Insulin-dependent phosphorylation of a 70-kDa protein in light microsomes from rat adipocytes

In order to discover possibly novel insulin receptor substrates and/or down stream targets in the insulin signaling pathway, we established a cell-free system for this purpose using purified insulin receptor and subcellular fr actions fi om rat adipocytes as a sourse of cellular substrates. Under thes e conditions, we have found a 70-kDa protein (pp70) in fat cells that is ty rosine-phosphorylated by the activated insulin receptor. Using sucrose velo city gradient sedimentation we also show that pp70 cofractionate a particul ate fraction containing IRS-1 but not with GLUT-4 vesicle-enriched fraction s. Our results suggest that pp70 may be an endogenous substrate for the ins ulin receptor tyrosine kinase. (C) 2000 Academic Press.