Ras binding to a C‐terminal region of GAP

[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.

[学科分类] 生物化学/生物物理

[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