Ras binding to a C-terminal region of GAP.
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 p120GAP protein and provide the first direct demonstration of Ras interaction with GAP in the region 715-753.
Molloy
D P
DP
CRC Institute for Cancer Studies, Medical School, University of Birmingham, Edgbaston, UK.
Owen
D
D
Grand
R J
RJ
D000595:Amino Acid Sequence; D005456:Fluorescent Dyes; D020558:GTP Phosphohydrolases / Q000378:metabolism; D020690:GTPase-Activating Proteins; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D009282:Naphthalenesulfonates; D015689:Oncogene Protein p21(ras) / Q000235:genetics / Q000378:metabolism*; D010446:Peptide Fragments / Q000138:chemical synthesis; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D020703:ras GTPase-Activating Proteins
D005456:Fluorescent Dyes; D020690:GTPase-Activating Proteins; D009282:Naphthalenesulfonates; D010446:Peptide Fragments; D011506:Proteins; D011993:Recombinant Fusion Proteins; D020703:ras GTPase-Activating Proteins; D006153:Guanosine Diphosphate; C006421:1,5-I-AEDANS; D006160:Guanosine Triphosphate; D020558:GTP Phosphohydrolases; D015689:Oncogene Protein p21(ras); D008274:Magnesium