Human vitamin D receptor is selectively phosphorylated by protein kinase C on serine 51, a residue crucial to its trans-activation function

Jui Cheng Hsieh, Peter W. Jurutka, Michael A. Galligan, Christopher M. Terpening, Carol A. Haussler, D. Scott Samuels, Yoshiko Shimizu, Nobuyoshi Shimizu, Mark R. Haussler

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207 Scopus citations

Abstract

The vitamin D receptor (VDR) is known to be a phosphoprotein and inspection of the deduced amino acid sequence of human VDR (hVDR) reveals the conservation of three potential sites of phosphorylation by protein kinase C (PKC) - namely, Ser-51, Ser-119, and Ser-125. Immunoprecipitated extracts derived from a rat osteoblast-like osteosarconia cell line that contains the VDR in high copy number were incubated with the α, β, and γ isozymes of PKC, and VDR proved to be an effective substrate for PKC-β, in vitro. When hVDR cDNAs containing single, double, and triple mutations of Ser-51, Ser-119, and Ser-125 were expressed in CV-1 monkey kidney cells, immunoprecipitated and phosphorylated by PKC-β, in vitro, the mutation of Ser-51 selectively abolished phosphorylation. Furthermore, when transfected CV-1 cells were treated with phorbol 12-myristate 13-acetate, a PKC activator, phosphorylation of wild-type hVDR was enhanced, whereas that of the Ser-51 mutant hVDR was unaffected. Therefore, Ser-51 is the site of hVDR phosphorylation by PKC, both in vitro and in vivo. To evaluate the functional role of Ser-51 and its potential phosphorylation, hVDR-mediated transcription was tested using cotransfection with expression plasmids and a reporter gene that contained a vitamin D response element. Mutation of Ser-51 markedly inhibited transcriptional activation by the vitamin D hormone, suggesting that phosphorylation of Ser-51 by PKC could play a significant role in vitamin D-dependent transcriptional activation. Therefore, the present results link the PKC signal transduction pathway of growth regulation and tumor promotion to the phosphorylation and function of VDR.

Original languageEnglish
Pages (from-to)9315-9319
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Volume88
Issue number20
DOIs
StatePublished - Oct 15 1991

Keywords

  • 1,25-dihydroxyvitamin D
  • Site-directed mutagenesis
  • Steroid hormone receptors
  • Tumor promoter

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