Abstract
Ras proteins possess multiple downstream effectors of distinct structures. We and others demonstrated that Ha-Ras carrying certain effector region mutations could interact differentially with its effectors, implying that significant differences exist in their Ras recognition mechanisms. Here, by employing the fluorescence polarization method, we measured the activity of effector region synthetic peptides bearing various amino acid substitutions to inhibit association of Ras with the effectors human Raf-1 and Schizosaccharomyces pombe Byr2. The effect of these peptides on association with another effector Saccharomyces cerevisiae adenylyl cyclase was also examined by measuring inhibition of the Ras-dependent adenylyl cyclase activity. The peptide corresponding to the residues 17-44 competitively inhibited Ras association with all the three effectors at the Ki values of 1 approximately 10 microM, and the inhibition was considerably attenuated by the D38A mutation. The peptide with the D38N mutation inhibited association of Ha-Ras with Byr2 but not with the others, whereas that with the P34G mutation inhibited association of Ha-Ras with Raf-1 and Byr2 but not with adenylyl cyclase. Thus, the specificity observed with the whole Ras protein was retained in the effector region peptide. These results suggest that the effector region residues constitute a major determinant for differential recognition of the effector molecules, raising a possibility for selective inhibition of a particular Ras function.
Highlights
The ras genes are widely conserved from yeasts to mammals
Inhibition of Ras Effector Association by Synthetic Peptides— We examined the ability of synthetic peptides encompassing the Ha-Ras effector region with various single amino acid substitutions (Table I) to interfere with the Ha-Ras-Raf-1 and Ha-Ras-Byr2 associations
Synthetic peptides encompassing the Ras effector region, in particular the one corresponding to the residues 17– 44, were used successfully to competitively inhibit association of Ras with its effector proteins including Raf-1 [46] and the isoform of protein kinase C [7]
Summary
The peptides were dissolved in buffer A (20 mM Tris/HCl (pH 7.4), 50 mM NaCl, 5 mM MgCl2, 1 mM EDTA) or in dimethylformamide and centrifuged at 100,000 ϫ g for 30 min to remove insoluble materials Their concentrations were determined spectroscopically by measurement of absorbance at 293 nm at pH 11.6. After centrifugation at 100,000 ϫ g for 1 h at 4 °C, the resulting supernatant was used for the measurement of adenylyl cyclase activity in the presence of varying concentrations of the GTP␥Sbound form of posttranslationally fully modified Ha-Ras as described before [19, 40]. For the measurement of inhibition of adenylyl cyclase activities by the synthetic peptides, varying amounts of the peptides were added to the reaction mixtures before commencing the reaction
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
