Publications by authors named "Mathieu Bollen"

129 Publications

Protein phosphatase-1: dual activity regulation by Inhibitor-2.

Biochem Soc Trans 2020 Oct;48(5):2229-2240

Laboratory of Biosignaling and Therapeutics, KU Leuven Department of Cellular and Molecular Medicine, University of Leuven, B-3000 Leuven, Belgium.

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.
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http://dx.doi.org/10.1042/BST20200503DOI Listing
October 2020

Towards Dissecting the Mechanism of Protein Phosphatase-1 Inhibition by Its C-Terminal Phosphorylation.

Chembiochem 2021 Mar 17;22(5):834-838. Epub 2020 Nov 17.

Genome Biology Unit, European Molecular Biology Laboratory, Meyerhofstraße 1, 69117, Heidelberg, Germany.

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.
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http://dx.doi.org/10.1002/cbic.202000669DOI Listing
March 2021

RepoMan stimulates the chromosome-dependent pathway of microtubule assembly.

Cell Cycle 2020 Nov 15;19(22):3029-3041. Epub 2020 Oct 15.

Department of Cellular and Molecular Medicine, Laboratory of Biosignaling & Therapeutics, KU Leuven , Leuven, Belgium.

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.
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http://dx.doi.org/10.1080/15384101.2020.1830607DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7747800PMC
November 2020

De Novo Variants in LMNB1 Cause Pronounced Syndromic Microcephaly and Disruption of Nuclear Envelope Integrity.

Am J Hum Genet 2020 10 9;107(4):753-762. Epub 2020 Sep 9.

Center for Human Genetics, University Hospitals Leuven, Herestraat 49, 3000 Leuven, Belgium; Laboratory for the Genetics of Cognition, Department of Human Genetics, KU Leuven, Herestraat 49, 3000 Leuven, Belgium. Electronic address:

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.
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http://dx.doi.org/10.1016/j.ajhg.2020.08.015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7536573PMC
October 2020

Enhanced DNA-repair capacity and resistance to chemically induced carcinogenesis upon deletion of the phosphatase regulator NIPP1.

Oncogenesis 2020 Mar 2;9(3):30. Epub 2020 Mar 2.

Laboratory of Biosignaling & Therapeutics, KU Leuven Department of Cellular and Molecular Medicine, University of Leuven, Leuven, Belgium.

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.
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http://dx.doi.org/10.1038/s41389-020-0214-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7051951PMC
March 2020

Phosphatase Regulator NIPP1 Restrains Chemokine-Driven Skin Inflammation.

J Invest Dermatol 2020 08 21;140(8):1576-1588. Epub 2020 Jan 21.

Laboratory of Biosignaling & Therapeutics, KU Leuven Department of Cellular and Molecular Medicine, University of Leuven, Leuven, Belgium. Electronic address:

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.
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http://dx.doi.org/10.1016/j.jid.2020.01.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7371497PMC
August 2020

Co-regulation of the antagonistic RepoMan:Aurora-B pair in proliferating cells.

Mol Biol Cell 2020 03 22;31(6):419-438. Epub 2020 Jan 22.

Laboratory of Biosignaling & Therapeutics, KU Leuven, B-3000 Leuven, Belgium.

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 SCF. 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.
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http://dx.doi.org/10.1091/mbc.E19-12-0698DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7185888PMC
March 2020

The antibiotic robenidine exhibits guanabenz-like cytoprotective properties by a mechanism independent of protein phosphatase PP1:PPP1R15A.

J Biol Chem 2019 09 23;294(36):13478-13486. Epub 2019 Jul 23.

Laboratory of Biosignaling and Therapeutics, KU Leuven Department of Cellular and Molecular Medicine, University of Leuven, 3000 Leuven, Belgium

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.
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http://dx.doi.org/10.1074/jbc.RA119.008857DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6737217PMC
September 2019

Coordination of Timers and Sensors in Cell Signaling.

