MAPPING OF THE P56(LCK)-MEDIATED PHOSPHORYLATION OF GAP AND ANALYSIS OF ITS INFLUENCE ON P21(RAS)-GTPASE ACTIVITY IN-VITRO

The protein tyrosine kinase p56(lck) and other members of the src fami ly can transduce signals from activated cell-surface receptors. As we showed earlier the GTPase-activating protein (GAP), a regulator of p21 (ras), is a substrate of p56(lck). Here, tryptic peptides of p56(lck)- phosphorylated GAP were generated and analyzed by two-dimensional thin layer chromatography and mass spectroscopy. Results revealed that p56 (lck) phosphorylates GAP specifically on Tyr-460 in vitro and in vivo. The effect of tyrosine phosphorylation of GAP on its GTPase-activatin g activity versus p21(ras) was then tested using a p21(ras)-dependent GTPase assay system. Our results demonstrate that p56(lck)-mediated ty rosine phosphorylation of GAP is not sufficient to change directly its effect on the intrinsic GTPase activity of p21(ras).