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The effects of imeglimin, a novel antidiabetes agent, on β-cell function remain unclear. Here, we unveiled the impact of imeglimin on β-cell survival. Treatment with imeglimin augmented mitochondrial function, enhanced insulin secretion, promoted β-cell proliferation, and improved β-cell survival in mouse islets. Imeglimin upregulated the expression of endoplasmic reticulum (ER)-related molecules, including Chop (Ddit3), Gadd34 (Ppp1r15a), Atf3, and Sdf2l1, and decreased eIF2α phosphorylation after treatment with thapsigargin and restored global protein synthesis in β-cells under ER stress. Imeglimin failed to protect against ER stress-induced β-cell apoptosis in CHOP-deficient islets or in the presence of GADD34 inhibitor. Treatment with imeglimin showed a significant decrease in the number of apoptotic β-cells and increased β-cell mass in Akita mice. Imeglimin also protected against β-cell apoptosis in both human islets and human pluripotent stem cell-derived β-like cells. Taken together, imeglimin modulates the ER homeostasis pathway, which results in the prevention of β-cell apoptosis both in vitro and in vivo., (© 2022 by the American Diabetes Association.)

The unfolded protein response (UPR) is a direct consequence of cellular endoplasmic reticulum (ER) stress and a key disease driving mechanism in IPF. The resolution of the UPR is directed by PPP1R15A (GADD34) and leads to the restoration of normal ribosomal activity. While the role of PPP1R15A has been explored in lung epithelial cells, the role of this UPR resolving factor has yet to be explored in lung mesenchymal cells. The objective of the current study was to determine the expression and role of PPP1R15A in IPF fibroblasts and in a bleomycin-induced lung fibrosis model. A survey of IPF lung tissue revealed that PPP1R15A expression was markedly reduced. Targeting PPP1R15A in primary fibroblasts modulated TGF-β-induced fibroblast to myofibroblast differentiation and exacerbated pulmonary fibrosis in bleomycin-challenged mice. Interestingly, the loss of PPP1R15A appeared to promote lung fibroblast senescence. Taken together, our findings demonstrate the major role of PPP1R15A in the regulation of lung mesenchymal cells, and regulation of PPP1R15A may represent a novel therapeutic strategy in IPF., (© 2021. The Author(s).)

Very limited progress has been made in the management of advanced melanoma, especially melanoma of uveal origin. Lactamase β (LACTB) is a novel tumor suppressor; however, its biological function in melanoma remains unknown. Herein we demonstrated markedly lower LACTB expression levels in melanoma tissues and cell lines. Overexpression of LACTB suppressed the proliferation, migration and invasion of melanoma cells in vitro. Mechanistically, LACTB inhibited the activity of yes-associated protein (YAP). We showed that the level of phospho-YAP (Serine 127) was increased upon LACTB overexpression, which prevented the translocation of YAP to the nucleus. Further, LACTB could directly bind to PP1A and attenuate the interaction between PP1A and YAP, resulting in decreased YAP dephosphorylation and inactivation in a LATS1-independent manner. Additionally, transfection of phosphorylation-defective YAP mutants reversed LACTB-induced tumor suppression. Upstream, we demonstrated that SOX10 binds to the LACTB promoter and negatively regulates its transcription. Overexpression of LACTB also suppressed the tumorigenicity and lung metastasis of MUM2B uveal melanoma cells in vivo. Taken together, our findings indicate a novel SOX10/LACTB/PP1A signaling cascade that renders YAP inactive and modulates melanoma progression, offering a new therapeutic target for melanoma treatment., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Protein phosphatases, by counteracting protein kinases, regulate the reversible phosphorylation of many substrates involved in synaptic plasticity, a cellular model for learning and memory. A prominent phosphatase regulating synaptic plasticity and neurologic disorders is the serine/threonine protein phosphatase 1 (PP1). PP1 has three isoforms (α, β, and γ, encoded by three different genes), which are regulated by a vast number of interacting subunits that define their enzymatic substrate specificity. In this review, we discuss evidence showing that PP1 regulates synaptic transmission and plasticity, as well as presenting novel models of PP1 regulation suggested by recent experimental evidence. We also outline the required targeting of PP1 by neurabin and spinophilin to achieve substrate specificity at the synapse to regulate AMPAR and NMDAR function. We then highlight the role of inhibitor-2 in regulating PP1 function in plasticity, including its positive regulation of PP1 function in vivo in memory formation. We also discuss the distinct function of the three PP1 isoforms in synaptic plasticity and brain function, as well as briefly discuss the role of inhibitory phosphorylation of PP1, which has received recent emphasis in the regulation of PP1 activity in neurons., (Copyright © 2021 the authors.)