Bioessays 2019 03 7;41(3):e1800217. Epub 2019 Feb 7.

Laboratory of Biosignaling & Therapeutics, Department of Cellular and Molecular Medicine, KU Leuven, 3000, Leuven, Belgium.

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.
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http://dx.doi.org/10.1002/bies.201800217DOI Listing
March 2019

Structure-Guided Exploration of SDS22 Interactions with Protein Phosphatase PP1 and the Splicing Factor BCLAF1.

Structure 2019 03 17;27(3):507-518.e5. Epub 2019 Jan 17.

Laboratory of Biosignaling & Therapeutics, Department of Cellular and Molecular Medicine, KU Leuven, Leuven, Belgium. Electronic address:

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.
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http://dx.doi.org/10.1016/j.str.2018.12.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6538287PMC
March 2019

Ubiquitin-Independent Disassembly by a p97 AAA-ATPase Complex Drives PP1 Holoenzyme Formation.

Mol Cell 2018 11 18;72(4):766-777.e6. Epub 2018 Oct 18.

Centre for Medical Biotechnology, Faculty of Biology, University of Duisburg-Essen, 45117 Essen, Germany. Electronic address:

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.
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http://dx.doi.org/10.1016/j.molcel.2018.09.020DOI Listing
November 2018

The deletion of the protein phosphatase 1 regulator NIPP1 in testis causes hyperphosphorylation and degradation of the histone methyltransferase EZH2.

J Biol Chem 2018 11 10;293(47):18031-18039. Epub 2018 Oct 10.

From the Laboratory of Biosignaling & Therapeutics, KU Leuven Department of Cellular and Molecular Medicine, University of Leuven, B-3000 Leuven, Belgium and

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.
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http://dx.doi.org/10.1074/jbc.AC118.005577DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6254331PMC
November 2018

A substrate-trapping strategy for protein phosphatase PP1 holoenzymes using hypoactive subunit fusions.

J Biol Chem 2018 09 16;293(39):15152-15162. Epub 2018 Aug 16.

From the Laboratory of Biosignaling and Therapeutics,

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.
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http://dx.doi.org/10.1074/jbc.RA118.004132DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6166715PMC
September 2018

KNL1 Binding to PP1 and Microtubules Is Mutually Exclusive.

Structure 2018 10 9;26(10):1327-1336.e4. Epub 2018 Aug 9.

Department of Chemistry and Biochemistry, University of Arizona, Tucson, AZ 85721, USA. Electronic address:

The kinetochore scaffold 1 (KNL1) protein coordinates the spindle assembly checkpoint (SAC), a signaling pathway that delays chromosome segregation until all sister chromatids are properly attached to spindle microtubules. Recently, microtubules and protein phosphatase 1 (PP1), which both bind the N-terminal domain of KNL1, have emerged as regulators of the SAC; however, how these proteins interact to contribute to SAC signaling is unknown. Here, we use X-ray crystallography, nuclear magnetic resonance spectroscopy, and biochemical assays to show how KNL1 binds both PP1 and microtubules. Unexpectedly, we discovered that PP1 and microtubules bind KNL1 via overlapping binding sites. Further, we showed that Aurora B kinase phosphorylation results in distinct patterns of KNL1 complex disruption. Finally, combining this data with co-sedimentation assays unequivocally demonstrated that microtubules and PP1 binding to KNL1 is mutually exclusive, with preferential formation of the KNL1:PP1 holoenzyme in the presence of PP1.
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http://dx.doi.org/10.1016/j.str.2018.06.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6601351PMC
October 2018

Functions and therapeutic potential of protein phosphatase 1: Insights from mouse genetics.

Biochim Biophys Acta Mol Cell Res 2019 01 26;1866(1):16-30. Epub 2018 Jul 26.

Laboratory of Biosignaling & Therapeutics, KU Leuven Department of Cellular and Molecular Medicine, University of Leuven, B-3000 Leuven, Belgium. Electronic address:

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.
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http://dx.doi.org/10.1016/j.bbamcr.2018.07.019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7114192PMC
January 2019

Overexpression of PP1-NIPP1 limits the capacity of cells to repair DNA double-strand breaks.

