A cAMP-independent serine/threonine kinase activity is associated with the mos sequences of ts110 Moloney murine sarcoma virus-encoded P85gag-mos.
Two proteins, termed P85gag-mos and P58gag, are encoded by the temperature-sensitive transformation mutant, ts110 Moloney murine sarcoma virus (MuSV). Based on temperature-shift studies, P85gag-mos is believed to be important for the transforming potential of ts110 MuSV and has been found to be associated with a thermolabile kinase activity that phosphorylates both P85gag-mos and P58gag in immune complexes. Modifications of the original kinase assay conditions are reported here that have allowed a 30-fold increase in the specific activity of P85gag-mos phosphorylated in vitro. The in vitro P85gag-mos-phosphorylating activity was found to be unresponsive to 10 microM-cAMP or 10 microM-cGMP. Addition of 1 mM-pyrophosphate, a known phosphatase inhibitor, to the reaction mixture resulted in an increased yield of phosphorylated P85gag-mos and P58gag; the molar phosphate incorporation per mole of P85gag-mos increased from 0.032 to 0.9, whereas the specific activity of in vitro-phosphorylated P58gag increased 18-fold, from 0.013 to 0.234. pH curves of the in vitro kinase reaction further confirmed the presence of phosphatase activity; in the absence of pyrophosphate, a sharp optimum at pH 4 to 5 was observed, whereas it shifted broadly to pH 7.0 in the presence of pyrophosphate. Under the latter conditions, several experiments were performed in order to determine if the kinase was associated with either gag or mos sequences of P85gag-mos. Antisera directed against p15, p12 and p30 sequences of the gag protein region of P85gag-mos yielded immune complexes that allowed phosphorylation in vitro of P85gag-mos. No phosphorylating activity was detected in immune complexes containing MuSV-124-encoded P62gag. An anti-mos serum generated against a synthetic peptide representing the predicted v-mos amino acid residues 37 to 55 recognizes P85gag-mos and allowed phosphorylation of P85gag-mos in vitro in the absence of P58gag. Peptide mapping of both phosphorylated P85gag-mos and P58gag, by using a combination of Cleveland and Western/immunoperoxidase techniques, demonstrated that P85gag-mos became phosphorylated not only on gag sequences, but also at the N-terminal portion of v-mos. Phosphoamino acid analyses of P85gag-mos and P58gag phosphorylated in vitro under these modified conditions yielded predominantly phosphoserine and lesser amounts of phosphothreonine. Metabolically 32P-labelled P85gag-mos and P58gag were also found to contain phosphoserine and phosphothreonine. Based on these results, we conclude that a cAMP-independent, serine/threonine protein kinase activity is associated with the mos sequences of P85gag-mos.
D000936:Antigen-Antibody Complex; D002472:Cell Transformation, Viral; D000242:Cyclic AMP / Q000494:pharmacology; D006152:Cyclic GMP / Q000494:pharmacology; D011756:Diphosphates / Q000494:pharmacology; D015683:Gene Products, gag; D008980:Moloney murine sarcoma virus / Q000201:enzymology* / Q000235:genetics; D009154:Mutation; D010766:Phosphorylation; D010768:Phosphoserine / Q000032:analysis; D010769:Phosphothreonine / Q000032:analysis; D011494:Protein Kinases / Q000378:metabolism*; D011794:Quercetin / Q000494:pharmacology; D012191:Retroviridae Proteins / Q000032:analysis / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D009053:Sarcoma Viruses, Murine / Q000201:enzymology*; D013696:Temperature
D000936:Antigen-Antibody Complex; D011756:Diphosphates; D015683:Gene Products, gag; D012191:Retroviridae Proteins; D010769:Phosphothreonine; D010768:Phosphoserine; D011794:Quercetin; D000242:Cyclic AMP; D011494:Protein Kinases; D006152:Cyclic GMP