Single amino acid substitution, from Glu1025 to Asp, of the fps oncogenic protein causes temperature sensitivity in transformation and kinase activity.
We have sequenced 2.7 kilobases of v-fps DNA encoding the transforming protein, p140, of the temperature-sensitive (ts) FL-15 clone of avian Fujinami sarcoma virus. Ten single nucleotide differences were found when compared with the v-fps sequence of the temperature-resistant (tr) clone, FSV-2. Of these differences, five encoded altered amino acids within the 5' fps domain, only one encoded an altered amino acid in the 3' kinase domain, and four were silent. Among the five amino acid changes in the 5' fps domain, four were identical to the corresponding residues of c-fps, and the remaining one, a change from His to Arg at amino acid number 559, was located in the middle of a stretch of five consecutive histidine residues. These sequence comparisons suggested that only two amino acid changes, His to Arg at amino acid 559 and Glu to Asp at amino acid 1025, were likely to be responsible for the temperature sensitivity of the v-fps protein. Two recombinants, pFL-11 containing the 5' alterations and pFL-12 containing the single 3' mutation, were constructed in vitro to determine the precise ts lesion. It was found that both the recombinant pFL-12 and the parental pFL-5 were ts by three criteria: cell morphology, colony formation, and kinase activity. In contrast, the recombinant pFL-11 was ts in morphology, but not in colony formation, and was partially ts in kinase activity. pFSV 2-2 itself was temperature resistant by these criteria. We conclude that, first, the mutation of Glu to Asp at amino acid number 1025 can cause a complete ts phenotype, implying that this residue is located at a critical position of the v-fps oncogenic protein. Secondly, the change from His to Arg at amino acid position 559 results in a partial temperature sensitivity, providing the genetic evidence for a second functional domain of the v-fps oncogenic protein.
D000595:Amino Acid Sequence; D001483:Base Sequence; D002472:Cell Transformation, Viral; D009856:Oncogene Proteins, Viral / Q000235:genetics* / Q000378:metabolism; D009857:Oncogenes; D012150:Polymorphism, Restriction Fragment Length; D011487:Protein Conformation; D011505:Protein-Tyrosine Kinases / Q000235:genetics*; D005253:Sarcoma Viruses, Feline; D013329:Structure-Activity Relationship; D013696:Temperature
D009856:Oncogene Proteins, Viral; D011505:Protein-Tyrosine Kinases