The Rsu-1-PINCH1-ILK complex is regulated by Ras activation in tumor cells

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Abstract

The link between Ras transformation and enhanced cell migration due to altered integrin signaling is well established in tumorigenesis, however there remain gaps in our understanding of its mechanism. The Ras suppressor, Rsu-1, has recently been linked to the IPP (integrin-linked kinase {ILK}, PINCH-1/LIMS1, parvin) focal adhesion complex based on its interaction with the LIM 5 domain of PINCH1. Defining the role of the Rsu1-PINCH1-ILK-parvin complex in tumorigenesis is important because both ILK and PINCH1 are elevated in certain tumors while ectopic expression of Rsu-1 blocks tumorigenesis. Our studies previously identified an alternatively spliced isoform of Rsu-1 in high-grade gliomas. We report here the detection of a truncated (p29) Rsu-1 protein, which correlates with the presence of the alternatively spliced Rsu-1 RNA. This RNA and the respective protein were detected in human tumor cell lines that contain high levels of activated Ras, and inhibitor studies demonstrate that the Mek-ERK pathway regulates expression of this truncated Rsu-1 product. We also show that Rsu-1 co-localizes with ILK at focal contacts and co-immunoprecipitates with the ILK-PINCH1 complex in non-transformed cells, but following Ras transformation the association of Rsu-1 with the PINCH1-ILK complex is greatly reduced. Using a human breast cancer cell line, our in vitro studies demonstrate that the depletion of Rsu-1 full-length protein enhances cell migration coincident with an increase in Rac-GTP while the depletion of the p29 Rsu-1 truncated protein inhibits migration. These findings indicate that Rsu-1 may inhibit cell migration by stabilizing the IPP adhesion complex and that Ras activation perturbs this inhibitory function by modulating both Rsu-1 splicing and association of full-length Rsu-1 with IPP. Hence, our findings demonstrate that Rsu-1 links the Ras pathway with the IPP complex and the perturbations of cell attachment-dependent signaling that occur in the malignant process.

Introduction

The loss of integrin engagement and detachment of cells from the extracellular matrix can lead to apoptosis in a process referred to as anoikis (Frisch and Francis, 1994). Many tumor cells are insensitive to detachment-induced cell death and this insensitivity contributes to their motility and metastatic potential as well as the capacity to grow in an anchorage-independent manner. Oncogene activation enhances the resistance to anoikis observed in tumor cells, and the ectopic expression of tumor-suppressor genes can restore sensitivity to detachment in some circumstances (Davies et al., 1998; Khwaja et al., 1997; Koul et al., 2001; Lu et al., 1999; Rosen et al., 2000). Hence, the regulation of cell attachment signaling is critical for control of tumor growth.

Studies in our laboratory have focused on characterization of the Rsu-1 protein, which was originally isolated in an expression cloning assay based on its ability to suppress transformation by the Ras oncogene (Cutler et al., 1992). Rsu-1 is a highly conserved, ubiquitously expressed single-copy gene that encodes an LRR (leucine-rich repeat) protein (Cutler et al., 1992; Tsuda and Cutler, 1993). Ectopic expression of Rsu-1 prevented Ras oncogene-induced phenotypic transformation, inhibited anchorage-independent growth of rodent and human tumor cell lines and blocked tumor formation in a nude mouse xenograft model (Cutler et al., 1992; Tsuda et al., 1995; Vasaturo et al., 2000). The human Rsu-1 locus maps to10p13, a region that is deleted in high-grade gliomas, and an alternatively spliced Rsu-1 mRNA that encodes a truncated and unstable protein product occurs in 30% of high-grade gliomas (Chunduru et al., 2002). Ectopic Rsu-1 expression altered actin cytoskeleton organization and blocked the activation of Jun kinase and ROCK, but not ERK, by growth factor stimulation (Masuelli and Cutler, 1996; Vasaturo et al., 2000).

