Abstract
ErbB2 amplification and overexpression in breast cancer correlates with aggressive disease and poor prognosis. To find novel ErbB2-interacting proteins, we used stable isotope labeling of amino acids in cell culture followed by peptide affinity pull-downs and identified specific binders using relative quantification by mass spectrometry. Copine-III, a member of a Ca2+-dependent phospholipid-binding protein family, was identified as binding to phosphorylated Tyr1248 of ErbB2. In breast cancer cells, Copine-III requires Ca2+ for binding to the plasma membrane, where it interacts with ErbB2 upon receptor stimulation, an interaction that is dependent on receptor activity. Copine-III also binds receptor of activated C kinase 1 and colocalizes with phosphorylated focal adhesion kinase at the leading edge of migrating cells. Importantly, knockdown of Copine-III in T47D breast cancer cells causes a decrease in Src kinase activation and ErbB2-dependent wound healing. Our data suggest that Copine-III is a novel player in the regulation of ErbB2-dependent cancer cell motility. In primary breast tumors, high CPNE3 RNA levels significantly correlate with ERBB2 amplification. Moreover, in an in situ tissue microarray analysis, we detected differential protein expression of Copine-III in normal versus breast, prostate and ovarian tumors, suggesting a more general role for Copine-III in carcinogenesis.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 50 print issues and online access
$259.00 per year
only $5.18 per issue
Buy this article
- Purchase on Springer Link
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Akiyama T, Matsuda S, Namba Y, Saito T, Toyoshima K, Yamamoto T . (1991). The transforming potential of the c-erbB-2 protein is regulated by its autophosphorylation at the carboxyl-terminal domain. Mol Cell Biol 11: 833–842.
Benes CH, Wu N, Elia AE, Dharia T, Cantley LC, Soltoff SP . (2005). The C2 domain of PKCdelta is a phosphotyrosine binding domain. Cell 121: 271–280.
Blagoev B, Kratchmarova I, Ong SE, Nielsen M, Foster LJ, Mann M . (2003). A proteomics strategy to elucidate functional protein-protein interactions applied to EGF signaling. Nat Biotechnol 21: 315–318.
Brunton VG, Avizienyte E, Fincham VJ, Serrels B, Metcalf 3rd CA, Sawyer TK et al. (2005). Identification of Src-specific phosphorylation site on focal adhesion kinase: dissection of the role of Src SH2 and catalytic functions and their consequences for tumor cell behavior. Cancer Res 65: 1335–1342.
Church DL, Lambie EJ . (2003). The promotion of gonadal cell divisions by the Caenorhabditis elegans TRPM cation channel GON-2 is antagonized by GEM-4 copine. Genetics 165: 563–574.
Citri A, Yarden Y . (2006). EGF-ERBB signalling: towards the systems level. Nat Rev Mol Cell Biol 7: 505–516.
Cox EA, Bennin D, Doan AT, O'Toole T, Huttenlocher A . (2003). RACK1 regulates integrin-mediated adhesion, protrusion, and chemotactic cell migration via its Src-binding site. Mol Biol Cell 14: 658–669.
Creutz CE, Tomsig JL, Snyder SL, Gautier MC, Skouri F, Beisson J et al. (1998). The copines, a novel class of C2 domain-containing, calcium-dependent, phospholipid-binding proteins conserved from Paramecium to humans. J Biol Chem 273: 1393–1402.
Dankort D, Maslikowski B, Warner N, Kanno N, Kim H, Wang Z et al. (2001). Grb2 and Shc adapter proteins play distinct roles in Neu (ErbB-2)-induced mammary tumorigenesis: implications for human breast cancer. Mol Cell Biol 21: 1540–1551.
Dankort DL, Wang Z, Blackmore V, Moran MF, Muller WJ . (1997). Distinct tyrosine autophosphorylation sites negatively and positively modulate neu-mediated transformation. Mol Cell Biol 17: 5410–5425.
Farmer P, Bonnefoi H, Becette V, Tubiana-Hulin M, Fumoleau P, Larsimont D et al. (2005). Identification of molecular apocrine breast tumours by microarray analysis. Oncogene 24: 4660–4671.
Gharbi S, Gaffney P, Yang A, Zvelebil MJ, Cramer R, Waterfield MD et al. (2002). Evaluation of two-dimensional differential gel electrophoresis for proteomic expression analysis of a model breast cancer cell system. Mol Cell Proteomics 1: 91–98.
Gottschalk A, Almedom RB, Schedletzky T, Anderson SD, Yates 3rd JR, Schafer WR . (2005). Identification and characterization of novel nicotinic receptor-associated proteins in Caenorhabditis elegans. EMBO J 24: 2566–2578.
Graus-Porta D, Beerli RR, Daly JM, Hynes NE . (1997). ErbB-2, the preferred heterodimerization partner of all ErbB receptors, is a mediator of lateral signaling. EMBO J 16: 1647–1655.
