Phosphorylation of the Drosophila melanogaster RNA–Binding Protein HOW by MAPK/ERK Enhances Its Dimerization and Activity

Drosophila melanogaster Held Out Wings (HOW) is a conserved RNA–binding protein (RBP) belonging to the STAR family, whose closest mammalian ortholog Quaking (QKI) has been implicated in embryonic development and nervous system myelination. The HOW RBP modulates a variety of developmental processes by controlling mRNA levels and the splicing profile of multiple key regulatory genes; however, mechanisms regulating its activity in tissues have yet to be elucidated. Here, we link receptor tyrosine kinase (RTK) signaling to the regulation of QKI subfamily of STAR proteins, by showing that HOW undergoes phosphorylation by MAPK/ERK. Importantly, we show that this modification facilitates HOW dimerization and potentiates its ability to bind RNA and regulate its levels. Employing an antibody that specifically recognizes phosphorylated HOW, we show that HOW is phosphorylated in embryonic muscles and heart cardioblasts in vivo, thus documenting for the first time Serine/Threonine (Ser/Thr) phosphorylation of a STAR protein in the context of an intact organism. We also identify the sallimus/D-titin (sls) gene as a novel muscle target of HOW–mediated negative regulation and further show that this regulation is phosphorylation-dependent, underscoring the physiological relevance of this modification. Importantly, we demonstrate that HOW Thr phosphorylation is reduced following muscle-specific knock down of Drosophila MAPK rolled and that, correspondingly, Sls is elevated in these muscles, similarly to the HOW RNAi effect. Taken together, our results provide a coherent mechanism of differential HOW activation; MAPK/ERK-dependent phosphorylation of HOW promotes the formation of HOW dimers and thus enhances its activity in controlling mRNA levels of key muscle-specific genes. Hence, our findings bridge between MAPK/ERK signaling and RNA regulation in developing muscles

Origins of Context-Dependent Gene Repression by Capicua

© 2015 Forés et al. Receptor Tyrosine Kinase (RTK) signaling pathways induce multiple biological responses, often by regulating the expression of downstream genes. The HMG-box protein Capicua (Cic) is a transcriptional repressor that is downregulated in response to RTK signaling, thereby enabling RTK-dependent induction of Cic targets. In both Drosophila and mammals, Cic is expressed as two isoforms, long (Cic-L) and short (Cic-S), whose functional significance and mechanism of action are not well understood. Here we show that Drosophila Cic relies on the Groucho (Gro) corepressor during its function in the early embryo, but not during other stages of development. This Gro-dependent mechanism requires a short peptide motif, unique to Cic-S and designated N2, which is distinct from other previously defined Gro-interacting motifs and functions as an autonomous, transferable repressor element. Unexpectedly, our data indicate that the N2 motif is an evolutionary innovation that originated within dipteran insects, as the Cic-S isoform evolved from an ancestral Cic-L-type form. Accordingly, the Cic-L isoform lacking the N2 motif is completely inactive in early Drosophila embryos, indicating that the N2 motif endowed Cic-S with a novel Gro-dependent activity that is obligatory at this stage. We suggest that Cic-S and Gro coregulatory functions have facilitated the evolution of the complex transcriptional network regulated by Torso RTK signaling in modern fly embryos. Notably, our results also imply that mammalian Cic proteins are unlikely to act via Gro and that their Cic-S isoform must have evolved independently of fly Cic-S. Thus, Cic proteins employ distinct repressor mechanisms that are associated with discrete structural changes in the evolutionary history of this protein family.This work was funded by research grants from the Spanish Government (BFU2008-01875 and BFU2011-23611), Generalitat de Catalunya (2009SGR-1075) and Fundació La Marató de TV3 (20131730). GJ is an ICREA investigator. ZP is supported by grants from the National Institute of General Medical Sciences (NIH R01GM086537), the Israel Science Foundation (Center of Excellence; 1772/13) and the Król Charitable Foundation. ZP is an incumbent of The Lady Davis Chair in Experimental Medicine and Cancer ResearchPeer Reviewe

HOW(L) phosphorylation correlates with its homo-dimerization.

