ABCMdb

PMID: 18250151

Torra IP, Ismaili N, Feig JE, Xu CF, Cavasotto C, Pancratov R, Rogatsky I, Neubert TA, Fisher EA, Garabedian MJ
Phosphorylation of liver X receptor alpha selectively regulates target gene expression in macrophages.
Mol Cell Biol. 2008 Apr;28(8):2626-36. Epub 2008 Feb 4., [PubMed]

Sentences

No. Mutations Sentence Comment

7 ABCA1 p.Ser198Ala Expression of some (AIM and LPL), but not other (ABCA1 or SREBPc1) established LXR target genes is increased in RAW 264.7 cells expressing the LXRॷ S198A phosphorylation-deficient mutant compared to those with WT receptors. Login to comment 8 ABCA1 p.Ser198Ala ABCA1 p.Ser198Ala Expression of some (AIM and LPL), but not other (ABCA1 or SREBPc1) established LXR target genes is increased in RAW 264.7 cells expressing the LXR␣ S198A phosphorylation-deficient mutant compared to those with WT receptors. Login to comment 9 ABCA1 p.Ser198Ala ABCA1 p.Ser198Ala Surprisingly, a gene normally not expressed in macrophages, the chemokine CCL24, is activated specifically in cells expressing LXR␣ S198A. Login to comment 10 ABCA1 p.Ser198Ala Furthermore, inhibition of S198 phosphorylation by 9cRA or by a CK2 inhibitor similarly promotes CCL24 expression, thereby phenocopying the S198A mutation. Login to comment 45 ABCA1 p.Ser198Ala LZRSpBMN-GFP retroviral vectors carrying the FLAG-tagged wild-type (WT) or S198A mutant LXRॷ cDNA were generated. Login to comment 46 ABCA1 p.Ser198Ala LZRSpBMN-GFP retroviral vectors carrying the FLAG-tagged wild-type (WT) or S198A mutant LXR␣ cDNA were generated. Login to comment 55 ABCA1 p.Ser198Ala Recombinant retroviruses were produced by transfecting LZRSpBMN-GFP, LZRSpBMN-GFP/LXRॷ, or LZRSpBMN-GFP/S198A into 293GP cells as described previously (66). Login to comment 56 ABCA1 p.Ser198Ala Recombinant retroviruses were produced by transfecting LZRSpBMN-GFP, LZRSpBMN-GFP/LXR␣, or LZRSpBMN-GFP/S198A into 293GP cells as described previously (66). Login to comment 57 ABCA1 p.Ser198Ala ABCA1 p.Ser198Ala Cells infected with either the retroviral vector devoid of an LXRॷ sequence (RAW-VO [vector only]), the FLAG-tagged WT LXRॷ (RAW-LXRॷ), or mutant S198A (RAW-S198A) were sorted for green fluorescent protein expression by fluorescence-activated cell sorting. Login to comment 58 ABCA1 p.Ser198Ala ABCA1 p.Ser198Ala Cells infected with either the retroviral vector devoid of an LXR␣ sequence (RAW-VO [vector only]), the FLAG-tagged WT LXR␣ (RAW-LXR␣), or mutant S198A (RAW-S198A) were sorted for green fluorescent protein expression by fluorescence-activated cell sorting. Login to comment 81 ABCA1 p.Ser198Ala For nuclear receptor corepressor (NCoR) coimmunoprecipitation assays, RAW-LXRॷ or RAW-S198A cells treated with vehicle or T0901317 for 2 h were cross-linked for 20 min in phosphate-buffered saline with 1.6 mM DSP [dithiobis(succinimidyl)propionate] (Pierce) prior to whole-cell extract preparation. Login to comment 82 ABCA1 p.Ser198Ala For nuclear receptor corepressor (NCoR) coimmunoprecipitation assays, RAW-LXR␣ or RAW-S198A cells treated with vehicle or T0901317 for 2 h were cross-linked for 20 min in phosphate-buffered saline with 1.6 mM DSP [dithiobis(succinimidyl)propionate] (Pierce) prior to whole-cell extract preparation. Login to comment 136 ABCA1 p.Ser198Ala Specific immunoreactivity of the phospho-S198 antibody was demonstrated by its ability to recognize WT phosphorylated LXRॷ, but not phosphorylation-deficient S198A LXRॷ (see Fig. S2A in the supplemental material). Login to comment 137 ABCA1 