The differential effects of the Gly-60 to Ala mutation on the interaction of H-Ras p21 with different downstream targets.
We examined the effects of the Gly-60 to Ala mutation on the interaction of H-Ras with Ras GTPase activating protein (GAP), neurofibromin 1 (NF1), Raf-1, and ral guanine nucleotide dissociation stimulator (ralGDS), factors that interact with GTP-bound form of H-Ras. Previous study has shown that the G60A mutation perturbs GTP-induced conformational changes of H-Ras. We found that the G60A mutation decreases GTPase activity of H-Ras without significantly affecting GTP/GDP binding. The reduction in GTPase activity is most dramatic in the presence of GAP or NF1. Interestingly, the G60A mutation does not appear to alter the affinity of H-Ras for GAP or NF1. The G60A mutation moderately reduces the binding of H-Ras to Raf-1 Ras binding domain; however, the binding of H-Ras to ralGDS Ras binding domain was more significantly affected by the same mutation. These results indicate that although GAP, NF1, Raf-1, and ralGDS all interact with H-Ras in a GTP-dependent manner and they are able to compete against each other for binding to H-Ras, these factors share overlapping but not identical binding domains on H-Ras. The significance of our findings is discussed in the light of the GTP-induced conformational change model.
Hwang
M C
MC
Molecular Biology Department, New York State Institute for Basic Research in Developmental Disabilities, Staten Island, New York 10314, USA.
Sung
Y J
YJ
Hwang
Y W
YW
D000409:Alanine / Q000737:chemistry; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D017931:DNA Primers / Q000737:chemistry; D020558:GTP Phosphohydrolases / Q000737:chemistry*; D020690:GTPase-Activating Proteins; D005998:Glycine / Q000737:chemistry; D006160:Guanosine Triphosphate / Q000378:metabolism; D046911:Macromolecular Substances; D051379:Mice; D008969:Molecular Sequence Data; D025542:Neurofibromin 1; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D011506:Proteins / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D019908:Proto-Oncogene Proteins c-raf; D016283:Proto-Oncogene Proteins p21(ras) / Q000737:chemistry*; D015398:Signal Transduction; D013329:Structure-Activity Relationship; D020703:ras GTPase-Activating Proteins
D017931:DNA Primers; D020690:GTPase-Activating Proteins; D046911:Macromolecular Substances; D025542:Neurofibromin 1; D011506:Proteins; D011518:Proto-Oncogene Proteins; D020703:ras GTPase-Activating Proteins; D006160:Guanosine Triphosphate; D017346:Protein Serine-Threonine Kinases; D019908:Proto-Oncogene Proteins c-raf; D020558:GTP Phosphohydrolases; D016283:Proto-Oncogene Proteins p21(ras); D000409:Alanine; D005998:Glycine