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

<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

<i>Ubc6</i>, <i>poe</i>, and <i>Ufd4</i> dsRNAs alleviate <i>Usp47</i>’s impact on MAPK levels.

<p>(A) Selected hits from the <i>Usp47</i> co-depletion RNAi screen (qPCR validation in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002539#pbio.1002539.s016" target="_blank">S5 Table</a>). After validation with independent dsRNA reagents, our set of candidates included one E2 conjugating enzyme, <i>Ubc6</i>, and two E3 ligases, <i>poe</i> and <i>Ufd4</i>. The set also included proteasome components such as <i>Rpn2</i> (FBgn0028692) and ubiquitin genes such as <i>Ubi-p5E</i> (FBgn0086558). <i>Ufd1-like</i>, a proteasome-associated factor also linked to endoplasmic-reticulum-associated protein degradation (ERAD) was also present in our set (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002539#pbio.1002539.s013" target="_blank">S2 Table</a> for full validation screen data). Data shown here is from the RNAi validation experiment. All MAPK levels are normalized to <i>GFP</i> dsRNA controls. The numerical data presented in this panel can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002539#pbio.1002539.s013" target="_blank">S2 Table</a>. (B) Schematic representations of the UBC6, UFD4, and POE protein products drawn to scale with the position of identifiable domains and regions (UBCc: ubiquitin-conjugating enzyme E2 catalytic domain; ANK: ankyrin repeat motif; HECT: E3 ligase domain [HECT stands for “homologous to the E6-AP carboxyl terminus”]; UBR: ubiquitin protein ligase E3 component n-recognin domain [also known as UBR box motif]; CRD: cysteine rich domain). Amino acid lengths are also shown. (C) Western blot experiments confirm that <i>Ubc6</i>, <i>poe</i> and <i>Ufd4</i> depletion rescue endogenous MAPK levels in <i>Usp47</i> depleted cells. The rescue mediated by <i>Ufd4</i> depletion is weaker, possibly due to comparatively moderate depletion efficiency (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002539#pbio.1002539.s016" target="_blank">S5 Table</a>). (D) Co-depletion experiments conducted in third instar <i>Drosophila</i> wing discs show that <i>poe</i> and <i>Ubc6</i> can rescue <i>Usp47</i> depletion in vivo. The <i>engrailed-gal4</i> driver was used to drive expression of RNAi in the posterior segment (GFP-positive) of the disc. <i>Ubc6</i> RNAi expression caused extensive larval lethality. Those wing discs that could be recovered were of reduced size (cell lethality was also problematic in confirming <i>Ubc6</i> depletion by qPCR (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002539#pbio.1002539.s016" target="_blank">S5 Table</a>). The RNAi lines used in this experiment correspond to the following VDRC lines: <i>Usp47</i>, GD26027; <i>poe</i> (1), KK108296; <i>poe</i> (2), GD17648; <i>Ubc6</i>, GD23229. The depletion of the <i>Usp47</i>, <i>poe</i>, and <i>Ubc6</i> transcripts were also measured by qPCR (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002539#pbio.1002539.s016" target="_blank">S5 Table</a>). (E) The extra wing vein material induced by <i>mapk</i>^<i>Sem</i> expression under the control of <i>sal</i>^<i>EPv</i>-<i>Gal4</i> is suppressed by <i>Usp47</i> RNAi (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002539#pbio.1002539.g001" target="_blank">Fig 1D</a>). Co-expression of <i>poe</i> dsRNA counteracts <i>Usp47</i> RNAi and restores the extra wing vein phenotype. The RNAi lines used here are the same as in C. (F) Pre-translational downregulation of mapk expression is not rescued by <i>poe</i> RNAi. Knocking down the EJC component <i>eIF4AIII</i> reduces MAPK levels due to altered splicing of the <i>mapk</i> transcript. This, unlike the depletion produced by <i>Usp47</i> dsRNA, is not rescued by co-depleting POE. (G) Exogenous MAPK levels measured by immunoblot are rescued by co-depletion of <i>Usp47</i> with <i>poe</i> or <i>Ubc6</i>. An <i>HA-mapk</i> stable cell line was treated with the indicated dsRNA for 4 d. A rescue effect was observed on both endogenous and exogenous MAPK. (C, F, and G) Densitometry quantifications are provided for MAPK levels (normalized to the AKT loading controls). All experiments 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. “ns” denotes not significant. Numerical data for (A) can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002539#pbio.1002539.s013" target="_blank">S2 Table</a>. Raw data for (C, F, and G) 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

Predicted protein complexes have similar secondary screen functional profiles.

