The Rab4A Effector Protein Rabip4 Is Involved in Migration of NIH 3T3 Fibroblasts

Jelena Vukmirica,Pascale Monzo,Yannick Le Marchand-Brustel,Mireille Cormont

doi:10.1074/jbc.m602920200

Abstract

The small GTP-binding protein Rab4 has been involved in the recycling of alphavbeta3 integrins in response to platelet-derived growth factor (PDGF) stimulation suggesting a role for Rab4 in cell adhesion and migration. In this study, we explored the role of Rabip4 and Rabip4', two Rab4 effector proteins, in migration of NIH 3T3 fibroblasts. In these cells, Rabip4 and Rabip4', collectively named Rabip4s, were partially co-localized with the early endosomal marker EEA1. PDGF treatment re-distributed endogenous Rabip4s toward the cell periphery where they colocalized with F-actin. In cells expressing green fluorescent protein (GFP)-Rabip4 or GFP-Rabip4', constitutive appearance of GFP-Rabip4s at the cell periphery was accompanied by local increase in cortical F-actin in membrane ruffles at the leading edge. The expression of GFP-Rabip4 induced an increased migration compared with control cells expressing GFP alone, even in the absence of PDGF stimulation. On the contrary, in cells expressing a mutated form of Rabip4s unable to interact with Rab4, lack of typical leading edge was observed. Furthermore, PDGF treatment did not stimulate the migration of these cells. Furthermore, down-regulation of the expression of Rabip4s inhibited PDGF-stimulated cell migration. Endogenous Rabip4s were localized with alphav integrins at the leading edge following PDGF treatment, whereas in cells expressing GFP-Rabip4s, alphav integrins, together with GFP-Rabip4s, were constitutively localized at the leading edge. In contrast, reduction in Rabip4s expression levels using small interfering RNA was associated with impaired PDGF-induced translocation of alphav integrins toward the leading edge. Taken together, our data provide evidence that Rabip4s, possibly via their interaction with Rab4, regulate integrin trafficking and are involved in the migration of NIH 3T3 fibroblasts.

Highlights

Rab4a is a small GTPase implicated in endocytosis in multiple cell types [9, 10]
We characterized the intracellular distribution of endogenous Rabip4 isoforms, collectively named Rabip4s, in NIH 3T3 fibroblasts in the presence or absence of platelet-derived growth factor (PDGF) stimulation, and we studied the effects of Rabip4s using cells expressing green fluorescent protein (GFP)-Rabip4s or the mutated Rabip4s form that is unable to bind Rab4a
Rabip4s partially co-localized with a marker of early endosomes, endosome antigen 1 (EEA1) in this region, indicating that, in fibroblasts, Rabip4s are likely localized to early endosomes, as previously reported for the expressed Rabip4 in other cell types [11, 13, 21]

Summary

Introduction

Rab4a is a small GTPase implicated in endocytosis in multiple cell types [9, 10]. We identified Rabip4, a ubiquitous 69-kDa protein, as an effector of Rab4a on the basis of its ability to bind to the GTP-bound form of Rab4a in a yeast twohybrid system [11]. In adhering cells that were subsequently stimulated with PDGF for 60 min, Rabip4s detected at the periphery of the cell were co-localized with F-actin, indicating that Rabip4s are present at the PDGF-induced leading edge of migrating fibroblasts (Fig. 2B).

Results

Conclusion