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Protein phosphatase 1 (PP1) is expressed in all eukaryotic cells and catalyzes a sizable fraction of protein Ser/Thr dephosphorylation events. It is tightly regulated in space and time through association with a wide array of regulatory interactors of protein phosphatase one (RIPPOs). Suppressor-of-Dis2-number 2 (SDS22) and Inhibitor-3 (I3), which form a ternary complex with PP1, are the first two evolved and most widely expressed RIPPOs. Their deletion causes mitotic-arrest phenotypes and is lethal in some organisms. The role of SDS22 and I3 in PP1 regulation has been a mystery for decades as they were independently identified as both activators and inhibitors of PP1. This conundrum has largely been solved by recent reports showing that SDS22 and I3 control multiple steps of the life course of PP1. Indeed, they contribute to (a) the stabilization and activation of newly translated PP1, (b) the translocation of PP1 to the nucleus, and (c) the storage of PP1 as a reserve for holoenzyme assembly. Preliminary evidence suggests that SDS22 and I3 may also function as scavengers of released or aged PP1 for re-use in holoenzyme assembly or proteolytical degradation, respectively. Hence, SDS22 and I3 are emerging as master regulators of the life course of PP1.

Nuclear Inhibitor of PP1 (NIPP1) is a conserved regulatory subunit of protein phosphatase PP1. The selective deletion of NIPP1 in mouse liver parenchymal cells or skin epidermal cells culminates in a late-onset hyperproliferation of a subset of resident progenitor cells. Although a hyperplastic phenotype is usually tumor promoting, we show here that the absence of NIPP1 conferred a strong resistance to chemically induced hepatocellular or skin carcinoma. The ablation of NIPP1 did not affect the metabolism of the administered mutagens (diethylnitrosamine or 7,12-dimethylbenz[a]anthracene), but reduced the conversion of mutagen-induced covalent DNA modifications into cancer-initiating mutations. This reduced sensitivity to mutagens correlated with an enhanced DNA-damage response and an augmented expression of rate-limiting DNA-repair proteins (MGMT in liver, XPD and XPG in skin), hinting at an increased DNA-repair capacity. Our data identify NIPP1 as a repressor of DNA repair and as a promising target for novel cancer prevention and treatment therapies. ispartof: ONCOGENESIS vol:9 issue:3 ispartof: location:United States status: published

Phosphoprotein phosphatase‐1 (PP1) is a key player in the regulation of phospho‐serine (pSer) and phospho‐threonine (pThr) dephosphorylation and is involved in a large fraction of cellular signaling pathways. Aberrant activity of PP1 has been linked to many diseases, including cancer and heart failure. Besides a well‐established activity control by regulatory proteins, an inhibitory function for phosphorylation (p) of a Thr residue in the C‐terminal intrinsically disordered tail of PP1 has been demonstrated. The associated phenotype of cell‐cycle arrest was repeatedly proposed to be due to autoinhibition of PP1 through either conformational changes or substrate competition. Here, we use PP1 variants created by mutations and protein semisynthesis to differentiate between these hypotheses. Our data support the hypothesis that pThr exerts its inhibitory function by mediating protein complex formation rather than by a direct mechanism of structural changes or substrate competition., Interrogating a mechanistic black box: Phosphoprotein phosphatase 1 (PP1) is among the major serine/threonine phosphatases and is involved in numerous pathways. Autoinhibition by phosphorylation of Thr320 at the C‐terminus leads to cell‐cycle arrest and neuronal defects. However, the mechanism underlying this regulatory phosphorylation site is unknown. Herein, we investigate potential mechanisms.

PP1 is a conserved eukaryotic serine/threonine phosphatase that regulates many aspects of mitosis and meiosis, often working in concert with other phosphatases, such as CDC14 and CDC25. The proliferative stages of the malaria parasite life cycle include sexual development within the mosquito vector, with male gamete formation characterized by an atypical rapid mitosis, consisting of three rounds of DNA synthesis, successive spindle formation with clustered kinetochores, and a meiotic stage during zygote to ookinete development following fertilization. It is unclear how PP1 is involved in these unusual processes. Using real-time live-cell and ultrastructural imaging, conditional gene knockdown, RNA-seq and proteomic approaches, we show thatPlasmodiumPP1 is implicated in both mitotic exit and, potentially, establishing cell polarity during zygote development in the mosquito midgut, suggesting that small molecule inhibitors of PP1 should be explored for blocking parasite transmission.

PP1 is a conserved eukaryotic serine/threonine phosphatase that regulates many aspects of mitosis and meiosis, often working in concert with other phosphatases, such as CDC14 and CDC25. The proliferative stages of the malaria parasite life cycle include sexual development within the mosquito vector, with male gamete formation characterized by an atypical rapid mitosis, consisting of three rounds of DNA synthesis, successive spindle formation with clustered kinetochores, and a meiotic stage during zygote to ookinete development following fertilization. It is unclear how PP1 is involved in these unusual processes. Using real-time live-cell and ultrastructural imaging, conditional gene knockdown, RNA-seq and proteomic approaches, we show that Plasmodium PP1 is implicated in both mitotic exit and, potentially, establishing cell polarity during zygote development in the mosquito midgut, suggesting that small molecule inhibitors of PP1 should be explored for blocking parasite transmission., Zeeshan et al. used real-time live-cell and ultrastructural imaging, conditional gene knockdown, RNA-seq and proteomic approaches to implicate Plasmodium PP1 in both mitotic exit and, potentially, establishing cell polarity during zygote development in the mosquito midgut. They suggest that small molecule inhibitors of PP1 should be explored for blocking parasite transmission.

