Phosphorylation of human vitamin D receptor serine-182 by PKA suppresses 1,25(OH)2D3-dependent transactivation

• Published in: Biochemical and biophysical research communications Vol. 324; no. 2; pp. 801 - 809
• Main Authors: Hsieh, Jui-Cheng, Dang, Hope T.L., Galligan, Michael A., Whitfield, G. Kerr, Haussler, Carol A., Jurutka, Peter W., Haussler, Mark R.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 12.11.2004
• Subjects: 1,25-Dihydroxyvitamin D3; Animals; Binding Sites; Calcium - metabolism; Calcium homeostasis; Catalysis; COS Cells; Cyclic AMP-Dependent Protein Kinases - chemistry; Cyclic AMP-Dependent Protein Kinases - metabolism; DNA, Complementary - metabolism; Electrophoresis, Polyacrylamide Gel; Humans; Immunoblotting; Immunoprecipitation; Ligands; Mutagenesis, Site-Directed; Mutation; Nonconsensus site; Phosphorylation; Plasmids - metabolism; Protein kinase A; Receptors, Calcitriol - chemistry; Retinoid X receptor; Retinoid X Receptors - metabolism; Serine - chemistry; Transcriptional Activation; Transfection; Two-Hybrid System Techniques; Protein kinase A; Nonconsensus site; Retinoid X receptor; Transcriptional activation; 1,25-Dihydroxyvitamin D3; Calcium homeostasis
• ISSN: 0006-291X; 1090-2104
• DOI: 10.1016/j.bbrc.2004.09.139

The human vitamin D receptor (hVDR), which is a substrate for several protein kinases, mediates the actions of its 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) 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 [γ-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)2D3 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)2D3 target genes.