The 1,25-dihydroxy-vitamin D3 receptor is phosphorylated in response to 1,25-dihydroxy-vitamin D3 and 22-oxacalcitriol in rat osteoblasts, and by casein kinase II, in vitro.
We analyzed the endogenous nuclear 1,25-dihydroxy-vitamin D3 (1,25(OH)2D3) receptor (VDR) in rat osteosarcoma (ROS 17/2.8) cells and present biochemical evidence that it is a phosphoprotein. When ROS 17/2.8 cells are labeled metabolically with [35S]methionine, treatment with 10(-8) M 1,25(OH)2D3 elicits a decrease in the electrophoretic mobility of immunoprecipitated VDR in denaturing polyacrylamide gels, a property characteristic of phosphorylated proteins. Similar labeling of cells with [32P]orthophosphate results in a rapid (< or = 30 min), 1,25(OH)2D3-dependent incorporation of 32P into a 54-kDa VDR species that comigrates with the slower migrating receptor species extracted from [35S]methionine-labeled ROS 17/2.8 cells that have been exposed to 1,25(OH)2D3. Alkaline phosphatase treatment of immunoprecipitated VDR from 1,25(OH)2D3-treated cells converts the form of the VDR with reduced mobility to the faster migrating species present in 1,25(OH)2D3-deficient cells. Incubation of ROS 17/2.8 cells with the non-hypercalcemic 1,25(OH)2D3 analog, 22-oxacalcitriol (OCT), produces a level of VDR phosphorylation similar to that elicited by 1,25(OH)2D3 treatment. Transient transfection of osteosarcoma cells with a reporter vector containing a vitamin D responsive element derived from the rat osteocalcin gene yields equivalent transcriptional activation in the presence of either 1,25(OH)2D3 or OCT. Further experiments performed at various 1,25(OH)2D3 concentrations to assess the relationship between receptor phosphorylation and transcriptional activity in intact cells showed a positive correlation between these two parameters, indicating that the 1,25(OH)2D3 hormone stimulates VDR phosphorylation and transcriptional activation in parallel. Finally, highly purified casein kinase II (CK-II) phosphorylates the VDR in a 1,25(OH)2D3-independent, in vitro reaction.(ABSTRACT TRUNCATED AT 250 WORDS)
Jurutka
P W
PW
Department of Biochemistry, College of Medicine, University of Arizona, Tucson 85724.
Terpening
C M
CM
Haussler
M R
MR
D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D002117:Calcitriol / Q000031:analogs & derivatives* / Q000494:pharmacology*; D047390:Casein Kinase II; D007163:Immunosorbent Techniques; D007700:Kinetics; D008715:Methionine / Q000378:metabolism; D010006:Osteoblasts / Q000187:drug effects / Q000378:metabolism*; D015675:Osteocalcin / Q000235:genetics; D012516:Osteosarcoma; D010710:Phosphates / Q000378:metabolism; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D051381:Rats; D018167:Receptors, Calcitriol; D011987:Receptors, Steroid / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D014158:Transcription, Genetic; D014162:Transfection; D014407:Tumor Cells, Cultured
D010710:Phosphates; D018167:Receptors, Calcitriol; D011987:Receptors, Steroid; D011994:Recombinant Proteins; D015675:Osteocalcin; D008715:Methionine; D047390:Casein Kinase II; D017346:Protein Serine-Threonine Kinases; D000469:Alkaline Phosphatase; D002117:Calcitriol; C051883:maxacalcitol