A synthetic peptide corresponding to a sequence in the GTPase activating protein inhibits p21ras stimulation and promotes guanine nucleotide exchange

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Abstract
Amino acid sequence homology between the GTPase Activating Protein (GAP) and the GTP‐binding regulatory protein, Gsα, suggests that a specific region of GAP primary structure (residues 891–898) may be involved in its stimulation of p21ras GTP hydrolytic activity (McCormick, F. [1989] Nature340, 678–679). A peptide, designated p891, corresponding to GAP residues 891–906 (M891RTRVVSGFVFLRLIC906) was synthesized and tested for its ability to inhibit GAP‐stimulated p21ras GTPase activity. At a concentration of 25 μm, p891 inhibited GAP activity approximately 50%. Unexpectedly, p891 also stimulated GTP binding to p21N‐ras independent of GAP. This stimulation correlated with an enhancement of p21N‐ras GDP dissociation; an approximate 15‐fold increase in the presence of 10 μm p891. In contrast, dissociation of the p21N‐ras GTPγS complex was unaffected by 10μM p891. The p21N‐ras GDP complex was unresponsive to 100μM mastoparan, a peptide toxin shown previously to accelerate GDP dissociation from the guanine nucleotide regulatory proteins, Gi and Go. p21H‐ras, as well as the two p21H‐ras effector mutants, Ala‐38, and Ala‐35, Leu‐36, also exhibited increased rates of GDP dissociation in the presence of p891. Also tested were three ras‐related GTP‐binding proteins; rap, G25K and rac. The rap GDP complex was unaffected by 10 μm p891. Dissociation of the G25K‐ and rac GDP complexes were enhanced slightly; approximately 1.3‐and 1.8‐fold over control, respectively. Thus, the inhibitory effect of p891 on GAP stimulation of p21ras suggests that amino acids within the region 891–906 of GAP may be essential for interaction with p21ras. In addition, p891 independently affects the nucleotide exchange properties of p21ras.