Glucose Stimulates the Tyrosine Phosphorylation of Crk-associated Substrate in Pancreatic β-Cells

Several years ago, we demonstrated that glucose induced tyrosine phosphorylation of a 125-kDa protein (p125) in pancreatic β-cells (Konrad, R. J., Dean, R. M., Young, R. A., Bilings, P. C., and Wolf, B. A. (1996) J. Biol. Chem. 271, 24179–24186). Glucose induced p125 tyrosine phosphorylation in β-TC...

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Veröffentlicht in:The Journal of biological chemistry 2003-07, Vol.278 (30), p.28116-28122
Hauptverfasser: Konrad, Robert J., Gold, Gerald, Lee, Thomas N., Workman, Robert, Broderick, Carol L., Knierman, Michael D.
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Sprache:eng
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Zusammenfassung:Several years ago, we demonstrated that glucose induced tyrosine phosphorylation of a 125-kDa protein (p125) in pancreatic β-cells (Konrad, R. J., Dean, R. M., Young, R. A., Bilings, P. C., and Wolf, B. A. (1996) J. Biol. Chem. 271, 24179–24186). Glucose induced p125 tyrosine phosphorylation in β-TC3 insulinoma cells, β-HC9 cells, and in freshly isolated rat islets, whereas increased tyrosine phosphorylation was not observed with other fuel secretagogues. Initial efforts to identify p125 were unsuccessful, so a new approach was taken. The protein was purified from βTC6,F7 cells via an immunodepletion method. After electrophoresis and colloidal Coomassie Blue staining, the area of the gel corresponding to p125 was excised and subjected to tryptic digestion. Afterward, mass spectrometry was performed and the presence of Crk-associated substrate (Cas) was detected. Commercially available antibodies against Cas were obtained and tested directly in β-cells, confirming glucose-induced tyrosine phosphorylation of Cas. Further experiments demonstrated that in β-cells the glucose-induced increase in Cas tyrosine phosphorylation occurs immediately and is not accompanied by increased focal adhesion kinase tyrosine phosphorylation. Finally, it is also demonstrated via Western blotting that Cas is present in normal isolated rat islets. Together, these results show that the identity of the previously described p125 β-cell protein is Cas and that Cas undergoes rapid glucose-induced tyrosine phosphorylation in β-cells.
ISSN:0021-9258
1083-351X
DOI:10.1074/jbc.M212899200