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.
Department of Biochemistry and Molecular Biophysics, College of Medicine, University of Arizona, Tucson, AZ 85724, USA.
Jui-Cheng
JC
Hsieh
Hope T L
HT
Dang
Michael A
MA
Galligan
G Kerr
GK
Whitfield
Carol A
CA
Haussler
Peter W
PW
Jurutka
Mark R
MR
Haussler
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