RepoMan is a chromosome-associated scaffold protein that integrates signaling of multiple kinases and phosphatases to coordinate spindle-kinetochore interactions, chromosome (de)condensation and nuclear envelope (dis)assembly during mitosis. Another key mitotic event is the assembly of a microtubule-based spindle, which involves redundant pathways emanating from the centrosomes, microtubules and chromosomes. Here we describe a novel mitotic function of RepoMan in regulating chromosome-dependent microtubule assembly. At limiting concentrations of microtubule-destabilizing agents, RepoMan-depleted cells showed enhanced chromosome clustering. This clustering was completely dependent on the partial inhibition of microtubule growth originating from the chromosome-dependent pathway. We also demonstrated that RepoMan interacts with prime regulators of the chromosome-dependent spindle assembly such as NuSAP1, NuMA, and TPX2. In addition, RepoMan was required to enable or maintain phosphorylation of NuSAP1 at CDK sites, thereby enabling activation of NuSAP1 through dissociation of inhibitory importin β/7. Our data identify RepoMan as an enhancer of microtubule assembly at chromosomes. ispartof: CELL CYCLE vol:19 issue:22 pages:3029-3041 ispartof: location:United States status: published

Lamin B1 plays an important role in the nuclear envelope stability, the regulation of gene expression, and neural development. Duplication of LMNB1, or missense mutations increasing LMNB1 expression, are associated with autosomal-dominant leukodystrophy. On the basis of its role in neurogenesis, it has been postulated that LMNB1 variants could cause microcephaly. Here, we confirm this hypothesis with the identification of de novo mutations in LMNB1 in seven individuals with pronounced primary microcephaly (ranging from -3.6 to -12 SD) associated with relative short stature and variable degree of intellectual disability and neurological features as the core symptoms. Simplified gyral pattern of the cortex and abnormal corpus callosum were noted on MRI of three individuals, and these individuals also presented with a more severe phenotype. Functional analysis of the three missense mutations showed impaired formation of the LMNB1 nuclear lamina. The two variants located within the head group of LMNB1 result in a decrease in the nuclear localization of the protein and an increase in misshapen nuclei. We further demonstrate that another mutation, located in the coil region, leads to increased frequency of condensed nuclei and lower steady-state levels of lamin B1 in proband lymphoblasts. Our findings collectively indicate that de novo mutations in LMNB1 result in a dominant and damaging effect on nuclear envelope formation that correlates with microcephaly in humans. This adds LMNB1 to the growing list of genes implicated in severe autosomal-dominant microcephaly and broadens the phenotypic spectrum of the laminopathies.

Inhibitor-2 (I2) ranks amongst the most ancient regulators of protein phosphatase-1 (PP1). It is a small, intrinsically disordered protein that was originally discovered as a potent inhibitor of PP1. However, later investigations also characterized I2 as an activator of PP1 as well as a chaperone for PP1 folding. Numerous studies disclosed the importance of I2 for diverse cellular processes but did not describe a unifying molecular principle of PP1 regulation. We have re-analyzed the literature on I2 in the light of current insights of PP1 structure and regulation. Extensive biochemical data, largely ignored in the recent I2 literature, provide substantial indirect evidence for a role of I2 as a loader of active-site metals. In addition, I2 appears to function as a competitive inhibitor of PP1 in higher eukaryotes. The published data also demonstrate that several segments of I2 that remain unstructured in the PP1 : I2 complex are in fact essential for PP1 regulation. Together, the available data identify I2 as a dynamic activity-modulator of PP1.

Protein phosphatase 1 (PP1) catalyzes more than half of all phosphoserine/threonine dephosphorylation reactions in mammalian cells. In vivo PP1 does not exist as a free catalytic subunit but is always associated with at least one regulatory PP1-interacting protein (PIP) to generate a large set of distinct holoenzymes. Each PP1 complex controls the dephosphorylation of only a small subset of PP1 substrates. We screened the literature for genetically engineered mouse models and identified models for all PP1 isoforms and 104 PIPs. PP1 itself and at least 49 PIPs were connected to human disease-associated phenotypes. Additionally, phenotypes related to 17 PIPs were clearly linked to altered PP1 function, while such information was lacking for 32 other PIPs. We propose structural reverse genetics, which combines structural characterization of proteins with mouse genetics, to identify new PP1-related therapeutic targets. The available mouse models confirm the pleiotropic action of PP1 in health and diseases., Highlights • Four protein phosphatases 1 (PP1) isoforms and >200 PP1-interacting proteins (PIPs) • Genetically engineered mice of 49 PIPs display human disease-associated phenotypes. • The use of structural reverse genetics to identify the PP1-dependent phenotypes • Various interaction sites in PP1:PIP complexes suit for therapeutic targeting.

Chromosome segregation during mitosis is antagonistically regulated by the Aurora-B kinase and RepoMan (recruits PP1 onto mitotic chromatin at anaphase)-associated phosphatases PP1/PP2A. Aurora B is overexpressed in many cancers but, surprisingly, this only rarely causes lethal aneuploidy. Here we show that RepoMan abundance is regulated by the same mechanisms that control Aurora B, including FOXM1-regulated expression and proteasomal degradation following ubiquitination by APC/C-CDH1 or SCFFBXW7. The deregulation of these mechanisms can account for the balanced co-overexpression of Aurora B and RepoMan in many cancers, which limits chromosome segregation errors. In addition, Aurora B and RepoMan independently promote cancer cell proliferation by reducing checkpoint--induced cell-cycle arrest during interphase. The co-up-regulation of RepoMan and Aurora B in tumors is inversely correlated with patient survival, underscoring its potential importance for tumor progression. Finally, we demonstrate that high RepoMan levels sensitize cancer cells to Aurora-B inhibitors. Hence, the co-up-regulation of RepoMan and Aurora B is associated with tumor aggressiveness but also exposes a vulnerable target for therapeutic intervention. ispartof: MOLECULAR BIOLOGY OF THE CELL vol:31 issue:6 pages:419-438 ispartof: location:United States status: published