Recently we and others reported that Rsu-1 binds to the LIM-domain protein PINCH1 (Dougherty et al., 2005; Kadrmas and Beckerle, 2004). PINCH1 contains five LIM domains (LIM 1–5) and functions as a scaffolding protein. The LIM 1 domain of PINCH1 binds to the aminoterminal ankyrin repeat domain of the integrin-linked kinase (ILK) and can modulate ILK activity (Tu et al., 1999). The LIM 4 domain of PINCH1 binds to the SH2-SH3 protein Nck2 (Tu et al., 1998). Our data revealed that the LIM 5 domain of PINCH1 binds to Rsu-1, and that Rsu-1 colocalized with PINCH1 in focal adhesions (Dougherty et al., 2005). PINCH1, in association with ILK and α-parvin, mediates cell matrix-adhesion functions in part by linking focal adhesion contacts to the actin cytoskeleton (Herreros et al., 2000; Nikolopoulos and Turner, 2000, Nikolopoulos and Turner, 2001; Tu et al., 1999; Zhang et al., 2002a, Zhang et al., 2002b). ILK binds to the cytoplasmic domain of the β-integrins (Hannigan et al., 1996) through the respective carboxyterminal domain. The aminoterminal ankyrin repeat region of ILK interacts with the LIM-domain proteins PINCH1 and 2 (Tu et al., 1999, Tu et al., 2001) and paxillin (Nikolopoulos and Turner, 2001). Parvin, which also binds to ILK, can bind F-actin as well as paxillin, providing another mechanism to localize ILK to focal adhesions (Legate et al., 2006; Nikolopoulos and Turner, 2002). Studies demonstrated that both PINCH1 and ILK are required for the localization of the complex to focal adhesions (Tu et al., 2001), and the inhibition of PINCH–ILK interaction in mammalian cells inhibited spreading and reduced motility, suggesting that PINCH is required for ILK activity (Zhang et al., 2002a, Zhang et al., 2002b). In this study, we examined the relationship of Rsu-1 to the IPP complex in the context of Ras activation. We demonstrate that expression of a truncated form, p29 Rsu-1, initially observed in high-grade gliomas, correlates with Ras activation in tumor cell lines. We also demonstrate that the p29 Rsu-1 does not bind to PINCH1. The association of full-length Rsu-1 with the IPP complex is reduced by Ras transformation, and the Ras-dependent effects on Rsu-1 can be partially restored by blocking the Mek-Erk pathway. Additionally, MDA-MB-468 breast cancer cells depleted of p33 Rsu-1 showed enhanced cell migration and Rac activation. We conclude that Rsu-1 promotes adhesion and inhibition of migration as a component of the IPP complex. This function is actively altered by Ras signaling and these data lend insight to the tumor-suppressor effects of Rsu-1.

Section snippets

Cell lines

The human breast cancer cell lines used in the study (MCF7, T47D, MDA-MB-231, MDA-MB-468) were obtained from the American Type Culture Collection. The immortalized human astrocytes (E6/E7/hTERT) and the Ras-transformed version (E6/E7/hTERT/Ras) were provided by Dr. Russell Pieper and the cells were propagated as described (Sonoda et al., 2001a, Sonoda et al., 2001b). The A7r5 (rat vascular smooth muscle) cell line was cultured as previously described (Burgstaller and Gimona, 2004). Cos-1 cells

A truncated p29 Rsu-1 protein is specifically detected in Ras-activated tumor cell lines

Rsu-1 was isolated based on its ability to suppress transformation by activated Ras. The main Rsu-1-interacting protein, PINCH1, is an adaptor protein that binds to ILK. Since both PINCH1 and ILK exhibit increased expression in tumors and in tumor stroma (Ahmed et al., 2003; Dai et al., 2003; Gao et al., 2004; Graff et al., 2001; Ito et al., 2003; Marotta et al., 2001, Marotta et al., 2003; Wang-Rodriguez et al., 2002), we examined both the level of Rsu-1 expression in tumor cell lines and its

Discussion

Previous work demonstrated that Rsu-1 binds to PINCH1 and is required for cell adhesion (Dougherty et al., 2005). The results reported here indicate that Rsu-1 can also function to inhibit cell migration. It appears that the association of p33 Rsu-1 with PINCH1 is required for inhibition of migration, as the reduction of p33 Rsu-1 association with the IPP complex correlates with Rac1 activation and increased migration. This is further supported by our observation that Rsu-1 localizes to focal

Acknowledgments

The studies were supported by grants from the NIH (R01CA90908) and the USUHS to M.L. Cutler, and from the European Union (Marie Curie Excellence Grant 002573) to M. Gimona.

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