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The cytoskeleton and cell-matrix adhesions constitute a dynamic network that controls cellular behavior during development and cancer. The Focal Adhesion Kinase (FAK) is a central actor of these cell dynamics, promoting cell-matrix adhesion turnover and active membrane fluctuations. However, the initial steps leading to FAK activation and subsequent promotion of cell dynamics remain elusive. Here, we report that the serine/threonine kinase PKCθ participates in the initial steps of FAK activation. PKCθ, which is strongly expressed in aggressive human breast cancers, controls the dynamics of cell-matrix adhesions and active protrusions through direct FAK activation, thereby promoting cell invasion and lung metastases. Using various tools for in vitro and live cell studies, we precisely decipher the molecular mechanisms of FAK activation. PKCθ directly interacts with the FAK FERM domain to open FAK conformation through PKCθ’s specific V3 domain, while phosphorylating FAK at newly identified serine/threonine residues within nascent adhesions, inducing cell dynamics and aggressive behavior. This study thus places PKCθ-directed FAK opening and phosphorylations as an original mechanism controlling dynamic, migratory, and invasive abilities of aggressive breast cancer cells, further strengthening the emerging oncogenic function of PKCθ.

BackgroundRNase E has crucial roles in the initiation of mRNA degradation, the processing of ‘stable’ transcripts such as rRNA and tRNA, and the quality control of ribosomes. With over 20’000 potential cleavage sites, RNase E is a low specificity endoribonuclease with the capacity to cleave multiple times nearly every transcript in the cell. A large noncatalytic region in the C-terminal half of RNase E is the scaffold for assembly of the multienzyme RNA degradosome. The components of the RNA degradosome cooperate in the degradation of mRNA to oligoribonucleotides, which are then degraded to nucleotides by oligoribonuclease. Over the past decade, compelling evidence has emerged that the RNA degradosome is attached to the phospholipid bilayer of the inner cytoplasmic membrane by the Membrane Targeting Sequence (MTS), which is a 15-residue amphipathic alpha-helix located in the noncatalytic region of RNase E. Systematic proteomic analyses have identified RNase E as an inner membrane protein that can only be solubilized by disrupting the phospholipid bilayer with detergent. Important components of the mRNA degradation machinery are therefore membrane-attached. The reason for this cellular localization has until now been a mystery.ResultsWe have constructed and characterized the rneΔMTS strain expressing ncRNase E (nucleo-cytoplasmic-RNase E), which is a soluble variant that is uniformly distributed in the interior of the cell. In the mutant strain, there is a slowdown in the rates of growth and mRNA degradation. Surprisingly, we have identified aberrant 20S and 40S ribosomal particles in the rneΔMTS strain that contain, respectively, precursors of 16S and 23S rRNA that have been ‘nicked’ by ncRNase E. Although intact ribosomes are resistant to RNase E cleavage in vitro, protein-free rRNA is readily degraded by RNase E. Partially unfolded ribosomes are susceptible to nicking by RNase E in vitro. We have mapped rRNA cleavage sites cRACE. In vivo and in vitro rRNA cleavages map to the same sites. The sequence of the cleavage sites matches the RNase E consensus sequence previously determined in a transcriptomic analysis that did not include rRNA. Construction of additional mutant strains demonstrated in vivo that fragments of 16S and 23S rRNA as well as a precursor of 5S rRNA are degraded in a pathway involving 3’ oligoadenylation and exonucleolytic digestion. A proteomic analysis showed that 17 small subunit proteins and 21 large subunit proteins are underrepresented in the 20S and 40S particles, respectively.ConclusionsRibosome biogenesis is a complex process involving co-transcriptional rRNA folding and r-protein binding in the nucleoid. Ribonucleoprotein intermediates are released from chromatin by RNase III cleavage. Maturation continues with the addition of ‘late’ proteins resulting in the compact rRNA structures found in mature 30S and 50S ribosomal subunits. Considering our experimental results, we propose that the physical separation of rRNA transcription in the nucleoid from the RNA degradosome on the inner cytoplasmic membrane protects intermediates in ribosome assembly from degradation. A corollary is that ribosome quality control normally occurs when defective ribosomal particles interact with the membrane-attached RNA degradosome. The rRNA degradation pathway described here is the same as described previously for RNase E-dependent degradation of mRNA. Since the pathway for rRNA degradation is the same as the pathway for mRNA degradation, the slowdown of mRNA degradation in the rneΔMTS strain could be due to competition by rRNA degradation. Since growth rate is limited by ribosome synthesis rate, the slow growth of the rneΔMTS strain is likely due to wasteful degradation of a proportion of newly synthesized rRNA. If r-proteins released by rRNA degradation are not recycled, then this would be an additional burden on cell growth. Avoiding a futile cycle in which rRNA intermediates in ribosome assembly are degraded likely explains why localization of RNase E homologues to the inner cytoplasmic membrane is conserved throughout the β- and γ-Proteobacteria.ImportanceIn E. coli, transcription in the nucleoid, translation in the cytoplasm and initiation of mRNA degradation on the inner cytoplasmic membrane are physically separated. Despite the lack of internal membranes, this separation can be viewed as a compartmentalization of the bacterial cell. Our work shows that the inner membrane localization of the RNA degradosome restricts access of RNase E to intermediates in ribosome assembly. Thus, as in the eukaryotic cell, the architecture of the bacterial cell has an important role in the organization of cellular processes such as ribosome biogenesis, ribosome quality control, and mRNA degradation.

