Phosphorylated retinoid X receptor alpha loses its heterodimeric activity with retinoic acid receptor beta

Abstract

A malfunction in retinoid X receptor (RXR) alpha due to phosphorylation is associated with the development of hepatocellular carcinoma. However, the precise mechanisms by which phosphorylated RXRalpha loses its physiological function remain unclear. In the present study we examined whether phosphorylation of RXRalpha affects its dimeric activity. Fluorescence resonance energy transfer studies and immunoprecipitation assays showed that the physical interaction between RXRalpha and retinoic acid receptor beta was impaired when 293T cells were transfected with phosphomimic mutant RXRalpha (T82D/S260D), whereas this interaction was activated at a level similar to wild-type RXRalpha-transfected cells when the cells were transfected with an unphosphorylated mutant RXRalpha (T82A/S260A). Treating the T82A/S260A-transfected cells with retinoid resulted in a significant increase in the transcriptional activities of the retinoic acid receptor responsive element and RXR responsive element promoters, whereas these transcriptional activities did not increase in the T82D/S260D-transfected cells. Transfection with T82A/S260A enhanced both the inhibition of cell growth and the induction of apoptosis caused by retinoid, although the T82D/S260D-transfected cells lost their responsiveness to retinoid. Moreover, transfection with T82A/S260A caused an inhibition of cell growth and a reduction of colony-forming ability in soft agar in HuH7 human hepatocellular carcinoma cells. These findings suggest that phosphorylation of RXRalpha abolishes its ability to form homodimers and heterodimers with RXR and retinoic acid receptor beta, thus resulting in the loss of cell growth control and the acceleration of cancer development. In conclusion, the inhibition of RXRalpha phosphorylation and the restoration of its original function as a master regulator of nuclear receptors might therefore be an effective strategy for controlling cancer cell growth.

Figure 1

Schematic structure of wild‐type retinoid X receptor (RXR) α and the RXRα mutants (T82A/S260A and T82D/S260D). T82A/S260A, in which threonine 82 and serine 260 were mutated to alanine, is an unphosphorylated form of RXRα. T82D/S260D, in which threonine 82 and serine 260 were mutated to aspartate, is a phosphomimic of RXRα.

Figure 2

Heterodimerization of RXR α mutants and retinoic acid receptor (RAR) β in living 293T cells as demonstrated by FRET. The 293T cells were cotransfected with (g–i) YFP‐tagged WT‐RXRα, or the RXRα mutants (m–o) T82A/S260A and (j–l) T82D/S260D using CFP‐tagged RARβ plasmids. (d–f) Cells overexpressing the YFP–CFP fusion protein were used as a positive control. (a–c) As a negative control, 293T cells were cotransfected with pCS2‐Venus plasmid and CFP‐tagged RARβ plasmid. Cells were visualized using the donor filter set (CFP filter) and the acceptor filter set (YFP filter). Corrected fluorescence resonance energy transfer values are shown in pseudocolor.

Figure 3

The physical interaction between phosphorylated retinoid X receptor (RXR) α (p‐RXRα) and retinoic acid receptor (RAR) β proteins as demonstrated by immunoprecipitation assay. The wild‐type or mutant RXRα‐transfected 293T cells were treated with 9‐cis‐retinoic acid for 24 h and the total cellular protein was extracted. The expression levels of the RXRα and p‐RXRα proteins that physically interacted with the RARβ protein were determined using an immunoprecipitation western blot analysis. An antibody to glyceraldehyde‐3‐phosphate dehydrogenase was used as a loading control. The intensities of the blots were quantitated by densitometry and the values are shown in a bar graph in the lower panel. Similar results were obtained in a repeat experiment. Asterisks represent significant differences (P < 0.05). Bars, SD.

Figure 4

Effects of overexpression of the phosphomimic or unphosphorylated mutants of retinoid X receptor (RXR) α on retinoic acid receptor responsive element (RARE) promoter activity in 293T cells. The 293T cells were cotransfected with wild‐type or mutant RXRα‐expressing plasmids (T82A/S260A or T82D/S260D) using the RARE‐Luc and CMV‐Luc plasmids. The transfected cells were then treated with vehicle or 1.0 µM 9‐cis‐retinoic acid for 24 h and the extracts were examined for luciferase activity. Values are the mean ± SD. Asterisks represent significant differences (P < 0.05). Representative results from three independent experiments are shown.

Figure 5

Effects of overexpression of the phosphomimic or unphosphorylated mutants of retinoid X receptor (RXR) α on the induction of apoptosis and the inhibition of 293T cell growth. Wild‐type or mutant RXRα‐transfected cells were treated with vehicle or 1.0 µM 9‐cis‐retinoic acid for 24 h. (a) The cell lysates were then extracted and DNA fragmentation was determined using an ELISA assay. (b) Cell growth in replica plates was examined using the WST‐1 assay system. Values are the mean ± SD. Asterisks represent significant differences (P < 0.05). Representative results from three independent experiments with similar results are shown.

Figure 6

Effects of the overexpression of the unphosphorylated mutant of retinoid X receptor (RXR) α on retinoic acid receptor responsive element (RARE) and RXR responsive element (RXRE) promoter activities in HuH7 cells. The cells were cotransfected with wild‐type or T82A/S260A mutant RXRα‐expressing plasmid using the RARE‐Luc or RXRE‐Luc plasmids. After the transfected cells were treated with vehicle or 1.0 µM 9‐cis‐retinoic acid for 24 h, the extracts were then examined for (a) RARE or (b) RXRE luciferase activities. Values are the mean ± SD. Asterisks represent significant differences (P < 0.05). The results presented are the average of the triplicate findings from three independent experiments.

Figure 7

Effects of overexpression of the unphosphorylated or phosphorylated mutants of retinoid X receptor (RXR) α on cell growth and anchorage‐dependent colony‐forming ability in HuH7 cells. (a) Wild‐type (WT) or mutant RXRα (T82A/S260A or T82D/S260D)‐transfected HuH7 cells were treated with 1.0 µM 9‐cis‐retinoic acid for 6 days and the cell growth in replica plates was examined using the trypan blue dye exclusion method. (b) The WT and T82A/S260A mutant RXRα‐transfected HuH7 cells were plated in soft agar and the number of macroscopic colonies per plate was counted after 3 weeks. Values are the mean ± SD. Asterisks represent significant differences (P < 0.05). The results presented are the average of triplicate findings from three independent experiments.