Different structural requirements within the switch II region of the Ras protein for interactions with specific downstream targets.
Ras proteins function through the formation of specific complexes with Raf-1, B-raf, PI-3 kinase and RalGDS. These interactions all require Ras-GTP with an intact effector binding domain (Switch I region). We have examined the requirements of the Switch II region (amino acids 60-72) for the production of stable interactions between Ras and its downstream effectors. A point mutation at position 65 or 64 combined with additional mutations at either position 65 or 71 rendered nucleotide-free Ras protein unable to stably interact with Ras specific guanine nucleotide exchange factors. Ha-Ras containing point mutations at positions 65 and 71 possessed a twofold higher affinity for B-raf and consequently MEK1. The point mutation at 64, in combination with additional point mutations at either position 65 or 71, resulted in a protein which failed to interact with either PI-3 kinase or neurofibromin, though these Ras mutants effectively bound both Raf-1 and B-raf. An activated form of Ras, Q61L-Ras, associated with all effector proteins independent of the bound guanine nucleotide. Q61L-Ras-GDP was almost as effective as wild type Ras-GMPPNP in the in vitro activation of MEK1 and MAP kinase. Competitive studies with the catalytic domain if neurofibromin, NF1-GRD, demonstrated that its interaction with Ras-GMPPNP is mutually exclusive with both Raf-1 and B-raf. These data suggest that rasGAP and neurofibromin are unable to downregulate Ras-GTP complexed to Raf-1 or B-raf.
Moodie
S A
SA
Dept. Mole. Biology, Bristol Myers & Squibb Research Institute, Princeton, New Jersey 08543, USA.
Paris
M
M
Villafranca
E
E
Kirshmeier
P
P
Willumsen
B M
BM
Wolfman
A
A
D000595:Amino Acid Sequence; D000818:Animals; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D048369:MAP Kinase Kinase 1; D020929:Mitogen-Activated Protein Kinase Kinases; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D025542:Neurofibromin 1; D011485:Protein Binding; D011487:Protein Conformation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D011505:Protein-Tyrosine 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*; D051381:Rats; D015398:Signal Transduction; D013329:Structure-Activity Relationship
D025542:Neurofibromin 1; D011506:Proteins; D011518:Proto-Oncogene Proteins; D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate; D011505:Protein-Tyrosine Kinases; D017346:Protein Serine-Threonine Kinases; D019908:Proto-Oncogene Proteins c-raf; D048369:MAP Kinase Kinase 1; D020929:Mitogen-Activated Protein Kinase Kinases; D016283:Proto-Oncogene Proteins p21(ras)