La régulation du gène P450aromatase dans les cellules de granulosa bovine in vitro

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal

A KSR/CNK complex mediated by HYP, a novel SAM domain-containing protein, regulates RAS-dependent RAF activation in Drosophila

RAF is a critical effector of the small GTPase RAS in normal and malignant cells. Despite intense scrutiny, the mechanism regulating RAF activation remains partially understood. Here, we show that the scaffold KSR (kinase suppressor of RAS), a RAF homolog known to assemble RAF/MEK/ERK complexes, induces RAF activation in Drosophila by a mechanism mediated by its kinase-like domain, but which is independent of its scaffolding property or putative kinase activity. Interestingly, we found that KSR is recruited to RAF prior to signal activation by the RAF-binding protein CNK (connector enhancer of KSR) in association with a novel SAM (sterile α motif) domain-containing protein, named Hyphen (HYP). Moreover, our data suggest that the interaction of KSR to CNK/HYP stimulates the RAS-dependent RAF-activating property of KSR. Together, these findings identify a novel protein complex that controls RAF activation and suggest that KSR does not only act as a scaffold for the MAPK (mitogen-activated protein kinase) module, but may also function as a RAF activator. By analogy to catalytically impaired, but conformationally active B-RAF oncogenic mutants, we discuss the possibility that KSR represents a natural allosteric inducer of RAF catalytic function

The PP2C Alphabet Is a Negative Regulator of Stress-Activated Protein Kinase Signaling in Drosophila

The Jun N-terminal kinase and p38 pathways, also known as stress-activated protein kinase (SAPK) pathways, are signaling conduits reiteratively used throughout the development and adult life of metazoans where they play central roles in the control of apoptosis, immune function, and environmental stress responses. We recently identified a Drosophila Ser/Thr phosphatase of the PP2C family, named Alphabet (Alph), which acts as a negative regulator of the Ras/ERK pathway. Here we show that Alph also plays an inhibitory role with respect to Drosophila SAPK signaling during development as well as under stress conditions such as oxidative or genotoxic stresses. Epistasis experiments suggest that Alph acts at a step upstream of the MAPKKs Hep and Lic. Consistent with this interpretation, biochemical experiments identify the upstream MAPKKKs Slpr, Tak1, and Wnd as putative substrates. Together with previous findings, this work identifies Alph as a general attenuator of MAPK signaling in Drosophila

<i>Usp47</i> RNAi impacts RTK-RAS signaling downstream of MEK.

<p>(A) Schematic representation of the USP47 protein product with the position of the USP domain (ubiquitin specific protease catalytic domain) represented along with the amino acid length. (B) Epistasis analysis in <i>Drosophila</i> S2 cells employing constitutively active forms of RAS, RAF, and MEK to induce MAPK activation [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002539#pbio.1002539.ref008" target="_blank">8</a>]. Phosphorylated MAPK was measured by quantitative microscopy and normalized to a <i>GFP</i> dsRNA control [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002539#pbio.1002539.ref007" target="_blank">7</a>]. (C) MAPK activation is induced by three different receptor tyrosine kinases (RTKs) acting upstream: InR through insulin stimulation; EGFR-expressing cells stimulated with the Spitz ligand; or a heat-shock inducible constitutively activated form of Sevenless (Sev^S11). JNK pathway activation induced by RAC1^V12 is used as a negative control. (B-C) A dsRNA targeting the Exon Junction Complex (EJC) component <i>mago</i>, a factor also positioned downstream of MEK and known to reduce MAPK expression [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002539#pbio.1002539.ref007" target="_blank">7</a>], is shown for comparison. The <i>cnk</i> dsRNA is a control for a factor known to act at the level of RAF [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002539#pbio.1002539.ref024" target="_blank">24</a>]. (D) The <i>sal</i>^<i>EPv</i><i>-Gal4</i> drives expression in the wing pouch, which corresponds to a segment of the wing blade extending from the L2 wing vein to the L4-5 intervein with a weaker expression area extending in the periphery of this region [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002539#pbio.1002539.ref025" target="_blank">25</a>,<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002539#pbio.1002539.ref026" target="_blank">26</a>]. A <i>mapk</i> gain of function <i>Sevenmaker</i> (<i>Sem</i>) mutant induced the production of extra wing vein material. Two different <i>Usp47</i> RNAi lines suppressing the extra wing vein material generated by <i>mapk</i>^<i>Sem</i> are shown (these correspond to the following fly lines: <i>Usp47</i> (1), VDRC line GD26027; <i>Usp47</i> (2), NIG line 5486R-3). Raw data for (B and C) can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002539#pbio.1002539.s001" target="_blank">S1 Data</a>.</p

