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

Research output: Contribution to journalArticle

180 Citations (Scopus)

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

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Calcitriol Receptors
Serine
Protein Kinase C
Phosphorylation
Vitamin D
Mutation
Transcriptional Activation
Vitamin D Response Element
Phosphoproteins
Osteoblasts
Reporter Genes
Isoenzymes
Haplorhini
Amino Acid Sequence
Signal Transduction
Acetates
Plasmids
Complementary DNA
Hormones
Kidney

Keywords

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

ASJC Scopus subject areas

  • General
  • Genetics

Cite this

Human vitamin D receptor is selectively phosphorylated by protein kinase C on serine 51, a residue crucial to its trans-activation function. / Hsieh, Jui-Cheng; Jurutka, Peter W.; Galligan, Michael A.; Terpening, Christopher M.; Haussler, Carol A.; Samuels, D. Scott; Shimizu, Yoshiko; Shimizu, Nobuyoshi; Haussler, Mark R.

In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 88, No. 20, 15.10.1991, p. 9315-9319.

Research output: Contribution to journalArticle

Hsieh, Jui-Cheng ; Jurutka, Peter W. ; Galligan, Michael A. ; Terpening, Christopher M. ; Haussler, Carol A. ; Samuels, D. Scott ; Shimizu, Yoshiko ; Shimizu, Nobuyoshi ; Haussler, Mark R. / Human vitamin D receptor is selectively phosphorylated by protein kinase C on serine 51, a residue crucial to its trans-activation function. In: Proceedings of the National Academy of Sciences of the United States of America. 1991 ; Vol. 88, No. 20. pp. 9315-9319.
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title = "Human vitamin D receptor is selectively phosphorylated by protein kinase C on serine 51, a residue crucial to its trans-activation function",
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.",
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author = "Jui-Cheng Hsieh and Jurutka, {Peter W.} and Galligan, {Michael A.} and Terpening, {Christopher M.} and Haussler, {Carol A.} and Samuels, {D. Scott} and Yoshiko Shimizu and Nobuyoshi Shimizu and Haussler, {Mark R}",
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T1 - Human vitamin D receptor is selectively phosphorylated by protein kinase C on serine 51, a residue crucial to its trans-activation function

AU - Hsieh, Jui-Cheng

AU - Jurutka, Peter W.

AU - Galligan, Michael A.

AU - Terpening, Christopher M.

AU - Haussler, Carol A.

AU - Samuels, D. Scott

AU - Shimizu, Yoshiko

AU - Shimizu, Nobuyoshi

AU - Haussler, Mark R

PY - 1991/10/15

Y1 - 1991/10/15

N2 - 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.

AB - 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.

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KW - Site-directed mutagenesis

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