DIFFERENTIAL ANTAGONISM OF RAS BIOLOGICAL-ACTIVITY BY CATALYTIC AND SRC HOMOLOGY DOMAINS OF RAS GTPASE ACTIVATION PROTEIN

Ras p120 GTPase activation protein (GAP), a cytosolic protein, is a ne gative mediator and potential down-stream effector of Ras function. Si nce membrane association is critical for Ras function, we introduced t he Ras membrane-targeting signal (a 19-residue peptide ending in CAAX, where C = cysteine, A = aliphatic amino acid, and X = any amino acid) onto the GAP N-terminal Src homology 2 and 3 and the C-terminal catal ytic domains (designated nGAP/CAAX and cGAP/CAAX, respectively) to det ermine the role of membrane association in GAP function. cGAP/CAAX and full-length GAP/CAAX, but not GAP or nGAP/CAAX, exhibited potent grow th inhibitory activity. Whereas both oncogenic and normal Ras activity were inhibited by cGAP/CAAX, nGAP/CAAX, despite lacking the Ras bindi ng domain, inhibited the activity of oncogenic Ras without affecting t he action of normal Ras. Altogether, these results demonstrate that me mbrane association potentiates GAP catalytic activity, support an effe ctor function for GAP, and suggest that normal and oncogenic Ras posse ss different downstream interactions.