The Deubiquitinase USP47 Stabilizes MAPK by Counteracting the Function of the N-end Rule ligase POE/UBR4 in <i>Drosophila</i>

<div><p>RAS-induced MAPK signaling is a central driver of the cell proliferation apparatus. Disruption of this pathway is widely observed in cancer and other pathologies. Consequently, considerable effort has been devoted to understanding the mechanistic aspects of RAS-MAPK signal transmission and regulation. While much information has been garnered on the steps leading up to the activation and inactivation of core pathway components, comparatively little is known on the mechanisms controlling their expression and turnover. We recently identified several factors that dictate <i>Drosophila</i> MAPK levels. Here, we describe the function of one of these, the deubiquitinase (DUB) USP47. We found that USP47 acts post-translationally to counteract a proteasome-mediated event that reduces MAPK half-life and thereby dampens signaling output. Using an RNAi-based genetic interaction screening strategy, we identified UBC6, POE/UBR4, and UFD4, respectively, as E2 and E3 enzymes that oppose USP47 activity. Further characterization of POE-associated factors uncovered KCMF1 as another key component modulating MAPK levels. Together, these results identify a novel protein degradation module that governs MAPK levels. Given the role of UBR4 as an N-recognin ubiquitin ligase, our findings suggest that RAS-MAPK signaling in <i>Drosophila</i> is controlled by the N-end rule pathway and that USP47 counteracts its activity.</p></div

USP47 acts post-translationally on MAPK.

<p>(A) Pulse-chase experiments of [35S]-methionine labeled MAPK show an impact on MAPK half-life. S2 cells were treated with the indicated dsRNAs for 5 d followed by 6 h of [35S]-methionine labeling. Cell lysates were then prepared at the indicated time following the labeling period. (B) Densitometry quantification of three separate replicates of the experiment presented in A. MAPK levels were normalized to that of three bands from the input gel. Based on these results, MAPK half-life was reduced from 13.68 h (<i>Rluc</i> dsRNA controls) to 10.34 h (<i>Usp47</i> dsRNA). (C) <i>Usp47</i> dsRNA was added to cells stably expressing wild-type exogenous HA-tagged MAPK, causing a reduction in MAPK levels. Similar cell lines were established in which the 10 predicted surface exposed lysines (MAPK^KextR; external lysines were selected based on the structure of ERK2 and correspond to residues 67, 68, 92, 127, 151, 164, 177, 216, 220, 283, and 313 of <i>Drosophila</i> MAPK) or all lysines (MAPK^noK) were switched to arginines. Only mutation of all of the lysines abrogated the sensitivity of MAPK levels to <i>Usp47</i> depletion. (D) Depletion of the <i>Drosophila</i> E1 ligase, <i>Uba1</i> (FBgn0023143; see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002539#pbio.1002539.s016" target="_blank">S5 Table</a> for qPCR validation of RNAi reagent), rescues the impact of <i>Usp47</i> RNAi on MAPK levels. (E) Treating S2 cells with the epoxomicin proteasome inhibitor restored MAPK levels in a <i>Usp47</i> depleted context. (C–E) Densitometry quantifications are provided for MAPK levels (normalized to the AKT loading controls). All immunoblots were performed in triplicate or more, and <i>p</i>-values were calculated (paired two tailed Student’s <i>t</i> test) comparing values to the <i>Rluc</i> control (*: <i>p</i> < 0.05; **: <i>p</i> < 0.01). <i>t</i> tests performed on other samples are shown in red. Raw data for (B–E) can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002539#pbio.1002539.s001" target="_blank">S1 Data</a>.</p

An RNAi screen to identify factors that modify the impact of USP47 on MAPK levels.

