Human Vitamin D Receptor is Selectively Phosphorylated by Protein Kinase C on Serine 51, a Residue Crucial to Its Trans-Activation Function

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 88; no. 20; pp. 9315 - 9319
• Main Authors: Hsieh, Jui-Cheng, Jurutka, Peter W., Galligan, Michael A., Terpening, Christopher M., Haussler, Carol A., Samuels, D. Scott, Shimizu, Yohiko, Shimizu, Nobuyoshi, Haussler, Mark R.
• Format: Journal Article
• Language: English
• Published: Washington, DC National Academy of Sciences of the United States of America 15-10-1991; National Acad Sciences
• Subjects: Amino Acid Sequence; Animals; Biological and medical sciences; Brain - enzymology; Calcitriol - metabolism; Cell Line; Cell receptors; Cell structures and functions; Cellular immunity; Cloning, Molecular; Epithelial cells; Escherichia coli - genetics; Fundamental and applied biological sciences. Psychology; Genetic Vectors; Hormone receptors. Growth factor receptors. Cytokine receptors. Prostaglandin receptors; Hormones; Humans; Immunoprecipitation; Mice; Molecular and cellular biology; Mutagenesis, Site-Directed; Phosphorylation; Plasmids; protein kinase C; Protein Kinase C - isolation & purification; Protein Kinase C - metabolism; Proteins; Receptors; Receptors, Calcitriol; Receptors, Steroid - genetics; Receptors, Steroid - metabolism; Recombinant Proteins - metabolism; Serine; Transactivation; Transcription, Genetic; Transcriptional Activation; Transfection; Vitamin D; Human; Phosphorylation; Protein kinase C; Enzyme; Steroid hormone; Cholecalciferol(1,25-dihydroxy); Isozyme; Vitamin; Site directed mutagenesis; Structure function relationship; Site specificity; Substrate; Signal transduction; Hormonal receptor
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.88.20.9315

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 osteosarcoma 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.