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

J Biol Chem. 2017 Jan 27;292(4):1490-1509. doi: 10.1074/jbc.M116.758185. Epub 2016 Nov 16.

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 D3 To 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.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Active Transport, Cell Nucleus / drug effects
  • Amino Acid Substitution
  • Calcitriol / pharmacology*
  • Cell Line, Transformed
  • Cell Nucleus / genetics
  • Cell Nucleus / metabolism*
  • Extracellular Signal-Regulated MAP Kinases / genetics
  • Extracellular Signal-Regulated MAP Kinases / metabolism
  • Humans
  • Keratinocytes / metabolism*
  • Mutation, Missense
  • Phosphorylation / drug effects
  • Phosphorylation / genetics
  • Retinoid X Receptor alpha / genetics
  • Retinoid X Receptor alpha / metabolism*
  • Serine
  • ras Proteins / genetics
  • ras Proteins / metabolism

Substances

  • Retinoid X Receptor alpha
  • Serine
  • Extracellular Signal-Regulated MAP Kinases
  • ras Proteins
  • Calcitriol

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