<p>(A) The rescue effect of <i>Uba1</i> depletion can be robustly measured in a plate-based quantitative microscopy assay suitable for large-scale screening. <i>Usp47</i> and <i>Uba1</i> co-depletion significantly restored MAPK levels (***: <i>p</i> < 0.001) compared to a <i>Usp47</i> single depletion (**: <i>p</i> < 0.01). <i>Uba1</i> single depletion did not significantly alter MAPK levels compared to <i>GFP</i> dsRNA treated control cells (used for normalization). In this experiment, S2 cells were pre-incubated with either <i>GFP</i> dsRNA (first and third sample) or with <i>Usp47</i> dsRNA (second and fourth sample) for 3 d. The cells were then distributed in 384 well plates containing the indicated dsRNAs and incubated for another 3 d. Following dsRNA treatment, anti-MAPK stained cells were imaged and analyzed by high-content microscopy. (B) Screening strategy for a large-scale RNAi screen focused on ubiquitin-proteasome associated factors. RNAi treatment and MAPK quantification for the ubiquitin-proteasome dsRNA set was performed as in A. Each condition was tested in quadruplicate. (C) Histogram showing the distribution of results from the <i>Usp47</i> RNAi screen. The frequency (number of dsRNAs) is displayed on the <i>y</i>-axis. MAPK levels (<i>x</i>-axis) were normalized to GFP dsRNA treated cells. The <i>Usp47</i> co-depleted cells show a clear shift towards a reduction in MAPK levels. The <i>Uba1</i> dsRNA control stands out from the lot, as it completely counteracts the <i>Usp47</i> dsRNA. (D) Distribution of <i>Usp47</i> genetic interaction scores (Δ<i>m</i>; <i>x</i>-axis) for the candidate dsRNAs (frequency displayed on <i>y</i>-axis) tested in the <i>Usp47</i> RNAi screen. The negative Δ<i>m</i> obtained for Uba1 is consistent with the observed alleviation of the MAPK level reduction. Raw data for (A) can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002539#pbio.1002539.s001" target="_blank">S1 Data</a>. Numerical data for (C and D) can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002539#pbio.1002539.s012" target="_blank">S1 Table</a>.</p

Models for regulation of MAPK stability by USP47.

<p>(A) Direct ubiquitination of MAPK. In this model, UBC6, POE, UFD4, and KCMF1 would participate in the direct ubiquitination of MAPK. Ubiquitinated MAPK could then be targeted to the proteasome for degradation. In the presence of USP47, MAPK would be stabilized as USP47 acts to deubiquitinate MAPK, thus counteracting the activity of the ubiquitin ligases. (B) Indirect regulation of MAPK stability. In this alternative model, UBC6, the E3s, and USP47 act on a yet unidentified factor whose proposed role is to stabilize MAPK. In this scenario, the unidentified factor is the direct target of (de)ubiquitination, whereas MAPK is destabilized through ubiquitin-independent means. The unidentified factor may be degraded by the proteasome following its ubiquitination. MAPK might be degraded through a process that does not implicate the proteasome. Alternatively, even though it is not directly ubiquitinated, MAPK might be degraded by the proteasome through ubiquitin-independent degradation. One possibility in this latter case would be that the unidentified factor acts as a chaperone, bringing MAPK to the proteasome. (A,B) Arrow colors are used to indicate positive (green) or negative (red) regulatory impact on MAPK.</p