Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Original Article
  • Published:

Annexin A6 inhibits Ras signalling in breast cancer cells

Oncogene volume 28, pages 363–377 (2009)Cite this article

Abstract

Overexpression of epidermal growth factor receptor (EGFR) is associated with enhanced activation of wild-type (hyperactive) Ras in breast cancer. Little is known about the regulation of Ras inactivation and GTPase-activating proteins (GAPs), such as p120GAP, in cells with hyperactive Ras. Recently, we showed that in EGFR-overexpressing A431 cells, which lack endogenous Annexin A6 (AnxA6), ectopic expression of AnxA6 stimulates membrane recruitment of p120GAP to modulate Ras signalling. We now demonstrate that, AnxA6 is downregulated in a number of EGFR-overexpressing and estrogen receptor (ER)-negative breast cancer cells. In these cells, AnxA6 overexpression promotes Ca2+- and EGF-inducible membrane targeting of p120GAP. In ER-negative MDA-MB-436 cells, overexpression of p120GAP, but not CAPRI or a p120GAP mutant lacking the AnxA6-binding domain inhibits Ras/MAPK activity. AnxA6 knockdown in MDA-MB-436 increases Ras activity and cell proliferation in anchorage-independent growth assays. Furthermore, AnxA6 co-immunoprecipitates with H-Ras in a Ca2+- and EGF-inducible manner and fluorescence resonance energy transfer (FRET) microscopy confirmed that AnxA6 is in close proximity of active (G12V), but not inactive (S17N) H-Ras. Thus, association of AnxA6 with H-Ras-containing protein complexes may contribute to regulate p120GAP/Ras assembly in EGFR-overexpressing and ER-negative breast cancer cells.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7

Similar content being viewed by others

References

  • Agazie YM, Hayman MJ . (2003). Molecular mechanism for a role of SHP2 in epidermal growth factor receptor signaling. Mol Cell Biol 23: 7875–7886.

    Article  CAS  Google Scholar 

  • Bernards A, Settleman J . (2005). GAPs in growth factor signalling. Growth Factors 23: 143–149.

    Article  CAS  Google Scholar 

  • 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.

    Article  CAS  Google Scholar 

  • Boon LM, Mulliken JB, Vikkula M . (2005). RASA1: variable phenotype with capillary and arteriovenous malformations. Curr Opin Genet Dev 15: 265–269.

    Article  CAS  Google Scholar 

  • Chetcuti A, Margan SH, Russell P, Mann S, Millar DS, Clark SJ et al. (2001). Loss of annexin II heavy and light chains in prostate cancer and its precursors. Cancer Res 61: 6331–6334.

    CAS  PubMed  Google Scholar 

  • Chow A, Gawler D . (1999). Mapping the site of interaction between annexin VI and the p120GAP C2 domain. FEBS Lett 460: 166–172.

    Article  CAS  Google Scholar 

  • Cook SJ, Lockyer PJ . (2006). Recent advances in Ca(2+)-dependent Ras regulation and cell proliferation. Cell Calcium 39: 101–112.

    Article  CAS  Google Scholar 

  • Cooper JA, Kashishian A . (1993). In vivo binding properties of SH2 domains from GTPase-activating protein and phosphatidylinositol 3-kinase. Mol Cell Biol 13: 1737–1745.

    Article  CAS  Google Scholar 

  • Cubells L, de Muga SV, Tebar F, Bonventre JV, Balsinde J, Pol A et al. (2008). Annexin A6-induced inhibition of cytoplasmic phospholipase A2 is linked to caveolin-1 export from the Golgi. J Biol Chem 283: 10174–10183.

    Article  CAS  Google Scholar 

  • Cubells L, Vila de Muga S, Tebar F, Wood P, Evans R, Ingelmo-Torres M et al. (2007). Annexin A6-induced alterations in cholesterol transport and caveolin export from the Golgi complex. Traffic 8: 1568–1589.

    Article  CAS  Google Scholar 

  • Cullen PJ . (2006). Decoding complex Ca2+ signals through the modulation of Ras signaling. Curr Opin Cell Biol 18: 157–161.

    Article  CAS  Google Scholar 

  • Cullen PJ, Lockyer PJ . (2002). Integration of calcium and Ras signalling. Nat Rev Mol Cell Biol 3: 339–348.

