A SINGLE SERINE RESIDUE AT POSITION-375 OF VP16 IS CRITICAL FOR COMPLEX ASSEMBLY WITH OCT-1 AND HCF AND IS A TARGET OF PHOSPHORYLATION BY CASEIN KINASE-II

We show that VP16 is phosphorylated by cellular kinases in vivo and in vitro and map the major sites of phosphorylation to be on serines tow ards the C-terminus, downstream of position 370 in both cases, Deletio n of the acidic activation domain had no effect on phosphorylation, re fining the sites to between position 370 and 411, Within VP16, the C-t erminal boundary for complex formation with Oct-1 and HCF lies at posi tion 388, and between 370 and 388 lies one serine, at position 375. Th is is a consensus casein kinase II (CKII) site and, using purified wil d-type and mutant proteins, we show that it is the main CKII site in t he body of the N-terminal complex-forming region, This site is also ph osphorylated in nuclear extracts, Although other sites, mainly Ser411, are also phosphorylated by nuclear kinase(s), the single substitution of Ser375 to alanine abolishes CKII phosphorylation in vitro and virt ually eliminates complex formation, This serine lies in a surface-expo sed region of VP16 and, although complex formation is disrupted, other activities of the mutant are unaffected, Ser375 is also required in v ivo where substitution to alanine abolishes transactivation, while rep lacement with threonine restores normal levels of activity.