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The Guanine Nucleotide Exchange Factor Arhgef5 Plays Crucial Roles in Src-Induced Podosome Formation

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The Guanine Nucleotide Exchange Factor Arhgef5 Plays Crucial Roles in Src-Induced Podosome Formation 1726 Research Article The guanine nucleotide exchange factor Arhgef5 plays crucial roles in Src-induced podosome formation Miho Kuroiwa, Chitose Oneyama, Shigeyuki Nada and Masato Okada* Department of Oncogene Research, Research institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita, Osaka 565-0871, Japan *Author for correspondence ([email protected]) Accepted 19 January 2011 Journal of Cell Science 124, 1726-1738 © 2011. Published by The Company of Biologists Ltd doi:10.1242/jcs.080291 Summary Podosomes and invadopodia are actin-rich membrane protrusions that play a crucial role in cell adhesion and migration, and extracellular matrix remodeling in normal and cancer cells. The formation of podosomes and invadopodia is promoted by upregulation of some oncogenic molecules and is closely related to the invasive potential of cancer cells. However, the molecular mechanisms underlying the podosome and invadopodium formation still remain unclear. Here, we show that a guanine nucleotide exchange factor (GEF) for Rho family GTPases (Arhgef5) is crucial for Src-induced podosome formation. Using an inducible system for Src activation, we found that Src-induced podosome formation depends upon the Src SH3 domain, and identified Arhgef5 as a Src SH3-binding protein. RNA interference (RNAi)-mediated depletion of Arhgef5 caused robust inhibition of Src-dependent podosome formation. Overexpression of Arhgef5 promoted actin stress fiber remodeling through activating RhoA, and the activation of RhoA or Cdc42 was required for Src-induced podosome formation. Arhgef5 was tyrosine-phosphorylated by Src and bound to Src to positively regulate its activity. Furthermore, the pleckstrin homology (PH) domain of Arhgef5 was required for podosome formation, and Arhgef5 formed a ternary complex with Src and phosphoinositide 3-kinase when Src and/or Arhgef5 were upregulated. These findings provide novel insights into the molecular mechanisms of podosome and invadopodium formation induced by Src upregulation. Key words: Podosome, Invadopodium, Src, Guanine nucleotide exchange factor (GEF), SH3 domain Introduction essential for the formation and function of podosomes (Erikson et Invasion is one of the most characteristic features of cancer al., 1980; Frame, 2004; Miyazaki et al., 2004). Furthermore, Src- malignancy. Cancer invasion is the process in which tumor cells knockout mice develop severe osteopetrosis as a result of defective degrade the adjacent extracellular matrix (ECM) and migrate into podosome formation in osteoclasts (Tiffee et al., 1999). These Journal of Cell Science surrounding tissues. Accumulating evidence suggests that the cancer lines of evidence suggest that Src contributes to cancer malignancy, invasion depends upon invadopodia (Chen, 1989; Monsky et al., including both invasion and metastasis, by upregulating podosome 1994). Invadopodia are actin-rich membrane protrusions associated and invadopodium formation and function. However, the underlying with high levels of proteases that degrade ECM. They form on the molecular mechanism regulating these events remains elusive. ventral surface of the plasma membrane that is perpendicular to Podosomes and invadopodia are associated with a multi-protein and in contact with the ECM (Bowden et al., 1999; Oikawa et al., complex consisting of adhesion molecules, actin-modulating 2008). The structural and morphological features of invadopodia proteins, tyrosine kinases and matrix metalloproteinases (MMPs) are similar to those of podosomes, which are actin-rich ECM- (Chan et al., 2009; Luxenburg et al., 2006a; Yamaguchi et al., degrading membrane protrusions seen in osteoclasts, macrophages, 2005). The compositions of this complex are similar to those smooth muscle cells and Src-transformed cells (Davies and Stossel, associated with focal adhesions (FAs), although there are apparent 1977; Kaverina et al., 2003; Tarone et al., 1985; Zambonin-Zallone differences in their ECM-degrading activity and how they associate et al., 1988). Despite their physiological and pathological with stress fibers. Stress fibers are disrupted in cells with podosomes importance, the mechanisms underlying podosome and and invadopodia, whereas FAs associate and assemble with stress invedopodium formation still need to be clarified. fibers (Carragher and Frame, 2004; Gimona and Buccione, 2006; The Src family kinases (SFKs) are non-receptor tyrosine kinases, Schoenwaelder and Burridge, 1999). Furthermore, podosomes and which were originally identified as proto-oncogene products invadopodia specifically contain cortactin, N-WASP and Tks5 (also (Hunter and Sefton, 1980; Jove