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 towards the C-terminus, downstream of position 370 in both cases. Deletion of the acidic activation domain had no effect on phosphorylation, refining the sites to between position 370 and 411. Within VP16, the C-terminal boundary for complex formation with Oct-1 and HCF lies at position 388, and between 370 and 388 lies one serine, at position 375. This is a consensus casein kinase II (CKII) site and, using purified wild-type and mutant proteins, we show that it is the main CKII site in the body of the N-terminal complex-forming region. This site is also phosphorylated 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 virtually eliminates complex formation. This serine lies in a surface-exposed region of VP16 and, although complex formation is disrupted, other activities of the mutant are unaffected. Ser375 is also required in vivo where substitution to alanine abolishes transactivation, while replacement with threonine restores normal levels of activity.
O'Reilly
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Marie Curie Research Institute, The Chart, Oxted, Surrey, UK.
Hanscombe
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O'Hare
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D000595:Amino Acid Sequence; D000818:Animals; D001224:Aspartic Acid / Q000378:metabolism; D001665:Binding Sites; D019556:COS Cells; D047390:Casein Kinase II; D016384:Consensus Sequence; D004268:DNA-Binding Proteins / Q000378:metabolism*; D018698:Glutamic Acid / Q000378:metabolism; D006367:HeLa Cells; D017405:Herpes Simplex Virus Protein Vmw65 / Q000235:genetics / Q000378:metabolism*; D051863:Host Cell Factor C1; D006801:Humans; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D050811:Octamer Transcription Factor-1; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D012694:Serine / Q000378:metabolism*; D013912:Threonine / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism*; D015533:Transcriptional Activation
D004268:DNA-Binding Proteins; C497692:HCFC1 protein, human; D017405:Herpes Simplex Virus Protein Vmw65; D051863:Host Cell Factor C1; D046911:Macromolecular Substances; D050811:Octamer Transcription Factor-1; C494165:POU2F1 protein, human; D014157:Transcription Factors; D013912:Threonine; D001224:Aspartic Acid; D018698:Glutamic Acid; D012694:Serine; D047390:Casein Kinase II; D017346:Protein Serine-Threonine Kinases