Glucose stimulates the tyrosine phosphorylation of Crk-associated substrate in pancreatic beta-cells.
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.
Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, Indiana 46285, USA. konrad.robert@lilly.com
Robert J
RJ
Konrad
Gerald
G
Gold
Thomas N
TN
Lee
Robert
R
Workman
Carol L
CL
Broderick
Michael D
MD
Knierman
D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D015153:Blotting, Western; D002217:Carbachol / Q000378:metabolism; D002460:Cell Line; D002478:Cells, Cultured; D003599:Cytoskeleton / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; D005947:Glucose / Q000378:metabolism / Q000494:pharmacology*; D007074:Immunoglobulin G / Q000378:metabolism; D007515:Islets of Langerhans / Q000378:metabolism*; D013058:Mass Spectrometry; D010455:Peptides / Q000737:chemistry; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D011233:Precipitin Tests; D011485:Protein Binding; D011487:Protein Conformation; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D013997:Time Factors; D014443:Tyrosine / Q000378:metabolism*; D018909:src Homology Domains
D048868:Adaptor Proteins, Signal Transducing; D007074:Immunoglobulin G; C099942:NEDD9 protein, human; D010455:Peptides; D010750:Phosphoproteins; D014443:Tyrosine; D002217:Carbachol; D011505:Protein-Tyrosine Kinases; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; C496052:Ptk2 protein, rat; D005947:Glucose