Differential structural requirements for interaction of Ras protein with its distinct downstream effectors.
Ras proteins have multiple effectors of distinct structures that do not share significant structural homology at their Ras interaction sites. To prove possible differences in their recognition mechanisms of Ras, we screened 44 human Ha-Ras proteins carrying mutations in the effector region and its flanking sequences for interaction with human Raf-1, Schizosaccharomyces pombe Byr2, and Saccharomyces cerevisiae adenylyl cyclase. The Ras binding specificities were largely shared between Raf-1 and Byr2 although Ras mutants, Y32F, T35S, and A59E, had their affinities for Byr2 selectively reduced. The only exception was Ras(D38N), which lost the ability to bind Raf-1 while retaining the activity to bind Byr2 and complement the Byr2- phenotype of S. pombe. On the other hand, adenylyl cyclase had quite distinct requirements for Ras residues; mutations P34G and T58A selectively abolished the ability to bind and activate it without considerably affecting the interaction with Raf-1 and Byr2. Y32F mutant, whereas losing the ability to activate Raf-1 and Byr2, could activate adenylyl cyclase efficiently. In addition, V45E mutation was found to impair the ability of Ras to activate both Raf-1 and adenylyl cyclase without significantly affecting the binding affinities for them. These results demonstrate that significant differences exist in the recognition mechanisms by which the three effector molecules associate with Ras and suggest that a region of Ras required for activation of the effectors in general may exist separately from that for binding the effectors.
Akasaka
K
K
Department of Physiology II, Kobe University School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650, Japan.
Tamada
M
M
Wang
F
F
Kariya
K
K
Shima
F
F
Kikuchi
A
A
Yamamoto
M
M
Shirouzu
M
M
Yokoyama
S
S
Kataoka
T
T
D000262:Adenylyl Cyclases / Q000378:metabolism*; D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal; D001483:Base Sequence; D001665:Binding Sites; D017931:DNA Primers; D005816:Genetic Complementation Test; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D019908:Proto-Oncogene Proteins c-raf; D016283:Proto-Oncogene Proteins p21(ras) / Q000737:chemistry* / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D012568:Schizosaccharomyces / Q000235:genetics; D013329:Structure-Activity Relationship; D018631:ras Proteins / Q000737:chemistry* / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D017931:DNA Primers; D011518:Proto-Oncogene Proteins; D017346:Protein Serine-Threonine Kinases; D019908:Proto-Oncogene Proteins c-raf; C501009:HRAS protein, human; D016283:Proto-Oncogene Proteins p21(ras); D018631:ras Proteins; D000262:Adenylyl Cyclases