Phosphorylation-driven cell signaling governs most biological functions and is widely studied using mass-spectrometry-based phosphoproteomics. Identifying the peptides and localizing the phosphorylation sites within them from the raw data is challenging and can be performed by several algorithms that return scores that are not directly comparable. This increases the heterogeneity among published phosphoproteomics data sets and prevents their direct integration. Here we compare 22 pipelines implemented in the main software tools used for bottom-up phosphoproteomics analysis (MaxQuant, Proteome Discoverer, PeptideShaker). We test six search engines (Andromeda, Comet, Mascot, MS Amanda, SequestHT, and X!Tandem) in combination with several localization scoring algorithms (delta score, D-score, PTM-score, phosphoRS, and Ascore). We show that these follow very different score distributions, which can lead to different false localization rates for the same threshold. We provide a strategy to discriminate correctly from incorrectly localized phosphorylation sites in a consistent manner across the tested pipelines. The results presented here can help users choose the most appropriate pipeline and cutoffs for their phosphoproteomics analysis.

X-linked myotubular myopathy (XLMTM) is a fatal neuromuscular disorder caused by loss of function mutations in MTM1. At present, there are no directed therapies for XLMTM, and incomplete understanding of disease pathomechanisms. To address these knowledge gaps, we performed a drug screen in mtm1 mutant zebrafish and identified four positive hits, including valproic acid, which functions as a potent suppressor of the mtm1 zebrafish phenotype via HDAC inhibition. We translated these findings to a mouse XLMTM model, and showed that valproic acid ameliorates the murine phenotype. These observations led us to interrogate the epigenome in Mtm1 knockout mice; we found increased DNA methylation, which is normalized with valproic acid, and likely mediated through aberrant 1-carbon metabolism. Finally, we made the unexpected observation that XLMTM patients share a distinct DNA methylation signature, suggesting that epigenetic alteration is a conserved disease feature amenable to therapeutic intervention.

T cells recognize a few high-affinity antigens among a vast array of lower affinity antigens. According to the kinetic proofreading model, antigen discrimination properties could be explained by the gradual amplification of small differences in binding affinities as the signal is transduced downstream of the T cell receptor. Which early molecular events are affected by ligand affinity, and how, has not been fully resolved. Here, we used time-resolved high-throughput proteomic analyses to identify and quantify the phosphorylation events and protein–protein interactions encoding T cell ligand discrimination in antigen-experienced T cells. Although low-affinity ligands induced phosphorylation of the Cd3 chains of the T cell receptor and the interaction of Cd3 with the Zap70 kinase as strongly as high-affinity ligands, they failed to activate Zap70 to the same extent. As a result, formation of the signalosome of the Lat adaptor was severely impaired with low- compared with high-affinity ligands, whereas formation of the signalosome of the Cd6 receptor was affected only partially. Overall, this study provides a comprehensive map of molecular events associated with T cell ligand discrimination.

Ribosome biogenesis lies at the nexus of various signaling pathways coordinating protein synthesis with cell growth and proliferation. This process is regulated by well-described transcriptional mechanisms, but a growing body of evidence indicates that other levels of regulation exist. Here we show that the Ras/mitogen-activated protein kinase (MAPK) pathway stimulates post-transcriptional stages of human ribosome synthesis. We identify RIOK2, a pre-40S particle assembly factor, as a new target of the MAPK-activated kinase RSK. RIOK2 phosphorylation by RSK stimulates cytoplasmic maturation of late pre-40S particles, which is required for optimal protein synthesis and cell proliferation. RIOK2 phosphorylation facilitates its release from pre-40S particles and its nuclear re-import, prior to completion of small ribosomal subunits. Our results bring a detailed mechanistic link between the Ras/MAPK pathway and the maturation of human pre-40S particles, which opens a hitherto poorly explored area of ribosome biogenesis., Author summary Ribosomes are universal molecular machines composed of ribosomal RNAs (rRNAs) and ribosomal proteins (RPs) responsible for the synthesis of cellular proteins. In eukaryotic cells, ribosome biogenesis relies on the assembly of large precursor particles (pre-ribosomes) containing, in addition to rRNAs and RPs, more than 200 accessory proteins named assembly and maturation factors (AMFs). Ribosome biogenesis is a highly energy demanding process which is tightly regulated to accommodate protein synthesis with cell growth requirements. In humans, the Ras/MAPK regulatory pathway is one of the main systems controlling cell growth and proliferation. Under favorable growth conditions, the Ras/MAPK pathway activates several aspects of ribosome production, such as synthesis of rRNAs and RPs. Our study highlights that the MAPK pathway also directly stimulates the assembly and maturation of pre-ribosomes. We identify the kinase RIOK2, an AMF involved in the synthesis of the small (40S) ribosomal subunit, as a new target of the MAPK-activated kinase RSK. We describe molecular events promoted by the MAPK pathway to finely tune the activity of RIOK2 during pre-40S particle maturation. This work is particularly important because of the current lack of mechanistic connections between growth-promoting signaling pathways and the assembly and maturation of pre-ribosomes in human cells.

