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The retinoid X receptor α (RXRα) is a member of the nuclear receptor superfamily that regulates transcription of target genes through heterodimerization with several partners, including peroxisome proliferator-activated receptor, retinoic acid receptor, thyroid receptor, and vitamin D receptor (VDR). We have shown previously that signaling through VDR·RXRα heterodimers was attenuated in ras-transformed keratinocytes due to phosphorylation of serine 260 of the RXRα via the activated Ras-Raf-MAPK cascade in these cells. In this study we demonstrate that phosphorylation at serine 260, a site located in the omega loop-AF-2 interacting domain of RXRα, inhibits signaling through several heterodimeric partners of the RXRα. The inhibition of signaling results in reduced transactivational response to ligand presentation and the reduced physiological response of growth inhibition not only of 1,25-dihydroxyvitamin D3 but also of retinoic acid receptor a ligands and LG1069 (an RXRα ligand). This partial resistance to ligands could be reversed by inhibition of MAPK activity or by overexpression of a non-phosphorylable RXRα mutant at serine 260 (RXRα Ser-260 → Ala). Importantly, phosphorylation of RXRα at serine 260 impaired the recruitment of DRIP205 and other coactivators to the VDR·RXRα complex. Chromatin immunoprecipitation and pulldown assays further demonstrated that coactivator recruitment to the VDR·RXR complex could be restored by treatment with a MAPK inhibitor. Our data suggest that phosphorylation at serine 260 plays a critical role in inducing hormone resistance of RXRα-mediated signaling likely through structural changes in the H1-H3 omega loop-AF2 coactivator(s) interacting domain. [ABSTRACT FROM AUTHOR]

Endothelial nitric-oxide synthase (eNOS) plays a central role in cardiovascular regulation. eNOS function is critically modulated by Ca2+ and protein phosphorylation, but the interrelationship between intracellular Ca2~ mobilization and eNOS phosphorylation is poorly understood. Here we show that endoplasmic reticulum (ER) Ca2+ release activates eNOS by selectively promoting its Ser-635/633 (bovine/human) phosphorylation. With bovine endothelial cells, thapsigargin-induced ER Ca2+ release caused a dose-dependent increase in eNOS Ser-635 phosphorylation, leading to elevated NO production. ER Ca2~ release also promoted eNOS Ser-633 phosphorylation in mouse vessels in vivo. This effect was independent of extracellular Ca~+ and selective to Ser-635 because the phosphorylation status of other eNOS sites, including Ser-1179 or Thr-497, was unaffected in thapsigargin-treated cells. Blocking ERK1/2 abolished ER Ca2~ release-induced eNOS Ser-635 phosphorylation, whereas inhibiting protein kinase A or Ca2~/calmodulin-dependent protein kinase II had no effect. Protein phosphorylation assay confirmed that ERK1/2 directly phosphorylated the eNOS Ser-635 residue in vitro. Further studies demonstrated that ER Ca2~ release-induced ERK1/2 activation mediated the enhancing action of purine or bradykinin receptor stimulation on eNOS Ser-635/633 phosphorylation in bovine/human endothelial cells. Mutating the Ser-635 to nonphosphorylatable alanine prevented ATP from activating eNOS in cells. Taken together, these studies reveal that ER Ca2+ release enhances eNOS Ser-635 phosphorylation and function via ERK1/2 activation. Because ER Ca2+ is commonly mobilized by agonists or physicochemical stimuli, the identified ER Ca2+-ERK1/2-eNO5 Ser-635 phosphorylation pathway may have a broad role in the regulation of endothelial function. [ABSTRACT FROM AUTHOR]