Post-translational Control of the Temporal Dynamics of Transcription Factor Activity Regulates Neurogenesis.

Neurogenesis is initiated by the transient expression of the highly conserved proneural proteins, bHLH transcriptional regulators. Here, we discover a conserved post-translational switch governing the duration of proneural protein activity that is required for proper neuronal development. Phosphorylation of a single Serine at the same position in Scute and Atonal proneural proteins governs the transition from active to inactive forms by regulating DNA binding. The equivalent Neurogenin2 Threonine also regulates DNA binding and proneural activity in the developing mammalian neocortex. Using genome editing in Drosophila, we show that Atonal outlives its mRNA but is inactivated by phosphorylation. Inhibiting the phosphorylation of the conserved proneural Serine causes quantitative changes in expression dynamics and target gene expression resulting in neuronal number and fate defects. Strikingly, even a subtle change from Serine to Threonine appears to shift the duration of Atonal activity in vivo, resulting in neuronal fate defects.

Restricted Location of PSEN2/γ-Secretase Determines Substrate Specificity and Generates an Intracellular Aß Pool.

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 Aß that contains longer Aß; familial AD (FAD)-associated mutations in PSEN2 increased the levels of longer Aß 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 Aß42 in intracellular acidic compartments. The findings reveal potentially important roles for specific intracellular, localized reactions contributing to AD pathogenesis.

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

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.

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

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.

Emerging roles of the Protein Phosphatase 1 (PP1) in the context of viral infections.

Protein Phosphatase 1 (PP1) is a major serine/threonine phosphatase in eukaryotes, participating in several cellular processes and metabolic pathways. Due to their low substrate specificity, PP1's catalytic subunits do not exist as free entities but instead bind to Regulatory Interactors of Protein Phosphatase One (RIPPO), which regulate PP1's substrate specificity and subcellular localization. Most RIPPOs bind to PP1 through combinations of short linear motifs (4-12 residues), forming highly specific PP1 holoenzymes. These PP1-binding motifs may, hence, represent attractive targets for the development of specific drugs that interfere with a subset of PP1 holoenzymes. Several viruses exploit the host cell protein (de)phosphorylation machinery to ensure efficient virus particle formation and propagation. While the role of many host cell kinases in viral life cycles has been extensively studied, the targeting of phosphatases by viral proteins has been studied in less detail. Here, we compile and review what is known concerning the role of PP1 in the context of viral infections and discuss how it may constitute a putative host-based target for the development of novel antiviral strategies.

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

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.

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

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.

Coordination of Timers and Sensors in Cell Signaling.

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.

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

Adeno-associated virus (AAV) is a small human Dependovirus 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 rep 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.

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

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.

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

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.

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

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.

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

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.

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

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 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 eIF2αP 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 eIF2αP 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.

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

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.

Inhibitor-3 ensures bipolar mitotic spindle attachment by limiting association of SDS22 with kinetochore-bound protein phosphatase-1.

Faithful chromosome segregation during mitosis is tightly regulated by opposing activities of Aurora B kinase and protein phosphatase-1 (PP1). PP1 function at kinetochores has been linked to SDS22, but the exact localization of SDS22 and how it affects PP1 are controversial. Here, we confirm that SDS22 is required for PP1 activity, but show that SDS22 does not normally localize to kinetochores. Instead, SDS22 is kept in solution by formation of a ternary complex with PP1 and inhibitor-3 (I3). Depletion of I3 does not affect the amount of PP1 at kinetochores but causes quantitative association of SDS22 with PP1 on KNL1 at the kinetochore. Such accumulation of SDS22 at kinetochores interferes with PP1 activity and inhibits Aurora B threonine-232 dephosphorylation, which leads to increased Aurora B activity in metaphase and persistence in anaphase accompanied with segregation defects. We propose a model in which I3 regulates an SDS22-mediated PP1 activation step in solution that precedes SDS22 dissociation and transfer of PP1 to kinetochores, and which is required for PP1 to efficiently antagonize Aurora B.