    Article  CAS  Google Scholar 

  • Daly RJ, Gu H, Parmar J, Malaney S, Lyons RJ, Kairouz R et al. (2002). The docking protein Gab2 is overexpressed and estrogen regulated in human breast cancer. Oncogene 21: 5175–5181.

    Article  CAS  Google Scholar 

  • Davies AA, Moss SE, Crompton MR, Jones TA, Spurr NK, Sheer D et al. (1989). The gene coding for the p68 calcium-binding protein is localised to bands q32-q34 of human chromosome 5, and to mouse chromosome 11. Hum Genet 82: 234–238.

    Article  CAS  Google Scholar 

  • Davis AJ, Butt JT, Walker JH, Moss SE, Gawler DJ . (1996). The Ca2+-dependent lipid binding domain of P120GAP mediates protein-protein interactions with Ca2+-dependent membrane-binding proteins. Evidence for a direct interaction between annexin VI and P120GAP. J Biol Chem 271: 24333–24336.

    Article  CAS  Google Scholar 

  • de Diego I, Schwartz F, Siegfried H, Dauterstedt P, Heeren J, Beisiegel U et al. (2002). Cholesterol modulates the membrane binding and intracellular distribution of annexin 6. J Biol Chem 277: 32187–32194.

    Article  CAS  Google Scholar 

  • deFazio A, Chiew YE, Sini RL, Janes PW, Sutherland RL . (2000). Expression of c-erbB receptors, heregulin and oestrogen receptor in human breast cell lines. Int J Cancer 87: 487–498.

    Article  CAS  Google Scholar 

  • Downward J . (2003). Targeting RAS signalling pathways in cancer therapy. Nat Rev Cancer 3: 11–22.

    Article  CAS  Google Scholar 

  • Edwards HC, Moss SE . (1995). Functional and genetic analysis of annexin VI. Mol Cell Biochem 149–150: 293–299.

    Article  Google Scholar 

  • Fleet A, Ashworth R, Kubista H, Edwards H, Bolsover S, Mobbs P et al. (1999). Inhibition of EGF-dependent calcium influx by annexin VI is splice form-specific. Biochem Biophys Res Commun 260: 540–546.

    Article  CAS  Google Scholar 

  • Fridman M, Maruta H, Gonez J, Walker F, Treutlein H, Zeng J et al. (2000). Point mutants of c-raf-1 RBD with elevated binding to v-Ha-Ras. J Biol Chem 275: 30363–30371.

    Article  CAS  Google Scholar 

  • Gawler DJ, Zhang LJ, Reedijk M, Tung PS, Moran MF . (1995). CaLB: a 43 amino acid calcium-dependent membrane/phospholipid binding domain in p120 Ras GTPase-activating protein. Oncogene 10: 817–825.

    CAS  PubMed  Google Scholar 

  • Gideon P, John J, Frech M, Lautwein A, Clark R, Scheffler JE et al. (1992). Mutational and kinetic analyses of the GTPase-activating protein (GAP)-p21 interaction: the C-terminal domain of GAP is not sufficient for full activity. Mol Cell Biol 12: 2050–2056.

    Article  CAS  Google Scholar 

  • Grewal T, Enrich C . (2006). Molecular mechanisms involved in Ras inactivation: the annexin A6-p120GAP complex. Bioessays 28: 1211–1220.

    Article  CAS  Google Scholar 

  • Grewal T, Evans R, Rentero C, Tebar F, Cubells L, de Diego I et al. (2005). Annexin A6 stimulates the membrane recruitment of p120GAP to modulate Ras and Raf-1 activity. Oncogene 24: 5809–5820.

    Article  CAS  Google Scholar 

  • Grewal T, Heeren J, Mewawala D, Schnitgerhans T, Wendt D, Salomon G et al. (2000). Annexin VI stimulates endocytosis and is involved in the trafficking of low density lipoprotein to the prelysosomal compartment. J Biol Chem 275: 33806–33813.

    Article  CAS  Google Scholar 

  • Grewal T, Tebar F, Pol A, Enrich C . (2006). Involvement of targeting and scaffolding proteins in the regulation of the EGFR/Ras/MAPK pathway in oncogenesis. Curr Signal Transduct Ther 1: 147–167.

