Moon Sun Young, Zang Heesuk, Zheng Yi
Division of Experimental Hematology, Children's Hospital Research Foundation, Cincinnati, Ohio 45229, USA.
J Biol Chem. 2003 Feb 7;278(6):4151-9. doi: 10.1074/jbc.M207789200. Epub 2002 Nov 25.
The Rho GTPase-activating proteins (RhoGAPs) are a family of multifunctional molecules that transduce diverse intracellular signals by regulating Rho GTPase activities. A novel RhoGAP family member, p200RhoGAP, is cloned in 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 (Arg-58) contributed to the GAP activity. Expression of the RhoGAP domain of p200RhoGAP in Swiss 3T3 fibroblasts inhibited actin stress fiber formation stimulated by lysophosphatidic acid and platelet-derived growth factor-induced membrane ruffling but not Bradykinin-induced filopodia formation. Endogenous p200RhoGAP was localized to cortical actin in naive N1E-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. Finally, p200RhoGAP was capable of binding to the Src homology 3 domains of Src, Crk, and phospholipase Cgamma in vitro and became tyrosine-phosphorylated upon association with activated Src in cells. These results suggest that p200RhoGAP is involved in the regulation of neurite outgrowth by exerting its RhoGAP activity and that its cellular activity may be regulated through interaction with Src-like tyrosine kinases.
Rho GTP酶激活蛋白（RhoGAPs）是一类多功能分子，通过调节Rho GTP酶的活性来转导多种细胞内信号。一个新的RhoGAP家族成员p200RhoGAP已在人和小鼠中克隆出来。小鼠p200RhoGAP与人类同源物的序列同一性为86%。除了在N端有一个保守的RhoGAP结构域外，在该分子的C端区域还发现了多个富含脯氨酸的基序。Northern印迹分析显示p200RhoGAP具有脑特异性表达模式。p200RhoGAP的RhoGAP结构域在体外和体内均能刺激Rac1和RhoA的GTP酶活性，保守的催化精氨酸残基（Arg-58）对GAP活性有贡献。p200RhoGAP的RhoGAP结构域在瑞士3T3成纤维细胞中的表达抑制了溶血磷脂酸刺激的肌动蛋白应力纤维形成和血小板衍生生长因子诱导的膜皱襞，但不抑制缓激肽诱导的丝状伪足形成。内源性p200RhoGAP在未分化的N1E-115神经母细胞瘤细胞中定位于皮质肌动蛋白，在分化的N1E-115细胞的延伸神经突边缘也有定位。RhoGAP结构域和全长分子的瞬时表达，而不是催化精氨酸突变体，能在N1E-115细胞中轻易诱导分化表型。最后，p200RhoGAP在体外能够与Src、Crk和磷脂酶Cγ的Src同源3结构域结合，并在细胞中与活化的Src结合后发生酪氨酸磷酸化。这些结果表明p200RhoGAP通过发挥其RhoGAP活性参与神经突生长的调节，并且其细胞活性可能通过与Src样酪氨酸激酶的相互作用来调节。
