Phosphorylation of human vitamin D receptor serine-182 by PKA suppresses 1,25(OH) 2D 3-dependent transactivation

Jui-Cheng Hsieh, Hope T L Dang, Michael A. Galligan, G Kerr Whitfield, Carol A. Haussler, Peter W. Jurutka, Mark R Haussler

Research output: Contribution to journalArticle

14 Citations (Scopus)

Abstract

The human vitamin D receptor (hVDR), which is a substrate for several protein kinases, mediates the actions of its 1,25-dihydroxyvitamin D 3 (1,25(OH) 2D 3) ligand to regulate gene expression. To determine the site, and functional impact, of cAMP-dependent protein kinase (PKA)-catalyzed phosphorylation of hVDR, we generated a series of C-terminally truncated and point mutant receptors. Incubation of mutant hVDRs with PKA and [gamma;- 32P]ATP, in vitro, or overexpressing them in COS-7 kidney cells labeled with [ 32P]orthophosphate, revealed that serine-182 is the predominant residue in hVDR phosphorylated by PKA. An aspartate substituted mutant (S182D), incorporating a negative charge to mimic phosphorylation, displayed only 50% of the transactivation capacity in response to 1,25(OH) 2D 3 of either wild-type or an S182A-altered hVDR. When the catalytic subunit of PKA was overexpressed, a similar reduction in wild-type but not S182D hVDR transactivity was observed. In a mammalian two-hybrid system, S182D bound less avidly than wild-type or S182A hVDR to the retinoid X receptor (RXR) heterodimeric partner that co-mediates vitamin D responsive element recognition and transactivation. These data suggest that hVDR serine-182 is a primary site for PKA phosphorylation, an event that leads to an attenuation of both RXR heterodimerization and resultant transactivation of 1,25(OH) 2D 3 target genes.

Original languageEnglish (US)
Pages (from-to)801-809
Number of pages9
JournalBiochemical and Biophysical Research Communications
Volume324
Issue number2
DOIs
StatePublished - Nov 12 2004

Fingerprint

Calcitriol Receptors
Phosphorylation
Protein-Serine-Threonine Kinases
Protein Kinases
Serine
Transcriptional Activation
Retinoid X Receptors
COS Cells
Cyclic AMP-Dependent Protein Kinases
Hybrid systems
Gene expression
Aspartic Acid
Vitamin D
Catalytic Domain
Genes
Adenosine Triphosphate
Phosphates
Ligands
Kidney
Gene Expression

Keywords

  • 1,25-Dihydroxyvitamin D
  • Calcium homeostasis
  • Nonconsensus site
  • Protein kinase A
  • Retinoid X receptor
  • Transcriptional activation

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology

Cite this

Phosphorylation of human vitamin D receptor serine-182 by PKA suppresses 1,25(OH) 2D 3-dependent transactivation. / Hsieh, Jui-Cheng; Dang, Hope T L; Galligan, Michael A.; Whitfield, G Kerr; Haussler, Carol A.; Jurutka, Peter W.; Haussler, Mark R.

In: Biochemical and Biophysical Research Communications, Vol. 324, No. 2, 12.11.2004, p. 801-809.

Research output: Contribution to journalArticle

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abstract = "The human vitamin D receptor (hVDR), which is a substrate for several protein kinases, mediates the actions of its 1,25-dihydroxyvitamin D 3 (1,25(OH) 2D 3) ligand to regulate gene expression. To determine the site, and functional impact, of cAMP-dependent protein kinase (PKA)-catalyzed phosphorylation of hVDR, we generated a series of C-terminally truncated and point mutant receptors. Incubation of mutant hVDRs with PKA and [gamma;- 32P]ATP, in vitro, or overexpressing them in COS-7 kidney cells labeled with [ 32P]orthophosphate, revealed that serine-182 is the predominant residue in hVDR phosphorylated by PKA. An aspartate substituted mutant (S182D), incorporating a negative charge to mimic phosphorylation, displayed only 50{\%} of the transactivation capacity in response to 1,25(OH) 2D 3 of either wild-type or an S182A-altered hVDR. When the catalytic subunit of PKA was overexpressed, a similar reduction in wild-type but not S182D hVDR transactivity was observed. In a mammalian two-hybrid system, S182D bound less avidly than wild-type or S182A hVDR to the retinoid X receptor (RXR) heterodimeric partner that co-mediates vitamin D responsive element recognition and transactivation. These data suggest that hVDR serine-182 is a primary site for PKA phosphorylation, an event that leads to an attenuation of both RXR heterodimerization and resultant transactivation of 1,25(OH) 2D 3 target genes.",
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AB - The human vitamin D receptor (hVDR), which is a substrate for several protein kinases, mediates the actions of its 1,25-dihydroxyvitamin D 3 (1,25(OH) 2D 3) ligand to regulate gene expression. To determine the site, and functional impact, of cAMP-dependent protein kinase (PKA)-catalyzed phosphorylation of hVDR, we generated a series of C-terminally truncated and point mutant receptors. Incubation of mutant hVDRs with PKA and [gamma;- 32P]ATP, in vitro, or overexpressing them in COS-7 kidney cells labeled with [ 32P]orthophosphate, revealed that serine-182 is the predominant residue in hVDR phosphorylated by PKA. An aspartate substituted mutant (S182D), incorporating a negative charge to mimic phosphorylation, displayed only 50% of the transactivation capacity in response to 1,25(OH) 2D 3 of either wild-type or an S182A-altered hVDR. When the catalytic subunit of PKA was overexpressed, a similar reduction in wild-type but not S182D hVDR transactivity was observed. In a mammalian two-hybrid system, S182D bound less avidly than wild-type or S182A hVDR to the retinoid X receptor (RXR) heterodimeric partner that co-mediates vitamin D responsive element recognition and transactivation. These data suggest that hVDR serine-182 is a primary site for PKA phosphorylation, an event that leads to an attenuation of both RXR heterodimerization and resultant transactivation of 1,25(OH) 2D 3 target genes.

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