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

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

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

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

Phosphorylation of the Retinoic Acid Receptor Alpha Induces a Mechanical Allosteric Regulation and Changes in Internal Dynamics

<div><p>Nuclear receptor proteins constitute a superfamily of proteins that function as ligand dependent transcription factors. They are implicated in the transcriptional cascades underlying many physiological phenomena, such as embryogenesis, cell growth and differentiation, and apoptosis, making them one of the major signal transduction paradigms in metazoans. Regulation of these receptors occurs through the binding of hormones, and in the case of the retinoic acid receptor (RAR), through the binding of retinoic acid (RA). In addition to this canonical scenario of RAR activity, recent discoveries have shown that RAR regulation also occurs as a result of phosphorylation. In fact, RA induces non-genomic effects, such as the activation of kinase signaling pathways, resulting in the phosphorylation of several targets including RARs themselves. In the case of RARα, phosphorylation of Ser369 located in loop L9–10 of the ligand-binding domain leads to an increase in the affinity for the protein cyclin H, which is part of the Cdk-activating kinase complex of the general transcription factor TFIIH. The cyclin H binding site in RARα is situated more than 40 Å from the phosphorylated serine. Using molecular dynamics simulations of the unphosphorylated and phosphorylated forms of the receptor RARα, we analyzed the structural implications of receptor phosphorylation, which led to the identification of a structural mechanism for the allosteric coupling between the two remote sites of interest. The results show that phosphorylation leads to a reorganization of a local salt bridge network, which induces changes in helix extension and orientation that affects the cyclin H binding site. This results in changes in conformation and flexibility of the latter. The high conservation of the residues implicated in this signal transduction suggests a mechanism that could be applied to other nuclear receptor proteins.</p></div

Distribution of the angle values in the unphosphorylated (in black) and the phosphorylated (in grey) simulations of RARα between H9–H10 (A) and H4–H9 (B).

<p>Distribution of the angle values in the unphosphorylated (in black) and the phosphorylated (in grey) simulations of RARα between H9–H10 (A) and H4–H9 (B).</p

Backbone RMS fluctuations as a function of residue number calculated from the last 40 ns of the molecular dynamics simulations (A) and from the ten lowest frequency modes of the quasi-harmonic analysis (B).

<p>Black lines correspond to the average over the three unphosphorylated RARα, dashed lines to the three phosphorylated RARα. In <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003012#pcbi-1003012-g007" target="_blank">Figure 7.A</a>, dotted lines correspond to the experimental B-factor values. Similar behavior is observed for the fluctuations calculated from simulations and the experimental values. The RMS fluctuations are averaged and displayed by residue. Fluctuations of loop L8–9 are highlighted in purple color.</p

Distribution of the radius of the circle fitted to the α-carbons (Å) of helix H9 in the unphosphorylated (in black) and the phosphorylated (in grey) simulations of RARα.

<p>A decrease in the radius of the circle corresponds to an increase in the bend of the helix.</p