1,25-DIHYDROXYVITAMIN-D(3) MODULATES PHOSPHORYLATION OF SERINE-205 INTHE HUMAN VITAMIN-D RECEPTOR - SITE-DIRECTED MUTAGENESIS OF THIS RESIDUE PROMOTES ALTERNATIVE PHOSPHORYLATION

The vitamin D receptor (VDR) from a variety of animal species is a hor mone-modulated substrate for phosphorylation in vivo. In this report, we utilize an expression vector to produce recombinant human VDR(hVDR) in 1,25-dihydroxyvitamin D3-treated COS-1 cells. Immunoprecipitation o f the phosphorylated hVDR followed by gel purification and phosphoamin o acid analysis revealed modification exclusively on one or more serin e residues, consistent with previous studies of the VDR in other speci es. To identify the region of phosphorylation, immunoprecipitated and gel-purified hVDR from COS-1 cells was first mixed with purified hVDR isolated to homogeneity from Saccharomyces cerevisiae and then digeste d with trypsin or V8 protease, and the peptides were resolved on HPLC. The single phosphate-containing peptides were recovered and subjected to amino acid sequence analysis, revealing the modification to reside in a region extending from residue 171 to residue 206 common to both the tryptic- and the V8 protease-derived peptides. Sequential cleavage of similar VDR mixtures using trypsin and then CNBr, alpha-chymotryps in, or thermolysin demonstrated an amino-terminal boundary of the phos phorylated peptide at 202. Selective manual Edman degradation of phosp horylated peptides beginning at 171, 195, and 200 revealed phosphate r elease only at serine 205. This peptide contained an average of 8-fold less radioactive phosphate in the absence of prior treatment of the c ulture cells wit h 1,25(OH)2D3. Site-directed modification of VDR seri ne 205 to alanine, aspartate, or glutamate each led to fully functiona l proteins when assessed in a transactivation assay using several VDRE -linked natural promoters. Unexpectedly, evaluation of the serine 205 to alanine hVDR mutant revealed that this protein continued to be phos phorylated in a hormone-dependent manner on an alternative site. These studies show directly that hVDR serine residue 205, a consensus site for casein kinase II, is modified in vivo in response to hormone.