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Cortactin Overexpression Inhibits Ligand-Induced Down-Regulation of the Epidermal Growth Factor Receptor

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Cortactin Overexpression Inhibits Ligand-Induced Down-Regulation of the Epidermal Growth Factor Receptor Research Article Cortactin Overexpression Inhibits Ligand-Induced Down-regulation of the Epidermal Growth Factor Receptor Paul Timpson,1 Danielle K. Lynch,1 Daniel Schramek,1 Francesca Walker,2 and Roger J. Daly1 1Cancer Research Program, Garvan Institute of Medical Research, St. Vincent’s Hospital, Sydney, New South Wales and 2Ludwig Institute for Cancer Research, Melbourne Tumour Biology Branch, Royal Melbourne Hospital, Victoria, Australia Abstract factor receptor signaling, thereby avoiding aberrant cellular Ligand-induced receptor down-regulation by endocytosis is a stimulation (3, 4). In brief, ligand-induced activation of the EGFR critical process regulating the intensity and duration of leads to the assembly of protein signaling complexes that relay receptor tyrosine kinase signaling. Ubiquitylation of specific various biological signals. Concomitantly, recruitment of the receptor tyrosine kinases, for example, the epidermal growth endocytic machinery occurs and subsequently leads to endosomal factor receptor (EGFR) by the E3 ubiquitin ligase c-Cbl, sorting which can result in lysosomal degradation of the receptor provides a sorting signal for lysosomal degradation and leads and termination of the signal or recycling of the receptor back to to termination of receptor signaling. Cortactin, which couples the cell surface (5–8). The relative efficiency of lysosomal the endocytic machinery to dynamic actin networks, is degradation versus recycling determines the potency of EGFR signaling (9). encoded by EMS1, a gene commonly amplified in breast and head and neck cancers. One mechanism whereby cortactin The recruitment of c-Cbl, a ubiquitin ligase that directs activated overexpression contributes to tumor progression is by receptor tyrosine kinases to lysosomal degradation by tagging them enhancing tumor cell invasion and metastasis. However, in with multi-monoubiquitin chains, plays a fundamental role in this study, we show that overexpression of cortactin in HeLa ligand-induced down-regulation of various receptors (10, 11). cells markedly inhibits ligand-induced down-regulation of the Tyrosines 1045 and 1068 in the carboxy terminal region of EGFR. This is independent of alterations in receptor auto- the EGFR are responsible for the direct and indirect recruitment phosphorylation and correlates with impaired c-Cbl phos- of c-Cbl, respectively (12, 13). Subsequently c-Cbl is tyrosine phorylation and association with the EGFR, reduced EGFR phosphorylated, which stimulates its ubiquitin ligase activity (13). ubiquitylation, and sustained EGF-induced extracellular sig- Receptor ubiquitylation via c-Cbl then regulates endocytic trafficking and degradation of the EGFR (5, 6, 14, 15). In addition nal-regulated kinase activation. Furthermore, analysis of a panel of head and neck squamous cell carcinoma (HNSCC) cell to its role as an ubiquitin ligase, c-Cbl functions as an adaptor, lines revealed that cortactin overexpression is associated with binding the scaffolding proteins Cbl-interacting protein of 85 kDa attenuated ligand-induced EGFR down-regulation. Impor- (CIN85) or CD2-associated protein (CD2AP) in a phosphorylation- tantly, RNAi-mediated reduction of cortactin expression in dependent manner (7, 8, 16). In turn, these scaffolds bind an 11q13-amplified HNSCC cell line accelerates EGFR degra- endophilin, which is thought to induce negative curvature of the dation. This represents the first demonstration of modulation plasma membrane. of growth factor receptor signaling by cortactin. Moreover, Importantly, it has recently become evident that the trans- enhanced EGFR signaling due to cortactin overexpression may forming potential of several oncogenic receptor tyrosine kinases is provide an alternative explanation for EMS1 gene amplifica- partly explained by their ability to avoid c-Cbl recruitment and tion in human cancers. (Cancer Res 2005; 65(8): 3273-80) therefore ligand-induced receptor down-regulation (1–3). Various oncogenic versions of the EGFR including the v-erbB protein of the avian erythroblastosis virus and the EGFR mutant EGFRvV, harbor Introduction deletions spanning the c-Cbl docking site (1). Furthermore, a point Control of growth factor receptor signaling is fundamental to a mutation in the direct c-Cbl binding site of the EGFR impairs wide variety of cellular processes including cell proliferation, down-regulation of the receptor and elicits enhanced mitogenic differentiation, motility, and survival. Therefore, growth factor signaling (12). Also, the most oncogenic member of the EGFR receptors are subject to tight regulation in order to induce