Internal ribosome entry sites (IRESs) drive translation initiation during stress. In response to hypoxia, (lymph)angiogenic factors responsible for tissue revascularization in ischemic diseases are induced by the IRES-dependent mechanism. Here, we searched for IRES

In Proteobacteria, integral outer membrane proteins (OMPs) are crucial for the maintenance of the envelope permeability barrier to some antibiotics and detergents. In Enterobacteria, envelope stress caused by unfolded OMPs activates the sigmaE (σE) transcriptional response. σE upregulates OMP biogenesis factors, including the β-barrel assembly machinery (BAM) that catalyses OMP folding. Here we report that DolP (formerly YraP), a σE-upregulated and poorly understood outer membrane lipoprotein, is crucial for fitness in cells that undergo envelope stress. We demonstrate that DolP interacts with the BAM complex by associating with outer membrane-assembled BamA. We provide evidence that DolP is important for proper folding of BamA that overaccumulates in the outer membrane, thus supporting OMP biogenesis and envelope integrity. Notably, mid-cell recruitment of DolP had been linked to regulation of septal peptidoglycan remodelling by an unknown mechanism. We now reveal that, during envelope stress, DolP loses its association with the mid-cell, thereby suggesting a mechanistic link between envelope stress caused by impaired OMP biogenesis and the regulation of a late step of cell division.

SUMMARYInternal ribosome entry sites (IRESs) drive translation initiation during stress. In response to hypoxia, (lymph)angiogenic factors responsible for tissue revascularization in ischemic diseases are induced by the IRES-dependent mechanism. Here we searched for IRES trans-acting factors (ITAFs) active in early hypoxia in mouse cardiomyocytes. Using knock-down and proteomics approaches, we show a link between a stressed-induced nuclear body, the paraspeckle, and IRES-dependent translation. Furthermore, smiFISH experiments demonstrate the recruitment of IRES-containing mRNA into paraspeckle during hypoxia. Our data reveal that the long non-coding RNA Neat1, an essential paraspeckle component, is a key translational regulator, active on IRESs of (lymph)angiogenic and cardioprotective factor mRNAs. In addition, paraspeckle proteins p54nrb and PSPC1 as well as nucleolin and Rps2, two p54nrb-interacting proteins identified by mass spectrometry, are ITAFs for IRES subgroups. Paraspeckle thus appears as a platform to recruit IRES-containing mRNAs and possibly host IRESome assembly. Polysome PCR array shows that Neat1 isoforms regulate IRES-dependent translation and, more widely, translation of mRNAs involved in stress response.HighlightsParaspeckle formation correlates with activation of translation via internal ribosome entry sites (IRES) in mouse hypoxic cardiomyocytes as well as in tumoral cells.The long non-coding RNA Neat1, an essential paraspeckle component, is a key translational regulator of (lymph)angiogenic and cardioprotective factor expression in this process.IRES-containing mRNA is recruited into paraspeckles during hypoxia.Paraspeckle proteins p54nrb and PSPC1 as well as two p54nrb-interacting proteins, nucleolin and RPS2, contribute to this process.Paraspeckle appears as a platform for IRESome formation in the nucleus.The Neat1 isoforms widely regulate the translation of mRNAs containing IRESs and of genes involved in the stress response.GRAPHICAL ABSTRACT

Preventing premature interaction of pre-ribosomes with the translation apparatus is essential for translational accuracy. Hence, the final maturation step releasing functional 40S ribosomal subunits, namely processing of the 18S ribosomal RNA 3′ end, is safeguarded by the protein DIM2, which both interacts with the endoribonuclease NOB1 and masks the rRNA cleavage site. To elucidate the control mechanism that unlocks NOB1 activity, we performed cryo-electron microscopy analysis of late human pre-40S particles purified using a catalytically inactive form of the ATPase RIO1. These structures, together with in vivo and in vitro functional analyses, support a model in which ATP-loaded RIO1 cooperates with ribosomal protein RPS26/eS26 to displace DIM2 from the 18S rRNA 3′ end, thereby triggering final cleavage by NOB1; release of ADP then leads to RIO1 dissociation from the 40S subunit. This dual key lock mechanism requiring RIO1 and RPS26 guarantees the precise timing of pre-40S particle conversion into translation-competent ribosomal subunits., eLife, 10, ISSN:2050-084X

G protein-coupled receptors (GPCRs) are notoriously difficult to detect in native tissues. In an effort to resolve this problem, we have developed a novel mouse model by fusing the hemagglutinin (HA)-epitope tag sequence to the amino-terminus of the µ-opioid receptor (MOP). This approach allows for highly efficient immunodetection of low abundant GPCR targets. We also show that the HA-tag facilitates both high-resolution imaging and immunoisolation of MOP. Mass spectrometry (MS) confirmed post-translational modifications, most notably agonist-selective phosphorylation of carboxyl-terminal serine and threonine residues. MS also unequivocally identified the carboxyl-terminal 387LENLEAETAPLP398 motif, which is part of the canonical MOP sequence. Unexpectedly, MS analysis of brain lysates failed to detect any of the 15 MOP isoforms that have been proposed to arise from alternative splicing of the MOP carboxyl-terminus. For quantitative analysis, we performed multiple successive rounds of immunodepletion using the well-characterized rabbit monoclonal antibody UMB-3 that selectively detects the 387LENLEAETAPLP398 motif. We found that >98% of HA-tagged MOP contain the UMB-3 epitope indicating that virtually all µ-opioid receptors expressed in the mouse brain exhibit the canonical amino acid sequence.

