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ABSTRACT: Previously, we demonstrated glucose-induced beta-cell tyrosine phosphorylation of p130Cas, a protein containing 15 YXXP repeats that can become tyrosine phosphorylated and bind Src-homology 2 (SH2)-containing proteins. In light of the importance of p130Cas in other cell types, we determined which beta-cell proteins exhibited glucose-induced association with p130Cas.
beta cells were stimulated with glucose and/or the muscarinic agonist carbachol to determine which SH2-containing adapter proteins underwent glucose-induced association with p130Cas.
The SH2-containing adapter protein Crk underwent glucose-induced association with p130Cas, while other SH2-containing proteins such as grb2, PI3 kinase, Shp-2, paxillin, and pyk2 did not. Glucose-induced Crk-p130Cas association was rapid and sustained and was maximal with the combination of glucose and carbachol, paralleling insulin secretion. There was no increased tyrosine phosphorylation of Crk itself. The expression of Crk in isolated rat islets was also demonstrated.
beta cells contain the SH2-containing adapter protein Crk, which undergoes glucose-induced association with p130Cas.
Pancreas 12/2004; 29(4):e100-5. · 2.39 Impact Factor
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ABSTRACT: Several years ago, we demonstrated that glucose induced tyrosine phosphorylation of a 125-kDa protein (p125) in pancreatic beta-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 beta-TC3 insulinoma cells, beta-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 betaTC6,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 beta-cells, confirming glucose-induced tyrosine phosphorylation of Cas. Further experiments demonstrated that in beta-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 beta-cell protein is Cas and that Cas undergoes rapid glucose-induced tyrosine phosphorylation in beta-cells.
Journal of Biological Chemistry 08/2003; 278(30):28116-22. · 4.77 Impact Factor
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ABSTRACT: Amylin, an islet amyloid peptide secreted by the pancreatic beta cell, has been proposed as a humoral regulator of islet insulin secretion. Four separate preparations of amylin were tested for effects on hormone secretion in both freshly isolated and cultured rat islets and in HIT-T15, hamster insulinoma cells. With all three experimental models, exposure to human amylin acid and human and rat amylin at concentrations as high as 100 nM had no significant effect on rates of insulin or glucagon secretion. These observations suggest that amylin, even at concentrations appreciably higher than those measured in peripheral plasma, is not a significant humoral regulator of islet hormone secretion.
Biochemical and Biophysical Research Communications 07/1991; · 2.48 Impact Factor
