Characterization and evaluation of the functional significance of phosphorylation of the vitamin D receptor.

Abstract

The hormonal actions of 1,25-dihydroxyvitamin D3 (1,25(OH)₂D₃) are mediated by a nuclear 1,25(OH)₂D₃ receptor phosphoprotein (VDR). The phosphorylation of VDR was characterized in rat osteoblast-like cells (ROS 1712.S). In these bone cells, rat VDR (rVDR) was phosphorylated in the absence of the 1,25(OH)₂D₃ ligand and became hyperphosphorylated upon binding the sterol hormone or a novel, noncalcemic analog, 22-oxa-calcitriol. Treatment of ROS 17/2.S cells, transfected with a 1,25(OH)₂D₃-responsive reporter vector, with increasing concentrations of 1 ,25(OH)₂D₃ indicated a positive correlation between the level of receptor phosphorylation and rVDR transcriptional activity. Analysis of the deduced amino acid sequence of the rVDR and the human VDR (h VDR) revealed several consensus recognition sites for casein kinase II (CK-II), a nuclear-localized enzyme. Both of these receptors served as efficient substrates for CK-II, in vitro, in 1,25(OH)₂D₃ independent reactions. Evaluation of CK-IIcatalyzed phosphorylation of truncated and point-mutated human receptor species, in vitro, and of deletion and site-directed h VDR mutants in transfected ROS 17/2.8 and COS-7 cells revealed that a major site of CK-II phosphorylation is Ser208, a classic CK-II site located in the hormone binding domain of the receptor. The hVDR is also hyperphosphorylated at Ser208 in COS-7 cells overexpressing human CK-II. Replacement of Ser208 with either glycine or alanine does not disrupt the ability of h VDR to bind 1,25(OH)₂D₃, localize to the nucleus, associate specifically with the vitamin D responsive element (VDRE) or activate transcription. Significantly, phosphorylation of hVDR at the Ser208 target site by CK-II overexpressed in COS-7 cells stimulates 1,25(OH)₂D₃ mediated transcriptional activation of a reporter gene without affecting other receptor activities. Therefore, CK-II phosphorylation of h VDR appears to playa modulatory rather than obligatory role in receptor function. Conversely, protein kinase C (PK-C) phosphorylation ofhVDR at Ser51, located in the DNA binding domain, while also not required for receptor functions, results in an attenuation of VDRE binding. Finally, cAMP-dependent protein kinase (PK-A) phosphorylates h VDR in the "hinge" region of the receptor producing a decrease in 1,25(OH)₂D₃ stimulated gene activation. Thus, the hVDR appears to be a substrate for several protein kinases within distinct functional domains and the composite activity of the receptor may be dependent on the convergence of integrated inputs from several signal transduction pathways, including those involving CK-II, PK-C and PK-A. These results could explain how the transcriptional activity of VDR is governed during various stages of cell growth and differentiation as well as by "cross-talk" from other hormonal signals in specific vitamin D target organs.