RESUMEN
There is a class of GTPase activating proteins for the Rho family GTPases (RhoGAPs) that contain the steroidogenic acute regulatory protein (STAR)-related lipid transfer (START) domain. In mammals three genes encode such proteins and they are designated START-GAP1-3 or deleted in liver cancer 1-3 (DLC1-3). In this study, we examined the intracellular localization and roles of START-GAP1/DLC1 in cell motility. Immunofluorescence microscopic analysis of NRK cells and HeLa cells revealed that START-GAP1 was localized in focal adhesions. Amino acid residues 265-459 of START-GAP1 were found to be necessary for focal adhesion targeting and we name the region "the focal adhesion-targeting (FAT) domain." It was previously known that ectopic expression of START-GAP1 induced cell rounding. We demonstrated that the FAT domain of START-GAP1 was partially required for this morphological change. Furthermore, expression of this domain in HeLa cells resulted in dissociation of endogenous START-GAP1 from focal adhesions as a dominant negative modulator, reducing cell migration and spreading. Taken together, START-GAP1 is targeted to focal adhesions via the FAT domain and regulates actin rearrangement through down-regulation of active RhoA and Cdc42. Its absence from focal adhesions could, therefore, cause abnormal cell motility and spreading.
Asunto(s)
Movimiento Celular , Forma de la Célula , Adhesiones Focales/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Proteínas Supresoras de Tumor/fisiología , Movimiento Celular/fisiología , Forma de la Célula/fisiología , Células Cultivadas , Adhesiones Focales/fisiología , Proteínas Activadoras de GTPasa , Células HeLa , Humanos , Modelos Biológicos , Estructura Terciaria de Proteína/genética , Estructura Terciaria de Proteína/fisiología , Distribución Tisular , Transfección , Proteínas Supresoras de Tumor/química , Proteínas Supresoras de Tumor/genética , Proteína de Unión al GTP cdc42/metabolismo , Proteína de Unión al GTP rhoA/metabolismoRESUMEN
START-GAP2, also termed as DLC2, is a START domain-containing RhoGAP and a negative regulator of RhoA and Cdc42. Although it was reported as a tumor suppresser gene product, the molecular basis for function of START-GAP2 remains to be clarified. Here, we demonstrate that START-GAP2 is localized in focal adhesions through a "FAT (focal adhesion targeting)" region in the N-terminal half. START-GAP2 competes with START-GAP1/DLC1, another START domain-containing RhoGAP, in focal adhesion targeting. Moreover, the C-terminus of tensin2, one of focal adhesion components and reported to bind START-GAP1, also directly interacts with START-GAP2. These results suggest that START-GAP2 and START-GAP1 share the same molecular mechanism in targeting to focal adhesions.