<p>A. Phospho-mutant HOW(L)^TTAA oligomerizes to a lesser extent than HOW(L)^WT. S2R+ cells were co-transfected with expression vectors for the following: <i>gfp- how(l)^WT</i> and <i>how(l)^WT-HA</i> (1) <i>gfp- how(l)^TTAA</i> with <i>how(l)^TTAA-HA</i> (2); <i>gfp-how(l)^EG</i> with <i>how(l)^EG-HA</i> (3); or <i>gfp</i> with <i>how(l)^WT-HA</i> (4) as a control. IP was carried out using anti-GFP antibody and Western blotting was performed with anti-HOW. Crude lysate was reacted with anti-HOW (2^nd panel from the top), anti-GFP (to visualize GFP-HOW (lanes 1–3) and the GFP control (lane 4)), as well as with anti-Actin. A′. Quantification of lanes 1–3 from three experiments as described in A. For each sample, the ratio between the Co-immunoprecipitated HA protein and its total crude level was normalized to the amount of the GFP protein that pulled it down. HOW^TTAA (2) exhibited partial dimerization (0.56±0.14, P = 0.039 unpaired t-test, n = 3). The HOW^EG (3) is almost completely non-dimerized (0.13±0.04, P<0.0001, unpaired t-test, n = 3). B. HOW(L) protein that is unable to homo-dimerize undergoes significantly less phosphorylation. S2R+ cells were transfected with either <i>how(l)^WT</i>, <i>how(l)^TTAA</i> or the <i>how(l)^EG</i> mutant construct. Proteins were immunoprecipitated using anti-HOW antibody. The amount of lysate used for the IP of the HOW(L)^EG protein was doubled, in order to obtain a comparable amount of immunoprecipitated HOW protein (since it does not dimerize, less protein precipitates). B′. Quantification of two of several experiments in which the total levels of the different proteins were comparable. For each sample, a ratio between the pTP band measurement and the total HOW band was calculated, and normalized to the HOW(L)^WT ratio. Error bars indicate SEM (HOW(L)^EG 0.12±0.10).</p

Phosphorylated HOW is detected in the nuclei of somatic muscles and cardioblasts.

<p>A. Anti-pHOW (pT64) antibody reacts with HOW, in a phosphorylation-dependent manner. HOW protein was immunoprecipitated with an anti-HOW antibody from S2R+ cells transfected with either HOW(L)^WT, treated (or not) with U0126 (lanes 3, 2 respectively), with HOW(L)^TTAA (lane 4), or no HOW (lane 1). The IP was reacted with anti-pHOW (pT64) antibody (top) or with anti-HOW antibody (middle). The crude extract was reacted with anti-pERK antibody (bottom), confirming U0126 inhibition. Note specific reactivity of the anti-pHOW antibody. B. Anti-pHOW (pT64) antibody reacts with HOW protein overexpressed in embryonic muscles and heart. Transgenic UAS-HOW(L)^WT (lane 2) and UAS-HOW(L)^TTAA (lane 3) flies or wild-type controls (lane 1) were crossed to the <i>mef2</i>-GAL4 driver line. Embryos were collected, and protein extracts were subjected to IP with an anti-HA antibody followed by Western with anti-pHOW (pT64) antibody (top), and anti-HOW antibody (2^nd from top). Crude extracts were reacted with anti-HOW (2^nd from bottom), and anti-Actin (bottom) as a loading control. C-F. <i>Drosophila</i> embryos stained with anti-pHOW (pT64) exhibit specific staining in somatic muscles and heart cardioblasts. Stage 16 WT (C,D) and <i>how ^stru</i> mutant (E,F) embryos were stained with anti-pHOW (pT64) (red, C–F) and anti-HOW (green in merge D,F). Embryos are oriented dorsal to the top and anterior to the left. (C,D) Note the staining in muscles (hollow arrow), heart cardioblasts (white arrow) and the lack of pHOW staining in the muscle attachment sites (arrowhead). (E,F) Weak staining in cardioblasts in <i>how ^stru−/−</i> mutants. G–I. <i>Drosophila</i> embryos expressing CD8-GFP, which localizes at the plasma membrane, but also often concentrates in the ER surrounding the nuclei, in muscles under <i>mef2</i>-GAL4, were stained with anti-pHOW (red, G) shown merged with GFP (green, H) and with HOW (blue, I). J–M. HOW(L) overexpressed in embryonic tendon cells reacts with the anti-pHOW (pT64) antibody. Embryos expressing UAS-HOW(L)^WT-HA (J,K) or UAS-HOW(L)^TTAA–HA (L,M) in the tendon cells under <i>sr</i>–GAL4 were stained with anti-pHOW (pT64) (red, J–M) and anti-HA (green, K, M, merged with the anti-pHOW staining). Arrows mark tendon cells reactive with anti-pHOW upon expression of HOW(L)^WT (J,K), or non-reactive upon expression of the phospho-mutant (L,M).</p

<i>Drosophila</i> MAPK <i>rolled</i> is directing HOW phosphorylation in muscles and regulation of Sallimus levels.

