The Ca2+-dependent Lipid Binding Domain of P120GAP Mediates Protein-Protein Interactions with Ca2+-dependent Membrane-binding Proteinss

doi:10.1074/jbc.271.40.24333

The Ca²⁺-dependent Lipid Binding Domain of P120^GAP Mediates Protein-Protein Interactions with Ca²⁺-dependent Membrane-binding Proteinss

EVIDENCE FOR A DIRECT INTERACTION BETWEEN ANNEXIN VI AND P120^GAP*

From the ^‡ Department of Pharmacology and the
^§ Department of Biochemistry & Molecular Biology, University of Leeds, Leeds LS2 9JT, United Kingdom and the
^¶ Department of Physiology, University College London, Gower Street, London WC1E 6BT, United Kingdom

^∥ To whom correspondence should be addressed:
Dept. of Pharmacology, Worsley Medical & Dental Bldg., University of Leeds, Leeds LS2 9JT, UK
. Tel.: 4413-233-4312; Fax: 4413-233-4331; E-mail: D.J.gawler{at}leeds.ac.uk

Abstract

The CaLB domain is a 43-amino acid sequence motif found in a number of functionally diverse signaling proteins including three Ras-specific GTPase activating proteins (GAPs). In the Ras GTPase activating protein, P120^GAP, this domain has the ability to confer membrane association in response to intracellular Ca²⁺ elevation. Here we have isolated three proteins, p55, p70, and p120, which interact with the P120^GAP CaLB domain in vitro. We identify p70 as the Ca²⁺-dependent phospholipid-binding protein annexin VI. Using co-immunoprecipitation studies, we have shown that the interaction between P120^GAP and annexin VI is also detectable in rat fibroblasts, suggesting that this interaction may have a physiological role in vivo. Thus, the CaLB domain in P120^GAP appears to have the ability to direct specific protein-protein interactions with Ca²⁺-dependent membrane-associated proteins. In addition, annexin VI is known to have tumor suppressor activity. Therefore, it is possible that the interaction of annexin VI with P120^GAP may be important in the subsequent modulation of p21^ras activity.

Footnotes

↵* This work was supported by the North of England Cancer Research Campaign and the Academic Development Fund (University of Leeds). The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

GAP

GTPase activating proteins

CaLB

Ca²⁺-dependent lipid binding

GST

glutathione S-transferase

PBS

phosphate-buffered saline

PAGE

polyacrylamide gel electrophoresis

HRP

horseradish peroxidase.
- Received July 8, 1996.
- Revision received August 8, 1996.