T-ANTIGEN KINASE INHIBITS SIMIAN-VIRUS 40 DNA-REPLICATION BY PHOSPHORYLATION OF INTACT T-ANTIGEN ON SERINE-120 AND SERINE-123

Simian virus 40 (SV40) DNA replication begins after two large T-antige n hexamers assemble on the viral minimal origin of replication and loc ally unwind the template DNA. The activity of T antigen in this reacti on is regulated by its phosphorylation state. A form of casein kinase I purified from HeLa nuclear extracts (T-antigen kinase) phosphorylate s T antigen on physiologic sites and inhibits its activity in the unwi nding reaction (A. Cegielska and D. M. Virshup, Mel. Cell. Biol. 13:12 02-1211, 1993). Using a series of mutant T antigens expressed by recom binant baculoviruses in Sf9 cells, we find that the origin unwinding a ctivities of both T-S677-->A and T-S677,T-679-->A are inhibited by the T-antigen kinase, as is wild-type T antigen. In contrast, mutants T-S 120-->A and T-S123,T-679-->A resistant to inhibition by the kinase, Th us, 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 am ino 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 P-i per mol to full-length bacteria lly produced T antigen and that it inhibits the unwinding activity of p34(cdc2)-activated bacterially produced T antigen. Since no prior pho sphorylation 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 th e recognition signals for T-antigen kinase. In support of this conclus ion, we find that while T-antigen kinase phosphorylates amino-terminal residues in bacterially produced full-length T antigen, it cannot pho sphorylate bacterially produced truncated T antigen containing amino a cids 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 critica l amino-terminal sites by the T-antigen kinase. This conclusion is con sistent with previous studies suggesting close apposition of amino- an d carboxy-terminal domains of T antigen in the native protein. The thr ee-dimensional conformation of the substrate appears to make a signifi cant contribution to T antigen kinase substrate specificity.