J Cell Sci 2018 07 9;131(13). Epub 2018 Jul 9.

Laboratory of Biosignaling & Therapeutics, KU Leuven Department of Cellular and Molecular Medicine, University of Leuven, B-3000 Leuven, Belgium

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.
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http://dx.doi.org/10.1242/jcs.214932DOI Listing
July 2018

A Sephin1-insensitive tripartite holophosphatase dephosphorylates translation initiation factor 2α.

J Biol Chem 2018 05 4;293(20):7766-7776. Epub 2018 Apr 4.

From the Cambridge Institute for Medical Research, University of Cambridge, Cambridge CB2 0XY, United Kingdom,

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 eIF2α-directed holophosphatase, a therapeutic target in diseases of protein misfolding, is comprised of a regulatory subunit, PPP1R15, and a catalytic subunit, protein phosphatase 1 (PP1). In mammals, there are two isoforms of the regulatory subunit, PPP1R15A and PPP1R15B, with overlapping roles in the essential function of eIF2α dephosphorylation. However, conflicting reports have appeared regarding the requirement for an additional co-factor, G-actin, in enabling substrate-specific dephosphorylation by PPP1R15-containing PP1 holoenzymes. An additional concern relates to the sensitivity of the holoenzyme to the [(o-chlorobenzylidene)amino]guanidines Sephin1 or guanabenz, putative 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 the requirement for G-actin and sensitivity of the holoenzyme to inhibitors. We found that eIF2α dephosphorylation by PP1 was moderately stimulated by repeat-containing PPP1R15A in an unphysiological low ionic strength buffer, whereas stimulation imparted by the co-presence of PPP1R15A and G-actin was observed under a broad range of conditions, low and physiological ionic strength, regardless of whether the PPP1R15A regulatory subunit had or lacked the N-terminal repeat-containing region and whether it was paired with native PP1 purified from rabbit muscle or recombinant PP1 purified from bacteria. Furthermore, none of the PPP1R15A-containing holophosphatases tested were inhibited by Sephin1 or guanabenz.
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http://dx.doi.org/10.1074/jbc.RA118.002325DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5961032PMC
May 2018

Adeno-associated virus Rep proteins antagonize phosphatase PP1 to counteract KAP1 repression of the latent viral genome.

Proc Natl Acad Sci U S A 2018 04 26;115(15):E3529-E3538. Epub 2018 Mar 26.

Department of Infectious Diseases, School of Immunology and Microbial Sciences, King's College London, SE1 9RT London, United Kingdom;

Adeno-associated virus (AAV) is a small human whose 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 the ORF, 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.
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http://dx.doi.org/10.1073/pnas.1721883115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5899473PMC
April 2018

Correction: Substrate-specifying determinants of the nucleotide pyrophosphatases/phosphodiesterases NPP1 and NPP2.

Biochem J 2017 12 14;474(24):4273-4274. Epub 2017 Dec 14.

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http://dx.doi.org/10.1042/BCJ-2004-0465_CORDOI Listing
December 2017

An Attachment-Independent Biochemical Timer of the Spindle Assembly Checkpoint.

Mol Cell 2017 Nov 9;68(4):715-730.e5. Epub 2017 Nov 9.

Laboratory of Biosignaling & Therapeutics, Department of Cellular and Molecular Medicine, University of Leuven, 3000 Leuven, Belgium. Electronic address:

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.
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http://dx.doi.org/10.1016/j.molcel.2017.10.011DOI Listing
November 2017

The Importance of Kinase-Phosphatase Integration: Lessons from Mitosis.

Trends Cell Biol 2018 01 28;28(1):6-21. Epub 2017 Oct 28.

