PHOSPHORYLATION SITES IN POLYOMAVIRUS LARGE T-ANTIGEN THAT REGULATE ITS FUNCTION IN VIRAL, BUT NOT CELLULAR, DNA-SYNTHESIS

Polgomavirus large T antigen (large T) is a highly phosphorylated prot ein that can be separated by proteolysis into two domains that have in dependent function, A cluster of phosphorylation sites was found in th e protease-sensitive region connecting the N-terminal and C-terminal d omains, Edman degradation of P-32-labeled protein identified serines 2 67, 271, and 274 and threonine 278 as sites of phosphorylation. Analys is of site-directed mutants confirmed directly that residues 271, 274, and 278 were phosphorylated. Threonine 278, shown here to be phosphor ylated by cyclin/cyclin-dependent kinase activity, is required for vir al DNA replication in either the full-length large T or C-terminal dom ain context, The serine phosphorylations are unimportant in the C-term inal domain context even though their mutations activates viral DNA re plication in full-length large T. This finding suggests that these sit es may function in relating the two domains to each other, Although th e phosphorylation sites were involved in viral DNA replication, none w as important for the ability of large T to drive cellular DNA replicat ion as measured by bromodeoxyuridine incorporation, and they did not a ffect large T interactions with the Rb tumor suppressor family.