PHOSPHORYLATION OF THE HUMAN VITAMIN-D RECEPTOR BY PROTEIN-KINASE-C -BIOCHEMICAL AND FUNCTIONAL-EVALUATION OF THE SERINE-51 RECOGNITION SITE

We have reported previously that the human vitamin D receptor (hVDR) i s selectively phosphorylated by protein kinase C-beta (PKC-beta), in v itro, on a serine residue in the sequence RRS51MKRK, which is located between the two zinc fingers of hVDR and is potentially important to i ts transacting function (Hsieh, J.C., Jurutka, P. W., Galligan, M. A., Terpening, C. M., Haussler, C. A., Samuels, D. S., Shimizu, Y., Shimi zu, N., and Haussler, M. R. (1991) Proc. Natl. Acad. Sci. U.S.A. 88,93 15-9319). In the present experiments we evaluated this phosphorylation event using a series of hVDR mutants in which serine 51 or its flanki ng residues were modified. Alteration of serine 51 to a nonphosphoryla table residue resulted in an approximately 60% reduction in basal hVDR phosphorylation in intact cells but did not diminish 1,25-dihydroxyvi tamin D3-stimulated phosphorylation. Such mutations also abolished sub sequent phosphorylation of immunoprecipitated hVDR by purified PKC-bet a, in vitro, as did replacement of basic residues on either side of se rine 51. Mutation of serine 51 to glycine (S51G) or to aspartic acid ( S51D), as well as altering the basic residues flanking serine 51, abol ished the interaction of hVDR with the vitamin D-responsive element (V DRE) as monitored by gel mobility shift analysis. Thus, we conclude th at unmodified serine 51 and its surrounding basic residues are crucial not only for PKC-beta substrate recognition but also for the optimal VDRE binding of native hVDR. In transactivation assays, S51G and S51D possessed only 35 and 10% of wild-type hVDR activity, respectively. Mu tation of serine 51 to threonine (S51T) restored phosphorylation by PK C-beta, in vitro, to about 40% of wild-type and transactivation to 45% of that of wild-type hVDR. Alteration of serine 51 to alanine, which is the residue in the corresponding position of the glucocorticoid, pr ogesterone, mineralocorticoid, and androgen receptors, eliminated PKC- beta phosphorylation but completely preserved the specific DNA binding activity and transactivation capacity of hVDR. Thus, phosphorylation of hVDR at serine 51 is not required for either VDRE binding or transa ctivation. Finally, incubation of Escherichia coli-expressed hVDR with PKC-beta elicits marked phosphorylation of the receptor and significa ntly inhibits its ability to complex with the VDRE. We therefore specu late that posttranslational modification of hVDR at serine 51 may cons titute a negative regulatory loop which could be operative when target cells are subject to PKC activation events.