<p>(A) Unsupervised hierarchical clustering of secondary screen results. The 13 secondary assays are listed at the top of the clustering diagram. <i>Bona fide</i> MAPK pathway components are identified with an asterisk. (B) Protein interaction network (PIN) assembled using interaction data from <i>Drosophila</i> and homologs from other species. Edge color represents the source of the interaction data and edge width denotes the number of distinct experimental evidences for a given interaction. The coloring of the node border represents RAS^V12 screen results while the coloring of the node center reflects MAPK protein levels. Node shapes reflect the functional category of the hits and specificity results are represented by the size of nodes. In both panels, epistasis results are represented by the coloring of gene symbols in orange (RAS-RAF), magenta (RAF-MEK), dark blue (MEK-MAPK), or grey (ambiguous). MAPK pathway components, STRIPAK (shaded area 1) and splicing factors (shaded area 2) group together in both the clustering analysis and PIN. Note that the full name is used for the gene raspberry, instead of <i>ras</i>, to avoid confusion with <i>Ras85D</i>.</p

A Functional Screen Reveals an Extensive Layer of Transcriptional and Splicing Control Underlying RAS/MAPK Signaling in Drosophila

<div><p>The small GTPase RAS is among the most prevalent oncogenes. The evolutionarily conserved RAF-MEK-MAPK module that lies downstream of RAS is one of the main conduits through which RAS transmits proliferative signals in normal and cancer cells. Genetic and biochemical studies conducted over the last two decades uncovered a small set of factors regulating RAS/MAPK signaling. Interestingly, most of these were found to control RAF activation, thus suggesting a central regulatory role for this event. Whether additional factors are required at this level or further downstream remains an open question. To obtain a comprehensive view of the elements functionally linked to the RAS/MAPK cascade, we used a quantitative assay in Drosophila S2 cells to conduct a genome-wide RNAi screen for factors impacting RAS-mediated MAPK activation. The screen led to the identification of 101 validated hits, including most of the previously known factors associated to this pathway. Epistasis experiments were then carried out on individual candidates to determine their position relative to core pathway components. While this revealed several new factors acting at different steps along the pathway—including a new protein complex modulating RAF activation—we found that most hits unexpectedly work downstream of MEK and specifically influence MAPK expression. These hits mainly consist of constitutive splicing factors and thereby suggest that splicing plays a specific role in establishing MAPK levels. We further characterized two representative members of this group and surprisingly found that they act by regulating mapk alternative splicing. This study provides an unprecedented assessment of the factors modulating RAS/MAPK signaling in Drosophila. In addition, it suggests that pathway output does not solely rely on classical signaling events, such as those controlling RAF activation, but also on the regulation of MAPK levels. Finally, it indicates that core splicing components can also specifically impact alternative splicing.</p></div

Epistasis analysis.

<p>(A) MAPK pathway models depicting the secondary screen assays used to conduct the epistasis analysis. See <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001809#pbio.1001809.s020" target="_blank">Text S1</a> for full secondary assay information. (B) Epistasis screen results are shown for <i>bona fide</i> MAPK pathway components as well as a selection of candidates. Results are presented as pMAPK values normalized to <i>GFP</i> dsRNA treated controls. <i>Bona fide </i>MAPK pathway components are identified with an asterisk (*). Genes to which we have associated new gene symbols are marked with a cross (†). (C) Known pathway components (labeled) and experimental candidates are assigned to specific epistasis intervals. The calculated Pearson correlation <i>r</i> between the epistasis screen data profiles and the three predetermined profiles for epistasis intervals (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001809#s4" target="_blank">Methods</a>) are represented on three axes (<i>x</i>-axis, RAS-RAF; <i>y</i>-axis, MEK-MAPK; <i>z</i>-axis, RAF-MEK). Values near 0 represent poor correlation while values near 1 (negative regulators) or −1 (positive regulators) indicate high correlation with a given epistasis profile. Candidates were assigned to the RAS-RAF (orange), RAF-MEK (magenta), or MEK-MAPK (dark blue) interval on the basis of the highest distance value. Candidates that could not be assigned to a specific interval (distance values within [−0.5, 0.5]) are shown in grey. Detailed epistasis screen results are available in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001809#pbio.1001809.s014" target="_blank">Table S3</a>.</p

RNAi screen candidates interact genetically with RAS/MAPK pathway components.

<p>(A–J) The <i>Ras^V12</i> rough eye phenotype is dominantly suppressed by heterozygous mutations in <i>Cka</i>, <i>gfzf</i>, <i>CG1603</i>, <i>Fip1</i>, <i>Prp19</i>, <i>Caper</i>, and a trans-heterozygous mutation in <i>CG4936</i>. Fly eyes of the indicated genotypes were imaged by stereomicroscopy. The <i>mapk</i> alleles <i>mapk^E1171</i> and <i>rl¹</i> are used as positive controls. All fly eye images are from female flies except <i>CG4936^DG10305</i>/<i>CG4936^EY10172</i>, which is from a male fly; the rough eye phenotype was observed to be similar in males and females except in this case where males displayed a stronger genetic interaction. (K–N) Genetic interactions with <i>rl¹</i> wing vein deletion phenotypes. <i>rl¹</i>/<i>rl¹</i> flies display a slight deletion of the mid-section of the L4 wing vein that is not fully penetrant. The L4 deletion is enhanced, sometimes extending to the posterior cross vein (pcv) in <i>Prp19^CE162</i> and <i>Caper^f07714</i> heterozygous backgrounds (pictures shown served to illustrate detailed scoring results in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001809#pbio.1001809.s008" target="_blank">Figure S8H</a>). (O–R) Genetic interactions with <i>rl¹</i> rough-eye phenotypes. The weak rough eye phenotype observed in <i>rl¹</i> homozygotes is shown. The severity of this phenotype is increased in heterozygous mutant backgrounds for <i>Prp19</i> and <i>Caper</i>; these flies display a further decrease in eye size and an increased eye roughness.</p