Phosphoproteomic analysis identifies supervillin as an ERK3 substrate regulating cytokinesis and cell ploidy

Extracellular signal-regulated kinase 3 (ERK3) is a poorly characterized member of the mitogen-activated protein (MAP) kinase family. Functional analysis of the ERK3 signaling pathway has been hampered by a lack of knowledge about the substrates and downstream effectors of the kinase. Here, we used large-scale quantitative phosphoproteomics and targeted gene silencing to identify direct ERK3 substrates and gain insight into its cellular functions. Detailed validation of one candidate substrate identified the gelsolin/villin family member supervillin (SVIL) as a bona fide ERK3 substrate. We show that ERK3 phosphorylates SVIL on Ser245 to regulate myosin II activation and cytokinesis completion in dividing cells. Depletion of SVIL or ERK3 leads to increased cytokinesis failure and multinucleation, a phenotype rescued by wild type SVIL but not by the non-phosphorylatable S245A mutant. Our results unveil a new function of the atypical MAP kinase ERK3 in cell division and the regulation of cell ploidy

Rôle de la Reptine in vivo dans la physiopathologie hépatique

Previous studies of the laboratory have shown that Reptin, an AAA+ ATPase, is overexpressed in hepatocellular carcinoma where it is necessary for proliferation and cell survival. It is known that Reptin plays a critical role in the stabilization of the mTOR kinase, but its pathophysiological role in vivo remains unknown. The objectives of my thesis were to study the role of Reptin in liver metabolism and regeneration using a new hepato-specific Reptin knock-out murine model (Reptin LKO). We have shown that hepatic Reptin maintains mTOR protein level in vivo through its ATPase activity. Unexpectedly, loss or pharmacological inhibition of Reptin induces an inhibition of mTORC1 activity and an increase of mTORC2 activity, associated with inhibition of lipogenesis and hepatic glucose production. The deletion of Reptin completely rescued pathological phenotypes associated with the metabolic syndrome induced by a high fat diet. Thus, inhibition of Reptin ATPase could represent a new therapeutic perspective for the metabolic syndrome. In Reptin LKO model, we have observed a progressive loss of Reptin invalidation associated with a liver regeneration phenomenon. Our preliminary data suggest that Reptin is necessary for hepatocyte survival and is required for hepatocyte proliferation during liver regeneration after partial hepatectomy. To conclude, altogether our results suggest that Reptin plays a crucial role in glucose and lipid metabolism in the liver, and in hepatocyte proliferation and survival.Les travaux antérieurs du laboratoire ont montré que la Reptine, une AAA+ ATPase, est surexprimée dans le carcinome hépatocellulaire où elle est nécessaire à la prolifération et la survie cellulaire. Il est connu que la Reptine joue un rôle crucial dans la stabilité de la kinase mTOR, mais son rôle physiopathologique in vivo reste inconnu. Les objectifs de ma thèse étaient d’étudier le rôle de la Reptine dans le métabolisme et la régénération hépatique grâce à un nouveau modèle murin d’invalidation hépato-spécifique de la Reptine (Reptin LKO). Nous avons montré que la Reptine régule la stabilité de la protéine mTOR in vivo, via son activité ATPase. De manière inattendue, la délétion ou l’inhibition pharmacologique de la Reptine induisent une inhibition de l’activité mTORC1 et une augmentation de l’activité mTORC2, associées à une inhibition de la lipogenèse et de la production de glucose hépatique. La délétion de la Reptine supprime complètement les phénotypes pathologiques associés au syndrome métabolique induit par un régime riche en graisses. Ainsi, l’inhibition de l’ATPase Reptine pourrait représenter une nouvelle stratégie thérapeutique pour le syndrome métabolique. Dans le modèle Reptin LKO, nous avons observé une perte progressive de l’invalidation de la Reptine associée à un phénomène de régénération hépatique. Nos résultats préliminaires suggèrent que la Reptine est nécessaire à la survie des hépatocytes et est requise pour la prolifération des hépatocytes durant la régénération hépatique après hépatectomie partielle. Pour conclure, l’ensemble de nos résultats suggèrent que la Reptine joue un rôle crucial dans l’homéostasie glucido-lipidique du foie, ainsi que dans la prolifération et la survie des hépatocytes