family, ErbB2/HER2, is poorly coupled to c-Cbl-mediated degra- signaling of an appropriate magnitude for the task required. dation (17). The EGFR can also escape c-Cbl-mediated down- Inappropriate activation and aberrant signaling by particular regulation by its heterodimerization with ErbB2, which impairs its growth factor receptors has been linked with a number of human normal recruitment of c-Cbl and results in both receptor diseases including cancer (1–3). recycling to the cell surface and enhanced, sustained signaling Multiple mechanisms serve to control signaling by the epidermal (18–20). growth factor receptor (EGFR), including the action of protein Cortactin is a multidomain protein consisting of an NH2- tyrosine phosphatases (3). However, a number of studies indicate terminal acidic region, which binds the actin related protein that ligand-induced receptor down-regulation by endocytosis plays (Arp)2/3 complex, a repeat region, which associates with a key role in regulating the propagation and duration of growth filamentous actin, and a COOH-terminal src homology (SH)3 domain, which recruits a variety of cellular proteins (21). Cortactin can stimulate the actin-nucleation activity of the Requests for reprints: Roger J. Daly, Cancer Research Program, Garvan Institute of Arp2/3 complex alone or in combination with N-WASP (22, 23). Medical Research, St. Vincent’s Hospital, Sydney, New South Wales 2010, Australia. Phone: 61-2-9295-8333; Fax: 61-2-9295-8321; E-mail: [email protected]. In addition, cortactin also inhibits debranching and disassembly I2005 American Association for Cancer Research. of dendritic actin networks (24). One cellular role for cortactin is www.aacrjournals.org 3273 Cancer Res 2005; 65: (8). April 15, 2005 Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 2005 American Association for Cancer Research. Cancer Research to bridge the endocytic machinery with components and Growth factor treatment of cells. Cells were starved overnight in regulators of the actin cytoskeleton. For example, cortactin serum-free medium and then simulated for the indicated time period with binds the mechanochemical GTPase dynamin 2, which regulates 100 ng/mL EGF (R&D Systems, Minneapolis, MN). the fission of endocytic vesicles. This interaction has recently Cell lysis, immunoprecipitation, and immunoblotting. Cell lysates were prepared using radioimmunoprecipitation assay buffer (38) containing been shown to participate in receptor-mediated endocytosis (25– 10 Ag/mL aprotinin, 10 Ag/mL leupeptin, 1 mmol/L sodium orthovanadate, 27). Furthermore, a close relative of CIN85, CD2AP, binds to and 1 mmol/L phenylmethylsulfonyl fluoride. Protein concentrations were cortactin in an endocytic complex containing c-Cbl, the EGFR, determined using Protein Assay Reagent (Bio-Rad, Hercules, CA). and endophilin (16). Finally, cortactin and the Arp2/3 complex Immunoprecipitations were done by incubation with the indicated antibody have been shown to colocalize and associate with endosomal for a minimum of 2 hours at 4jC with constant mixing. Protein A- or vesicles, suggesting a role for the actin nucleation activity of G-sepharose [25 Ag, Zymed, San Francisco, CA; prewashed twice with 20 cortactin in vesicle trafficking (28). mmol/L HEPES (pH 7.5)] was then added to the lysate-antibody mixture for Importantly, cortactin is implicated in the progression of certain 1 hour. Immunoprecipitates were then washed thrice with radioimmuno- human cancers. The gene encoding cortactin, EMS1, localizes to precipitation assay buffer and denatured in SDS-PAGE sample buffer by j chromosomal locus 11q13, a region commonly amplified in breast heating for 3 minutes at 100 C. Samples were analyzed by SDS-PAGE, transferred to a polyvinylidene difluoride membrane (Millipore, Bedford, cancers and head and neck squamous cell carcinoma (HNSCC; refs. MA) and subjected to Western blot analysis. Detection of bound antibodies 29, 30). Since cortactin overexpression increases the motility of was by enhanced chemiluminescence (Amersham Biosciences, Pty. Ltd., fibroblasts and endothelial cells (31, 32) and enhances bone Castle Hill, New South Wales, Australia). Densitometry was done using IP metastasis of MDA-MB-231 breast cancer cells in a nude mouse Lab Gel software (Signal Analytics Corp., Vienna, VA). model (33), the explanation to date for EMS1 amplification in Antibodies. Polyclonal EGFR 1005 (for Western blotting), EGFR (1045), human cancers has been that cortactin overexpression promotes CD2AP (H-290) antibodies, and monoclonal EGFR 528 (for immunopreci- tumor cell invasion or metastasis. pitations), EGFR (1068), and growth factor receptor binding protein 2 The overexpression of cortactin in specific cancers, coupled (Grb2) antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). with its involvement in endocytic events and vesicle trafficking, The cortactin monoclonal 4F11
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