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 (≤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. Comparison of the migration in denaturing gel electrophoresis of in vivo and in vitro phosphorylated VDR reveals a different electrophoretic mobility of the CK-II-phosphorylated receptor, suggesting that CK-II-mediated phosphorylation may be distinct from that occurring in response to 1,25(OH)₂D₃ or OCT. We speculate that hormone-dependent phosphorylation of the VDR may be required for transcriptional activation, while hormone-independent, CK-II-mediated phosphorylation may play a role in the modulation of receptor activity.