title: Identification of the sites of interaction between c-Raf-1 and Ras-GTP.
abstract: Specific sites of protein-protein interaction were identified in the 51-149 region of c-Raf-1 using contact epitope scanning and site-directed mutagenesis. Nineteen overlapping peptides based upon the primary sequence of the Ras binding domain of c-Raf-1 were tested for the ability to competitively inhibit complex formation between Ras-GTP and the c-Raf-1 N-terminus. A peptide containing c-Raf-1 residues 91-105 as well as five overlapping peptides covering a region extending from residues 118 to 143 interfered with Ras association, defining these sites as potential contact surfaces with Ras. Alanine scanning mutagenesis was used as a second probe for sites of Ras interaction with the c-Raf-1 N-terminus. Raf residues 64-67 and 80-103 were demonstrated as important for association with Ras-GTP with residues 66, 67, 84, 87, 89 and 91 identified as the most critical individual points of contact with the Ras protein. Alanine substitution of residues between 118-143 suggested only one potentially weak site of interaction defined by residues 120-125. The combined results of both peptide and mutagenic analyses suggest that the primary site of c-Raf-1 interaction with Ras maps to Raf residues 80-103, with secondary interactions occurring with residues 66 and 67 and possibly 120-125. Contact epitope scanning of the Ras effector region found maximum inhibition of Ras/Raf association with a peptide corresponding to Ras amino acids 37-51. A model is proposed for the GTP-dependent association of Ras and Raf.
authors.affiliation: Department of Medicine, Indiana University, Indianapolis 46202, USA.
authors.forename: D
authors.initials: D
authors.lastname: Barnard
authors.forename: B
authors.initials: B
authors.lastname: Diaz
authors.forename: L
authors.initials: L
authors.lastname: Hettich
authors.forename: E
authors.initials: E
authors.lastname: Chuang
authors.forename: X F
authors.initials: XF
authors.lastname: Zhang
authors.forename: J
authors.initials: J
authors.lastname: Avruch
authors.forename: M
authors.initials: M
authors.lastname: Marshall
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D018604:Epitope Mapping; D006160:Guanosine Triphosphate / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; 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) / Q000378:metabolism*; D013329:Structure-Activity Relationship
chemical_list: D010455:Peptides; D011518:Proto-Oncogene Proteins; D006160:Guanosine Triphosphate; D017346:Protein Serine-Threonine Kinases; D019908:Proto-Oncogene Proteins c-raf; C501009:HRAS protein, human; D016283:Proto-Oncogene Proteins p21(ras)