Phosphorylation of serine 208 in the human vitamin D receptor: The predominant amino acid phosphorylated by casein kinase II, in vitro, and identification as a significant phosphorylation site in intact cells

The human 1,25-dihydroxyvitamin D₃ receptor (hVDR), like other members of the steroid/thyroid receptor superfamily, has been observed to undergo rapid phosphorylation. We report here that the hVDR is a substrate for casein kinase II (CK-II), a regulatory enzyme of significance in the function of nuclear proteins. Intact hVDRs produced by in vitro transcription/ translation or in a baculovirus overexpression system served as efficient substrates for purified bovine CK-II, and the magnitude of this phosphorylation was not affected by the addition of 1,25-dihydroxyvitamin D₃. CK-II-catalyzed phosphorylation of truncated hVDRs suggested that phosphorylated residues may occur between Arg¹²¹ and Asp²³², including the region of hVDR which we have previously demonstrated to contain a major site(s) of phosphorylation in intact cells (Jones, B. B., Jurutka, P. W., Haussler, C. A., Haussler, M. R., and Whitfield, G. K. (1991) Mol. Endocrinol. 5, 1137-1146). Site-directed mutagenesis of serine/threonine residues in this region now reveals a site of phosphorylation at Ser²⁰⁸ contained within the sequence -S²⁰⁸(P)EEDSDD-, a classic CK-II consensus recognition site. Mutation of this serine to a glycine drastically reduces phosphorylation of hVDR by CK-II, in vitro. The Ser²⁰⁸ mutant receptor also shows a dramatic decrease in [³²P]orthophosphate incorporation when transfected into COS-7 cells. We therefore propose that phosphorylation of hVDR at Ser²⁰⁸ in target cells is mediated by casein kinase II or a similar enzyme, and that this quantitatively significant post-translational modification is a potential mechanism for the modulation of the activity of hVDR in controlling gene transcription.