Germ cell proliferation is epigenetically controlled, mainly through DNA methylation and histone modifications. However, the pivotal epigenetic regulators of germ cell self-renewal and differentiation in postnatal testis are still poorly defined. The histone methyltransferase enhancer of zeste homolog 2 (EZH2) is the catalytic subunit of Polycomb repressive complex 2, represses target genes through trimethylation of histone H3 at Lys-27 (H3K27me3), and interacts (in)directly with both protein phosphatase 1 (PP1) and nuclear inhibitor of PP1 (NIPP1). Here, we report that postnatal, testis-specific ablation of NIPP1 in mice results in loss of EZH2 and reduces H3K27me3 levels. Mechanistically, the NIPP1 deletion abrogated PP1-mediated EZH2 dephosphorylation at two cyclin-dependent kinase sites (Thr-345/487), thereby generating hyperphosphorylated EZH2, which is a substrate for proteolytic degradation. Accordingly, alanine mutation of these residues prolonged the half-life of EZH2 in male germ cells. Our study discloses a key role for the PP1:NIPP1 holoenzyme in stabilizing EZH2 and maintaining the H3K27me3 mark on genes that are important for germ cell development and spermatogenesis. ispartof: JOURNAL OF BIOLOGICAL CHEMISTRY vol:293 issue:47 pages:18031-18039 ispartof: location:United States status: published

Protein phosphatase 1 (PP1) is expressed in all eukaryotic cells and catalyzes a substantial fraction of phosphoserine/threonine dephosphorylation reactions. It forms stable complexes with PP1-interacting proteins (PIPs) that guide the phosphatase throughout its life cycle and control its fate and function. The diversity of PIPs is huge (≈200 in vertebrates), and most of them combine short linear motifs to form large and unique interaction interfaces with PP1. Many PIPs have separate domains for PP1 anchoring, PP1 regulation, substrate recruitment and subcellular targeting, which enable them to direct associated PP1 to a specific subset of substrates and mediate acute activity control. Hence, PP1 functions as the catalytic subunit of a large number of multimeric holoenzymes, each with its own subset of substrates and mechanism(s) of regulation.

Nuclear inhibitor of protein phosphatase 1 (NIPP1) is a ubiquitously expressed nuclear protein that regulates functions of protein serine/threonine phosphatase-1 in cell proliferation and lineage specification. The role of NIPP1 in tissue homeostasis is not fully understood. This study shows that the selective deletion of NIPP1 in mouse epidermis resulted in epidermal hyperproliferation, a reduced adherence of basal keratinocytes, and a gradual decrease in the stemness of hair follicle stem cells, culminating in hair loss. This complex phenotype was associated with chronic sterile skin inflammation and could be partially rescued by dexamethasone treatment. NIPP1-deficient keratinocytes massively expressed proinflammatory chemokines and immunomodulatory proteins in a cell-autonomous manner. Chemokines subsequently induced the recruitment and activation of immune cells, in particular conventional dendritic cells and Langerhans cells, accounting for the chronic inflammation phenotype. The data identifies NIPP1 as a key regulator of epidermal homeostasis and as a potential target for the treatment of inflammatory skin diseases. ispartof: Journal Of Investigative Dermatology vol:140 issue:8 pages:1576-1588 ispartof: location:United States status: published

The aminoguanidine compound robenidine is widely used as an antibiotic for the control of coccidiosis, a protozoal infection in poultry and rabbits. Interestingly, robenidine is structurally similar to guanabenz (analogs), which are currently undergoing clinical trials as cytoprotective agents for the management of neurodegenerative diseases. Here we show that robenidine and guanabenz protect cells from a tunicamycin-induced unfolded protein response to a similar degree. Both compounds also reduced the tumor necrosis factor α-induced activation of NF-κB. The cytoprotective effects of guanabenz (analogs) have been explained previously by their ability to maintain eIF2α phosphorylation by allosterically inhibiting protein phosphatase PP1:PPP1R15A. However, using a novel split-luciferase-based protein-protein interaction assay, we demonstrate here that neither robenidine nor guanabenz disrupt the interaction between PPP1R15A and either PP1 or eIF2α in intact cells. Moreover, both drugs also inhibited the unfolded protein response in cells that expressed a nonphosphorylatable mutant (S51A) of eIF2α. Our results identify robenidine as a PP1:PPP1R15A-independent cytoprotective compound that holds potential for the management of protein misfolding-associated diseases. ispartof: JOURNAL OF BIOLOGICAL CHEMISTRY vol:294 issue:36 pages:13478-13486 ispartof: location:United States status: published

