Phosphorylation sites in polyomavirus large T antigen that regulate its function in viral, but not cellular, DNA synthesis.
Polyomavirus large T antigen (large T) is a highly phosphorylated protein that can be separated by proteolysis into two domains that have independent function. A cluster of phosphorylation sites was found in the protease-sensitive region connecting the N-terminal and C-terminal domains. Edman degradation of 32P-labeled protein identified serines 267, 271, and 274 and threonine 278 as sites of phosphorylation. Analysis of site-directed mutants confirmed directly that residues 271, 274, and 278 were phosphorylated. Threonine 278, shown here to be phosphorylated by cyclin/cyclin-dependent kinase activity, is required for viral DNA replication in either the full-length large T or C-terminal domain context. The serine phosphorylations are unimportant in the C-terminal domain context even though their mutations activates viral DNA replication in full-length large T. This finding suggests that these sites may function in relating the two domains to each other. Although the phosphorylation sites were involved in viral DNA replication, none was important for the ability of large T to drive cellular DNA replication as measured by bromodeoxyuridine incorporation, and they did not affect large T interactions with the Rb tumor suppressor family.
Chatterjee
A
A
Department of Biochemistry, Sackler School of Graduate Biomedical Sciences, Tufts University School of Medicine, Boston, Massachusetts 02111, USA.
Bockus
B J
BJ
Gjørup
O V
OV
Schaffhausen
B S
BS
D016475:3T3 Cells; D000818:Animals; D000952:Antigens, Polyomavirus Transforming / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D001665:Binding Sites; D016203:CDC2 Protein Kinase / Q000378:metabolism; D004247:DNA / Q000096:biosynthesis; D004279:DNA, Viral / Q000096:biosynthesis*; D006801:Humans; D051379:Mice; D016296:Mutagenesis; D010766:Phosphorylation; D011120:Polyomavirus / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012694:Serine / Q000378:metabolism; D018411:Spodoptera / Q000166:cytology; D013912:Threonine / Q000378:metabolism; D014407:Tumor Cells, Cultured; D014779:Virus Replication / Q000502:physiology*
D000952:Antigens, Polyomavirus Transforming; D004279:DNA, Viral; D011993:Recombinant Fusion Proteins; D013912:Threonine; D012694:Serine; D004247:DNA; D016203:CDC2 Protein Kinase