T‐cell receptor (TCR) ligation‐mediated protein phosphorylation regulates the activation, cellular responses, and fates of T cells. Here, we used time‐resolved high‐resolution phosphoproteomics to identify, quantify, and characterize the phosphorylation dynamics of thousands of phosphorylation sites in primary T cells during the first 10 min after TCR stimulation. Bioinformatic analysis of the data revealed a coherent orchestration of biological processes underlying T‐cell activation. In particular, functional modules associated with cytoskeletal remodeling, transcription, translation, and metabolic processes were mobilized within seconds after TCR engagement. Among proteins whose phosphorylation was regulated by TCR stimulation, we demonstrated, using a fast‐track gene inactivation approach in primary lymphocytes, that the ITSN2 adaptor protein regulated T‐cell effector functions. This resource, called LymphoAtlas, represents an integrated pipeline to further decipher the organization of the signaling network encoding T‐cell activation. LymphoAtlas is accessible to the community at: https://bmm-lab.github.io/LymphoAtlas., The study presents LymphoAtlas, a phosphoproteomic dataset enabling the identification and visualization of phosphorylation dynamics during the first 10 min after TCR stimulation of primary mouse T cells.

In the context of human evolution, the study of proteins may overcome the limitation of the high degradation of ancient DNA over time to provide biomolecular information useful for the phylogenetic reconstruction of hominid taxa. In this study, we used a shotgun proteomics approach to compare the tooth proteomes of extant human and non-human primates (gorilla, chimpanzee, orangutan and baboon) in order to search for a panel of peptides able to discriminate between taxa and further help reconstructing the evolutionary relationships of fossil primates. Among the 25 proteins shared by the five genera datasets, we found a combination of peptides with sequence variations allowing to differentiate the hominid taxa in the proteins AHSG, AMBN, APOA1, BGN, C9, COL11A2, COL22A1, COL3A1, DSPP, F2, LUM, OMD, PCOLCE and SERPINA1. The phylogenetic tree confirms the placement of the samples in the appropriate genus branches. Altogether, the results provide experimental evidence that a shotgun proteomics approach on dental tissue has the potential to detect taxonomic variation, which is promising for future investigations of uncharacterized and/or fossil hominid/hominin specimens. SIGNIFICANCE: A shotgun proteomics approach on human and non-human primate teeth allowed to identify peptides with taxonomic interest, highlighting the potential for future studies on hominid fossils.

The phosphatase CDC25A is a key regulator of cell cycle progression by dephosphorylating and activating cyclin-CDK complexes. CDC25A is an unstable protein expressed from G1 until mitosis. CDC25A overexpression, which can be caused by stabilization of the protein, accelerates the G1/S and G2/M transitions, leading to genomic instability and promoting tumorigenesis. Thus, controlling CDC25A protein levels by regulating its stability is a critical mechanism for timing cell cycle progression and to maintain genomic integrity. Herein, we show that CDC25A is phosphorylated on Ser40 throughout the cell cycle and that this phosphorylation is established during the progression from G1 to S phase. We demonstrate that CyclinD-CDK4/CDK6 complexes mediate the phosphorylation of CDC25A on Ser40 during G1 and that these complexes directly phosphorylate this residue in vitro. Importantly, we also find that CyclinD1-CDK4 decreases CDC25A stability in a ßTrCP-dependent manner and that Ser40 and Ser88 phosphorylations contribute to this regulation. Thus our results identify cyclinD-CDK4/6 complexes as novel regulators of CDC25A stability during G1 phase, generating a negative feedback loop allowing control of the G1/S transition.

International audience; FLG2, a member of the S100-fused type protein family, is a 250-kDa protein expressed mainly in differentiated keratinocytes of the epidermis (Hsu et al.,2011; Wu et al., 2009). Its expression isreduced in atopic dermatitis (Pellerinet al., 2013), and FLG2 gene heterozygous non-sense mutations are associated with amore persistent formof the disease in African-American populations (Margolis et al., 2014). Loss of FLG2 due to homozygous mutations is responsible for peeling skin syndrome type A (Bolling et al., 2018; Mohamad et al., 2018).

International audience; Filaggrin (FLG) and corneodesmosin (CDSN) are two key proteins of the human epidermis. FLG loss-of-function mutations are the strongest genetic risk factors for human atopic dermatitis. Studies of the epidermal distribution of canine FLG and CDSN are limited. Our aim was to better characterize the distribution of FLG and CDSN in canine skin. Using immunohistochemistry on beagle skin, we screened a series of monoclonal antibodies (mAbs) specific for human FLG and CDSN. The cross-reactive mAbs were further used using immunoelectron microscopy and Western blotting. The structure of canine CDSN and FLG was determined using publicly available databases. In the epidermis, four anti-FLG mAbs stained keratohyalin granules in the granular keratinocytes and corneocyte matrix of the lower cornified layer. In urea-extracts of dog epidermis, several bands corresponding to proFLG and FLG monomers were detected. One anti-CDSN mAb stained the cytoplasm of granular keratinocytes and cells of both the inner root sheath and medulla of hair follicles. Dog CDSN was located in lamellar bodies, in the extracellular parts of desmosomes and in corneodesmosomes. A protein of 52 kDa was immunodetected. Genomic DNA analysis revealed that the amino acid sequence and structure of canine and human CDSN were highly similar.

International audience; The study demonstrates the high potential of MS-based proteomics coupled to an iterative database search strategy for the in-depth investigation of ancient proteomes. An efficient targeted PRM MS-based approach, although limited to the detection of a single pair of sex-specific amelogenin peptides, allowed confirming the sex of individuals in ancient dental remains, an essential information for paleoanthropologists facing the issue of sex determination and dimorphism.