Extracellular 2',3'-cyclic GMP-AMP (cGAMP) has a critical immune-transmitter role in the tumor microenvironment (TME). Tumor cells produce and secrete cGAMP, which primes immune cells for tumor rejection through STING (stimulator of interferon genes) signalling (Marcus et al., Immunity 2018; Carozza et al., Nature Cancer 2020). Ecto-nucleotide pyrophosphatase/phosphodiesterase-1 (ENPP1) is the only enzyme known to hydrolyze extracellular cGAMP. In concert with the ectoATPase CD39 and the 5'-nucleotidase CD73, ENPP1 also contributes to the generation of an immunosuppressive TME by converting extracellular ATP into adenosine. ENPP1 is overexpressed in a number of tumor types, including breast cancer, liver cancer, thyroid cancer and sarcomas, and has been associated with poor outcome. Hence, inhibition of ENPP1 is an emerging strategy to augment anti-tumor immunity by stabilizing extracellular cGAMP and ATP, thereby turning cold tumors into immunologically hot tumors. Here, we report on the identification and characterization of novel chemical series of ENPP1 inhibitors. A drug-like small-molecule library (~160,000 compounds) was screened to identify inhibitors of ENPP1-mediated cGAMP hydrolysis. Hits were confirmed to also inhibit ATP hydrolysis by ENPP1, but not nucleotide hydrolysis mediated by the closely related ENPP2 (autotaxin) enzyme. A selection of hits, chemically distinct from previously reported ENPP1 inhibitors (such as those based on QS1; Carroza et al, Cell Chem Biol, 2020), were subjected to hit-to-lead optimization supported by structure-based guidance. This led to the identification of ENPP1 inhibitors with sub-nM potency on ENPP1 in biochemical assays, which maintained a >1000x selectivity window with respect to ENPP2 and to other phosphodiesterases. These compounds translated well to stabilization of nucleotides in the presence of ENPP1-overexpressing cancer cell lines, with IC50 values in the nM range. A selection of compounds was profiled for DMPK (drug metabolism and pharmacokinetic) parameters, and compounds with a suitable profile were prioritized for in vivo evaluation. Orally bio-available, metabolically stable compounds were assessed in mouse syngeneic tumor models, selected on the basis of high ENPP1 and high cGAS (cGAMP synthase) expression. Stabilization of cGAMP, activation of a STING-mediated cytokine response and immune-cell infiltration/activation in the TME were used as pharmacodynamic endpoints. An update on in vivo efficacy data in a range of ENPP1-positive tumor models will be provided during the presentation. These novel orally bioavailable ENPP1 inhibitors unleash local, TME-restricted, innate immune activation, and hold the promise to overcome the current limitations of direct STING agonists. Citation Format: Matthias Versele, Javier del Pino Garcia, Ilse Vandecaetsbeek, Karolien Castermans, Ranie Kellens, Wanda Haeck, Hugo Klaassen, Arnaud Bourin, Dries De Clercq, Sara Allasia, Sandro Boland, Arnaud Marchand, Patrick Chaltin, Mathieu Bollen. Discovery of novel potent and orally bioavailable small-molecule inhibitors of ENPP1 to stabilize cGAMP and ATP in the tumor microenvironment and boost anti-tumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 53.

Protein kinase MELK has oncogenic properties and is highly overexpressed in some tumors. In the present study, we show that a novel MELK inhibitor causes both the inhibition and degradation of MELK, culminating in replication stress and a senescence phenotype., Maternal embryonic leucine zipper kinase (MELK), a serine/threonine protein kinase, has oncogenic properties and is overexpressed in many cancer cells. The oncogenic function of MELK is attributed to its capacity to disable critical cell-cycle checkpoints and reduce replication stress. Most functional studies have relied on the use of siRNA/shRNA-mediated gene silencing. In the present study, we have explored the biological function of MELK using MELK-T1, a novel and selective small-molecule inhibitor. Strikingly, MELK-T1 triggered a rapid and proteasome-dependent degradation of the MELK protein. Treatment of MCF-7 (Michigan Cancer Foundation-7) breast adenocarcinoma cells with MELK-T1 induced the accumulation of stalled replication forks and double-strand breaks that culminated in a replicative senescence phenotype. This phenotype correlated with a rapid and long-lasting ataxia telangiectasia-mutated (ATM) activation and phosphorylation of checkpoint kinase 2 (CHK2). Furthermore, MELK-T1 induced a strong phosphorylation of p53 (cellular tumour antigen p53), a prolonged up-regulation of p21 (cyclin-dependent kinase inhibitor 1) and a down-regulation of FOXM1 (Forkhead Box M1) target genes. Our data indicate that MELK is a key stimulator of proliferation by its ability to increase the threshold for DNA-damage tolerance (DDT). Thus, targeting MELK by the inhibition of both its catalytic activity and its protein stability might sensitize tumours to DNA-damaging agents or radiation therapy by lowering the DNA-damage threshold.

The protein Ser/Thr phosphatase PP1 catalyzes an important fraction of protein dephosphorylation events and forms highly specific holoenzymes through an association with regulatory interactors of protein phosphatase one (RIPPOs). The functional characterization of individual PP1 holoenzymes is hampered by the lack of straightforward strategies for substrate mapping. Because efficient substrate recruitment often involves binding to both PP1 and its associated RIPPO, here we examined whether PP1-RIPPO fusions can be used to trap substrates for further analysis. Fusions of an hypoactive point mutant of PP1 and either of four tested RIPPOs accumulated in HEK293T cells with their associated substrates and were co-immunoprecipitated for subsequent identification of the substrates by immunoblotting or MS analysis. Hypoactive fusions were also used to study RIPPOs themselves as substrates for associated PP1. In contrast, substrate trapping was barely detected with active PP1-RIPPO fusions or with nonfused PP1 or RIPPO subunits. Our results suggest that hypoactive fusions of PP1 subunits represent an easy-to-use tool for substrate identification of individual holoenzymes. ispartof: JOURNAL OF BIOLOGICAL CHEMISTRY vol:293 issue:39 pages:15152-15162 ispartof: location:United States status: published

