TRIM24 mediates the interaction of the retinoic acid receptor alpha with the proteasome

The nuclear retinoic acid (RA) receptors (RARalpha, beta and gamma) are ligand-dependent regulators of transcription. Upon activation by RA, they are recruited at the promoters of target genes together with several coregulators. Then, they are degraded by the ubiquitin proteasome system. Here, we report that the degradation of the RARalpha subtype involves ubiquitination and the tripartite motif protein TRIM24, which was originally identified as a ligand-dependent corepressor of RARalpha. We show that in response to RA, TRIM24 serves as an adapter linking RARalpha to the proteasome for its degradation. In addition, TRIM24 and the proteasome are recruited with RARalpha to the promoters of target genes and thus are inherently linked to RARalpha transcriptional activity

TRIM

The nuclear retinoic acid (RA) receptors (RARalpha, beta and gamma) are ligand-dependent regulators of transcription. Upon activation by RA, they are recruited at the promoters of target genes together with several coregulators. Then, they are degraded by the ubiquitin proteasome system. Here, we report that the degradation of the RARalpha subtype involves ubiquitination and the tripartite motif protein TRIM24, which was originally identified as a ligand-dependent corepressor of RARalpha. We show that in response to RA, TRIM24 serves as an adapter linking RARalpha to the proteasome for its degradation. In addition, TRIM24 and the proteasome are recruited with RARalpha to the promoters of target genes and thus are inherently linked to RARalpha transcriptional activity

Phosphoproteome and Transcriptome of RA-Responsive and RA-Resistant Breast Cancer Cell Lines.

Retinoic acid (RA), the main active vitamin A metabolite, controls multiple biological processes such as cell proliferation and differentiation through genomic programs and kinase cascades activation. Due to these properties, RA has proven anti-cancer capacity. Several breast cancer cells respond to the antiproliferative effects of RA, while others are RA-resistant. However, the overall signaling and transcriptional pathways that are altered in such cells have not been elucidated. Here, in a large-scale analysis of the phosphoproteins and in a genome-wide analysis of the RA-regulated genes, we compared two human breast cancer cell lines, a RA-responsive one, the MCF7 cell line, and a RA-resistant one, the BT474 cell line, which depicts several alterations of the "kinome". Using high-resolution nano-LC-LTQ-Orbitrap mass spectrometry associated to phosphopeptide enrichment, we found that several proteins involved in signaling and in transcription, are differentially phosphorylated before and after RA addition. The paradigm of these proteins is the RA receptor α (RARα), which was phosphorylated in MCF7 cells but not in BT474 cells after RA addition. The panel of the RA-regulated genes was also different. Overall our results indicate that RA resistance might correlate with the deregulation of the phosphoproteome with consequences on gene expression

ChIP-qPCR analysis of RARα recruitment at the <i>Cy26a1</i> gene promoter.

<p>(A) Kinetic ChIP experiments performed with RA-treated MCF7 and BT474 cells and determining the recruitment of RARα to the R1 and R2 response elements of the <i>Cyp26a1</i> gene. Values correspond to a representative experiment among 3. (B) ChIP experiments performed with MEFs expressing RARαWT or RARαS77A and determining the recruitment of RARα to the R1 and R2 elements of the <i>Cyp26a1</i> gene. Values are the mean ±SD of three experiments.</p

Comparison of the RA-regulated genes in MCF7 and BT474 cells.

<p>(A) Venn diagram showing that 80% of the genes that are RA-regulated in MCF7 cells are not in BT474 cells. (B) and (C) 3D pie charts showing the categories of genes that are RA-regulated in MCF7 only and in BT474 cells only. The genes were selected using the Manteia GO statistical analysis on GO analysis with a P value <0,01. (D) Heatmaps showing the genes that are RA-regulated in both cell lines.</p

Workflow for the phosphoproteomics strategy.

<p>(A) Phosphoproteome. Nuclear and cytoplasmic extracts were prepared and divided in two: one half was digested with trypsin/Lys-C and the other half with chymotrypsin. A small fraction of the trypsin/Lys-C digests was analyzed directly without further purification. The remaining digests were subjected to phosphopeptide enrichment and MS analysis. (B) RARα phosphorylation. Whole cell extracts were prepared from MCF7 and BT474 cells with and without a 30 min RA treatment. RARα was immunoprecipitated and the eluates were thermolysin-digested. Phosphopeptides were enriched and analyzed by nano-LC-LTQ-Orbitrap MS.</p

Broussonetia kaempferi Sieb.

原著和名: ツルカウゾ科名: クワ科 = Moraceae採集地: 長崎県 多良岳麓 (肥前 多良岳麓)採集日: 1970/5/14採集者: 萩庭丈壽整理番号: JH045354国立科学博物館整理番号: TNS-VS-99535

Overview of phosphorylation in MCF7 and BT474 cells.

<p>(A) Relative frequency of mono- (1P), bi-(2P) and tri- (3P) phosphorylated peptides in the cytosolic and nuclear extracts of MCF7 and BT474 cells with and without RA treatment. T: total number of phosphopeptides. C: cytosolic extracts, N: nuclear extracts. (B) Relative phosphorylation of Serine (S), Thr (T) and Tyr (Y) residues (Ag: ambiguous). The values are the average ±SD of two experiments.</p

List of phosphoproteins, grouped per biological functions that were detected in the nuclear extracts of MCF7 and/or BT474 cells, in the two replicate experiments.

<p>For each protein, the phosphopeptides were analyzed manually. The site classes are assigned as acidic (A), basic (B) or proline-directed (P). Phosphoproteins detected in both cell lines (Black), in MCF7 cells only (Green) or in BT474 cells only (Blue). Indicated is whether phosphorylation occurs in the absence of RA only (-), in the presence of RA only (+) or both in the absence and presence of RA (±).</p

Single-cell analyses unravel cell type–specific responses to a vitamin D analog in prostatic precancerous lesions

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