The Cdc25A phosphatase is an essential activator of CDK-cyclin complexes at all steps of the eukaryotic cell cycle. The activity of Cdc25A is itself regulated in part by positive and negative feedback regulatory loops performed by its CDK-cyclin substrates that occur in G1 as well as during the G1/S and G2/M transitions. However, the regulation of Cdc25A during G2 phase progression before mitotic entry has not been intensively characterized. Here, we identify by mass spectrometry analysis a new phosphorylation event of Cdc25A on Serine283. Phospho-specific antibodies revealed that the phosphorylation of this residue appears in late S/G2 phase of an unperturbed cell cycle and is performed by CDK-cyclin complexes. Overexpression studies of wild-type and non-phosphorylatable mutant forms of Cdc25A indicated that Ser283 phosphorylation increases the G2/M-promoting activity of the phosphatase without impacting its stability or subcellular localization. Our results therefore identify a new positive regulatory loop between Cdc25A and its CDK-cyclin substrates which contributes to accelerate entry into mitosis through the regulation of Cdc25A activity in G2.

Abscission is the last step of cytokinesis, allowing the physical separation of daughter cells at the end of cell division. It has been considered a cell autonomous process, yet Jungas et al. report that Ephrin/Eph signaling controls the completion of abscission., Cytokinesis is the last step of cell division, culminating in the physical separation of daughter cells at the end of mitosis. Cytokinesis is a tightly regulated process that until recently was mostly viewed as a cell-autonomous event. Here, we investigated the role of Ephrin/Eph signaling, a well-known local cell-to-cell communication pathway, in cell division. We show that activation of Eph signaling in vitro leads to multinucleation and polyploidy, and we demonstrate that this is caused by alteration of the ultimate step of cytokinesis, abscission. Control of abscission requires Eph kinase activity, and Src and citron kinase (CitK) are downstream effectors in the Eph-induced signal transduction cascade. CitK is phosphorylated on tyrosines in neural progenitors in vivo, and Src kinase directly phosphorylates CitK. We have identified the specific tyrosine residues of CitK that are phosphorylated and show that tyrosine phosphorylation of CitK impairs cytokinesis. Finally, we show that, similar to CitK, Ephrin/Eph signaling controls neuronal ploidy in the developing neocortex. Our study indicates that CitK integrates intracellular and extracellular signals provided by the local environment to coordinate completion of cytokinesis.

Neuropeptide FF (NPFF) interacts with specific receptors to modulate opioid functions in the central nervous system. On dissociated neurons and neuroblastoma cells (SH-SY5Y) transfected with NPFF receptors, NPFF acts as a functional antagonist of μ-opioid (MOP) receptors by attenuating the opioid-induced inhibition of calcium conductance. In the SH-SY5Y model, MOP and NPFF(2) receptors have been shown to heteromerize. To understand the molecular mechanism involved in the anti-opioid activity of NPFF, we have investigated the phosphorylation status of the MOP receptor using phospho-specific antibody and mass spectrometry. Similarly to direct opioid receptor stimulation, activation of the NPFF(2) receptor by [D-Tyr-1-(NMe)Phe-3]NPFF (1DMe), an analog of NPFF, induced the phosphorylation of Ser-377 of the human MOP receptor. This heterologous phosphorylation was unaffected by inhibition of second messenger-dependent kinases and, contrarily to homologous phosphorylation, was prevented by inactivation of G(i/o) proteins by pertussis toxin. Using siRNA knockdown we could demonstrate that 1DMe-induced Ser-377 cross-phosphorylation and MOP receptor loss of function were mediated by the G protein receptor kinase GRK2. In addition, mass spectrometric analysis revealed that the phosphorylation pattern of MOP receptors was qualitatively similar after treatment with the MOP agonist Tyr-D-Ala-Gly (NMe)-Phe-Gly-ol (DAMGO) or after treatment with the NPFF agonist 1DMe, but the level of multiple phosphorylation was more intense after DAMGO. Finally, NPFF(2) receptor activation was sufficient to recruit β-arrestin2 to the MOP receptor but not to induce its internalization. These data show that NPFF-induced heterologous desensitization of MOP receptor signaling is mediated by GRK2 and could involve transphosphorylation within the heteromeric receptor complex.

The cornea is a transparent, avascular, and highly specialized connective tissue that provides the majority of light refraction in the optical system of the eye. The human cornea is composed of several layers interacting in a complex manner and possessing specific functions, like eye protection and optical clearness. Only few proteomic studies of mammalian cornea have been performed leading to the identification of less than 200 proteins in human corneas. The present study explores the proteome of the intact normal human cornea using a shot-gun nanoLC-MS/MS strategy and an LTQ Orbitrap mass spectrometer. A total of 2070 distinct corneal proteins were identified from five human cornea samples, which represents a 14-fold improvement in the number of proteins identified so far for human cornea. This enlarged dataset of human corneal proteins represents a valuable reference library for further studies on cornea homeostasis and pathophysiology. Network and gene ontology analyses were used to determine biological pathways specific of the human cornea. They allowed the identification of subnetworks of putative importance for corneal diseases, like a redox regulation and oxidative stress network constituted of aldehyde and alcohol dehydrogenases, most of them being described for the first time in human cornea.