    Article  CAS  Google Scholar 

  • Huang DC, Marshall CJ, Hancock JF . (1993). Plasma membrane-targeted ras GTPase-activating protein is a potent suppressor of p21ras function. Mol Cell Biol 13: 2420–2431.

    Article  CAS  Google Scholar 

  • Jaumot M, Hancock JF . (2001). Protein phosphatases 1 and 2A promote Raf-1 activation by regulating 14-3-3 interactions. Oncogene 20: 3949–3958.

    Article  CAS  Google Scholar 

  • Jin H, Wang X, Ying J, Wong AH, Cui Y, Srivastava G et al. (2007). Epigenetic silencing of a Ca(2+)-regulated Ras GTPase-activating protein RASAL defines a new mechanism of Ras activation in human cancers. Proc Natl Acad Sci USA 104: 12353–12358.

    Article  CAS  Google Scholar 

  • Johannsdottir HK, Jonsson G, Johannesdottir G, Agnarsson BA, Eerola H, Arason A et al. (2006). Chromosome 5 imbalance mapping in breast tumors from BRCA1 and BRCA2 mutation carriers and sporadic breast tumors. Int J Cancer 119: 1052–1060.

    Article  CAS  Google Scholar 

  • Jones RB, Gordus A, Krall JA, MacBeath G . (2006). A quantitative protein interaction network for the ErbB receptors using protein microarrays. Nature 439: 168–174.

    Article  CAS  Google Scholar 

  • Karpova TS, Baumann CT, He L, Wu X, Grammer A, Lipsky P et al. (2003). Fluorescence resonance energy transfer from cyan to yellow fluorescent protein detected by acceptor photobleaching using confocal microscopy and a single laser. J Microsc 209: 56–70.

    Article  CAS  Google Scholar 

  • Kolch W . (2005). Coordinating ERK/MAPK signalling through scaffolds and inhibitors. Nat Rev Mol Cell Biol 6: 827–837.

    Article  CAS  Google Scholar 

  • Kolfschoten IG, van Leeuwen B, Berns K, Mullenders J, Beijersbergen RL, Bernards R et al. (2005). A genetic screen identifies PITX1 as a suppressor of RAS activity and tumorigenicity. Cell 121: 849–858.

    Article  CAS  Google Scholar 

  • Kraus MH, Yuasa Y, Aaronson SA . (1984). A position 12-activated H-ras oncogene in all HS578 T mammary carcinosarcoma cells but not normal mammary cells of the same patient. Proc Natl Acad Sci USA 81: 5384–5388.

    Article  CAS  Google Scholar 

  • Kupzig S, Deaconescu D, Bouyoucef D, Walker SA, Liu Q, Polte CL et al. (2006). GAP1 family members constitute bifunctional Ras and Rap GTPase-activating proteins. J Biol Chem 281: 9891–9900.

    Article  CAS  Google Scholar 

  • Leighton X, Srikantan V, Pollard HB, Sukumar S, Srivastava M . (2004). Significant allelic loss of ANX7region (10q21) in hormone receptor negative breast carcinomas. Cancer Lett 210: 239–244.

    Article  CAS  Google Scholar 

  • Lockyer PJ, Kupzig S, Cullen PJ . (2001). CAPRI regulates Ca(2+)-dependent inactivation of the Ras-MAPK pathway. Curr Biol 11: 981–986.

    Article  CAS  Google Scholar 

  • Loo LW, Grove DI, Williams EM, Neal CL, Cousens LA, Schubert EL et al. (2004). Array comparative genomic hybridization analysis of genomic alterations in breast cancer subtypes. Cancer Res 64: 8541–8549.

    Article  CAS  Google Scholar 

  • Malaney S, Daly RJ . (2001). The ras signaling pathway in mammary tumorigenesis and metastasis. J Mammary Gland Biol Neoplasia 6: 101–113.

    Article  CAS  Google Scholar 

  • Moreto J, Llado A, Vidal-Quadras M, Calvo M, Pol A, Enrich C et al. (2008). Calmodulin modulates H-Ras mediated Raf-1 activation. Cell Signal 20: 1092–1103.

    Article  CAS  Google Scholar 

  • Moss SE, Crumpton MJ . (1990). Alternative splicing gives rise to two forms of the p68 Ca2(+)-binding protein. FEBS Lett 261: 299–302.

