Novel, activated RAS mutations alter protein-protein interactions.
Random RAS2 mutants of Saccharomyces cerevisiae were screened for activating traits. A total of 69 distinct mutations were identified, affecting 44 different amino acid residues. Many activated alleles do not bypass the requirement for the nucleotide exchange factor, CDC25, nor is the severity of RAS2 phenotypic traits strictly correlated with the capacity to bypass CDC25. In vivo interactions of mutant RAS2 proteins with RAS effectors (adenylate cyclase and RAF), CDC25 and GTPase activating proteins (IRA2 and NF1) were assayed to assess how the various amino acid substitutions influence interactions with regulatory and target proteins of RAS. Nearly all activated RAS2 proteins were observed to interact better with adenylate cyclase and RAF, although some distinct differences were found. Several amino acid substitutions that reduce the affinity of RAS2 for guanine nucleotides apparently elevate the fraction of nucleotide-free RAS2, which has greater CDC25 affinity. Amino acid alterations that reduce the affinity of RAS2 for GTPase activating proteins included substitutions both within the switch I/switch II domain and distinctly outside it. One mutant, RAS2-Y78F, bound a lower fraction of GTP in vivo than the wild-type protein. The Y78F substitution is localized to the switch II domain, a region of the RAS protein that undergoes guanine nucleotide-dependent conformational changes.
Dalley
B K
BK
Department of Molecular Microbiology and Immunology, University of Missouri, Columbia 65212, USA.
Cannon
J F
JF
D000262:Adenylyl Cyclases / Q000378:metabolism / Q000502:physiology; D000483:Alleles; D000595:Amino Acid Sequence; D018797:Cell Cycle Proteins / Q000378:metabolism / Q000502:physiology; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism* / Q000502:physiology*; D020558:GTP Phosphohydrolases / Q000378:metabolism / Q000502:physiology; D020690:GTPase-Activating Proteins; D015966:Gene Expression Regulation, Fungal; D011905:Genes, ras; D006160:Guanosine Triphosphate / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D025542:Neurofibromin 1; D010641:Phenotype; D010749:Phosphoprotein Phosphatases / Q000378:metabolism / Q000502:physiology; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism / Q000502:physiology; D011506:Proteins / Q000378:metabolism / Q000502:physiology; D011518:Proto-Oncogene Proteins / Q000378:metabolism / Q000502:physiology; D019908:Proto-Oncogene Proteins c-raf; D012441:Saccharomyces cerevisiae / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins; D018631:ras Proteins / Q000235:genetics / Q000378:metabolism* / Q000502:physiology*; D020666:ras-GRF1
D018797:Cell Cycle Proteins; D005656:Fungal Proteins; D020690:GTPase-Activating Proteins; C064960:IRA2 protein, S cerevisiae; D025542:Neurofibromin 1; D011506:Proteins; D011518:Proto-Oncogene Proteins; D029701:Saccharomyces cerevisiae Proteins; D020666:ras-GRF1; D006160:Guanosine Triphosphate; D017346:Protein Serine-Threonine Kinases; D019908:Proto-Oncogene Proteins c-raf; D010749:Phosphoprotein Phosphatases; D020558:GTP Phosphohydrolases; C489015:RAS2 protein, S cerevisiae; D018631:ras Proteins; D000262:Adenylyl Cyclases