βPIX and GIT1 regulate HGF-induced lamellipodia formation and WAVE2 transport

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Abstract

Formation of lamellipodia is the first step during cell migration, and involves actin reassembly at the leading edge of migrating cells through the membrane transport of WAVE2. However, the factors that regulate WAVE2 transport to the cell periphery for initiating lamellipodia formation have not been elucidated. We report here that in human breast cancer MDA-MB-231 cells, the hepatocyte growth factor (HGF) induced the association between the constitutive complex of βPIX and GIT1 with WAVE2, which was concomitant with the induction of lamellipodia formation and WAVE2 transport. Although depletion of βPIX by RNA interference abrogated the HGF-induced WAVE2 transport and lamellipodia formation, GIT1 depletion caused HGF-independent WAVE2 transport and lamellipodia formation. Collectively, we suggest that βPIX releases cells from the GIT1-mediated suppression of HGF-independent responses and recruits GIT1 to WAVE2, thereby facilitating HGF-induced WAVE2 transport and lamellipodia formation.

Introduction

Cell migration plays an important role in a variety of physiological processes, including tissue remodeling, wound healing, and immune surveillance [1]. Misregulated cell migration may contribute to cancer cell invasion and metastasis. The first step in cancer cell migration is the formation of cell protrusions at the leading edge of the cell toward chemoattractants present in the tumor microenvironment [2], [3]. Members of the small GTPase protein family, including Cdc42, Rac, and Rho, mediate actin cytoskeleton rearrangement, thereby functioning as key regulators of cell migration [4], [5]. Activation of Cdc42 or Rac1 promotes the formation of filopodia or lamellipodia [6]. During the process of actin polymerization, the Wiskott-Aldrich syndrome protein (WASP) family, which includes WASP, N-WASP, WAVE1, WAVE2, and WAVE3, plays a crucial role downstream of Cdc42 and Rac, through the activation of the Arp 2/3 complex [7], [8], [9]. Recently, several effector molecules of the Rho family GTPases have been identified [10]; among these effectors, PAKs are serine/threonine kinases and are activated by Cdc42 and Rac [11]. PAKs mediate a number of cellular activities pertinent to cell migration, including the organization of the actin cytoskeleton and microtubule network [12]. βPIX is the guanine nucleotide exchange factor (GEF) for Cdc42 and Rac, and has been identified as one of the proteins that interacts with PAK [13]. The PIX proteins are also associated with GIT1, an ADP-ribosylation factor GTPase-activating protein (ARFGAP), which specifically regulates ARF6 [14], [15]. GIT1 is a multidomain protein with conserved ARFGAP, three ankyrin repeats, Spa2 homology, and a paxillin binding domain [16]. An important function of GIT1 is that it serves as a signaling anchor between PIX, PAK, and the focal adhesion complex in migrating cells, and as a key regulator of spine morphology and synapse formation [17], [18], [19]. However, the exact role of βPIX and GIT1 in the regulation of actin polymerization in invasive cancer cells has not yet been completely elucidated.

Previously, we have shown that in MDA-MB-231 cells, lamellipodia formation induced by HGF required Rac1 and PAK1 for the kinesin-mediated membrane transport of WAVE2 along the microtubules [20], [21]. These findings prompted us to examine the possible contribution of βPIX and GIT1 to HGF-induced WAVE2 transport as well as lamellipodia formation. In this study, we describe the differential roles of βPIX and GIT1 in the regulation of lamellipodia formation and WAVE2 transport, by using RNA interference assays for βPIX and GIT1.

Section snippets

Materials and methods

Cell culture and HGF stimulation. Human breast cancer MDA-MB-231 cells (European Cell Culture Collection) were maintained in RPMI 1640 medium supplemented with 10% FBS. Prior to stimulation with HGF, cells were serum-starved by incubation in 0.1% FBS-containing medium (low-serum medium) for 16 h and then stimulated with 50 ng/ml human recombinant HGF (PeproTech, Rocky Hill, NJ) for 1 h.

Immunoprecipitation and immunoblot analysis. For immunoprecipitation, cells were lysed in ice-cold RIPA buffer (50

Complex formation of βPIX with WAVE2 and GIT1

To determine whether βPIX forms a complex with WAVE2 in MDA-MB-231 cells, WAVE2 was immunoprecipitated from cells (Fig. 1A). We found that βPIX coprecipitated with WAVE2 in HGF-stimulated cells, but not in serum-starved cells (Fig. 1A, left panels). Although βPIX binds directly to GIT1 to function as a signaling complex [16], GIT1 did not immunoprecipitate with WAVE2 in the present study. Following immunoblot analysis, GIT1 coprecipitated with βPIX (Fig. 1A, middle panels) and vice versa in

Discussion

Immunoprecipitation analysis indicating the coprecipitation of βPIX with WAVE2 in HGF-stimulated cells suggests the HGF-induced complex formation of βPIX and WAVE2. Although GIT1 was not detected in the WAVE2 immunoprecipitates in the present study, constitutive coprecipitation of βPIX and GIT1 in both serum-starved and HGF-stimulated cells suggests that GIT1 forms a stable complex with βPIX and that the βPIX–GIT1 complex binds to WAVE2 in response to HGF.

Moreover, immunofluorescence analysis

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