GUANOSINE 5'-O-(3-THIOTRIPHOSPHATE) (GTP-GAMMA-S) STIMULATION OF GLUT4 TRANSLOCATION IS TYROSINE KINASE-DEPENDENT

Guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) treatment of permeab ilized adipocytes results in GLUT4 translocation similar to that elici ted by insulin treatment. However, although the selective phosphatidyl inositol 3-kinase inhibitor, wortmannin, completely prevented insulin- stimulated GLUT4 translocation, it was without effect on GTP gamma S-s timulated GLUT4 translocation, In addition, insulin was an effective s timulant, whereas GTP gamma S was a very weak activator of the downstr eam Akt serine/threonine kinase, Consistent with an Akt-independent me chanism, guanosine 5'-O-2-(thio) diphosphate inhibited insulin-stimula ted GLUT4 translocation without any effect on the Akt kinase, Surprisi ngly, two functionally distinct tyrosine kinase inhibitors, genistein and herbimycin A, as well as microinjection of a monoclonal phosphotyr osine specific antibody, inhibited both GTP gamma S- and insulin-stimu lated GLUT4 translocation. Phosphotyrosine immunoblotting and specific immunoprecipitation demonstrated that GTP gamma S did not elicit tyro sine phosphorylation of insulin receptor or insulin receptor substrate -1. In contrast to insulin, proteins in the 120-130-kDa and 55-75-kDa range were tyrosine-phosphorylated following GTP gamma S stimulation. Several of these proteins were identified and include protein-tyrosine kinase 2 (also known as CAK beta, RAFTK, and CADTK), pp125 focal adhe sion tyrosine kinase, pp130 Crk-associated substrate, paxillin, and Cb l, These data demonstrate that the GTP gamma S-stimulated GLUT4 transl ocation utilizes a novel tyrosine kinase pathway that is independent o f both the phosphatidylinositol 3-kinase and the Akt kinase.