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Volume 66, Issue 10

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

Abstract

SUMMARY

Two proteins, termed P85 and P58, are encoded by the temperature-sensitive transformation mutant, ts110 Moloney murine sarcoma virus (MuSV). Based on temperature-shift studies, P85 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 and P58 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 phosphorylated . The P85-phosphorylating activity was found to be unresponsive to 10 µ-cAMP or 10 µ-cGMP. Addition of 1 m-pyrophosphate, a known phosphatase inhibitor, to the reaction mixture resulted in an increased yield of phosphorylated P85 and P58; the molar phosphate incorporation per mole of P85 increased from 0.032 to 0.9, whereas the specific activity of -phosphorylated P58 increased 18-fold, from 0.013 to 0.234. pH curves of the 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 or sequences of P85. Antisera directed against p15, p12 and p30 sequences of the protein region of P85 yielded immune complexes that allowed phosphorylation of P85. No phosphorylating activity was detected in immune complexes containing MuSV-124-encoded P62. An anti- serum generated against a synthetic peptide representing the predicted v- amino acid residues 37 to 55 recognizes P85 and allowed phosphorylation of P85 in the absence of P58. Peptide mapping of both phosphorylated P85 and P58, by using a combination of Cleveland and Western/immunoperoxidase techniques, demonstrated that P85 became phosphorylated not only on sequences, but also at the N-terminal portion of v-. Phosphoamino acid analyses of P85 and P58 phosphorylated under these modified conditions yielded predominantly phosphoserine and lesser amounts of phosphothreonine. Metabolically P-labelled P85 and P58 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 sequences of P85.

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Keyword(s): mos , MuSV and protein kinase
© Journal of General virology 1985
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1985-10-01
2024-04-18
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A cAMP-independent Serine/Threonine Kinase Activity Is Associated with the mos Sequences of ts110 Moloney Murine Sarcoma Virus-encoded P85gag-mos
J. Gen. Virol. 66, 2135 (1985); https://doi.org/10.1099/0022-1317-66-10-2135
/content/journal/jgv/10.1099/0022-1317-66-10-2135
/content/journal/jgv/10.1099/0022-1317-66-10-2135
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