ABCMdb

ABCA1 p.Ser198Ala

Predicted by SNAP2:	A: N (93%), C: N (87%), D: N (82%), E: N (87%), F: D (53%), G: N (82%), H: N (82%), I: N (82%), K: N (93%), L: N (82%), M: N (87%), N: N (93%), P: N (87%), Q: N (93%), R: N (87%), T: N (97%), V: N (82%), W: D (63%), Y: D (53%),
Predicted by PROVEAN:	A: N, C: N, D: N, E: N, F: N, G: N, H: N, I: N, K: N, L: N, M: N, N: N, P: N, Q: N, R: N, T: N, V: N, W: N, Y: N,

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[hide] A novel role for c-Jun N-terminal kinase and phosp... Cell Signal. 2011 Mar;23(3):542-9. Epub 2010 Nov 8. Huwait EA, Greenow KR, Singh NN, Ramji DP
A novel role for c-Jun N-terminal kinase and phosphoinositide 3-kinase in the liver X receptor-mediated induction of macrophage gene expression.
Cell Signal. 2011 Mar;23(3):542-9. Epub 2010 Nov 8., [PMID:21070853]

Abstract [show]
Liver X receptors (LXRs) are ligand-dependent transcription factors that are activated by metabolites of cholesterol, oxysterols, and a number of synthetic agonists. LXRs play potent anti-atherogenic roles in part by stimulating the efflux of cholesterol from macrophage foam cells. The LXR-induced expression of ATP-binding cassette transporter (ABC)-A1 and Apolipoprotein E (ApoE) in macrophages is essential for the stimulation of cholesterol efflux and the prevention of atherosclerotic development. Unfortunately, the signaling pathways underlying such regulation are poorly understood and were therefore investigated in human macrophages. The expression of ApoE and ABCA1 induced by synthetic or natural LXR ligands [TO901317, GW3965, and 22-(R)-hydroxycholesterol (22-(R)-HC), respectively] was attenuated by inhibitors of c-Jun N-terminal kinase (JNK) (curcumin and SP600125) and phosphoinositide 3-kinase (PI3K) (LY294002). Similar results were obtained with ABCG1 and LXR-alpha, two other LXR target genes. LXR agonists activated several components of the JNK pathway (SEK1, JNK and c-Jun) along with AKT, a downstream target for PI3K. In addition, dominant negative mutants of JNK and PI3K pathways inhibited the LXR-agonists-induced activity of the ABCA1 and LXR-alpha gene promoters in transfected cells. LXR agonists also induced the binding of activator protein-1 (AP-1), a key transcription factor family regulated by JNK, to recognition sequences present in the regulatory regions of the ApoE and ABCA1 genes. These studies reveal a novel role for JNK and PI3K/AKT signaling in the LXR-regulated expression in macrophages of several key genes implicated in atherosclerosis.

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No. Sentence Comment
142 The action of this phosphorylation was gene-specific as the expression of only some LXR targets was increased in cells expressing the S198A phosphorylation-deficient mutant compared to those for the wild-type receptor [27]. Login to comment
141 The action of this phosphorylation was gene-specific as the expression of only some LXR targets was increased in cells expressing the S198A phosphorylation-deficient mutant compared to those for the wild-type receptor [27]. Login to comment

[hide] Phosphorylation of liver X receptor alpha selectiv... Mol Cell Biol. 2008 Apr;28(8):2626-36. Epub 2008 Feb 4. 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., [PMID:18250151]

Abstract [show]
Dysregulation of liver X receptor alpha (LXRalpha) activity has been linked to cardiovascular and metabolic diseases. Here, we show that LXRalpha target gene selectivity is achieved by modulation of LXRalpha phosphorylation. Under basal conditions, LXRalpha is phosphorylated at S198; phosphorylation is enhanced by LXR ligands and reduced both by casein kinase 2 (CK2) inhibitors and by activation of its heterodimeric partner RXR with 9-cis-retinoic acid (9cRA). 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 LXRalpha S198A phosphorylation-deficient mutant compared to those with WT receptors. Surprisingly, a gene normally not expressed in macrophages, the chemokine CCL24, is activated specifically in cells expressing LXRalpha S198A. Furthermore, inhibition of S198 phosphorylation by 9cRA or by a CK2 inhibitor similarly promotes CCL24 expression, thereby phenocopying the S198A mutation. Thus, our findings reveal a previously unrecognized role for phosphorylation in restricting the repertoire of LXRalpha-responsive genes.

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No. Sentence Comment
8 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 Surprisingly, a gene normally not expressed in macrophages, the chemokine CCL24, is activated specifically in cells expressing LXR␣ S198A. Login to comment
10 Furthermore, inhibition of S198 phosphorylation by 9cRA or by a CK2 inhibitor similarly promotes CCL24 expression, thereby phenocopying the S198A mutation. Login to comment
46 LZRSpBMN-GFP retroviral vectors carrying the FLAG-tagged wild-type (WT) or S198A mutant LXR␣ cDNA were generated. Login to comment
56 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
58 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
82 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
137 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
142 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 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
156 (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
175 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 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
179 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 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
187 (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
195 (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
199 (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
213 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
221 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
232 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
235 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
239 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
294 We thus explored the possibility that activation of CCL24 gene expression by S198A is mediated by NCoR. Login to comment
296 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
307 that similar amounts of LXR␣ protein were immunoprecipitated and NCoR expression levels were similar in WT and S198A cells (not shown). Login to comment
312 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 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
328 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
330 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
341 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
343 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
7 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
45 LZRSpBMN-GFP retroviral vectors carrying the FLAG-tagged wild-type (WT) or S198A mutant LXRॷ cDNA were generated. Login to comment
55 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
57 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
81 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
136 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
141 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
155 (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
174 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
178 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
186 (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
194 (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
198 (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
212 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
220 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
231 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
234 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
238 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 We thus explored the possibility that activation of CCL24 gene expression by S198A is mediated by NCoR. Login to comment
295 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 that similar amounts of LXRॷ protein were immunoprecipitated and NCoR expression levels were similar in WT and S198A cells (not shown). Login to comment
311 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
327 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
329 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
340 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
342 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