p.Ser198Ala Specific immunoreactivity of the phospho-S198 antibody was demonstrated by its ability to recognize WT phosphorylated LXR␣, but not phosphorylation-deficient S198A LXR␣ (see Fig. S2A in the supplemental material). Login to comment 141 ABCA1 p.Ser198Ala Pools of cell clones stably expressing either WT or S198A-mutated LXRॷ were generated by retroviral infection of RAW 264.7 cells, which lack endogenous LXRॷ but express LXRbeta. Login to comment 142 ABCA1 p.Ser198Ala ABCA1 p.Ser198Ala Pools of cell clones stably expressing either WT or S198A-mutated LXR␣ were generated by retroviral infection of RAW 264.7 cells, which lack endogenous LXR␣ but express LXRbeta. Login to comment 143 ABCA1 p.Ser198Ala Nuclear extracts from LXR␣-expressing RAW 264.7 cells loaded with cholesterol showed enhanced WT (and not S198A) LXR␣ phosphorylation compared to untreated cultures (Fig. 1B). Login to comment 155 ABCA1 p.Ser198Ala (B) RAW 264.7 cells infected with a retroviral vector only (VO) or stably expressing WT or S198A FLAG-hLXRॷ were treated with 40 òe;g/ml cholesterol (Chol) in the form of soluble cholesterol-cyclodextrin complexes for 48 h. Login to comment 156 ABCA1 p.Ser198Ala (B) RAW 264.7 cells infected with a retroviral vector only (VO) or stably expressing WT or S198A FLAG-hLXR␣ were treated with 40 ␮g/ml cholesterol (Chol) in the form of soluble cholesterol-cyclodextrin complexes for 48 h. Login to comment 174 ABCA1 p.Ser198Ala Next, to determine the impact of S198 phosphorylation on LXRॷ function, we investigated LXR target gene expression in RAW 264.7 cells stably expressing the phosphorylation mutant S198A. Login to comment 175 ABCA1 p.Ser198Ala ABCA1 p.Ser198Ala Next, to determine the impact of S198 phosphorylation on LXR␣ function, we investigated LXR target gene expression in RAW 264.7 cells stably expressing the phosphorylation mutant S198A. Login to comment 176 ABCA1 p.Ser198Ala RAW-S198A macrophages showed a dramatically enhanced ligand-dependent expression of LPL and AIM compared to the WT LXR␣-transduced cells (Fig. 3A), indicating that S198 phosphorylation exerts an inhibitory effect on the LXR␣-regulated transcription of these genes. Login to comment 178 ABCA1 p.Ser198Ala Consistent with the gene expression data, the activities of WT and S198A receptors were similar when tested in transient-transfection experiments on an ABCA1 promoter fragment containing the LXRE (reference 10 and data not shown). Login to comment 179 ABCA1 p.Ser198Ala ABCA1 p.Ser198Ala Consistent with the gene expression data, the activities of WT and S198A receptors were similar when tested in transient-transfection experiments on an ABCA1 promoter fragment containing the LXRE (reference 10 and data not shown). Login to comment 180 ABCA1 p.Ser198Ala Furthermore, the S198A mutant displayed no major difference in expression (Fig. 3B) or subcellular localization (see Fig. S3C in the supplemental material) compared to the WT and thus cannot account for the observed differential gene expression. Login to comment 186 ABCA1 p.Ser198Ala (A) RAW cells stably expressing FLAG-hLXRॷ (WT or S198A) were incubated with or without 5 òe;M T0901317 ligand for 2 h. Login to comment 187 ABCA1 p.Ser198Ala (A) RAW cells stably expressing FLAG-hLXR␣ (WT or S198A) were incubated with or without 5 ␮M T0901317 ligand for 2 h. Login to comment 194 ABCA1 p.Ser198Ala (A) RAW-LXRॷ (WT or S198A) cells were incubated with vehicle or T0901317 ligand (T) at the indicated concentrations for 24 h. Login to comment 195 ABCA1 