Adeno-associated virus (AAV) is a small humanwhose low immunogenicity and capacity for long-term persistence have led to its widespread use as vector for gene therapy. Despite great recent successes in AAV-based gene therapy, further improvements in vector technology may be hindered by an inadequate understanding of various aspects of basic AAV biology. AAV is unique in that its replication is largely dependent on a helper virus and cellular factors. In the absence of helper virus coinfection, wild-type AAV establishes latency through mechanisms that are not yet fully understood. Challenging the currently held model for AAV latency, we show here that the corepressor Krüppel-associated box domain-associated protein 1 (KAP1) binds the latent AAV2 genome at theORF, leading to trimethylation of AAV2-associated histone 3 lysine 9 and that the inactivation of KAP1 repression is necessary for AAV2 reactivation and replication. We identify a viral mechanism for the counteraction of KAP1 in which interference with the KAP1 phosphatase protein phosphatase 1 (PP1) by the AAV2 Rep proteins mediates enhanced phosphorylation of KAP1-S824 and thus relief from KAP1 repression. Furthermore, we show that this phenomenon involves recruitment of the NIPP1 (nuclear inhibitor of PP1)-PP1α holoenzyme to KAP1 in a manner dependent upon the NIPP1 FHA domain, identifying NIPP1 as an interaction partner for KAP1 and shedding light on the mechanism through which PP1 regulates cellular KAP1 activity. ispartof: Proceedings of the National Academy of Sciences of the United States of America vol:115 issue:15 pages:E3529-E3538 ispartof: location:United States status: published

The ubiquitously expressed nuclear protein NIPP1 (also known as PPP1R8) recruits phosphoproteins for regulated dephosphorylation by the associated protein phosphatase PP1. To bypass the PP1 titration artifacts seen upon NIPP1 overexpression, we have engineered covalently linked fusions of PP1 and NIPP1, and demonstrate their potential to selectively explore the function of the PP1:NIPP1 holoenzyme. By using inducible stable cell lines, we show that PP1-NIPP1 fusions cause replication stress in a manner that requires both PP1 activity and substrate recruitment via the ForkHead Associated domain of NIPP1. More specifically, PP1-NIPP1 expression resulted in the build up of RNA-DNA hybrids (R-loops), enhanced chromatin compaction and a diminished repair of DNA double-strand breaks (DSBs), culminating in the accumulation of DSBs. These effects were associated with a reduced expression of DNA damage signaling and repair proteins. Our data disclose a key role for dephosphorylation of PP1:NIPP1 substrates in setting the threshold for DNA repair, and indicate that activators of this phosphatase hold therapeutic potential as sensitizers for DNA-damaging agents.

The functional diversity of protein phosphatase-1 (PP1), with its countless substrates, relies on the ordered assembly of alternative PP1 holoenzymes. Here, we show that newly synthesized PP1 is first held by its partners SDS22 and inhibitor-3 (I3) in an inactive complex, which needs to be disassembled by the p97 AAA-ATPase to promote exchange to substrate specifiers. Unlike p97-mediated degradative processes that require the Ufd1-Npl4 ubiquitin adapters, p97 is targeted to PP1 by p37 and related adapter proteins. Reconstitution with purified components revealed direct interaction of the p37 SEP domain with I3 without the need for ubiquitination, and ATP-driven pulling of I3 into the central channel of the p97 hexamer, which triggers dissociation of I3 and SDS22. Thus, we establish regulatory ubiquitin-independent protein complex disassembly as part of the functional arsenal of p97 and define an unanticipated essential step in PP1 biogenesis that illustrates the molecular challenges of ordered subunit exchange. ispartof: MOLECULAR CELL vol:72 issue:4 pages:766-+ ispartof: location:United States status: published

Cole disease is a genodermatosis of pigmentation following a strict dominant mode of inheritance. In this study, we investigated eight patients affected with an overlapping genodermatosis after recessive inheritance. The patients presented with hypo- and hyperpigmented macules over the body, resembling dyschromatosis universalis hereditaria in addition to punctuate palmoplantar keratosis. By homozygosity mapping and whole-exome sequencing, a biallelic p.Cys120Arg mutation in ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) was identified in all patients. We found that this mutation, like those causing dominant Cole disease, impairs homodimerization of the ENPP1 enzyme that is mediated by its two somatomedin-B-like domains. Histological analysis revealed structural and molecular changes in affected skin that were likely to originate from defective melanocytes because keratinocytes do not express ENPP1. Consistently, RNA-sequencing analysis of patient-derived primary melanocytes revealed alterations in melanocyte development and in pigmentation signaling pathways. We therefore conclude that germline ENPP1 cysteine-specific mutations, primarily affecting the melanocyte lineage, cause a clinical spectrum of dyschromatosis, in which the p.Cys120Arg allele represents a recessive and more severe form of Cole disease.

The ubiquitously expressed nuclear protein NIPP1 (also known as PPP1R8) recruits phosphoproteins for regulated dephosphorylation by the associated protein phosphatase PP1. To bypass the PP1 titration artifacts seen upon NIPP1 overexpression, we have engineered covalently linked fusions of PP1 and NIPP1, and demonstrate their potential to selectively explore the function of the PP1:NIPP1 holoenzyme. By using inducible stable cell lines, we show that PP1–NIPP1 fusions cause replication stress in a manner that requires both PP1 activity and substrate recruitment via the ForkHead Associated domain of NIPP1. More specifically, PP1–NIPP1 expression resulted in the build up of RNA–DNA hybrids (R-loops), enhanced chromatin compaction and a diminished repair of DNA double-strand breaks (DSBs), culminating in the accumulation of DSBs. These effects were associated with a reduced expression of DNA damage signaling and repair proteins. Our data disclose a key role for dephosphorylation of PP1:NIPP1 substrates in setting the threshold for DNA repair, and indicate that activators of this phosphatase hold therapeutic potential as sensitizers for DNA-damaging agents.