<p>A. HOW was immunoprecipitated from lysates of 3^rd instar larvae either expressing <i>rolled</i> RNAi in muscles under <i>mef2</i>-GAL4 (left) or of wild-type <i>mef2</i>-GAL4 heterozygotes (w−, right) and reacted with a pTP antibody to observe its phosphorylation level and with anti-HOW to compare total HOW levels. The crude extract was reacted with anti-HOW and anti-Tubulin as a loading control, showing that the total quantity of HOW is not significantly altered by MAPK/Rolled levels. Note the reduction in HOW phosphorylation following expression of <i>rolled</i> RNAi. B. Reduction in MAPK/Rolled levels elevates Sls protein levels. Same genotypes as in A were analyzed by Western with the following antibodies (from top to bottom): anti-Sls, anti-MHC, anti-MSP300, anti-pERK (to verify the reduction in activated MAPK in these larvae) and anti-α-Actinin as a loading control. C–H. Wild-type (C–E) and <i>mef2</i>-GAL4><i>rolled</i> RNAi (F–H) larvae were stained with anti-Sls antibody (red, C,F) and anti MSP-300 (blue, D,G). Merge is shown in E,H. Note the significant elevation in Sls levels following down regulation of MAPK/Rolled levels.</p

HOW phosphorylation strengthens its affinity to RNA, while not affecting its subcellular localization.

<p>A. <i>In vitro</i> RNA-protein binding assay. HA tagged HOW(L)^WT, HOW(L)^TTAA or HOW(L)^EG constructs were purified from S2R+ cells by immunoprecipitation of the HA tag, followed by elution with free HA peptide. HOW(L)^WT transfected cells were either treated with U0126 or not treated. Equal volumes of purified proteins were incubated with biotin labeled 12 nt oligomers that either bind HOW (top panel) or do not bind HOW (middle panel). The complexes were incubated with avidin beads, eluted by boiling in sample buffer and reacted with anti HOW in a Western blot. The amounts of HOW protein used for each reaction were comparable (bottom panel). B–C′. Localization of HOW(L) was not altered due to the mutations in the Thr residues. Cells expressing either HOW(L)^WT (B,B′) or HOW(L)^TTAA (C,C′) were stained for HOW (red, B–C). Merge images (B′–C′) also present GFP (green, marks transfected cells) and Lamin (blue).</p

HOW(L) is phosphorylated <i>in vitro</i> and in S2R+ cells by MAPK/ERK on one or more TP sites.

<p>A. ERK2 phosphorylates HOW <i>in vitro</i>. HOW(L)^WT and HOW(L)^TTAA (phospho mutant) constructs were <i>in vitro</i> translated (using S³⁵ labeling) and incubated with activated ERK2. The reaction was resolved on an SDS gel. The left part of the panel (lanes 1–4) shows phosphorylation of the Yan (Aop) protein as a control. HOW(L)^WT protein, but not the HOW(L)^TTAA mutant (lanes 7–8) exhibits a molecular weight shift of a fraction of the protein when incubated with the kinase (lane 5) relative to HOW without the kinase (lane 6). B. HOW(L), but not HOW(S), is phosphorylated in S2R+ cells. HOW proteins were immunoprecipitated using an anti-HOW antibody and reacted with anti-pTP (top panel) and anti-HOW (middle panel) antibodies. HOW proteins in the crude extract are shown in the bottom panel. From the left: HOW(S)^WT, HOW(L)^WT and HOW(L)^TTAA. C. Treatment with the MAPKK/MEK inhibitor U0126 decreases HOW phosphorylation. HOW proteins were immunoprecipitated with an anti-HOW antibody from cells transfected with <i>how(l)^WT</i> treated (or not) with U0126, or with <i>how(l)^TTAA</i> and reacted with anti-pTP antibody (top panel) or with anti-HOW antibody (2^nd from the top). The crude extract was reacted with anti-pERK antibody (2^nd from the bottom), to confirm the extent of U0126 inhibition, and anti-Actin (bottom panel) as a loading control. C′. Quantification of the reduction of phosphorylation following treatment with U0126. For each sample, the ratio between the pTP band measurement and the total HOW band was calculated, and normalized to the HOW(L)^WT (non-treated sample) ratio. Results shown are the average of three experiments; error bars indicate SEM. Following U0126 treatment, HOW phosphorylation was reduced to 0.47±0.07 relative to non-treated cells (P = 0.0021, unpaired t-test, n = 3). D. Same as in C, except cells were treated with TPA/PMA. A representative experiment is presented. D′. Quantification of three TPA treatment experiments, in which HOW phosphorylation was increased by 1.28±0.06 (P = 0.0076, unpaired t-test, n = 3).</p