Mutagenesis of the H-ras p21 at glycine-60 residue disrupts GTP-induced conformational change.
The function of Gly-60, the conserved glycine in the DXXG domain of v-H-ras, was examined by site-directed mutagenesis. It was found that while the G60A (Gly-60 to Ala substitution) mutation has little effect on the interaction of H-ras with guanine nucleotides, it completely abolishes the biological activity of v-H-ras. The G60A mutation also exerts little effect on the interaction of H-ras with SDC25C (a guanine nucleotide exchange factor) and GAP. However, the G60A mutation does lower the ability of H-ras to bind Raf. GTP induces an enhancement of fluorescence emission in complexes consisting of H-ras and the fluorescent dye 8-anilino-1-naphthalenesulfonic acid. This enhancement is blocked by the G60A mutation. On the basis of these observations, we propose that the GTP-induced conformational change of H-ras, a process required for H-ras activities, is impaired by the G60A mutation.
Sung
Y J
YJ
Molecular Biology Department, New York State Institute for Basic Research in Developmental Disabilities, Staten Island 10314.
Carter
M
M
Zhong
J M
JM
Hwang
Y W
YW
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D017104:Bacteriophage M13 / Q000235:genetics; D001483:Base Sequence; D004279:DNA, Viral / Q000235:genetics; D005260:Female; D020690:GTPase-Activating Proteins; D005998:Glycine / Q000737:chemistry / Q000235:genetics; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism / Q000494:pharmacology; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D015689:Oncogene Protein p21(ras) / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D027384:Oncogene Proteins v-raf; D009865:Oocytes / Q000378:metabolism; D011487:Protein Conformation / Q000187:drug effects; D011506:Proteins / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015682:Retroviridae Proteins, Oncogenic / Q000378:metabolism; D014982:Xenopus laevis; D020703:ras GTPase-Activating Proteins
D004279:DNA, Viral; D020690:GTPase-Activating Proteins; D011506:Proteins; D011993:Recombinant Fusion Proteins; D015682:Retroviridae Proteins, Oncogenic; D020703:ras GTPase-Activating Proteins; D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate; D027384:Oncogene Proteins v-raf; D015689:Oncogene Protein p21(ras); D005998:Glycine