Vitamin D receptor phosphorylation in transfected ROS 17/2.8 cells is localized to the N-terminal region of the hormone-binding domain.
The human 1,25-dihydroxyvitamin D3 receptor (hVDR) has been recently shown to be phosphorylated in vitro by casein kinase-II. Most of the residues phosphorylated by this enzyme were shown to reside between Asn160 and Asp232, a region near the N-terminal boundary of the hormone-binding domain. We report here that the hVDR is also phosphorylated in vivo after transfection into ROS 17/2.8 cells. In addition to testing full-length hVDR, we analyzed several internally deleted hVDR mutants. The expression and phosphorylation of full-length and mutated hVDRs were monitored in transfected cells by metabolic labeling with either [35S]methionine or [32P]orthophosphate, followed by immunopurification using monoclonal anti-VDR antibody linked to agarose beads. Transfected hVDR is distinguishable from the endogenous rat VDR when the immunoprecipitated proteins are resolved on sodium dodecyl sulfate-polyacrylamide gels. Significant phosphorylation of transfected full-length hVDR was observed in ROS 17/2.8 cells, and it was less dependent on the presence of 1,25-dihydroxyvitamin D3 than that of the endogenous rat receptor. Most importantly, the region of in vivo phosphorylation, as defined by internal deletion mutants, resides between Met197 and Val234. Therefore, we have localized the major site of phosphorylation of hVDR to residues in the N-terminal region of the hormone-binding domain. The boundaries of this region fall within the amino acid segment defined for phosphorylation of hVDR by casein kinase-II in vitro, suggesting that VDR is an in vivo substrate for casein kinase-II or a related protein kinase.
Jones
B B
BB
Department of Biochemistry, University of Arizona College of Medicine, Tucson 85724.
Jurutka
P W
PW
Haussler
C A
CA
Haussler
M R
MR
Whitfield
G K
GK
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002117:Calcitriol / Q000494:pharmacology; D047388:Casein Kinases; D006801:Humans; D007163:Immunosorbent Techniques; D008715:Methionine / Q000378:metabolism; D008969:Molecular Sequence Data; D016296:Mutagenesis; D012516:Osteosarcoma / Q000378:metabolism*; D010710:Phosphates / Q000378:metabolism; D010766:Phosphorylation; D011487:Protein Conformation; D011494:Protein Kinases / Q000378:metabolism; D051381:Rats; D018167:Receptors, Calcitriol; D011987:Receptors, Steroid / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D014407:Tumor Cells, Cultured
D010710:Phosphates; D018167:Receptors, Calcitriol; D011987:Receptors, Steroid; D008715:Methionine; D011494:Protein Kinases; D047388:Casein Kinases; D002117:Calcitriol