Role of Reptin in hepatic pathophysiology in vivo

Les travaux antérieurs du laboratoire ont montré que la Reptine, une AAA+ ATPase, est surexprimée dans le carcinome hépatocellulaire où elle est nécessaire à la prolifération et la survie cellulaire. Il est connu que la Reptine joue un rôle crucial dans la stabilité de la kinase mTOR, mais son rôle physiopathologique in vivo reste inconnu. Les objectifs de ma thèse étaient d’étudier le rôle de la Reptine dans le métabolisme et la régénération hépatique grâce à un nouveau modèle murin d’invalidation hépato-spécifique de la Reptine (Reptin LKO). Nous avons montré que la Reptine régule la stabilité de la protéine mTOR in vivo, via son activité ATPase. De manière inattendue, la délétion ou l’inhibition pharmacologique de la Reptine induisent une inhibition de l’activité mTORC1 et une augmentation de l’activité mTORC2, associées à une inhibition de la lipogenèse et de la production de glucose hépatique. La délétion de la Reptine supprime complètement les phénotypes pathologiques associés au syndrome métabolique induit par un régime riche en graisses. Ainsi, l’inhibition de l’ATPase Reptine pourrait représenter une nouvelle stratégie thérapeutique pour le syndrome métabolique. Dans le modèle Reptin LKO, nous avons observé une perte progressive de l’invalidation de la Reptine associée à un phénomène de régénération hépatique. Nos résultats préliminaires suggèrent que la Reptine est nécessaire à la survie des hépatocytes et est requise pour la prolifération des hépatocytes durant la régénération hépatique après hépatectomie partielle. Pour conclure, l’ensemble de nos résultats suggèrent que la Reptine joue un rôle crucial dans l’homéostasie glucido-lipidique du foie, ainsi que dans la prolifération et la survie des hépatocytes.Previous studies of the laboratory have shown that Reptin, an AAA+ ATPase, is overexpressed in hepatocellular carcinoma where it is necessary for proliferation and cell survival. It is known that Reptin plays a critical role in the stabilization of the mTOR kinase, but its pathophysiological role in vivo remains unknown. The objectives of my thesis were to study the role of Reptin in liver metabolism and regeneration using a new hepato-specific Reptin knock-out murine model (Reptin LKO). We have shown that hepatic Reptin maintains mTOR protein level in vivo through its ATPase activity. Unexpectedly, loss or pharmacological inhibition of Reptin induces an inhibition of mTORC1 activity and an increase of mTORC2 activity, associated with inhibition of lipogenesis and hepatic glucose production. The deletion of Reptin completely rescued pathological phenotypes associated with the metabolic syndrome induced by a high fat diet. Thus, inhibition of Reptin ATPase could represent a new therapeutic perspective for the metabolic syndrome. In Reptin LKO model, we have observed a progressive loss of Reptin invalidation associated with a liver regeneration phenomenon. Our preliminary data suggest that Reptin is necessary for hepatocyte survival and is required for hepatocyte proliferation during liver regeneration after partial hepatectomy. To conclude, altogether our results suggest that Reptin plays a crucial role in glucose and lipid metabolism in the liver, and in hepatocyte proliferation and survival

Reptin Regulates DNA Double Strand Breaks Repair in Human Hepatocellular Carcinoma

<div><p>Reptin/RUVBL2 is overexpressed in most hepatocellular carcinomas and is required for the growth and viability of HCC cells. Reptin is involved in several chromatin remodeling complexes, some of which are involved in the detection and repair of DNA damage, but data on Reptin involvement in the repair of DNA damage are scarce and contradictory. Our objective was to study the effects of Reptin silencing on the repair of DNA double-strand breaks (DSB) in HCC cells. Treatment of HuH7 cells with etoposide (25 μM, 30 min) or γ irradiation (4 Gy) increased the phosphorylation of H2AX by 1.94 ± 0.13 and 2.0 ± 0.02 fold, respectively. These values were significantly reduced by 35 and 65 % after Reptin silencing with inducible shRNA. Irradiation increased the number of BRCA1 (3-fold) and 53BP1 foci (7.5 fold). Depletion of Reptin reduced these values by 62 and 48%, respectively. These defects in activation and/or recruitment of repair proteins were not due to a decreased number of DSBs as measured by the COMET assay. All these results were confirmed in the Hep3B cell line. Protein expression of ATM and DNA-PKcs, the major H2AX kinases, was significantly reduced by 52 and 61 % after Reptin depletion whereas their mRNA level remained unchanged. Phosphorylation of Chk2, another ATM target, was not significantly altered. Using co-immunoprecipitation, we showed an interaction between Reptin and DNA-PKcs. The half-life of newly-synthesized DNA-PKcs was reduced when Reptin was silenced. Finally, depletion of Reptin was synergistic with etoposide or γ irradiation to reduce cell growth and colony formation. In conclusion, Reptin is an important cofactor for the repair of DSBs. Our data, combined with those of the literature suggests that it operates at least in part by regulating the expression of DNA-PKcs by a stabilization mechanism. Overexpression of Reptin in HCC could be a factor of resistance to treatment, consistent with the observed overexpression of Reptin in subgroups of chemo-resistant breast and ovarian cancers.</p></div

Reptin depletion reduces the recruitment on chromatin of BRCA1 and 53BP1 after gamma ray irradiation.

