Identification of nonessential disulfide bonds and altered conformations in the v-sis protein, a homolog of the B chain of platelet-derived growth factor.
The protein encoded by v-sis, the oncogene of simian sarcoma virus, is homologous to the B chain of platelet-derived growth factor (PDGF). There are eight conserved Cys residues between PDGF-B and the v-sis protein. Both native PDGF and the v-sis protein occur as disulfide-bonded dimers, probably containing both intramolecular and intermolecular disulfide bonds. Oligonucleotide-directed mutagenesis was used to change the Cys codons to Ser codons in the v-sis gene. Four single mutants lacked detectable biological activity, indicating that Cys-127, Cys-160, Cys-171, and Cys-208 are required for formation of a biologically active v-sis protein. The other four single mutants retained biological activity as determined in transformation assays, indicating that Cys-154, Cys-163, Cys-164, and Cys-210 are dispensable for biological activity. Double and triple mutants containing three of these altered sites were constructed, some of which were transforming as well. The v-sis proteins encoded by biologically active mutants displayed significantly reduced levels of dimeric protein compared with the wild-type v-sis protein, which dimerized very efficiently. Furthermore, a mutant with a termination codon at residue 209 exhibited partial transforming activity. This study thus suggests that the minimal region required for transformation consists of residues 127 to 208. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis indicated that the v-sis proteins encoded by some of the biologically active mutants exhibited an altered conformation when compared with the wild-type v-sis protein, and suggested that Cys-154 and Cys-163 participate in a nonessential disulfide bond.
Sauer
M K
MK
Department of Biology, University of California, San Diego, La Jolla 92093.
Donoghue
D J
DJ
D000595:Amino Acid Sequence; D000818:Animals; D055598:Chemical Phenomena; D002621:Chemistry; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D005786:Gene Expression Regulation; D005822:Genetic Vectors; D007118:Immunoassay; D008969:Molecular Sequence Data; D009154:Mutation; D009841:Oligonucleotides; D020575:Oncogene Proteins v-sis; D009857:Oncogenes; D010982:Platelet-Derived Growth Factor / Q000235:genetics*; D011487:Protein Conformation; D012190:Retroviridae / Q000235:genetics*; D012191:Retroviridae Proteins / Q000032:analysis / Q000235:genetics*; D012842:Sarcoma Virus, Woolly Monkey / Q000235:genetics*; D012689:Sequence Homology, Nucleic Acid
D009841:Oligonucleotides; D020575:Oncogene Proteins v-sis; D010982:Platelet-Derived Growth Factor; D012191:Retroviridae Proteins