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Annexin A6 stimulates the membrane recruitment of p120GAP to modulate Ras and Raf-1 activity

Oncogene volume 24, pages 5809–5820 (2005)Cite this article

Abstract

Annexin A6 is a calcium-dependent membrane-binding protein that interacts with signalling proteins, including the GTPase-activating protein p120GAP, one of the most important inactivators of Ras. Since we have demonstrated that annexin A6 inhibits EGF- and TPA-induced Ras signalling, we investigated whether modulation of Ras activity by annexin A6 was mediated via altered subcellular localization of p120GAP. First, we exploited our observation that high-density lipoproteins (HDL) can activate the Ras/MAP kinase pathway. Expression of annexin A6 caused a significant reduction in HDL-induced activation of Ras and Raf-1. Annexin A6 promoted membrane binding of p120GAP in vitro, and plasma membrane targeting of p120GAP in living cells, both in a Ca2+-dependent manner, which is consistent with annexin A6 promoting the Ca2+-dependent assembly of p120GAP-Ras at the plasma membrane. We then extended these studies to other cell types and stimuli. Expression of annexin A6 in A431 cells reduced, while RNAi-mediated suppression of annexin A6 in HeLa cells enhanced EGF-induced Ras and Erk activation. Importantly, the enhancement of Ras activation following RNAi-mediated reduction in p120GAP levels was more marked in annexin A6-expressing A431 cells than controls, indicating that the effect of annexin A6 on Ras was mediated via p120GAP. Finally, we demonstrated that annexin A6 promotes plasma membrane targeting of p120GAP in A431 cells in response to a variety of stimuli, resulting in colocalization with H-Ras. These findings demonstrate an important role for annexin A6 in regulating plasma membrane localization of p120GAP and hence Ras activity.

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Acknowledgements

We thank Dr Peter J Lockyer (Cambridge, UK) and Dr John F Hancock (Brisbane, Australia) for p120GAP, CAPRI, HRas and HRasG12V expression vectors. We are grateful to Dr Maria Calvo (Serveis Científic i Tècnics, Universitat de Barcelona/IDIBAPS) for the assistance in the calcium imaging experiments. This work was supported by grants to CE (BMC2003-04754 and GEN2003-20662 from Ministerio de Educación y Ciencia), TG (Gretl Raymond Foundation, G04S1650 from the National Heart Foundation of Australia and RM02987 from the Cure Cancer Australia Foundation). CE and TG are thankful to the Acciones Integradas collaborative research grant (HA2002-0055). ID was supported by a pre-doctoral fellowship from Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS, Barcelona). RJD is supported by the National Health and Medical Research Council (NHMRC, Australia) and the Cancer Council of New South Wales. AP and FT are supported by Ramon y Cajal Research Program Ministerio de Educación y Ciencia (Spain).

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Authors and Affiliations

  1. Centre for Immunology, St Vincent's Hospital. University of New South Wales, Australia

    Thomas Grewal & Rachael Evans

  2. Departament de Biologia Cel·lular, Facultat de Medicina, Universitat de Barcelona, Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Barcelona, Spain

    Carles Rentero, Francesc Tebar, Laia Cubells, Iñaki de Diego, Albert Pol & Carlos Enrich

  3. Department of Molecular Cell Biology, Institute for Medical Biochemistry and Molecular Biology, University Hospital Eppendorf, Hamburg, Germany

    Matthias F Kirchhoff & Joerg Heeren

  4. The Garvan Institute of Medical Research, Darlinghurst, Sydney, Australia

    William E Hughes & Roger J Daly

  5. Heart Research Institute, Camperdown, Australia

    Kerry-Anne Rye

  6. Department of Internal Medicine, University Hospital Eppendorf, Hamburg, Germany

    Franz Rinninger

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  1. Thomas Grewal
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Correspondence to Carlos Enrich.

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Grewal, T., Evans, R., Rentero, C. et al. Annexin A6 stimulates the membrane recruitment of p120GAP to modulate Ras and Raf-1 activity. Oncogene 24, 5809–5820 (2005). https://doi.org/10.1038/sj.onc.1208743

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