Ras binding to a C‐terminal region of GAP

FEBS Letters

Owen, Darerca¹  Grand, Roger J.A.¹  Molloy, David P.¹ 

[1] CRC Institute for Cancer Studies, Medical School, The University of Birmingham, Edgbaston, Birmingham B15 2TJ, UK

关键词: 5′-(2-((Iodoacetyl)amino)ethyl)naphthalene-1-sulphonic acid fluorescence;    GAP peptide;    Ras;    p120GAP;    GAP;    GTPase;    activating protein;    mGTP;    2′(3′)-O-(N-methylanthraniloyl) GTP;    mGDP;    is similarly abbreviated;    NF1;    the GAP related protein neurofibromin;    Ras;    the wild-type (G12) protein product of the N-Ras oncogene;    G12D;    Y32A;    Y40F;    mutant forms of N-Ras used in this study;    IAEDANS;    5′-(2-((iodoacetyl)amino)ethyl)naphthalene-1-sulphonic acid;    IAS;    4-(iodoacetamido)salicylic acid;    Y1345;    Y922 synthetic peptides comprising residues 700–726 and 715–753 of human p120GAP;    respectively;    C·IAE-DANS;    Y922 · IAEDANS and Y922 · IAS;    cysteine and Y922;    labelled with IAEDANS and Y922 labelled with IAS;    respectively;    ΔF max;    the maximum observed fluorescence change in IAEDANS emission intensity;   

DOI  :  10.1016/0014-5793(95)00657-U

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

Using fluorescence spectroscopy we have identified a binding region for Ras on the GTPase activating protein (GAP) lying within residues 715–753. A synthetic peptide Y922, corresponding to residues 716–753 of GAP binds to wild type Ras showing 3.3-fold higher affinity for the GTP- over the GDP-bound forms of Ras. Binding is stabilised by Mg²⁺, although Y922 does not stimulate the GTPase activity of Ras. Peptide binding to the Y32A and Y40F Ras mutants showed equal affinity for both GDP- and GTP-bound forms, with binding to Y32A · GDP abolished in the absence of Mg²⁺. These results suggest that Y922 mimics the in vivo interactions shown by the intact p120^GAP protein and provide the first direct demonstration of Ras interaction with GAP in the region 715–753.

【 授权许可】

Unknown