International audience; Mammalian proteasomes are macromolecular complexes formed of a catalytic 20S core associated to two regulatory complexes. The 20S core complex consists of four stacked rings of seven α or β subunits. Three β subunits contain a catalytic site and can be replaced by three interferon γ‐inducible counterparts to form the immunoproteasome. Cells may constitutively possess a mixture of both 20S proteasome types leading to a heterogeneous proteasome population. Purified rat 20S proteasome has been separated in several chromatographic fractions indicating an even higher degree of complexity in 20S proteasome subunit composition. This complexity may arise from the presence of subunit isoforms, as previously detected in purified human erythrocyte 20S proteasome. In this study, we have used a quantitative proteomic approach based on two‐dimensional gel electrophoresis and isotope‐coded affinity tag (ICAT™) labeling to quantify the variations in subunit composition, including subunit isoforms, of 20S proteasomes purified from different cells. The protocol has been adapted to the analysis of low quantities of 20S proteasome complexes. The strategy has then been validated using standard proteins and has been applied to the comparison of 20S proteasomes from erythrocytes and U937 cancer cells. The results obtained show that this approach represents a valuable tool for the study of 20S proteasome heterogeneity.

Aurora-A protein kinase, which is the product of an oncogene, is required for the assembly of a functional mitotic apparatus and the regulation of cell ploidy. Overexpression of Aurora-A in tumour cells has been correlated with cancer susceptibility and poor prognosis. Aurora-A activity is required for the recruitment of CDK1-cyclin B1 to the centrosome prior to its activation and the commitment of the cell to mitosis. In this report, we demonstrate that the CDC25B phosphatase, an activator of cyclin dependent kinases at mitosis, is phosphorylated both in vitro and in vivo by Aurora-A on serine 353 and that this phosphorylated form of CDC25B is located at the centrosome during mitosis. Knockdown experiments by RNAi confirm that the centrosome phosphorylation of CDC25B on S353 depends on Aurora-A kinase. Microinjection of antibodies against phosphorylated S353 results in a mitotic delay whilst overexpression of a S353 phosphomimetic mutant enhances the mitotic inducing effect of CDC25B. Our results demonstrate that Aurora-A phosphorylates CDC25B in vivo at the centrosome during mitosis. This phosphorylation might locally participate in the control of the onset of mitosis. These findings re-emphasise the role of the centrosome as a functional integrator of the pathways contributing to the triggering of mitosis.

Human dual-specificity phosphatases CDC25 (A, B and C) play an important role in the control of cell cycle progression by activating the cyclin-dependent kinases (CDKs). Regulation of these phosphatases during the cell cycle involves post-translational modifications such as phosphorylation and protein-protein interactions. Given the suspected involvement of the protein kinase CK2 at the G2/M transition, we have investigated its effects on the CDC25B phosphatase. We show that in vitro CK2 phosphorylates CDC25B, but not CDC25C. Mass spectrometry analysis demonstrates that at least two serine residues, Ser-186 and Ser-187, are phosphorylated in vivo. We also report that CDC25B interacts with CK2, and this interaction, mediated by the CK2beta regulatory subunit, involves domains that are located within the first 55 amino acids of CK2beta and between amino acids 122 and 200 on CDC25B. This association was confirmed in vivo, in Sf9 insect cells and in U(2)OS human cells expressing an HA epitope-tagged CDC25B. Finally, we demonstrate that phosphorylation of CDC25B by protein kinase CK2 increases the catalytic activity of the phosphatase in vitro as well as in vivo. We discuss the possibility that CDC25B phosphorylation by CK2 could play a role in the regulation of the activity of CDC25B as a starter of mitosis.

The neuropeptide FF2 (NPFF2) receptor belongs to the rhodopsin family of G protein-coupled receptors and mediates the effects of several related RFamide neuropeptides. One of the main pharmacological interests of this system resides in its ability to regulate endogenous opioid systems, making it a potential target to reduce the negative effects of chronic opioid use. Phosphorylation of intracellular residues is the most extensively studied post-translational modification regulating G protein-coupled receptor activity. However, until now, no information concerning NPFF2 receptor phosphorylation is available. In this study, we combined mass spectrometric analysis and site-directed mutagenesis to analyze for the first time the phosphorylation pattern of the NPFF2 receptor and the role of the various phosphorylation sites in receptor signaling, desensitization, and trafficking in a SH-SY5Y model cell line. We identified the major, likely GRK-dependent, phosphorylation cluster responsible for acute desensitization, (412)TNST(415) at the end of the C terminus of the receptor, and additional sites involved in desensitization ((372)TS(373)) and internalization (Ser(395)). We thus demonstrate the key role played by phosphorylation in the regulation of NPFF2 receptor activity and trafficking. Our data also provide additional evidence supporting the concept that desensitization and internalization are partially independent processes relying on distinct phosphorylation patterns.

We previously identified the heterogeneous ribonucleoprotein SAF-A/hnRNP U as a substrate for DNA-PK, a protein kinase involved in DNA damage response (DDR). Using laser micro-irradiation in human cells, we report here that SAF-A exhibits a two-phase dynamics at sites of DNA damage, with a rapid and transient recruitment followed by a prolonged exclusion. SAF-A recruitment corresponds to its binding to Poly(ADP-ribose) while its exclusion is dependent on the activity of ATM, ATR and DNA-PK and reflects the dissociation from chromatin of SAF-A associated with ongoing transcription. Having established that SAF-A RNA-binding domain recapitulates SAF-A dynamics, we show that this domain is part of a complex comprising several mRNA biogenesis proteins of which at least two, FUS/TLS and TAFII68/TAF15, exhibit similar biphasic dynamics at sites of damage. Using an original reporter for live imaging of DNA:RNA hybrids (R-loops), we show a transient transcription-dependent accumulation of R-loops at sites of DNA damage that is prolonged upon inhibition of RNA biogenesis factors exclusion. We propose that a new component of the DDR is an active anti-R-loop mechanism operating at damaged transcribed sites which includes the exclusion of mRNA biogenesis factors such as SAF-A, FUS and TAF15.

