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Abstract
A novel RhoGAP family member, p200RhoGAP, was cloned from human and mouse. The murine p200RhoGAP shares 86% sequence identity with the human homolog. In addition to a conserved RhoGAP domain at the N-terminus, multiple proline rich motifs are found in the C-terminal region of the molecules. Northern blot analysis revealed a brain specific expression pattern of p200RhoGAP. The RhoGAP domain of p200RhoGAP stimulated the GTPase activities of Rac1 and RhoA in vitro and in vivo, and the conserved catalytic arginine residue (R58) is essential for the GAP activity. Expression of the RhoGAP domain of p200RhoGAP in Swiss 3T3 fibroblasts inhibited actin stress fiber formation stimulated by lysophosphatidic acid (LPA) and PDGF-induced membrane ruffling, but not Bradykinin-induced filopodia formation. Endogenous p200RhoGAP was localized to cortical actin in naive NIE-115 neuroblastoma cells and to the edges of extended neurites of differentiated N1E-115 cells. Transient expression of the RhoGAP domain and the full-length molecule, but not the catalytic arginine mutants, readily induced a differentiation phenotype in N1E-115 cells. p200RhoGAP was capable of binding to the SH3 domains of Src, Crk, p85α subunit of PI3 kinase, and PLCγ in vitro . Activated Src specifically binds to p200RhoGAP in vivo and phosphorylates on multiple tyrosines located in amino acid residue 1053-1738. Tyrosine phosphorylated p200RhoGAP further interacts with p120RasGAP SH2 domains in vitro. Stably transfected full-length p200RhoGAP in NIH3T3 fibroblast cells induced the transforming phenotypes including foci formation and anchorage independent growth in soft agar. The RhoGAP activity and the extreme end of C-terminus of p200RhoGAP (amino acid residues 1498-1738) are cooperatively involved in the transforming processes caused by constitutive expression of p200 in NIH3T3 cells. These results suggest that p200RhoGAP play a role in neuronal differentiation and cell growth through its RhoGAP activity which can be modulated by the various molecular interactions through its C-terminal region.
