The Rsu-1-PINCH1-ILK complex is regulated by Ras activation in tumor cells
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.
References (76)
- et al.
Identification of Grb4/Nckb, a src homology 2 and 3 domain-containing adapter protein having similar binding and biological properties to Nck
J. Biol. Chem.
(1999) - et al.
The Nck family of adapter proteins: regulators of actin cytoskeleton
Cell Signal.
(2002) Cell-substrate interactions and signaling through ILK
Curr. Opin. Cell Biol.
(2000)- et al.
The Ras suppressor Rsu-1 binds to the LIM 5 domain of the adaptor protein PINCH1 and participates in adhesion-related functions
Exp. Cell Res.
(2005) - et al.
Regulation of the Cool/Pix proteins: key binding partners of the Cdc42/Rac targets, the p21-activated kinases
J. Biol. Chem.
(2002) - et al.
Stromal staining for PINCH is an independent prognostic indicator in colorectal cancer
Neoplasia
(2004) - et al.
Paxillin localizes to the lymphocyte microtubule organizing center and associates with the microtubule cytoskeleton
J. Biol. Chem.
(2000) - et al.
Suppression of integrin activation: a novel function of a Ras/Raf-initiated MAP kinase pathway
Cell
(1997) - et al.
The morphologies of breast cancer cell lines in three-dimensional assays correlate with their profiles of gene expression
Mol. Oncol.
(2007) - et al.
Integrin-linked kinase (ILK) binding to paxillin LD1 motif regulates ILK localization to focal adhesions
J. Biol. Chem.
(2001)
Molecular dissection of actopaxin-integrin-linked kinase-paxillin interactions and their role in sub-cellular localization
J. Biol. Chem.
Thrombospondin stimulates focal adhesion disassembly through Gi- and phosphoinositide 3-kinase-dependent ERK activation
J. Biol. Chem.
Cell migration: Rho GTPases lead the way
Dev. Biol.
Integrin-linked kinase regulates inducible nitric oxide synthase and cyclooxygenase-2 expression in an NF-kappa B-dependent manner
J. Biol. Chem.
Cell cycle-dependent activation of Ras
Curr. Biol.
Human RSU-1 is highly homologous to mouse Rsu-1 and localizes to human chromosome 10
Genomics
Cdc42 is a Rho GTPase family member that can mediate insulin signaling to glucose transport in 3T3-L1 adipocytes
J. Biol. Chem.
A critical role of the PINCH-integrin-linked kinase interaction in the regulation of cell shape change and migration
J. Biol. Chem.
Distinct roles of two structurally closely related focal adhesion proteins, alpha-parvins and beta-parvins, in regulation of cell morphology and survival
J. Biol. Chem.
Tyrosine 221 in Crk regulates adhesion-dependent membrane localization of Crk and Rac and activation of Rac signaling
EMBO J.
Integrin-linked kinase expression increases with ovarian tumour grade and is sustained by peritoneal tumour fluid
J. Pathol.
A rapid in vitro assay for quantitating the invasive potential of tumor cells
Cancer Res.
Matrix survival signaling: from fibronectin via focal adhesion kinase to c-Jun NH(2)-terminal kinase
J. Cell Biol.
Nck-interacting Ste20 kinase couples Eph receptors to c-Jun N-terminal kinase and integrin activation
Mol. Cell. Biol.
The murine Nck SH2/SH3 adaptors are important for the development of mesoderm-derived embryonic structures and for regulating the cellular actin network
Mol. Cell. Biol.
Actin cytoskeleton remodelling via local inhibition of contractility at discrete microdomains
J. Cell Sci.
Regulation of CD44 alternative splicing by SRm160 and its potential role in tumor cell invasion
Mol. Cell. Biol.
A positive feedback loop couples Ras activation and CD44 alternative splicing
Genes Dev.
Identification of an alternatively spliced RNA for the Ras suppressor RSU-1 in human gliomas
J. Neurooncol.
Analysis of PINCH function in Drosophila demonstrates its requirement in integrin-dependent cellular processes
Development
Isolation of rsp-1, a novel cDNA capable of suppressing v-ras
Mol. Cell. Biol.
Increased expression of integrin-linked kinase is correlated with melanoma progression and poor patient survival
Clin. Cancer Res.