<p>HuH7 cells stably expressing a doxycycline-inducible Reptin shRNA were treated with doxycycline (sh Reptin +) or left untreated (sh Reptin-) for 4 days. (A) Representative images of 53BP1 and BRCA1 foci in HuH7 cells detected using immunofluorescence 2h after gamma ray irradiation. (B) The bars represent the mean number of foci per cell from two independent experiments (>200 cells were counted per experiment). Expression levels of BRCA1 and 53BP1 were assessed by Western Blot on whole cell extracts 4 days after doxycycline treatment. A representative picture is shown in (C). The migration positions of molecular weight standards (in kDa) are indicated on the left. (D) Quantification of 3 Western blot experiments.</p

Reptin depletion and DNA damage cooperate to reduce cell viability.

<p>(A) HuH7 cells transfected with a control siRNA were either left untreated (yellow) or received etoposide (ETO, green) after 3 days. Similarly, cells transfected with an anti-Reptin siRNA (siR2) were untreated (blue) or received etoposide (red). The effect on cell number was determined at 1, 3, 4 and 5 days after seeding, using the MTS assay. The results are the mean ± SD of 3 independent experiments. After 5 days, the difference between siR2 and siControl without treatment was significant with a p value <0.001 and the difference between untreated siR2 and treated siR2 was significant with a p value <0.05 (Two-way ANOVA followed by Bonferroni test). (B) Same design as in (A) except that DNA damage was induced with γ-irradiation (IRR). The figure shows the mean of 2 independent experiments. (C) Clonogenic tests were carried out as described in Materials and Methods using HuH7 cells stably expressing a doxycycline-inducible Reptin shRNA that were either treated with doxycycline (sh Rep +) or untreated (sh Rep-). The graph shows the mean of 3 replicates (*** p<0.001 and ** p<0.01 by One-way ANOVA followed by Bonferroni test).</p

Reptin depletion impairs H2AX phosphorylation at serine 139 (γH2AX) after DNA damage.

<p>(A) and (B) HuH7 cells were transfected with a control or a Reptin siRNA. After 3 days, they were then treated with etoposide (ETO, 25 μM) (A) or exposed to gamma radiation (IRR, 4 Gy) (B), stained for γ-H2AX and analyzed by flow cytometry. The bar graphs show the mean of 3 experiments (*** p<0.001 by One-way ANOVA followed by Bonferroni test). (C) and (D) HuH7 cells stably expressing a doxycycline-inducible Reptin shRNA were treated with doxycycline (sh Reptin +) or left untreated (sh Reptin-) for 4 days. Extracts from HuH7 cells treated with etoposide as in (A) were analyzed by Western blot with an anti-phospho-H2AX antibody (C). Data were normalized relative to the Sypro Ruby staining of the membrane. The graph on the right shows the mean of 9 experiments (*** p<0.001 by One-way ANOVA followed by Bonferroni test). (D) Control or Reptin depleted HuH7 cells were treated with etoposide or irradiated, then fixed at different time points and immunostained for phospho-H2AX. The graphs below show for each treatment the evolution of the number of foci per cell (left) or the fractional decrease of the number of foci, setting the initial time point as 100% (right).</p

Reptin interacts with DNA-PKcs and regulates its stability.

<p>(A) Interaction between Reptin and DNA-PKcs was tested by immunoprecipitation. The migration positions of molecular weight standards (in kDa) are indicated on the left. The faint band seen in the IgG lane with the Reptin antibody corresponds to traces of IgG heavy chains. The picture is representative of 3 similar experiments. (B) Metabolic labeling and pulse chase. HuH7 cells stably expressing a doxycycline-inducible Reptin shRNA were treated (sh Reptin) or not (Control) with doxycycline. After 4 days, they were labeled with EXPRE³⁵S³⁵S as described in Materials and Methods. Following the indicated periods of chase, DNA-PKcs was immunoprecipitated and the eluates were separated on SDS-PAGE. The top panel shows the autoradiographic image, and the bottom one the Coomassie blue staining of the gel with DNA-PKcs. (C) The graph shows the quantitative analysis of the data following normalization of the autoradiographic signal on the amount of immunoprecipitated DNA-PKcs.</p

Reptin depletion does not reduce the number of double strand breaks.

<p>HuH7 cells were treated as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0123333#pone.0123333.g001" target="_blank">Fig 1</a>. The neutral COMET assay was used to assess DNA double-strand breaks that were quantified using calculation of the comet tail moment. Bars represent the mean ±SD from 3 independent experiments (>140 cells were counted per experiment). Left, etoposide treatment, right, γ irradiation. * p<0.05, ** p<0.01 and *** p<0.001 by One-way ANOVA followed by Bonferroni test. Representative COMET images are shown below.</p