The integrated stress response (ISR) is regulated by kinases that phosphorylate the α subunit of translation initiation factor 2 and phosphatases that dephosphorylate it. Genetic and biochemical observations indicate that the eIF2αP-directed holophosphatase - a therapeutic target in diseases of protein misfolding - is comprised of a regulatory, PPP1R15, and a catalytic, Protein Phosphatase 1 (PP1) subunit. In mammals, there are two isoforms of the regulatory subunit, PPP1R15A and PPP1R15B, with overlapping roles in promoting the essential function of eIF2αP dephosphorylation. However, conflicting reports have appeared regarding the requirement for an additional co-factor, G-actin, in enabling substrate-specific de-phosphorylation by PPP1R15-containing PP1 holoenzymes. An additional concern relates to the sensitivity of the PPP1R15A-containing PP1 holoenzyme to the [(ochlorobenzylidene)amino]guanidines (Sephin1 or Guanabenz), small molecule proteostasis modulators. It has been suggested that the source and method of purification of the PP1 catalytic subunit and the presence or absence of an N-terminal repeat-containing region in the PPP1R15A regulatory subunit might influence both the requirement for G-actin by the eIF2αP-directed holophosphatase and its sensitivity to inhibitors. Here we report that in the absence of G-actin, PPP1R15A regulatory subunits were unable to accelerate eIF2αP dephosphorylation beyond that affected by a catalytic subunit alone, whether PPP1R15A regulatory subunit had or lacked the N-terminal repeat-containing region and whether paired with native PP1 purified from rabbit muscle, or recombinant PP1 expressed in and purified from bacteria. Furthermore, none of the PPP1R15A-containing PP1c holophosphatases were inhibited by Sephin1 or Guanabenz.

NIPP1 is one of the major nuclear interactors of protein phosphatase PP1. The deletion of NIPP1 in mice is early embryonic lethal, which has precluded functional studies in adult tissues. Hence, we have generated an inducible NIPP1 knockout model using a tamoxifen-inducible Cre recombinase transgene. The inactivation of the NIPP1 encoding alleles (Ppp1r8) in adult mice occurred very efficiently in testis and resulted in a gradual loss of germ cells, culminating in a Sertoli-cell only phenotype. Before the overt development of this phenotype Ppp1r8 −/− testis showed a decreased proliferation and survival capacity of cells of the spermatogenic lineage. A reduced proliferation was also detected after the tamoxifen-induced removal of NIPP1 from cultured testis slices and isolated germ cells enriched for undifferentiated spermatogonia, hinting at a testis-intrinsic defect. Consistent with the observed phenotype, RNA sequencing identified changes in the transcript levels of cell-cycle and apoptosis regulating genes in NIPP1-depleted testis. We conclude that NIPP1 is essential for mammalian spermatogenesis because it is indispensable for the proliferation and survival of progenitor germ cells, including (un)differentiated spermatogonia.

Kinases and phosphatases work antagonistically to control the behaviour of individual substrate molecules. This can be incorrectly extrapolated to imply that they also work antagonistically on the signals or processes that these molecules control. In fact, in many situations kinases and phosphatases work together to positively drive signal responses. We explain how this 'cooperativity' is critical for setting the amplitude, localisation, timing, and shape of phosphorylation signals. We use mitosis to illustrate why these properties are important for controlling mitotic entry, sister chromatid cohesion, kinetochore-microtubule attachments, the spindle assembly checkpoint, mitotic spindle elongation, and mitotic exit. These examples provide a rationale to explain how complex signalling behaviour could rely on similar types of integration within many other biological processes. ispartof: Trends in Cell Biology vol:28 issue:1 pages:6-21 ispartof: location:England status: published

Maternal embryonic leucine zipper kinase (MELK) belongs to the subfamily of AMP-activated Ser/Thr protein kinases. The expression of MELK is very high in glioblastoma-type brain tumors, but it is not clear how this contributes to tumor growth. Here we show that the siRNA-mediated loss of MELK in U87 MG glioblastoma cells causes a G1/S phase cell cycle arrest accompanied by cell death or a senescence-like phenotype that can be rescued by the expression of siRNA-resistant MELK. This cell cycle arrest is mediated by an increased expression of p21WAF1/CIP1, an inhibitor of cyclin-dependent kinases, and is associated with the hypophosphorylation of the retinoblastoma protein and the down-regulation of E2F target genes. The increased expression of p21 can be explained by the consecutive activation of ATM (ataxia telangiectasia mutated), Chk2, and p53. Intriguingly, the activation of p53 in MELK-deficient cells is not due to an increased stability of p53 but stems from the loss of MDMX (mouse double minute-X), an inhibitor of p53 transactivation. The activation of the ATM-Chk2 pathway in MELK-deficient cells is associated with the accumulation of DNA double-strand breaks during replication, as demonstrated by the appearance of γH2AX foci. Replication stress in these cells is also illustrated by an increased number of stalled replication forks and a reduced fork progression speed. Our data indicate that glioblastoma cells have elevated MELK protein levels to better cope with replication stress during unperturbed S phase. Hence, MELK inhibitors hold great potential for the treatment of glioblastomas as such or in combination with DNA-damaging therapies. Background: Protein kinase MELK is expressed at very high levels in glioblastomas, but it is not understood how this benefits tumor growth. Results: A deficiency of MELK causes replication stress and is associated with cell cycle arrest and senescence. Conclusion: MELK is required for progression through unperturbed S phase. Significance: The inhibition of MELK emerges as an attractive cancer therapy.

