Cyclin-dependent kinase regulation of the replication functions of polyomavirus large T antigen.
The amino-terminal portion of polyomavirus (Py) large T antigen (T Ag) contains two phosphorylation sites, at T187 and T278, which are potential substrates for cyclin-dependent kinases (CDKs). Our experiments were designed to test whether either or both of these sites are involved in the origin DNA (ori DNA) replication function of Py T Ag. Mutations were generated in Py T Ag whereby either or both threonines were replaced with alanine, generating T187A, T278A, and double-mutants (DM [T187A T278A]) mutant T Ags. We found that the Py ori DNA replication functions of T278A and DM, but not T187A, mutant T Ags were abolished both in vivo and in vitro. Consistent with this finding, it was shown that the ori DNA binding and unwinding activities of mutant T278A Py T Ag were greatly impaired. Moreover, whereas wild-type Py T Ag is an efficient substrate for phosphorylation by cyclin A-CDK2 and cyclin B-cdc2 complexes, it is phosphorylated poorly by a cyclin E-CDK2 complex. In contrast to mutant T187A, which behaved similarly to the wild-type protein, T278A was only weakly phosphorylated by cyclin B-cdc2. These data thus suggest that T278 is an important site on Py T Ag for phosphorylation by CDKs and that loss of this site leads to its various defects in mediating ori DNA replication. S- and G2-phase-specific CDKs, but not a G1-specific CDK, can phosphorylate wild-type T Ag, which suggests yet another reason why DNA tumor viruses require actively cycling host cells.
Li
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H
Department of Biological Sciences, Columbia University, New York, New York 10027, USA.
Bhattacharyya
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S
Prives
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C
D000952:Antigens, Polyomavirus Transforming / Q000235:genetics / Q000502:physiology*; D016203:CDC2 Protein Kinase / Q000378:metabolism; D042846:CDC2-CDC28 Kinases; D017489:CDC28 Protein Kinase, S cerevisiae / Q000378:metabolism; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases / Q000378:metabolism*; D004279:DNA, Viral / Q000096:biosynthesis*; D016195:G2 Phase; D008938:Mitosis; D010766:Phosphorylation; D011120:Polyomavirus / Q000235:genetics / Q000502:physiology*; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D018741:Replication Origin; D016196:S Phase; D013912:Threonine; D014779:Virus Replication / Q000502:physiology*
D000952:Antigens, Polyomavirus Transforming; D004279:DNA, Viral; D013912:Threonine; D017346:Protein Serine-Threonine Kinases; D016203:CDC2 Protein Kinase; D042846:CDC2-CDC28 Kinases; D017489:CDC28 Protein Kinase, S cerevisiae; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases