Study on signal transduction mechanism of p120 RasGAP piotein

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GTPase-activating proteins (GAPs) play an important role in signal tiansduction pathways regulated by small GTP-binding proteins RasGAP is composed of several domains. The C-terminus shows GAP activity as a negative regulator of Ras. The N-terminus contains SH2-SH3-SH2 that appears to be involved in interactions with signaling proteins, and functions as a Ras effector. These two SH2 domains have been found to associate stably with a tyrosine-phosphorylated protein, p190 RhoGAP, which is thought to regulate actin cytoskeleton dynamics. Several studies suggest that the interaction between Ras-GAP and Rho-GAP is regulated by phosphorylation of tyrosine residues, Tyr1087 and/or Tyr1105 in the middle domain of Rho-GAP, however, mechanisms for RasGAP-RhoGAP complex formation have not yet been determined.To investigate these regulation mechanisms for molecular interactions, seven recombinant proteins composed of SH2(n)-SH3-SH2(c) domains of RasGAP have been prepared and subjected to binding assays against three kinds of tyrosine-phosphorylated synthetic peptides of p190 RhoGAP by means of biosensor (BIAcore) respectively. Results obtained from biosensor analysis revealed that both SH2(n) and SH2(c) domain could bind to each tyrosine-phosphorylated peptide. In addition, the protein containing both SH2(n) and SH2(c) domains synergistically bound to the dual tyrosine-phosphorylated peptide.