Volume 45 (1), pages 89–99 Owing to an error that occurred during the production process, Figures 2 and 5 in this article were published in greyscale in the print version. The online version is not affected. The colour versions of the figures are presented here. Figure 2. The hypothetical life cycle of PP1. During or shortly after the translation of PP1, the metals Fe2+ and Zn2+ are incorporated into …

The spindle assembly checkpoint (SAC) generates a diffusible protein complex that prevents anaphase until all chromosomes are properly attached to spindle microtubules. A key step in SAC initiation is the recruitment of MAD1 to kinetochores, which is generally thought to be governed by the microtubule-kinetochore (MT-KT) attachment status. However, we demonstrate that the recruitment of MAD1 via BUB1, a conserved kinetochore receptor, is not affected by MT-KT interactions in human cells. Instead, BUB1:MAD1 interaction depends on BUB1 phosphorylation, which is controlled by a biochemical timer that integrates counteracting kinase and phosphatase effects on BUB1 into a pulse-generating incoherent feedforward loop. We propose that this attachment-independent timer serves to rapidly activate the SAC at mitotic entry, before the attachment-sensing MAD1 receptors have become fully operational. The BUB1-centered timer is largely impervious to conventional anti-mitotic drugs, and it is, therefore, a promising therapeutic target to induce cell death through permanent SAC activation. ispartof: Molecular Cell vol:68 issue:4 pages:715-730 ispartof: location:United States status: published

SDS22 is an ancient regulator of protein phosphatase-1 (PP1). Our crystal structure of SDS22 shows that its twelve leucine-rich repeats adopt a banana-shaped fold that is shielded from solvent by capping domains at its extremities. Subsequent modeling and biochemical studies revealed that the concave side of SDS22 likely interacts with PP1 helices α5 and α6, which are distal from the binding sites of many previously described PP1 interactors. Accordingly, we found that SDS22 acts as a "third" subunit of multiple PP1 holoenzymes. The crystal structure of SDS22 also revealed a large basic surface patch that enables binding of a phosphorylated form of splicing factor BCLAF1. Taken together, our data provide insights into the formation of PP1:SDS22 and the recruitment of additional interaction proteins, such as BCLAF1. ispartof: STRUCTURE vol:27 issue:3 pages:507-+ ispartof: location:United States status: published

Timers and sensors are common devices that make our daily life safer, more convenient, and more efficient. In a cellular context, they arguably play an even more crucial role as they ensure the survival of cells in the presence of various extrinsic and intrinsic stresses. Biological timers and sensors generate distinct signaling profiles, enabling them to produce different types of cellular responses. Recent data suggest that they can work together to guarantee correct timing and responsiveness. By exploring examples of cellular stress signaling from mitosis, DNA damage, and hypoxia, the authors discuss the common architecture of timer-sensor integration, and how its added features contribute to the generation of desired signaling profiles when dealing with stresses of variable duration and strength. The authors propose timer-sensor integration as a widespread mechanism with profound biological implications and therapeutic potential. ispartof: BIOESSAYS vol:41 issue:3 ispartof: location:United States status: published

Faithful progression through mitosis is critically dependent on the timely phosphorylation and dephosphorylation of a host of proteins. The involved protein kinases and phosphatases are embedded in interconnected feedback and feedforward circuits that ensure swift and robust phase transitions. Here we review recent evidence showing that protein phosphatases are modulators of the mitotic entry but also organize the mitotic exit through an orderly dephosphorylation of their substrates. In addition, phosphatases spatiotemporally restrict the phosphorylation of key regulatory proteins and oppose kinases to control highly dynamic mitotic processes, including chromosome congression and checkpoint signaling. In accordance with their important role as nodes in phosphorylation networks, mitotic protein phosphatases are tightly regulated in four dimensions.

The biotin-identification (BioID) protocol uses a mutant of the biotin ligase BirA (BirA*) fused to a protein-of-interest to biotinylate proximate proteins in intact cells. Here, we show that two inactive halves of BirA* separately fused to a catalytic and regulatory subunit of protein phosphatase PP1 reconstitute a functional BirA* enzyme upon heterodimerization of the phosphatase subunits. We also demonstrate that this BirA*-fragment complementation approach, termed split-BioID, can be used to screen for substrates and other protein interactors of PP1 holoenzymes. Split-BioID is a novel and versatile tool for the identification of (transient) interactors of protein dimers. This article is protected by copyright. All rights reserved. ispartof: FEBS Letters vol:591 issue:2 pages:415-424 ispartof: location:England status: published

Specific interactions between proteins govern essential physiological processes including signaling. Many enzymes, especially the family of serine/threonine phosphatases (PSPs: PP1, PP2A and PP2B/calcineurin/CN), recruit substrates and regulatory proteins by binding Short Linear Motifs (SLiMs), short sequences found within intrinsically disordered regions (IDRs) that mediate specific protein:protein interactions. While tremendous progress had been made in identifying where and how SLiMs bind PSPs, especially PP1 and CN, essentially nothing is known about how SLiMs bind PP2A, a validated cancer drug target. Here we describe three structures of PP2A:SLiM interaction (B56: pS-RepoMan, B56:pS-BubR1 and B56:pSpS-BubR1), show that this PP2A-specific SLiM is defined as LSPIxE and then use this data to discover scores of likely PP2A regulators and substrates. Together, these data not only provide a powerful approach for dissecting PP2A interaction networks in cells but also for targeting PP2A diseases, such as cancer.

