

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Welsh, N., Makeeva, N. & Welsh, M. Overexpression of the Shb SH2 Domain-Protein in Insulin-Producing Cells Leads to Altered Signaling Through the IRS-1 and IRS-2 Proteins. Mol Med 8, 695–704 (2002). https://doi.org/10.1007/BF03402033

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