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A cAMP-independent Serine/Threonine Kinase Activity Is Associated with the mos Sequences of ts110 Moloney Murine Sarcoma Virus-encoded P85^gag-mos

Department of Molecular Biology, Scripps Clinic and Research Foundation, 10666 North Torrey Pines Road, La Jolla, California 92037, U.S.A.

Two proteins, termed P85^gag-mos and P58^gag, are encoded by the temperature-sensitive transformation mutant, ts110 Moloney murine sarcoma virus (MuSV). Based on temperature-shift studies, P85^gag-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 P85^gag-mos and P58^gag 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 P85^gag-mos phosphorylated in vitro. The in vitro P85^gag-mos-phosphorylating activity was found to be unresponsive to 10 µM-cAMP or 10 µM-cGMP. Addition of 1 mM-pyrophosphate, a known phosphatase inhibitor, to the reaction mixture resulted in an increased yield of phosphorylated P85^gag-mos and P58^gag; the molar phosphate incorporation per mole of P85^gag-mos increased from 0.032 to 0.9, whereas the specific activity of in vitro-phosphorylated P58^gag 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 P85^gag-mos. Antisera directed against p15, p12 and p30 sequences of the gag protein region of P85^gag-mos yielded immune complexes that allowed phosphorylation in vitro of P85^gag-mos. No phosphorylating activity was detected in immune complexes containing MuSV-124-encoded P62^gag. An anti-mos serum generated against a synthetic peptide representing the predicted v-mos amino acid residues 37 to 55 recognizes P85^gag-mos and allowed phosphorylation of P85^gag-mos in vitro in the absence of P58^gag. Peptide mapping of both phosphorylated P85^gag-mos and P58^gag, by using a combination of Cleveland and Western/immunoperoxidase techniques, demonstrated that P85^gag-mos became phosphorylated not only on gag sequences, but also at the N-terminal portion of v-mos. Phosphoamino acid analyses of P85^gag-mos and P58^gag phosphorylated in vitro under these modified conditions yielded predominantly phosphoserine and lesser amounts of phosphothreonine. Metabolically ³²P-labelled P85^gag-mos and P58^gag 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 P85^gag-mos.

Keywords: MuSV, protein kinase, mos

Received 21 February 1985; accepted 15 July 1985.