γ-Secretases are a family of intramembrane-cleaving proteases involved in various signaling pathways and diseases, including Alzheimer's disease (AD). Cells co-express differing γ-secretase complexes, including two homologous presenilins (PSENs). We examined the significance of this heterogeneity and identified a unique motif in PSEN2 that directs this γ-secretase to late endosomes/lysosomes via a phosphorylation-dependent interaction with the AP-1 adaptor complex. Accordingly, PSEN2 selectively cleaves late endosomal/lysosomal localized substrates and generates the prominent pool of intracellular Aβ that contains longer Aβ; familial AD (FAD)-associated mutations in PSEN2 increased the levels of longer Aβ further. Moreover, a subset of FAD mutants in PSEN1, normally more broadly distributed in the cell, phenocopies PSEN2 and shifts its localization to late endosomes/lysosomes. Thus, localization of γ-secretases determines substrate specificity, while FAD-causing mutations strongly enhance accumulation of aggregation-prone Aβ42 in intracellular acidic compartments. The findings reveal potentially important roles for specific intracellular, localized reactions contributing to AD pathogenesis. publisher: Elsevier articletitle: Restricted Location of PSEN2/γ-Secretase Determines Substrate Specificity and Generates an Intracellular Aβ Pool journaltitle: Cell articlelink: http://dx.doi.org/10.1016/j.cell.2016.05.020 content_type: article copyright: © 2016 Elsevier Inc. ispartof: Cell vol:166 issue:1 pages:193-208 ispartof: location:United States status: published

The histone methyltransferase EZH2 regulates cell proliferation and differentiation by silencing Polycomb group target genes. NIPP1, a nuclear regulator of serine/threonine protein phosphatase 1 (PP1), has been implicated in the regulation of EZH2 occupancy at target loci, but the underlying mechanism is not understood. Here, we demonstrate that the phosphorylation of EZH2 by cyclin-dependent kinases at Thr416 creates a docking site for the ForkHead-associated domain of NIPP1. Recruited NIPP1 enables the net phosphorylation of EZH2 by inhibiting its dephosphorylation by PP1. Accordingly, a NIPP1-binding mutant of EZH2 is hypophosphorylated, and the knockdown of NIPP1 results in a reduced phosphorylation of endogenous EZH2. Conversely, the loss of PP1 is associated with a hyperphosphorylation of EZH2. A genome-wide promoter-binding profiling in HeLa cells revealed that the NIPP1-binding mutant shows a deficient association with about a third of the Polycomb target genes, and these are enriched for functions in proliferation. Our data identify PP1 as an EZH2 phosphatase and demonstrate that the phosphorylation-regulated association of EZH2 with proliferation-related targets depends on associated NIPP1.

7 páginas, 5 figuras, material suplementario en versión online., AMP-activated protein kinase (AMPK) is a sensor of cellular energy status. In pancreatic beta cells, glucose induces the dephosphorylation of Thr172 within the catalytic subunit and the inactivation of the AMPK complex. Here we demonstrate that glucose also activates protein kinase A (PKA), leading to the phosphorylation of AMPKα at Ser485 and Ser497. However, these modifications do not impair the phosphorylation of Thr172 by upstream kinases, and phosphorylation of Thr172 does not affect the phosphorylation of AMPKα by PKA either. Thus, although phosphorylation of Thr172 and Ser485/Ser497 are inversely correlated in response to glucose, they follow an independent regulation., This work was supported by a grant from the Spanish Ministry of Education and Science (SAF2011-27442) and a grant from Generalitat Valenciana (Prometeo 2009/051).

SummaryEctonucleotide pyrophosphatase/phosphodiesterase-1 (NPP1) converts extracellular nucleotides into inorganic pyrophosphate, whereas its close relative NPP2/autotaxin hydrolyzes lysophospholipids. NPP1 regulates calcification in mineralization-competent tissues, and a lack of NPP1 function underlies calcification disorders. Here, we show that NPP1 forms homodimers via intramembrane disulfide bonding, but is also processed intracellularly to a secreted monomer. The structure of secreted NPP1 reveals a characteristic bimetallic active site and a nucleotide-binding groove, but it lacks the lipid-binding pocket and open tunnel present in NPP2. A loop adjacent to the nucleotide-binding site, which is disordered in NPP2, is well ordered in NPP1 and might promote nucleotide binding. Remarkably, the N-terminal somatomedin B-like domains of NPP1, unlike those in NPP2, are flexible and do not contact the catalytic domain. Our results provide a structural basis for the nucleotide pyrophosphatase activity of NPP1 and help to understand how disease-causing mutations may affect NPP1 structure and function.

Regulation of the Ser/Thr phosphatase protein phosphatase 1 (PP1) is controlled by a diverse array of regulatory proteins. However, how these proteins direct the specificity of PP1 is not well understood. More than 1/3 of the nuclear pool of PP1 forms a holoenzyme with the nuclear inhibitor of PP1, NIPP1, to regulate chromatin remodeling, among other essential biological functions. Here, we show that the PP1-binding domain of NIPP1 is an intrinsically disordered protein, which binds PP1 in an unexpected manner. NIPP1 forms an α-helix that engages PP1 at a novel interaction site, using polar rather than hydrophobic contacts. Importantly, the structure also reveals the first shared PP1 interaction site outside of the RVxF motif, the ΦΦ motif. Finally, we show that NIPP1:PP1 substrate selectivity is determined by altered electrostatics and enhanced substrate localization. Together, our results provide the molecular basis by which NIPP1 directs PP1 substrate specificity in the nucleus.