Guerrero-Valero M, Marin-Vicente C, Gomez-Fernandez JC, Corbalan-Garcia S . (2007). The C2 domains of classical PKCs are specific PtdIns(4,5)P2-sensing domains with different affinities for membrane binding. J Mol Biol 371: 608–621.
Hazan R, Margolis B, Dombalagian M, Ullrich A, Zilberstein A, Schlessinger J . (1990). Identification of autophosphorylation sites of HER2/neu. Cell Growth Differ 1: 3–7.
Hendrix ND, Wu R, Kuick R, Schwartz DR, Fearon ER, Cho KR . (2006). Fibroblast growth factor 9 has oncogenic activity and is a downstream target of Wnt signaling in ovarian endometrioid adenocarcinomas. Cancer Res 66: 1354–1362.
Holbro T, Beerli RR, Maurer F, Koziczak M, Barbas 3rd CF, Hynes NE . (2003). The ErbB2/ErbB3 heterodimer functions as an oncogenic unit: ErbB2 requires ErbB3 to drive breast tumor cell proliferation. Proc Natl Acad Sci USA 100: 8933–8938.
Holbro T, Hynes NE . (2004). ErbB receptors: directing key signaling networks throughout life. Annu Rev Pharmacol Toxicol 44: 195–217.
Hua J, Grisafi P, Cheng SH, Fink GR . (2001). Plant growth homeostasis is controlled by the Arabidopsis BON1 and BAP1 genes. Genes Dev 15: 2263–2272.
Hynes NE, Lane HA . (2005). ERBB receptors and cancer: the complexity of targeted inhibitors. Nat Rev Cancer 5: 341–354.
Keely PJ, Westwick JK, Whitehead IP, Der CJ, Parise LV . (1997). Cdc42 and Rac1 induce integrin-mediated cell motility and invasiveness through PI(3)K. Nature 390: 632–636.
Kenworthy AK . (2001). Imaging protein-protein interactions using fluorescence resonance energy transfer microscopy. Methods 24: 289–296.
Kheifets V, Mochly-Rosen D . (2007). Insight into intra- and inter-molecular interactions of PKC: design of specific modulators of kinase function. Pharmacol Res 55: 467–476.
Kiely PA, Baillie GS, Barrett R, Buckley DA, Adams DR, Houslay MD et al. (2009). Phosphorylation of RACK1 on tyrosine 52 by c-Abl is required for IGF-I-mediated regulation of focal adhesion kinase (FAK). J Biol Chem 284: 20263–20274.
Klemke RL, Cai S, Giannini AL, Gallagher PJ, de Lanerolle P, Cheresh DA . (1997). Regulation of cell motility by mitogen-activated protein kinase. J Cell Biol 137: 481–492.
Lane HA, Beuvink I, Motoyama AB, Daly JM, Neve RM, Hynes NE . (2000). ErbB2 potentiates breast tumor proliferation through modulation of p27(Kip1)-Cdk2 complex formation: receptor overexpression does not determine growth dependency. Mol Cell Biol 20: 3210–3223.
Marone R, Hess D, Dankort D, Muller WJ, Hynes NE, Badache A . (2004). Memo mediates ErbB2-driven cell motility. Nat Cell Biol 6: 515–522.
McCahill A, Warwicker J, Bolger GB, Houslay MD, Yarwood SJ . (2002). The RACK1 scaffold protein: a dynamic cog in cell response mechanisms. Mol Pharmacol 62: 1261–1273.
Meira M, Masson R, Stagljar I, Lienhard S, Maurer F, Boulay A et al. (2009). Memo is a cofilin-interacting protein that influences PLCgamma1 and cofilin activities, and is essential for maintaining directionality during ErbB2-induced tumor-cell migration. J Cell Sci 122: 787–797.
Miller LD, Lee KC, Mochly-Rosen D, Cartwright CA . (2004). RACK1 regulates Src-mediated Sam68 and p190RhoGAP signaling. Oncogene 23: 5682–5686.
Nagy P, Bene L, Balazs M, Hyun WC, Lockett SJ, Chiang NY et al. (1998). EGF-induced redistribution of erbB2 on breast tumor cells: flow and image cytometric energy transfer measurements. Cytometry 32: 120–131.
Nalefski EA, Falke JJ . (1996). The C2 domain calcium-binding motif: structural and functional diversity. Protein Sci 5: 2375–2390.
Patterson RL, van Rossum DB, Nikolaidis N, Gill DL, Snyder SH . (2005). Phospholipase C-gamma: diverse roles in receptor-mediated calcium signaling. Trends Biochem Sci 30: 688–697.
Perkins DN, Pappin DJ, Creasy DM, Cottrell JS . (1999). Probability-based protein identification by searching sequence databases using mass spectrometry data. Electrophoresis 20: 3551–3567.