and Hanafusa, 1987). They play known as Fish), all of which can serve as substrates for Src family pivotal roles in various signaling pathways involved in cell growth, kinases (Mizutani et al., 2002; Oser et al., 2009; Seals et al., 2005; differentiation, adhesion, migration, invasion and metastasis (Brown Suetsugu et al., 2002). Cortactin and N-WASP are required for and Cooper, 1996). In a wide variety of cancers, especially in actin remodeling during invadopodium formation (Luxenburg et mammary carcinoma, colon cancer, prostate cancer and squamous al., 2006b; Mizutani et al., 2002; Yamaguchi et al., 2005). Tks5 cell carcinomas, Src is often overexpressed and activated, which localizes specifically to podosomes through its Phox homology implies that it plays a role in cancer malignancy (Cartwright et al., (PX) domain, which associates with phosphatidylinositol 3- 1989; Chang et al., 2007; Lehrer et al., 1989; van Oijen et al., phosphate and phosphatidylinositol (3,4)-bisphosphate [PtdIns(3)P 1998). A recent expression profiling strategy revealed that the and PtdIns(3,4)P2] (Seals et al., 2005). Because PtdIns(3,4)P2 is expression level of Src correlates with poor prognosis in cancer highly concentrated within podosomes (Oikawa et al., 2008), (Aligayer et al., 2002; Creighton, 2008). Activation of Src is also production of PtdIns(3,4)P2 might be necessary for the assembly Arhgef5 in podosome formation 1727 of crucial molecules in the podosomes. Recently, Oikawa et al. of cortactin (Y421-P) in Src-MER cells greatly increased during proposed a sequential mechanism for podosome formation (Oikawa the formation of podosomes, whereas that in Fyn-MER cells was et al., 2008): podosome formation is initiated at FAs owing to substantially lower (Fig. 1B). Because it is known that changes in the phosphorylation status of FA proteins and in the phosphorylation of cortactin Y421 is important for invadopodium composition of phosphoinositides on the plasma membrane; the precursor formation (Oser et al., 2009), the lower affinity of Fyn- onset of actin polymerization is then triggered by Tks5 recruitment MER for cortactin might partly explain its lower efficacy in and subsequent N-WASP accumulation, which requires Tks5 to podosome formation. We also found that the intracellular localization interact with both phosphoinositides and Grb2. In this mechanism, of Src and Fyn was appreciably different. Src-MER was distributed phosphoinositide 3-kinase (PI3K) plays an essential role (Oikawa in the perinuclear region of cytoplasm before 4-OHT treatment but et al., 2008). once activated it accumulated in the plasma membrane (Fig. 1F; Rho family small GTPases, such as RhoA, Rac1 and Cdc42, are supplementary material Fig. S1A). By contrast, Fyn-MER was also important for podosome formation (Berdeaux et al., 2004; constantly localized to the plasma membrane before and after 4- Chellaiah, 2006). They are involved in podosome formation by OHT treatment. These data suggest that Src preferentially contributes regulating the actin cytoskeleton, which is under the control of a to podosome formation by interacting with some specific variety of guanine nucleotide exchange factors (GEFs), including components that can recruit Src to the plasma membrane. the Dbl family (Quilliam et al., 1995; Rossman et al., 2005; Schmidt and Hall, 2002). However, it remains unclear which types The function of the Src SH3 domain is essential for of GEFs are necessary for regulating of podosome formation and podosome formation how these GEFs are regulated by the upstream triggering factors, Src has SH2 and SH3 domains that mediate protein–protein such as Src. interactions, but they only become available when the activated Most studies on Src-induced podosome formation have been Src adopts an open conformation (Fig. 1A). To examine the performed with viral Src (v-Src) or a Src mutant lacking the potential contribution of these domains to Src-induced podosome negative regulatory site (Irby and Yeatman, 2000). However, the formation, we generated mutant Src-MER constructs that had a SRC gene is rarely mutated in human cancers (Biscardi et al., substitution at a crucial residue in either the SH3 domain (Src 2000; Irby et al., 1997; Ishizawar and Parsons, 2004; Oneyama et W120A-MER) or the SH2 domain (Src R177L-MER). By al., 2008) and the structure and function of the oncogenic forms of expressing these constructs in MDCK cells at comparable levels Src significantly differ from those of Src (Oneyama et al., 2008). (Fig. 2A), we examined their ability to form podosomes (Fig. 2B). Therefore, to elucidate the precise function of Src in podosome Interestingly, we found that activation of the Src SH3 (Src W120A- formation, it is necessary to evaluate the function of the activated MER) mutant could only moderately induce podosome formation Src. For this purpose, we developed an experimental
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