    Article  CAS  Google Scholar 

  • Murakoshi H, Iino R, Kobayashi T, Fujiwara T, Ohshima C, Yoshimura A et al. (2004). Single-molecule imaging analysis of Ras activation in living cells. Proc Natl Acad Sci USA 101: 7317–7322.

    Article  CAS  Google Scholar 

  • Nori M, Vogel US, Gibbs JB, Weber MJ . (1991). Inhibition of v-src-induced transformation by a GTPase-activating protein. Mol Cell Biol 11: 2812–2818.

    Article  CAS  Google Scholar 

  • Pena V, Hothorn M, Eberth A, Kaschau N, Parret A, Gremer L et al. (2008). The C2 domain of SynGAP is essential for stimulation of the Rap GTPase reaction. EMBO Rep 9: 350–355.

    Article  CAS  Google Scholar 

  • Pierga JY, Reis-Filho JS, Cleator SJ, Dexter T, Mackay A, Simpson P et al. (2007). Microarray-based comparative genomic hybridisation of breast cancer patients receiving neoadjuvant chemotherapy. Br J Cancer 96: 341–351.

    Article  CAS  Google Scholar 

  • Plowman SJ, Hancock JF . (2005). Ras signaling from plasma membrane and endomembrane microdomains. Biochim Biophys Acta 1746: 274–283.

    Article  CAS  Google Scholar 

  • Pons M, Grewal T, Rius E, Schnitgerhans T, Jackle S, Enrich C . (2001a). Evidence for the Involvement of annexin 6 in the trafficking between the endocytic compartment and lysosomes. Exp Cell Res 269: 13–22.

    Article  CAS  Google Scholar 

  • Pons M, Tebar F, Kirchhoff M, Peiro S, de Diego I, Grewal T et al. (2001b). Activation of Raf-1 is defective in annexin 6 overexpressing Chinese hamster ovary cells. FEBS Lett 501: 69–73.

    Article  CAS  Google Scholar 

  • Rentero C, Evans R, Wood P, Tebar F, Vila de Muga S, Cubells L et al. (2006). Inhibition of H-Ras and MAPK is compensated by PKC-dependent pathways in annexin A6 expressing cells. Cell Signal 18: 1006–1016.

    Article  CAS  Google Scholar 

  • Rual JF, Venkatesan K, Hao T, Hirozane-Kishikawa T, Dricot A, Li N et al. (2005). Towards a proteome-scale map of the human protein–protein interaction network. Nature 437: 1173–1178.

    Article  CAS  Google Scholar 

  • Sacks DB . (2006). The role of scaffold proteins in MEK/ERK signalling. Biochem Soc Trans 34: 833–836.

    Article  CAS  Google Scholar 

  • Smythe E, Smith PD, Jacob SM, Theobald J, Moss SE . (1994). Endocytosis occurs independently of annexin VI in human A431 cells. J Cell Biol 124: 301–306.

    Article  CAS  Google Scholar 

  • Strzelecka-Kiliszek A, Buszewska ME, Podszywalow-Bartnicka P, Pikula S, Otulak K, Buchet R et al. (2008). Calcium- and pH-dependent localization of annexin A6 isoforms in Balb/3T3 fibroblasts reflecting their potential participation in vesicular transport. J Cell Biochem 104: 418–434.

    Article  CAS  Google Scholar 

  • Tebar F, Villalonga P, Sorkina T, Agell N, Sorkin A, Enrich C . (2002). Calmodulin regulates intracellular trafficking of epidermal growth factor receptor and the MAPK signaling pathway. Mol Biol Cell 13: 2057–2068.

    Article  CAS  Google Scholar 

  • Theobald J, Hanby A, Patel K, Moss SE . (1995). Annexin VI has tumour-suppressor activity in human A431 squamous epithelial carcinoma cells. Br J Cancer 71: 786–788.

    Article  CAS  Google Scholar 

  • Theobald J, Smith PD, Jacob SM, Moss SE . (1994). Expression of annexin VI in A431 carcinoma cells suppresses proliferation: a possible role for annexin VI in cell growth regulation. Biochim Biophys Acta 1223: 383–390.

