Detection of the myristylated gag-raf transforming protein with raf-specific antipeptide sera.
The post-translational modifications of the gag-raf fusion proteins of the 3611 murine sarcoma virus (MSV) have been examined by inhibiting glycosylation with tunicamycin and by in vivo labeling with [3H]myristic acid. The results show that P75gag-raf is myristylated but not glycosylated and that P90gag-raf is glycosylated but not myristylated (and is now termed gP90gag-raf). gP90gag-raf expression appeared to become lost during passage of the transformed cells, and consequently does not appear to be necessary for the maintenance of transformation. raf-specific sera for detecting gag-raf fusion proteins have been obtained from synthetic peptides made from different regions of the predicted v-raf sequence. Immunoprecipitation of P75gag-raf with raf-specific sera directly confirmed the deduced v-raf sequence. The fact that P75gag-raf is both myristylated and precipitated by antiserum to a predicted carboxyl-terminal peptide of the v-raf gene established that the mature protein represents the entire coding region. The gP90gag-raf thus appears to be a glycosylated form of P75gag-raf specified by the gag sequences of the fusion protein, in analogy with Pr65gag and gPr80gag of murine leukemia viruses. Antiserum to the carboxyl-terminal P75gag-raf peptide was the most efficient in immunoprecipitation, and will be useful for detecting the product of the c-raf gene.
D000818:Animals; D000952:Antigens, Polyomavirus Transforming; D000957:Antigens, Viral, Tumor / Q000032:analysis* / Q000276:immunology / Q000378:metabolism; D002460:Cell Line; D002471:Cell Transformation, Neoplastic; D002472:Cell Transformation, Viral; D005814:Genes, Viral; D007106:Immune Sera; D051379:Mice; D008970:Molecular Weight; D019814:Myristic Acid; D009227:Myristic Acids / Q000378:metabolism; D009857:Oncogenes; D011499:Protein Processing, Post-Translational; D009053:Sarcoma Viruses, Murine / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D014759:Viral Envelope Proteins / Q000032:analysis* / Q000378:metabolism; D014760:Viral Fusion Proteins; D014764:Viral Proteins / Q000032:analysis* / Q000276:immunology / Q000378:metabolism
D000952:Antigens, Polyomavirus Transforming; D000957:Antigens, Viral, Tumor; D007106:Immune Sera; D009227:Myristic Acids; D014759:Viral Envelope Proteins; D014760:Viral Fusion Proteins; D014764:Viral Proteins; D019814:Myristic Acid