Cytomegalovirus (CMV) is the most common cause of congenital infection, and is a major cause of sensorineural hearing loss and neurological disabilities. Evaluating the risk for a CMV infected fetus to develop severe clinical symptoms after birth is crucial to provide appropriate guidance to pregnant women who might have to consider termination of pregnancy or experimental prenatal medical therapies. However, establishing the prognosis before birth remains a challenge. This evaluation is currently based upon fetal imaging and fetal biological parameters, but the positive and negative predictive values of these parameters are not optimal, leaving room for the development of new prognostic factors. Here, we compared the amniotic fluid peptidome between asymptomatic fetuses who were born as asymptomatic neonates and symptomatic fetuses who were either terminated in view of severe cerebral lesions or born as severely symptomatic neonates. This comparison allowed us to identify a 34-peptide classifier in a discovery cohort of 13 symptomatic and 13 asymptomatic neonates. This classifier further yielded 89% sensitivity, 75% specificity and an area under the curve of 0.90 to segregate 9 severely symptomatic from 12 asymptomatic neonates in a validation cohort, showing an overall better performance than that of classical fetal laboratory parameters. Pathway analysis of the 34 peptides underlined the role of viral entry in fetuses with severe brain disease as well as the potential importance of both beta-2-microglobulin and adiponectin to protect the injured fetal brain infected with CMV. The results also suggested the mechanistic implication of the T calcium channel alpha-1G (CACNA1G) protein in the development of seizures in severely CMV infected children. These results open a new field for potential therapeutic options. In conclusion, this study demonstrates that amniotic fluid peptidome analysis can effectively predict the severity of congenital CMV infection. This peptidomic classifier may therefore be used in clinical settings during pregnancy to improve prenatal counseling., Author Summary CMV is the most common cause of congenital infection, and can result in significant neonatal morbidity and neurological disabilities. The birth prevalence of congenital CMV is estimated at 0.7% worldwide, and 10 to 20% of these neonates develop severe symptoms. In such cases the outcome is generally poor. Therefore, identification of additional prognostic markers is crucial for prenatal counseling in cases with an infected fetus. This may influence the decision of continuing with the pregnancy or requesting its termination, but also the decision of starting experimental antiviral therapy. The pathophysiology of CMV brain injury is not completely understood, and the identification of new biomarkers of CMV infection might also pave the way towards the development of new therapeutic alternatives. Here, we apply a recently developed and modern non-targeted peptidomics approach to amniotic fluid obtained from symptomatic and asymptomatic CMV-infected fetuses/neonates, followed by network analysis of the peptides of interest in the context of fetal infection and in relation with outcome. Our study identified 34 amniotic fluid peptides that form new prognostic biomarkers that could be used in clinical settings to improve prenatal counseling. In addition, this study provides novel mechanistic insight into the pathobiology of CMV congenital disease.

International audience; Spb4 is a putative ATP-dependent RNA helicase that is required for proper processing of 27SB pre-rRNAs and therefore for 60S ribosomal subunit biogenesis. To define the timing of association of this protein with preribosomal particles, we have studied the composition of complexes that copurify with Spb4 tagged by tandem affinity purification (TAP-tagged Spb4). These complexes contain mainly the 27SB pre-rRNAs and about 50 ribosome biogenesis proteins, primarily components of early pre-60S ribosomal particles. To a lesser extent, some protein factors of 90S preribosomal particles and the 35S and 27SA pre-rRNAs also copurify with TAP-tagged Spb4. Moreover, we have obtained by site-directed mutagenesis an allele that results in the R360A substitution in the conserved motif VI of the Spb4 helicase domain. This allele causes a dominant-negative phenotype when overexpressed in the wild-type strain. Cells expressing Spb4(R360A) display an accumulation of 35S and 27SB pre-rRNAs and a net 40S ribosomal subunit defect. TAP-tagged Spb4(R360A) displays a greater steady-state association with 90S preribosomal particles than TAP-tagged wild-type Spb4. Together, our data indicate that Spb4 is a component of early nucle(ol)ar pre-60S ribosomal particles containing 27SB pre-rRNA. Apparently, Spb4 binds 90S preribosomal particles and dissociates from pre-60S ribosomal particles after processing of 27SB pre-rRNA.

Highlights: {yields} Phosphorylation of CDC25B by CDK1 enhances its substrate properties for PLK1 in vitro. {yields} Sequential phosphorylation of CDC25B is analyzed using {sup 16}O and {sup 18}O ATP. {yields} Thirteen sites phosphorylated by PLK1 have been identified. -- Abstract: CDC25 (A, B and C) phosphatases control cell cycle progression through the timely dephosphorylation and activation of cyclin-dependent kinases (CDK). At mitosis the CDC25B phosphatase activity is dependent on its phosphorylation by multiple kinases impinging on its localisation, stability and catalytic activity. Here we report that prior phosphorylation of CDC25B by CDK1 enhances its substrate properties for PLK1 in vitro, and we also show that phosphorylated S50 serves as a docking site for PLK1. Using a sophisticated strategy based on the sequential phosphorylation of CDC25B with {sup 16}O and {sup 18}O ATP prior to nanoLC-MS/MS analysis we identified 13 sites phosphorylated by PLK1. This study illustrates the complexity of the phosphorylation pattern and of the subsequent regulation of CDC25B activity.