p.Ser198Ala (A) RAW-LXR␣ (WT or S198A) cells were incubated with vehicle or T0901317 ligand (T) at the indicated concentrations for 24 h. Login to comment 198 ABCA1 p.Ser198Ala (B) Protein expression levels of LXRॷ in RAW 264.7 cells infected with a retroviral vector only (VO) or FLAG-hLXRॷ (WT or S198A) incubated in the presence or absence of T0901317, as in panel A. Login to comment 199 ABCA1 p.Ser198Ala (B) Protein expression levels of LXR␣ in RAW 264.7 cells infected with a retroviral vector only (VO) or FLAG-hLXR␣ (WT or S198A) incubated in the presence or absence of T0901317, as in panel A. Login to comment 212 ABCA1 p.Ser198Ala Remarkably, in RAW-S198A macrophages, CCL24 basal expression was markedly induced compared to WT LXRॷ-expressing cells (Fig. 5A) and was further dramatically increased by T0901317 in a dose-dependent manner (Fig. 5B). Login to comment 213 ABCA1 p.Ser198Ala Remarkably, in RAW-S198A macrophages, CCL24 basal expression was markedly induced compared to WT LXR␣-expressing cells (Fig. 5A) and was further dramatically increased by T0901317 in a dose-dependent manner (Fig. 5B). Login to comment 220 ABCA1 p.Ser198Ala The induction of CCL24 expression by DMAT was not observed in RAW-S198A cells, indicating that this effect is due to LXRॷ dephosphorylation by the CK2 inhibitor. Login to comment 221 ABCA1 p.Ser198Ala The induction of CCL24 expression by DMAT was not observed in RAW-S198A cells, indicating that this effect is due to LXR␣ dephosphorylation by the CK2 inhibitor. Login to comment 231 ABCA1 p.Ser198Ala RAW-VO (VO) and RAW-LXRॷ (WT or S198A) cells were incubated for 24 h in medium with vehicle or 1 òe;M (A) or the indicated concentrations (B) of T0901317 LXR ligand. Login to comment 232 ABCA1 p.Ser198Ala RAW-VO (VO) and RAW-LXR␣ (WT or S198A) cells were incubated for 24 h in medium with vehicle or 1 ␮M (A) or the indicated concentrations (B) of T0901317 LXR ligand. Login to comment 234 ABCA1 p.Ser198Ala Indeed, in the presence of the RXR ligand 9cRA, CCL24 induction by T0901317 in the RAW LXRॷ cells was markedly enhanced (Fig. 7A), reaching expression levels similar to those observed in untreated RAW-S198A cells (see Fig. S5 in the supplemental material). Login to comment 235 ABCA1 p.Ser198Ala Indeed, in the presence of the RXR ligand 9cRA, CCL24 induction by T0901317 in the RAW LXR␣ cells was markedly enhanced (Fig. 7A), reaching expression levels similar to those observed in untreated RAW-S198A cells (see Fig. S5 in the supplemental material). Login to comment 238 ABCA1 p.Ser198Ala ABCA1 p.Ser198Ala Importantly, the synergistic action of T0901317 and 9cRA on CCL24 expression was lost in RAW-S198A cells (16-fold versus 2.7-fold over T0901317 alone in WT and S198A cells, respectively), suggesting that it is S198 phosphorylation that mediates this effect (see Fig. S5A in the supplemental material). Login to comment 239 ABCA1 p.Ser198Ala ABCA1 p.Ser198Ala Importantly, the synergistic action of T0901317 and 9cRA on CCL24 expression was lost in RAW-S198A cells (16-fold versus 2.7-fold over T0901317 alone in WT and S198A cells, respectively), suggesting that it is S198 phosphorylation that mediates this effect (see Fig. S5A in the supplemental material). Login to comment 293 ABCA1 p.Ser198Ala We thus explored the possibility that activation of CCL24 gene expression by S198A is mediated by NCoR. Login to comment 294 ABCA1 p.Ser198Ala We thus explored the possibility that activation of CCL24 gene expression by S198A is mediated by NCoR. Login to comment 295 