title: Phosphorylation of Human Retinoid X Receptor α at Serine 260 Impairs Its Subcellular Localization, Receptor Interaction, Nuclear Mobility, and 1α,25-Dihydroxyvitamin D3-dependent DNA Binding in Ras-transformed Keratinocytes.
abstract: Human retinoid X receptor α (hRXRα) plays a critical role in DNA binding and transcriptional activity through heterodimeric association with several members of the nuclear receptor superfamily, including the human vitamin D receptor (hVDR). We previously showed that hRXRα phosphorylation at serine 260 through the Ras-Raf-MAPK ERK1/2 activation is responsible for resistance to the growth inhibitory effects of 1α,25-dihydroxyvitamin D3(1α,25(OH)2D3), the biologically active metabolite of vitamin D3To further investigate the mechanism of this resistance, we studied intranuclear dynamics of hVDR and hRXRα-tagged constructs in living cells together with endogenous and tagged protein in fixed cells. We find that hVDR-, hRXRα-, and hVDR-hRXRα complex accumulate in the nucleus in 1α,25(OH)2D3-treated HPK1A cells but to a lesser extent in HPK1ARas-treated cells. Also, by using fluorescence resonance energy transfer (FRET), we demonstrate increased interaction of the hVDR-hRXRα complex in 1α,25(OH)2D3-treated HPK1A but not HPK1ARas cells. In HPK1ARas cells, 1α,25(OH)2D3-induced nuclear localization and interaction of hRXRα are restored when cells are treated with the MEK1/2 inhibitor UO126 or following transfection of the non-phosphorylatable hRXRα Ala-260 mutant. Finally, we demonstrate using fluorescence loss in photobleaching and quantitative co-localization with chromatin that RXR immobilization and co-localization with chromatin are significantly increased in 1α,25(OH)2D3-treated HPK1ARas cells transfected with the non-phosphorylatable hRXRα Ala-260 mutant. This suggests that hRXRα phosphorylation significantly disrupts its nuclear localization, interaction with VDR, intra-nuclear trafficking, and binding to chromatin of the hVDR-hRXR complex.
authors.affiliation: From the Department of Medicine, Calcium Research Laboratory, Royal Victoria Hospital, McGill University, Montreal, Quebec H4A 3J1.|the Department of Medicine, Experimental Therapeutics and Metabolism Program, McGill University Health Center, Montreal, Quebec H4A 3J1, Canada.
authors.forename: Sylvester
authors.initials: S
authors.lastname: Jusu
authors.affiliation: the Department of Anatomy and Cell Biology, McGill University, Montreal, Quebec H3A 0C7, and.
authors.forename: John F
authors.initials: JF
authors.lastname: Presley
authors.affiliation: From the Department of Medicine, Calcium Research Laboratory, Royal Victoria Hospital, McGill University, Montreal, Quebec H4A 3J1, richard.kremer@mcgill.ca.|the Department of Medicine, Experimental Therapeutics and Metabolism Program, McGill University Health Center, Montreal, Quebec H4A 3J1, Canada.
authors.forename: Richard
authors.initials: R
authors.lastname: Kremer
mesh_terms: D021581:Active Transport, Cell Nucleus / Q000187:drug effects; D019943:Amino Acid Substitution; D002117:Calcitriol / Q000494:pharmacology*; D002461:Cell Line, Transformed; D002467:Cell Nucleus / Q000235:genetics / Q000378:metabolism*; D048049:Extracellular Signal-Regulated MAP Kinases / Q000235:genetics / Q000378:metabolism; D006801:Humans; D015603:Keratinocytes / Q000378:metabolism*; D020125:Mutation, Missense; D010766:Phosphorylation / Q000187:drug effects / Q000235:genetics; D047490:Retinoid X Receptor alpha / Q000235:genetics / Q000378:metabolism*; D012694:Serine; D018631:ras Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D047490:Retinoid X Receptor alpha; D012694:Serine; D048049:Extracellular Signal-Regulated MAP Kinases; D018631:ras Proteins; D002117:Calcitriol
keywords: fluorescence loss in photobleaching (FLIP); fluorescence resonance energy transfer (FRET); mitogen-activated protein kinase (MAPK); phosphorylation; retinoid; retinoid X receptor; vitamin D; vitamin D receptor