The selective inhibition of protein phosphatase-1 results in mitotic catastrophe and impaired tumor growth.

The serine/threonine protein phosphatase-1 (PP1) complex is a key regulator of the cell cycle. However, the redundancy of PP1 isoforms and the lack of specific inhibitors have hampered studies on the global role of PP1 in cell cycle progression in vertebrates. Here, we show that the overexpression of nuclear inhibitor of PP1 (NIPP1; also known as PPP1R8) in HeLa cells culminated in a prometaphase arrest, associated with severe spindle-formation and chromosome-congression defects. In addition, the spindle assembly checkpoint was activated and checkpoint silencing was hampered. Eventually, most cells either died by apoptosis or formed binucleated cells. The NIPP1-induced mitotic arrest could be explained by the inhibition of PP1 that was titrated away from other mitotic PP1 interactors. Consistent with this notion, the mitotic-arrest phenotype could be rescued by the overexpression of PP1 or the inhibition of the Aurora B kinase, which acts antagonistically to PP1. Finally, we demonstrate that the overexpression of NIPP1 also hampered colony formation and tumor growth in xenograft assays in a PP1-dependent manner. Our data show that the selective inhibition of PP1 can be used to induce cancer cell death through mitotic catastrophe.

Biogenesis and activity regulation of protein phosphatase 1.

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.

Brief Report: The Deletion of the Phosphatase Regulator NIPP1 Causes Progenitor Cell Expansion in the Adult Liver.

The Ppp1r8 gene encodes NIPP1, a nuclear interactor of protein phosphatase PP1. The deletion of NIPP1 is embryonic lethal at the gastrulation stage, which has hampered its functional characterization in adult tissues. Here, we describe the effects of a conditional deletion of NIPP1 in mouse liver epithelial cells. Ppp1r8(-/-) livers developed a ductular reaction, that is, bile-duct hyperplasia with associated fibrosis. The increased proliferation of biliary epithelial cells was at least partially due to an expansion of the progenitor cell compartment that was independent of liver injury. Gene-expression analysis confirmed an upregulation of progenitor cell markers in the liver knockout livers but showed no effect on the expression of liver-injury associated regulators of cholangiocyte differentiation markers. Consistent with an inhibitory effect of NIPP1 on progenitor cell proliferation, Ppp1r8(-/-) livers displayed an increased sensitivity to diet-supplemented 3,5-diethoxycarbonyl-1,4-dihydrocollidine, which also causes bile-duct hyperplasia through progenitor cell expansion. In contrast, the liver knockouts responded normally to injuries (partial hepatectomy, single CCl4 administration) that are restored through proliferation of differentiated parenchymal cells. Our data indicate that NIPP1 does not regulate the proliferation of hepatocytes but is a suppressor of biliary epithelial cell proliferation, including progenitor cells, in the adult liver. Stem Cells 2016;34:2256-2262.

Genome-wide promoter binding profiling of protein phosphatase-1 and its major nuclear targeting subunits.

Protein phosphatase-1 (PP1) is a key regulator of transcription and is targeted to promoter regions via associated proteins. However, the chromatin binding sites of PP1 have never been studied in a systematic and genome-wide manner. Methylation-based DamID profiling in HeLa cells has enabled us to map hundreds of promoter binding sites of PP1 and three of its major nuclear interactors, i.e. RepoMan, NIPP1 and PNUTS. Our data reveal that the α, ß and γ isoforms of PP1 largely bind to distinct subsets of promoters and can also be differentiated by their promoter binding pattern. PP1ß emerged as the major promoter-associated isoform and shows an overlapping binding profile with PNUTS at dozens of active promoters. Surprisingly, most promoter binding sites of PP1 are not shared with RepoMan, NIPP1 or PNUTS, hinting at the existence of additional, largely unidentified chromatin-targeting subunits. We also found that PP1 is not required for the global chromatin targeting of RepoMan, NIPP1 and PNUTS, but alters the promoter binding specificity of NIPP1. Our data disclose an unexpected specificity and complexity in the promoter binding of PP1 isoforms and their chromatin-targeting subunits.