    Article  CAS  Google Scholar 

  • Timpson P, Wilson AS, Lehrbach GM, Sutherland RL, Musgrove EA, Daly RJ . (2007). Aberrant expression of cortactin in head and neck squamous cell carcinoma cells is associated with enhanced cell proliferation and resistance to the epidermal growth factor receptor inhibitor gefitinib. Cancer Res 67: 9304–9314.

    Article  CAS  Google Scholar 

  • van der Geer P, Henkemeyer M, Jacks T, Pawson T . (1997). Aberrant Ras regulation and reduced p190 tyrosine phosphorylation in cells lacking p120-Gap. Mol Cell Biol 17: 1840–1847.

    Article  CAS  Google Scholar 

  • Vishwanatha JK, Salazar E, Gopalakrishnan VK . (2004). Absence of annexin I expression in B-cell non-Hodgkin's lymphomas and cell lines. BMC Cancer 4: 8.

    Article  Google Scholar 

  • Walker SA, Cullen PJ, Taylor JA, Lockyer PJ . (2003). Control of Ras cycling by Ca2+. FEBS Lett 546: 6–10.

    Article  CAS  Google Scholar 

  • Walker SA, Kupzig S, Bouyoucef D, Davies LC, Tsuboi T, Bivona TG et al. (2004). Identification of a Ras GTPase-activating protein regulated by receptor-mediated Ca2+ oscillations. EMBO J 23: 1749–1760.

    Article  CAS  Google Scholar 

  • Wang Z, Tung PS, Moran MF . (1996). Association of p120 ras GAP with endocytic components and colocalization with epidermal growth factor (EGF) receptor in response to EGF stimulation. Cell Growth Differ 7: 123–133.

    CAS  PubMed  Google Scholar 

  • Wang Z, Wilson GF, Griffith LC . (2002). Calcium/calmodulin-dependent protein kinase II phosphorylates and regulates the Drosophila eag potassium channel. J Biol Chem 277: 24022–24029.

    Article  CAS  Google Scholar 

  • Westbrook TF, Martin ES, Schlabach MR, Leng Y, Liang AC, Feng B et al. (2005). A genetic screen for candidate tumor suppressors identifies REST. Cell 121: 837–848.

    Article  CAS  Google Scholar 

  • Zhang K, DeClue JE, Vass WC, Papageorge AG, McCormick F, Lowy DR . (1990). Suppression of c-ras transformation by GTPase-activating protein. Nature 346: 754–756.

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank Dr Maria Calvo (SCT-UB) for her excellent assistance in confocal microscopy. This study was supported by grants to CE (BFU2006-01151, V-2006-RET2008-0 and fellowship PR-2006-0142 from Ministerio de Educación y Ciencia, Spain) and TG (510293 and 510294 from the National Health and Medical Research Council of Australia and G06S2559 from the National Heart Foundation of Australia). SV is thankful to Ministerio de Educación y Ciencia, Spain, (FPI mobility Program) for a short-term fellowship at the laboratory of TG (Sydney, Australia).

Author information

Authors and Affiliations

  1. Departament de Biologia Cel·lular, Facultat de Medicina, Universitat de Barcelona, Barcelona, Spain

    S Vilá de Muga, L Cubells, C Rentero, M Reverter, F Tebar & C Enrich

  2. The Garvan Institute of Medical Research, Sydney, NSW, Australia

    P Timpson & R J Daly

  3. Faculty of Pharmacy, University of Sydney, Sydney, Australia

    R Evans, T E Hayes, A Hegemann, J Leschner & T Grewal

  4. Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Facultat de Medicina, Universitat de Barcelona, Barcelona, Spain

    A Pol & C Enrich

Authors
  1. S Vilá de Muga
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. P Timpson
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. L Cubells
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. R Evans
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. T E Hayes
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. C Rentero
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. A Hegemann
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. M Reverter
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. J Leschner
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. A Pol
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. F Tebar
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. R J Daly
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. C Enrich
    View author publications

    You can also search for this author in PubMed Google Scholar

  14. T Grewal
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to C Enrich or T Grewal.

Rights and permissions

Reprints and permissions

About this article

Cite this article

de Muga, S., Timpson, P., Cubells, L. et al. Annexin A6 inhibits Ras signalling in breast cancer cells. Oncogene 28, 363–377 (2009). https://doi.org/10.1038/onc.2008.386

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/onc.2008.386

Keywords

This article is cited by

Search

Advanced search

Quick links