International audience; In Saccharomyces cerevisiae, ribosome biogenesis requires, in addition to rRNA and ribosomal proteins, a myriad of small nucleolar RNAs (snoRNAs) and over two hundred protein trans-acting factors. There are protein trans-acting factors predicted to participate in ribosome biogenesis that have not been so far characterized. Here, we report the functional analysis of the Nucleolar protein 6 (Nop6) in ribosome biogenesis. Our results show that Nop6 is needed for optimal 40S ribosomal subunit biogenesis. Deletion of NOP6 leads to an appropriate 20% reduction in 18S rRNA levels and therefore in 40S ribosomal subunits. This is due to mild inhibition of pre-rRNA processing at cleavage site A 2. Tandem affinity purification followed by mass spectrometry and northern blot analyses indicate that Nop6 is a component of 90S pre-ribosomal particles. rDNA chromatin immunoprecipitation experiments and analysis of the intracellular localisation of Nop6-eGFP after in vivo shut down of pre-rRNA transcription strongly suggest that Nop6 binds to the pre-rRNA early during transcription. Genetic data suggest that Nop6 and the snoRNA snR57 both interact similarly with the protein trans-acting factor Nep1. It has been proposed that snR57 and Nep1 participate in a pre-rRNA conformational switch that allows the proper assembly of 40S ribosomal protein S19. Our results strongly suggest that the role Nop6 might have in this conformational switch is independent of snR57.

International audience; Prp43p is a RNA helicase required for pre-mRNA splicing and for the synthesis of large and small ribosomal subunits. The molecular functions and modes of regulation of Prp43p during ribosome biogenesis remain unknown. We demonstrate that the G-patch protein Pfa1p, a component of pre-40S pre-ribosomal particles, directly interacts with Prp43p. We also show that lack of Gno1p, another G-patch protein associated with Prp43p, specifically reduces Pfa1p accumulation, whereas it increases the levels of the pre-40S pre-ribosomal particle component Ltv1p. Moreover, cells lacking Pfa1p and depleted for Ltv1p show strong 20S pre-rRNA accumulation in the cytoplasm and reduced levels of 18S rRNA. Finally, we demonstrate that Pfa1p stimulates the ATPase and helicase activities of Prp43p. Truncated Pfa1p variants unable to fully stimulate the activity of Prp43p fail to complement the 20S pre-rRNA processing defect of Deltapfa1 cells depleted for Ltv1p. Our results strongly suggest that stimulation of ATPase/helicase activities of Prp43p by Pfa1p is required for efficient 20S pre-rRNA-to-18S rRNA conversion.

Aiming to identify novel phosphorylation sites in response to DNA double-strand breaks (DSB) inducers, we have isolated a phosphorylation site on KU70. Unexpectedly, a rabbit antiserum raised against this site cross-reacted with a 120 kDa protein in cells treated by DNA DSB inducers. We identified this protein as SAF-A/hnRNP U, an abundant and essential nuclear protein containing regions binding DNA or RNA. The phosphorylation site was mapped at S59 position in a sequence context favoring a "S-hydrophobic" consensus model for DNA-PK phosphorylation site in vivo. This site was exclusively phosphorylated by DNA-PK in response to DNA DSB inducers. In addition, the extent and duration of this phosphorylation was in inverse correlation with the capacity of the cells to repair DSB by Nonhomologous End Joining. These results bring a new link between the hnRNP family and the DNA damage response. Addtionaly, the mapped phospho-site on SAF-A might serve as a potential bio-marker for DNA-PK activity in academic studies and clinical analyses of DNA-PK activators or inhibitors.

NanoLC-MS/MS analysis was used to characterize the phosphorylation pattern in vivo of CDC25B3 (phosphatase splice variant 1) expressed in a human cell line and to compare it to the phosphorylation of CDC25B3 by Cdk1/cyclin B and Chk1 in vitro. Cellular CDC25B3 was purified from U2OS cells conditionally overexpressing the phosphatase. Eighteen sites were detectably phosphorylated in vivo. Nearly all existing (S/T)P sites were phosphorylated in vivo and in vitro. Eight non(S/T)P sites were phosphorylated in vivo. All these sites could be phosphorylated by kinase Chk1, which phosphorylated a total of 11 sites in vitro, with consensus sequence (R/K) X(2-3) (S/P)-non P. Nearly half of the sites identified in this study were not previously described and were not homologous to sites reported to be phosphorylated in other CDC25 species. We also show that in vivo a significant part of CDC25B molecules can be hyperphosphorylated, with up to 13 phosphates per phosphatase molecule.

CDC25C phosphatase is a key actor in cell cycle progression that controls the activation of CDK1-cyclin B at mitosis. Its activity is known to be highly regulated by a number of signalling pathway-activated kinases resulting in its phosphorylation on multiple residues. In this study, we have purified CDC25C from cells and have used a proteomic approach to identify new regulatory phosphorylations. Here, we report the identification by mass spectrometry of a peptide monophosphorylated on serine 263. We demonstrate by cell imaging that mutation of S263 to alanine leads to a nuclear accumulation of CDC25C that is further reinforced by leptomycin-B. We propose that phosphorylation at S263 is part of the regulatory mechanism that modulates nuclear import of CDC25C, thus preventing cytoplasm to nucleus shuttling.