The replication functions of polyomavirus large tumor antigen are regulated by phosphorylation.
Polyomavirus (Py) large T antigen (T Ag) contains two clusters of phosphorylation sites within the amino-terminal half of the protein. To characterize possible regulatory effects of phosphorylation on viral DNA replication, Py T Ag was treated with calf intestinal alkaline phosphatase (CIAP). Incubation of the protein with a range of phosphatase concentrations caused progressive loss of phosphate without affecting its stability. Treatment with smaller quantities of CIAP stimulated the ability of the viral protein to mediate replication of constructs containing the viral replication origin, while higher concentrations of CIAP caused a marked diminution of this replication function. Several biochemical activities of Py T Ag were examined after CIAP treatment. Py T Ag DNA unwinding and nonspecific DNA binding were only slightly affected by dephosphorylation. However, as determined by DNase I footprinting experiments, treatment with smaller amounts of CIAP stimulated specific binding to the Py replication origin by Py T Ag, while treatment with larger amounts of CIAP caused marked inhibition of origin-specific binding by the viral protein. Phosphotryptic maps of Py T Ag before or after treatment with CIAP revealed changes in individual phosphopeptides that were uniquely associated with either the stimulation or the inhibition of replication. Our data therefore suggest that Py T Ag is regulated by both repressing and activating phosphates.
Wang
E H
EH
Department of Biological Sciences, Columbia University, New York, New York 10027.
Bhattacharyya
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S
Prives
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D000469:Alkaline Phosphatase; D000952:Antigens, Polyomavirus Transforming / Q000378:metabolism / Q000502:physiology*; D004265:DNA Helicases / Q000378:metabolism; D004279:DNA, Viral / Q000096:biosynthesis; D004268:DNA-Binding Proteins / Q000378:metabolism; D066298:In Vitro Techniques; D010449:Peptide Mapping; D010750:Phosphoproteins / Q000502:physiology; D010766:Phosphorylation; D013329:Structure-Activity Relationship; D014779:Virus Replication
D000952:Antigens, Polyomavirus Transforming; D004279:DNA, Viral; D004268:DNA-Binding Proteins; D010750:Phosphoproteins; D000469:Alkaline Phosphatase; D004265:DNA Helicases