T-antigen kinase inhibits simian virus 40 DNA replication by phosphorylation of intact T antigen on serines 120 and 123.
Simian virus 40 (SV40) DNA replication begins after two large T-antigen hexamers assemble on the viral minimal origin of replication and locally unwind the template DNA. The activity of T antigen in this reaction is regulated by its phosphorylation state. A form of casein kinase I purified from HeLa nuclear extracts (T-antigen kinase) phosphorylates T antigen on physiologic sites and inhibits its activity in the unwinding reaction (A. Cegielska and D. M. Virshup, Mol. Cell. Biol. 13:1202-1211, 1993). Using a series of mutant T antigens expressed by recombinant baculoviruses in Sf9 cells, we find that the origin unwinding activities of both TS677-->A and TS677,679-->A are inhibited by the T-antigen kinase, as is wild-type T antigen. In contrast, mutants TS120-->A and TS123,679-->A are resistant to inhibition by the kinase. Thus, phosphorylation of serines 120 and 123 is necessary for inhibition of T-antigen activity. Previous studies of casein kinase I substrate specificity have suggested that acidic residues or a phosphorylated amino acid amino terminal to the target residue are required to create a casein kinase I recognition site. However, we find that the T-antigen kinase can add more than 3 mol of Pi per mol to full-length bacterially produced T antigen and that it inhibits the unwinding activity of p34cdc2-activated bacterially produced T antigen. Since no prior phosphorylation is present in this bacterially produced T antigen, and no acidic residues are present immediately amino terminal to serines 120 and 123, other structural elements of T antigen must contribute to the recognition signals for T-antigen kinase. In support of this conclusion, we find that while T-antigen kinase phosphorylates amino-terminal residues in bacterially produced full-length T antigen, it cannot phosphorylate bacterially produced truncated T antigen containing amino acids 1 to 259, a 17-kDa amino-terminal tryptic fragment of T antigen, nor can it phosphorylate denatured T antigen. These findings strongly suggest that the carboxy-terminal domain of T antigen is an important modifier of the recognition signals for phosphorylation of the critical amino-terminal sites by the T-antigen kinase. This conclusion is consistent with previous studies suggesting close apposition of amino- and carboxy-terminal domains of T antigen in the native protein. The three-dimensional conformation of the substrate appears to make a significant contribution to T-antigen kinase substrate specificity.
Cegielska
A
A
Program in Human Molecular Biology and Genetics, University of Utah, Salt Lake City 84112.
Moarefi
I
I
Fanning
E
E
Virshup
D M
DM
D000595:Amino Acid Sequence; D000818:Animals; D000952:Antigens, Polyomavirus Transforming / Q000235:genetics / Q000378:metabolism*; D016367:Baculoviridae / Q000235:genetics; D016203:CDC2 Protein Kinase / Q000378:metabolism; D047388:Casein Kinases; D002478:Cells, Cultured; D004265:DNA Helicases / Q000378:metabolism; D004261:DNA Replication; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D009036:Moths / Q000166:cytology; D010766:Phosphorylation; D011487:Protein Conformation; D011494:Protein Kinases / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000378:metabolism; D013539:Simian virus 40 / Q000254:growth & development*; D013379:Substrate Specificity; D014779:Virus Replication
D000952:Antigens, Polyomavirus Transforming; D011994:Recombinant Proteins; D011494:Protein Kinases; D047388:Casein Kinases; D016203:CDC2 Protein Kinase; D004265:DNA Helicases