Division of Cancer Research, School of Medicine, Jacqui Wood Cancer Centre, Ninewells Hospital and Medical School, University of Dundee, Dundee, DD1 9SY, UK. Electronic address:

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.
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http://dx.doi.org/10.1016/j.tcb.2017.09.005DOI Listing
January 2018

The protein phosphatase 1 regulator NIPP1 is essential for mammalian spermatogenesis.

Sci Rep 2017 10 17;7(1):13364. Epub 2017 Oct 17.

Laboratory of Biosignaling & Therapeutics, KU Leuven Department of Cellular and Molecular Medicine, University of Leuven, Leuven, Belgium.

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.
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http://dx.doi.org/10.1038/s41598-017-13809-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5645368PMC
October 2017

ENPP1 Mutation Causes Recessive Cole Disease by Altering Melanogenesis.

J Invest Dermatol 2018 02 28;138(2):291-300. Epub 2017 Sep 28.

Laboratory of Human Genetics and Embryology, Institute of Medical Biology, A*STAR, Singapore, Singapore. Electronic address:

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.
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http://dx.doi.org/10.1016/j.jid.2017.08.045DOI Listing
February 2018

Correction: Biogenesis and activity regulation of protein phosphatase 1.

Biochem Soc Trans 2017 04;45(2):583-584

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http://dx.doi.org/10.1042/BST-2016-0154C_CORDOI Listing
April 2017

Biogenesis and activity regulation of protein phosphatase 1.

Biochem Soc Trans 2017 02;45(1):89-99

Laboratory of Biosignaling & Therapeutics, KU Leuven Department of Cellular and Molecular Medicine, University of Leuven, B-3000 Leuven, Belgium

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.
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http://dx.doi.org/10.1042/BST20160154DOI Listing
February 2017

Split-BioID: a proximity biotinylation assay for dimerization-dependent protein interactions.

FEBS Lett 2017 01 12;591(2):415-424. Epub 2017 Jan 12.

Laboratory of Biosignaling & Therapeutics, KU Leuven Department of Cellular and Molecular Medicine, University of Leuven, Belgium.

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.
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http://dx.doi.org/10.1002/1873-3468.12548DOI Listing
January 2017

Expanding the PP2A Interactome by Defining a B56-Specific SLiM.

Structure 2016 12 27;24(12):2174-2181. Epub 2016 Oct 27.

Department of Molecular Biology, Cell Biology and Biochemistry, Brown University, Providence, RI 02912, USA. Electronic address:

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 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 a 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 these data to discover scores of likely PP2A regulators and substrates. Together, these data provide a powerful approach not only for dissecting PP2A interaction networks in cells but also for targeting PP2A diseases, such as cancer.
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http://dx.doi.org/10.1016/j.str.2016.09.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5180209PMC
December 2016

The Ki-67 and RepoMan mitotic phosphatases assemble via an identical, yet novel mechanism.

Elife 2016 08 30;5. Epub 2016 Aug 30.

Department of Molecular Biology, Cell Biology and Biochemistry, Brown University, Providence, United States.

Ki-67 and RepoMan have key roles during mitotic exit. Previously, we showed that Ki-67 organizes the mitotic chromosome periphery and recruits protein phosphatase 1 (PP1) to chromatin at anaphase onset, in a similar manner as RepoMan (Booth et al., 2014). Here we show how Ki-67 and RepoMan form mitotic exit phosphatases by recruiting PP1, how they distinguish between distinct PP1 isoforms and how the assembly of these two holoenzymes are dynamically regulated by Aurora B kinase during mitosis. Unexpectedly, our data also reveal that Ki-67 and RepoMan bind PP1 using an identical, yet novel mechanism, interacting with a PP1 pocket that is engaged only by these two PP1 regulators. These findings not only show how two distinct mitotic exit phosphatases are recruited to their substrates, but also provide immediate opportunities for the design of novel cancer therapeutics that selectively target the Ki-67:PP1 and RepoMan:PP1 holoenzymes.
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http://dx.doi.org/10.7554/eLife.16539DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5005033PMC
August 2016