1. ¹Centre for Immunology, St Vincent's Hospital. University of New South Wales, Australia
2. ²Departament de Biologia Cellular, Facultat de Medicina, Universitat de Barcelona, Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Barcelona, Spain
3. ³Institute for Medical Biochemistry and Molecular Biology, Department of Molecular Cell Biology, University Hospital Eppendorf, Hamburg, Germany
4. ⁴The Garvan Institute of Medical Research, Darlinghurst, Sydney, Australia
5. ⁵Heart Research Institute, Camperdown, Australia
6. ⁶Department of Internal Medicine, University Hospital Eppendorf, Hamburg, Germany

Correspondence: C Enrich, Departament de Biologia Cellular, Facultat de Medicina. Universitat de Barcelona, Casanova 143, 08036-Barcelona, Spain. E-mail: enrich@ub.edu

Received 25 October 2004; Revised 1 April 2005; Accepted 8 April 2005; Published online 6 June 2005.

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.