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Structure and phosphorylation of the Fujinami sarcoma virus gene product
Abstract:
The Fujinami avian sarcoma virus (FSV) transforming gene product, P140, is a fusion protein which contains both gag-related and FSV-specific methionine-containing tryptic peptides. The virion protease p15 cleaved p140 into two fragments: an N-terminal 33K fragment which contained all but one of the gag-related tryptic peptides and a C-terminal 120K fragment which contained all of the FSV-specific tryptic peptides. The 33K gag-related fragment from P140 phosphorylated in FSV-transformed cells contained only phosphoserine, whereas the 120K C-terminal FSV-specific fragments contained both phosphoserine and phosphotyrosine. P140 isolated from cells infected at the nonpermissive temperature with an isolate of FSV which is temperature sensitive for transformation had a normally phosphorylated 33K fragment, but a hypophosphorylated 120K fragment deficient in both phosphotyrosine and phosphoserine. When P140 was immunoprecipitated from cells and phosphorylated in vitro at tyrosine residues in the immune complex kinase reaction, only the FSV-specific fragment was labeled. These data define the structure of FSV P140 and locate the phosphorylated amino acids within the two regions of the polypeptide.
Insights
Fujinami avian sarcoma virus (FSV) P140 protein has gag-related and FSV-specific regions. Protease cleavage yields fragments, with phosphorylation sites differing between gag-related (phosphoserine) and FSV-specific (phosphotyrosine, phosphoserine) regions.
Area of Science:
- * Molecular Biology
- * Virology
- * Oncogenesis
Background:
- * Fujinami avian sarcoma virus (FSV) encodes a P140 transforming protein.
- * P140 is a fusion protein with gag-related and FSV-specific domains.
- * Understanding P140's structure and phosphorylation is key to FSV-induced transformation.
Purpose of the Study:
- * To define the structural organization of the FSV P140 protein.
- * To identify the specific phosphorylated amino acid residues within P140 domains.
- * To investigate the role of phosphorylation in P140 function.
Main Methods:
- * Tryptic peptide mapping to analyze P140 composition.
- * Proteolytic cleavage assays to determine fragment generation.
- * In vitro kinase assays to assess phosphorylation sites.
Main Results:
- * FSV P140 is cleaved by protease p15 into 33K (gag-related) and 120K (FSV-specific) fragments.
- * The 33K fragment is phosphorylated on serine; the 120K fragment on serine and tyrosine.
- * Temperature-sensitive FSV mutants show hypophosphorylation of the 120K fragment at nonpermissive temperatures.
- * In vitro phosphorylation specifically labels the FSV-specific 120K fragment at tyrosine residues.
Conclusions:
- * FSV P140's structure comprises distinct gag-related and FSV-specific regions.
- * Phosphorylation occurs on serine in the gag-related region and on serine and tyrosine in the FSV-specific region.
- * Tyrosine phosphorylation of the FSV-specific region is critical for P140's transforming activity.