ABSTRACT
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.
Subject(s)
Actins/metabolism , Carrier Proteins/metabolism , Cell Cycle Proteins , Endosomes/metabolism , GTPase-Activating Proteins/metabolism , ADP-Ribosylation Factors/genetics , ADP-Ribosylation Factors/physiology , Adaptor Proteins, Signal Transducing , Animals , Carrier Proteins/biosynthesis , Carrier Proteins/genetics , Carrier Proteins/physiology , Cell Compartmentation/genetics , Cell Membrane/genetics , Cell Membrane/metabolism , Cells, Cultured , Chick Embryo , Cytoskeletal Proteins/metabolism , Endosomes/genetics , Enzyme Activation/physiology , Fibroblasts/metabolism , GTPase-Activating Proteins/biosynthesis , GTPase-Activating Proteins/genetics , GTPase-Activating Proteins/physiology , Genetic Vectors/metabolism , Intracellular Fluid/metabolism , Paxillin , Peptide Fragments/genetics , Peptide Fragments/metabolism , Peptides/genetics , Peptides/metabolism , Phosphoproteins/metabolism , Protein Structure, Tertiary/genetics , Subcellular Fractions/metabolism , rac GTP-Binding Proteins/metabolism , rac GTP-Binding Proteins/physiologyABSTRACT
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.
