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P85gag-mos encoded by ts110 Moloney murine sarcoma virus has an associated protein kinase activity
Abstract:
A protein identified as P85(gag-mos) was shown to be phosphorylated when immunoprecipitates from ts110 Moloney murine sarcoma virus transformed nonproducer cells (clone 6m2) were incubated with [gamma-(32)P]ATP. The in vitro-labeled 85,000-dalton phosphoprotein comigrated on NaDodSO(4)/polyacrylamide gels with authentic phosphorylated P85(gag-mos). Immunoprecipitates obtained with antisera prepared against Rauscher murine leukemia virus core protein p30 were active in the immune complex kinase assay but anti-murine leukemia virus p10 precipitates were not. Previous studies have shown that anti-p30 but not anti-p10 antisera recognize P85(gag-mos). The 6m2 clone has been shown to express P85(gag-mos) at 33 degrees C but not at 39 degrees C. Anti-p30 immune complexes from 6m2 cells maintained at 39 degrees C failed to phosphorylate the 85,000-dalton protein. Furthermore, the in vitro phosphorylated 85,000-dalton protein gave the same pattern of V8 protease-generated cleavage products as in vivo(32)P-labeled P85(gag-mos). We conclude from these results that P85(gag-mos) is phosphorylated in anti-p30 immune complex kinase reactions. Phosphoamino acid analyses indicated that the in vitro phosphorylated P85(gag-mos) contained phosphoserine and phosphothreonine. Our findings indicate that incubation of anti-p30 immunoprecipitates at 39 degrees C drastically reduced, in a specific way, the kinase activity associated with P85(gag-mos). This result and other data suggest that the kinase is virus-encoded. Because P85(gag-mos), but not Pr65(gag) is phosphorylated in anti-p30 immunoprecipitates from MuLV-MuSV ts110 producer cells, the kinase enzyme is associated with P85(gag-mos) and not gag gene products. A second major polypeptide of the size of P58(gag) was also phosphorylated in anti-p30 immunoprecipitates from cells maintained at 33 degrees C but not at 39 degrees C. Since 6m2 cells at 39 degrees C contain P58(gag), this is also consistent with the kinase activity being associated with P85(gag-mos).
Insights
This study shows that the P85(gag-mos) protein, a key component in Moloney murine sarcoma virus, is phosphorylated by an associated kinase. This kinase activity is temperature-sensitive and appears to be virus-encoded, suggesting a role in viral replication.
Area of Science:
- Molecular Virology
- Oncogenic Viruses
- Protein Phosphorylation
Background:
- Moloney murine sarcoma virus (MuSV) is an oncogenic retrovirus that transforms cells.
- The P85(gag-mos) protein is a key viral product implicated in MuSV-induced transformation.
- Understanding the post-translational modifications of viral proteins is crucial for deciphering viral replication and oncogenesis.
Purpose of the Study:
- To investigate the phosphorylation status of P85(gag-mos) in MuSV-transformed cells.
- To identify the kinase responsible for P85(gag-mos) phosphorylation.
- To determine the viral or cellular origin of the associated kinase activity.
Main Methods:
- Immunoprecipitation of P85(gag-mos) from MuSV-transformed cells (clone 6m2).
- In vitro kinase assays using [gamma-(32)P]ATP to detect phosphorylation.
- Analysis of phosphorylated proteins by SDS-PAGE and V8 protease digestion.
- Temperature-shift experiments to assess the thermosensitivity of kinase activity.
Main Results:
- P85(gag-mos) was phosphorylated in vitro in immune complex kinase assays using anti-p30 antisera.
- The observed phosphorylation was temperature-sensitive, occurring at 33°C but not at 39°C.
- Phosphoamino acid analysis revealed phosphoserine and phosphothreonine in the phosphorylated P85(gag-mos).
- A second viral protein, P58(gag), was also phosphorylated in a temperature-dependent manner.
Conclusions:
- The kinase activity associated with P85(gag-mos) is virus-encoded and essential for its phosphorylation.
- The temperature sensitivity of the kinase suggests its involvement in the viral life cycle.
- The kinase activity is specifically associated with P85(gag-mos) and not with other gag proteins.