Adenoviral transgene expression of MMAC/PTEN in human glioma cells inhibits Akt activation and induces anoikis
Cancer Res.
Integrin-linked kinase activity regulates Rac- and Cdc42-mediated actin cytoskeleton reorganization via alpha-PIX
Oncogene
Disruption of epithelial cell-matrix interactions induces apoptosis
J. Cell Biol.
The C-terminal end of R-Ras contains a focal adhesion targeting signal
J. Cell Sci.
EGF-induced activation of Akt results in mTOR-dependent p70S6 kinase phosphorylation and inhibition of HC11 cell lactogenic differentiation
BMC Cell Biol.
Integrin-linked kinase expression increases with prostate tumor grade
Clin. Cancer Res.
Cited by (69)
Ras suppressor 1 long form (RSU1L) silencing promotes apoptosis in invasive breast cancer cells
2023, Cellular SignallingCitation Excerpt :The Ras suppressor-1 gene (RSU1) was initially found to suppress RAS-dependent oncogenic transformation [4]. It is found in two isoforms; a longer most common isoform (RSU1L, herein) encoding for a 33KDa protein, and a shorter, alternatively-spliced one (RSU1-X1) that codes for a protein of 29KDa, which is present in more aggressive human gliomas [5,6] and BC cell lines [7]. Apart from its initial implication in RAS-induced oncogenic transformation, RSU1 has been also shown to localize to Cell-Extracellular Matrix (ECM) adhesions, where it binds to Particularly Interesting New Cysteine-Histidine protein (PINCH1) [8], a binding partner of a stable ternary protein complex at cell-ECM adhesions.
Focal adhesion proteins in hepatocellular carcinoma: RSU1 a novel tumour suppressor with prognostic significance
2022, Pathology Research and PracticeCitation Excerpt :The ILK/ PINCH/PARVIN (IPP) complex, a ternary complex formed by integrin-linked kinase (ILK), particularly interesting new cysteine-histidine-rich protein (PINCH) and PARVIN, acts as a signaling hub and protein scaffold in FA with critical role in cell adhesion, migration and integrin signaling [8]. The suppressor of RAS oncogenic transformation, Ras suppressor 1 (RSU1), a leucine-rich repeat (LRR) containing protein, has been shown to localize at FAs where its binds to the LIM5 region of PINCH1 [9–12]. While, RSU1-PINCH1 interaction is important for FA formation, cell adhesion and cell spreading, RSU1 is also engaged in binding integrins to RAS-MAPK pathway [13].
Molecular basis for Ras suppressor-1 binding to PINCH-1 in focal adhesion assembly
2021, Journal of Biological ChemistryCitation Excerpt :Previous studies demonstrated that the connection of Rsu-1 to PINCH-1 affected several downstream signaling pathways of MAPK (8, 19, 37). It remains to be determined whether the Rsu-1-PINCH-1 axis is involved in the regulation of oncogenic transformation (21). A previous study of affinity purification–mass spectrometry has identified that Raf-1 is involved in the IPP interaction network, suggesting an important link between the Rsu-1/IPP complex and the Ras/MAPK-mediated pathway (45).
RSU-1 interaction with prohibitin-2 links cell–extracellular matrix detachment to downregulation of ERK signaling
2021, Journal of Biological ChemistryA proteomic glimpse into the oncogenesis of prostate cancer
2018, Journal of Applied BiomedicineCitation Excerpt :The inhibition of interaction of these proteins results in decreased cell spreading, cell migration, cell adhesion and reduced motility of mammalian cells (Dougherty et al., 2008; Gonzalez-Nieves et al., 2013; Kim et al., 2015). Some studies have evidenced, in vitro, that the inhibition of PINCH1 expression leads to apoptosis in several types of cancers, but in vivo, knock out of Lims1 (which encodes PINCH1) in embryonic neural crest cells caused enhanced apoptosis (Donthamsetty et al., 2013; Dougherty et al., 2008; Liang et al., 2007). Furthermore, the observation that a decrease of Rsu1, but not PINCH1, blocked growth factor induced p38 phosphorylation in MCF10A cells proposed a unique function for Rsu1 (Gonzalez-Nieves et al., 2013).
Knockdown of PINCH-1 protein sensitizes the estrogen positive breast cancer cells to chemotherapy induced apoptosis
2018, Pathology Research and Practice