CHARACTERIZATION OF THE C-TERMINAL DOMAIN OF RAS-GTPASE-ACTIVATING PROTEIN (RAS-GAP) AS SUBSTRATE FOR EPIDERMAL GROWTH-FACTOR RECEPTOR AND P60(C-SRC) KINASE

We describe in vitro tyrosine phosphorylation of the C-terminal 334 am ino acids of ras-GTPase-activating protein (ras-GAP)(1) that contains the activity domain for ras interaction. To date, there have been no o ther phosphorylation sites determined than the reported in N-terminal domain of ras-GAP Tyr-460, which is considered to be the major phospho rylation site of ms-GAP. In our assays some differences of the kinetic parameters were observed when the reaction was catalyzed by EGF-R com pared to p60(c-src). Enzyme specific regulation of activity is associa ted with autophosphorylation which leads to reduced (in case of EGF-R) or increased (in case of p60(c-src)) phosphorylation of the C-termina l 334 amino acids of ms-CAP (GAP334). Because of the characteristics o f these investigated reactions the phosphorylation of GAP334 seems to be independent from the presence of SH2 or SH3 domains - triggered off by complex mechanisms different from those regulating the phosphoryla tion at Tyr-460.