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.
Department of Biochemistry, College of Medicine, University of Arizona, Tucson 85724.
P W
PW
Jurutka
J C
JC
Hsieh
P N
PN
MacDonald
C M
CM
Terpening
C A
CA
Haussler
M R
MR
Haussler
G K
GK
Whitfield
D000595:Amino Acid Sequence; D000818:Animals; D002117:Calcitriol / Q000378:metabolism*; D047390:Casein Kinase II; D002460:Cell Line; D002522:Chlorocebus aethiops; D006801:Humans; D008969:Molecular Sequence Data; D009036:Moths; D009154:Mutation; D010766:Phosphorylation; D011233:Precipitin Tests; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D018167:Receptors, Calcitriol; D011987:Receptors, Steroid / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012694:Serine / Q000378:metabolism*; D014162:Transfection
D018167:Receptors, Calcitriol; D011987:Receptors, Steroid; D012694:Serine; D047390:Casein Kinase II; D017346:Protein Serine-Threonine Kinases; D002117:Calcitriol