ABCA1 p.Ser198Ala However, no detectable binding to the phosphorylation mutant S198A was observed, despite the fact FIG. 8. Identification of an LXR/RXR-responsive region in the CCL24 gene. Login to comment 296 ABCA1 p.Ser198Ala However, no detectable binding to the phosphorylation mutant S198A was observed, despite the fact FIG. 8. Identification of an LXR/RXR-responsive region in the CCL24 gene. Login to comment 306 ABCA1 p.Ser198Ala that similar amounts of LXRॷ protein were immunoprecipitated and NCoR expression levels were similar in WT and S198A cells (not shown). Login to comment 307 ABCA1 p.Ser198Ala that similar amounts of LXR␣ protein were immunoprecipitated and NCoR expression levels were similar in WT and S198A cells (not shown). Login to comment 311 ABCA1 p.Ser198Ala In agreement with this observation, NCoR was barely present at the d1.6 region in RAW-S198A cells compared to RAW-LXRॷ WT cells under similar conditions. Login to comment 312 ABCA1 p.Ser198Ala ABCA1 p.Ser198Ala In agreement with this observation, NCoR was barely present at the d1.6 region in RAW-S198A cells compared to RAW-LXR␣ WT cells under similar conditions. Login to comment 313 ABCA1 p.Ser198Ala These data also indicate that in the absence of ligand, this transcriptional corepressor is present at higher levels on this regulatory sequence in the CCL24 gene in WT-expressing cells than in the S198A cells, which could explain, at least in part, higher CCL24 levels in cells expressing the phosphorylation-deficient mutant. Login to comment 327 ABCA1 p.Ser198Ala The authors also present two forms of LXRॷ bearing double mutations (S195A and S196A, the latter being equivalent to S198A in the human sequence, and T290 and S291) that show resistance to PKA inhibition of SREBP-1c promoter FIG. 9. Login to comment 328 ABCA1 p.Ser198Ala The authors also present two forms of LXR␣ bearing double mutations (S195A and S196A, the latter being equivalent to S198A in the human sequence, and T290 and S291) that show resistance to PKA inhibition of SREBP-1c promoter FIG. 9. Login to comment 329 ABCA1 p.Ser198Ala ABCA1 p.Ser198Ala ChIP assays were performed using chromatin isolated from RAW-LXRॷ (WT) (A) or WT and RAW-S198A (S198A) cells (B) treated as described in the legend to Fig. 7. Login to comment 330 ABCA1 p.Ser198Ala ABCA1 p.Ser198Ala ChIP assays were performed using chromatin isolated from RAW-LXR␣ (WT) (A) or WT and RAW-S198A (S198A) cells (B) treated as described in the legend to Fig. 7. Login to comment 340 ABCA1 p.Ser198Ala Although we did not study phosphorylation of LXRॷ by PKA, our mass spectrometry assays detected S198A as the only phosphorylated residue in LXRॷ. Login to comment 341 ABCA1 p.Ser198Ala Although we did not study phosphorylation of LXR␣ by PKA, our mass spectrometry assays detected S198A as the only phosphorylated residue in LXR␣. Login to comment 342 ABCA1 p.Ser198Ala Concerning the effects of LXRॷ phosphorylation on DNA binding and its heterodimerization with RXR, we did not observe reduced binding of the S198A mutant to different LXREs or impaired interaction between RXR and LXRॷ upon changes in LXRॷ phosphorylation by gel shift assays (not shown). Login to comment 343 ABCA1 p.Ser198Ala Concerning the effects of LXR␣ phosphorylation on DNA binding and its heterodimerization with RXR, we did not observe reduced binding of the S198A mutant to different LXREs or impaired interaction between RXR and LXR␣ upon changes in LXR␣ phosphorylation by gel shift assays (not shown). Login to comment