Rhodes DR, Yu J, Shanker K, Deshpande N, Varambally R, Ghosh D et al. (2004). ONCOMINE: a cancer microarray database and integrated data-mining platform. Neoplasia 6: 1–6.
Ron D, Chen CH, Caldwell J, Jamieson L, Orr E, Mochly-Rosen D . (1994). Cloning of an intracellular receptor for protein kinase C: a homolog of the beta subunit of G proteins. Proc Natl Acad Sci USA 91: 839–843.
Ross JS, Fletcher JA . (1998). The HER-2/neu oncogene in breast cancer: prognostic factor, predictive factor, and target for therapy. Stem Cells 16: 413–428.
Schlessinger J . (2000). Cell signaling by receptor tyrosine kinases. Cell 103: 211–225.
Schulze WX, Deng L, Mann M . (2005). Phosphotyrosine interactome of the ErbB-receptor kinase family. Mol Syst Biol E-pub 25 May 2005.
Schulze WX, Mann M . (2004). A novel proteomic screen for peptide-protein interactions. J Biol Chem 279: 10756–10764.
Slamon DJ, Clark GM, Wong SG, Levin WJ, Ullrich A, McGuire WL . (1987). Human breast cancer: correlation of relapse and survival with amplification of the HER-2/neu oncogene. Science 235: 177–182.
Tomsig JL, Creutz CE . (2002). Copines: a ubiquitous family of Ca(2+)-dependent phospholipid-binding proteins. Cell Mol Life Sci 59: 1467–1477.
Tomsig JL, Snyder SL, Creutz CE . (2003). Identification of targets for calcium signaling through the copine family of proteins. Characterization of a coiled-coil copine-binding motif. J Biol Chem 278: 10048–10054.
Tomsig JL, Sohma H, Creutz CE . (2004). Calcium-dependent regulation of tumour necrosis factor-alpha receptor signalling by copine. Biochem J 378: 1089–1094.
Traxler P, Allegrini PR, Brandt R, Brueggen J, Cozens R, Fabbro D et al. (2004). AEE788: a dual family epidermal growth factor receptor/ErbB2 and vascular endothelial growth factor receptor tyrosine kinase inhibitor with antitumor and antiangiogenic activity. Cancer Res 64: 4931–4941.
White SL, Gharbi S, Bertani MF, Chan HL, Waterfield MD, Timms JF . (2004). Cellular responses to ErbB-2 overexpression in human mammary luminal epithelial cells: comparison of mRNA and protein expression. Br J Cancer 90: 173–181.
Yarden Y, Sliwkowski MX . (2001). Untangling the ErbB signalling network. Nat Rev Mol Cell Biol 2: 127–137.
Yu YP, Landsittel D, Jing L, Nelson J, Ren B, Liu L et al. (2004). Gene expression alterations in prostate cancer predicting tumor aggression and preceding development of malignancy. J Clin Oncol 22: 2790–2799.
Acknowledgements
We thank Elizabeth A Grimm for the polyclonal Copine-III antibody, Martin Spiess for the transferrin receptor antibody, Susanne Schenk for help in generating the Copine-III monoclonal antibody, Laurent Gelman for help with FRET, Michael Rebhan for some bioinformatic analyses and Gwen MacDonald and Julien Dey for helpful discussions. We also thank Daniel Hess and Florence Dalvai for help with establishing the ‘MRM buddy’. The work of CH, MV and MB was partially supported by TRANSFOG FP6 IP funding (LSHC-CT-2004-503438). The laboratories of NEH and JH are supported by the Novartis Research Foundation.
Author information
Authors and Affiliations
Corresponding author
Additional information
Supplementary Information accompanies the paper on the Oncogene website (http://www.nature.com/onc)
Supplementary information
Rights and permissions
About this article
Cite this article
Heinrich, C., Keller, C., Boulay, A. et al. Copine-III interacts with ErbB2 and promotes tumor cell migration. Oncogene 29, 1598–1610 (2010). https://doi.org/10.1038/onc.2009.456
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/onc.2009.456
Keywords
This article is cited by
-
Copine 3 “CPNE3” is a novel regulator for insulin secretion and glucose uptake in pancreatic β-cells
Scientific Reports (2021)
-
The regulation of CPNE1 ubiquitination by the NEDD4L is involved in the pathogenesis of non-small cell lung cancer
Cell Death Discovery (2021)
-
Upregulation of CPNE3 suppresses invasion, migration and proliferation of glioblastoma cells through FAK pathway inactivation
Journal of Molecular Histology (2021)
-
Proteomic analysis of cholera toxin adjuvant-stimulated human monocytes identifies Thrombospondin-1 and Integrin-β1 as strongly upregulated molecules involved in adjuvant activity
Scientific Reports (2019)
-
Cyclic AMP signaling in Dictyostelium promotes the translocation of the copine family of calcium-binding proteins to the plasma membrane
BMC Cell Biology (2018)