RESUMEN
Cell migration requires coordination between adhesion, actin organization and membrane traffic. Rac and ARF6 have been shown to cooperate for the organization of actin at the cell surface. Recently, the GIT family of ARF-GAPs has been identified, which includes proteins that can functionally interact with both ARF and Rac GTPases. The p95-APP1 protein is a member of this family, isolated as part of a multi-molecular complex interacting with GTP-Rac. Our previous work has indicated that this protein may be part of the machinery redirecting membrane recycling towards sites of protrusion during locomotion. By analyzing the distribution and the effects of truncated forms of p95-APP1, we show here that the lack of the ARF-GAP domain of p95-APP1 dramatically shifts its localization to large vesicles. The use of several markers of the endocytic pathway has revealed that the truncated p95-APP1 localizes specifically to a Rab11-, transferrin receptor-positive compartment. Other markers are excluded from the p95-APP1-positive vesicles, while known components of the multi-molecular complex colocalize with truncated p95-APP1 in this compartment. Coexpression of a constitutively active form of Rac induces the redistribution of the truncated constructs and of the associated PIX, PAK, and paxillin to peripheral sites of Rac-mediated actin organization, and the disassembly of the large Rab11-positive vesicles. Together, the data presented indicate that p95-APP1 is part of a complex that shuttles between the plasma membrane and the endocytic recycling compartment, and suggest that the dynamic redistribution of the p95-APP1-containing complex is mediated both by the ARF-GAP domain, and by the recruitment of the complex at the cell surface at sites of Rac activation.
Asunto(s)
Actinas/metabolismo , Proteínas Portadoras/metabolismo , Proteínas de Ciclo Celular , Endosomas/metabolismo , Proteínas Activadoras de GTPasa/metabolismo , Factores de Ribosilacion-ADP/genética , Factores de Ribosilacion-ADP/fisiología , Proteínas Adaptadoras Transductoras de Señales , Animales , Proteínas Portadoras/biosíntesis , Proteínas Portadoras/genética , Proteínas Portadoras/fisiología , Compartimento Celular/genética , Membrana Celular/genética , Membrana Celular/metabolismo , Células Cultivadas , Embrión de Pollo , Proteínas del Citoesqueleto/metabolismo , Endosomas/genética , Activación Enzimática/fisiología , Fibroblastos/metabolismo , Proteínas Activadoras de GTPasa/biosíntesis , Proteínas Activadoras de GTPasa/genética , Proteínas Activadoras de GTPasa/fisiología , Vectores Genéticos/metabolismo , Líquido Intracelular/metabolismo , Paxillin , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Péptidos/genética , Péptidos/metabolismo , Fosfoproteínas/metabolismo , Estructura Terciaria de Proteína/genética , Fracciones Subcelulares/metabolismo , Proteínas de Unión al GTP rac/metabolismo , Proteínas de Unión al GTP rac/fisiologíaRESUMEN
Motility requires protrusive activity at the cellular edge, where Rho family members regulate actin dynamics. Here we show that p95-APP1 (ArfGAP-putative, Pix-interacting, paxillin-interacting protein 1), a member of the GIT1/PKL family, is part of a complex that interacts with Rac. Wild-type and truncated p95-APP1 induce actin-rich protrusions mediated by Rac and ADP-ribosylation factor 6 (Arf6). Distinct p95-APP1-derived polypeptides have different distributions, indicating that p95-APP1 cycles between the cell surface and endosomes. Our results show that p95-APP1 functionally interacts with Rac and localizes to endosomal compartments, thus identifying p95-APP1 as a molecular link between actin organization, adhesion, and membrane transport during cell motility.