Phosphorylation of human vitamin D receptor serine-182 by PKA suppresses 1,25(OH)2D3-dependent transactivation.
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 [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)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.
Hsieh
Jui-Cheng
JC
Department of Biochemistry and Molecular Biophysics, College of Medicine, University of Arizona, Tucson, AZ 85724, USA.
Dang
Hope T L
HT
Galligan
Michael A
MA
Whitfield
G Kerr
GK
Haussler
Carol A
CA
Jurutka
Peter W
PW
Haussler
Mark R
MR
D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D002118:Calcium / Q000378:metabolism; D002384:Catalysis; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry* / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D015151:Immunoblotting; D047468:Immunoprecipitation; D008024:Ligands; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D018167:Receptors, Calcitriol / Q000737:chemistry*; D047488:Retinoid X Receptors / Q000378:metabolism; D012694:Serine / Q000737:chemistry*; D015533:Transcriptional Activation; D014162:Transfection; D020798:Two-Hybrid System Techniques
D018076:DNA, Complementary; D008024:Ligands; D018167:Receptors, Calcitriol; D047488:Retinoid X Receptors; D012694:Serine; D017868:Cyclic AMP-Dependent Protein Kinases; D002118:Calcium