Mapping the site of interaction between annexin VI and the p120GAP C2 domain

FEBS Letters

Gawler, Debra¹  Chow, Andrew¹ 

[1] School of Biomedical Sciences, University of Leeds, Woodhouse Lane, Leeds LS2 9JT, Yorkshire, UK

关键词: Conserved region 2;    GTPase activating protein;    Ras;    Annexin;    Ca2+;    GAP;    GTPase activating protein;    C2;    conserved region 2;    EGTA;    ethylene glycol-bis(β-amino ethyl ether)-N′N′N′;    N′-tetra-acetic acid;    PMSF;    phenylmethylsulfonyl fluoride;    SDS-PAGE;    sodium dodecyl sulfate-polyacrylamide gel electrophoresis;    PBS;    phosphate buffered saline;    FBS;    foetal bovine serum;    HRP;    horseradish peroxidase;    ECL;    enhanced chemiluminescence;   

DOI  :  10.1016/S0014-5793(99)01336-8

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

Annexin VI is a Ca²⁺-dependent membrane and phospholipid binding protein. It mediates a protein-protein interaction with the Ras p21 regulatory protein p120^GAP. In this study we have mapped the binding site of GAP within the annexin VI protein. Using Far Western overlay binding assays and cell lysate competition studies we have mapped the site of interaction to the inter-lobe linker region; amino acids 325–363. Finally, using a GST fusion protein corresponding to this linker region we have demonstrated that cellular loading of the fusion protein into Rat-1 fibroblasts by electroporation blocks the interaction and co-immunoprecipitation of annexin VI and GAP.

【 授权许可】

Unknown