Structural significance of the GTP-binding domain of ras p21 studied by site-directed mutagenesis.
Point mutations of p21 proteins were constructed by oligonucleotide-directed mutagenesis of the v-rasH oncogene, which substituted amino acid residues within the nucleotide-binding consensus sequence, GXG GXGK. When the glycine residue at position 10, 13, or 15 was substituted with valine, the viral rasH product p21 lost its GTP-binding and autokinase activities. Other substitutions at position 33, 51, or 59 did not impair its binding activity. G418-resistant NIH 3T3 cell lines were derived by transfection with constructs obtained by inserting the mutant proviral DNA into the pSV2neo plasmid. Clones with a valine mutation at position 13 or 15 were incapable of transforming cells, while all other mutants with GTP-binding activity were competent. A mutant with a substitution of valine for glycine at position 10 which had lost its ability to bind GTP and its autokinase activity was fully capable of transforming NIH 3T3 cells. These cells grew in soft agar and rapidly formed tumors in nude mice. The p21 of cell lines derived from tumor explants still lacked the autokinase activity. These findings suggest that the glycine-rich consensus sequence is important in controlling p21 activities and that certain mutations may confer to p21 its active conformation without participation of ligand binding.
D000818:Animals; D001665:Binding Sites; D002461:Cell Line, Transformed; D003001:Cloning, Molecular; D019204:GTP-Binding Proteins / Q000378:metabolism*; D005998:Glycine; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism*; D051379:Mice; D008819:Mice, Nude; D009154:Mutation; D009374:Neoplasms, Experimental / Q000235:genetics; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism*; D016283:Proto-Oncogene Proteins p21(ras)
D011518:Proto-Oncogene Proteins; D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate; D019204:GTP-Binding Proteins; D016283:Proto-Oncogene Proteins p21(ras); D005998:Glycine