We have recently reported that a specific pool of ceramide, located in the plasma membrane, mediated the effects of sublethal doses of the chemotherapeutic compound doxorubicin on enhancing cancer cell migration. We identified neutral sphingomyelinase 2 (nSMase2) as the enzyme responsible to generate this bioactive pool of ceramide. In this work, we explored the role of members of the protein phosphatases 1 family (PP1), and we identified protein phosphatase 1 alpha isoform (PP1 alpha) as the specific PP1 isoform to mediate this phenotype. Using a bioinformatics approach, we build a functional interaction network based on phosphoproteomics data on plasma membrane ceramide. This led to the identification of several ceramide-PP1 alpha downstream substrates. Studies on phospho mutants of ezrin (T567) and Scrib (S1378/S1508) demonstrated that their dephosphorylation is sufficient to enhance cell migration. In summary, we identified a mechanism where reduced doses of doxorubicin result in the dysregulation of cytoskeletal proteins and enhanced cell migration. This mechanism could explain the reported effects of doxorubicin worsening cancer metastasis in animal models., (© 2021 Federation of American Societies for Experimental Biology.)

By acting as a ligand-dependent transcription factor the glucocorticoid receptor (GR) mediates the actions of glucocorticoids and regulates many physiological processes. An impaired regulation of glucocorticoid action has been associated with numerous disorders. Thus, the elucidation of underlying signaling pathways is essential to understand mechanisms of disrupted glucocorticoid function and contribution to diseases. This study found increased GR transcriptional activity upon overexpression of protein phosphatase 1 alpha (PP1α) in HEK-293 cells and decreased expression levels of GR-responsive genes following PP1α knockdown in the endogenous A549 cell model. Mechanistic investigations revealed reduced phosphorylation of GR-Ser211 following PP1α silencing and provided a first indication for an involvement of glycogen synthase kinase 3 (GSK-3). Thus, the present study identified PP1α as a novel post-translational activator of GR signaling, suggesting that disruption of PP1α function could lead to impaired glucocorticoid action and thereby contribute to diseases., (Copyright © 2020 The Author(s). Published by Elsevier B.V. All rights reserved.)

Collective cell migration is central to many developmental and pathological processes. However, the mechanisms that keep cell collectives together and coordinate movement of multiple cells are poorly understood. Using the Drosophila border cell migration model, we find that Protein phosphatase 1 (Pp1) activity controls collective cell cohesion and migration. Inhibition of Pp1 causes border cells to round up, dissociate, and move as single cells with altered motility. We present evidence that Pp1 promotes proper levels of cadherin-catenin complex proteins at cell-cell junctions within the cluster to keep border cells together. Pp1 further restricts actomyosin contractility to the cluster periphery rather than at individual internal border cell contacts. We show that the myosin phosphatase Pp1 complex, which inhibits non-muscle myosin-II (Myo-II) activity, coordinates border cell shape and cluster cohesion. Given the high conservation of Pp1 complexes, this study identifies Pp1 as a major regulator of collective versus single cell migration., Competing Interests: YC, NK, GA, LC, CM, AB, KS, DR, XW, JM No competing interests declared, (© 2020, Chen et al.)

The essential and distinct functions of Protein Phosphatase type 1 (PP1) catalytic subunit in eukaryotes are exclusively achieved through its interaction with a myriad of regulatory partners. In this work, we report the molecular and functional characterization of Gametocyte EXported Protein 15 (GEXP15), a Plasmodium specific protein, as a regulator of PP1. In vitro interaction studies demonstrated that GEXP15 physically interacts with PP1 through the RVxF binding motif in P. berghei. Functional assays showed that GEXP15 was able to increase PP1 activity and the mutation of the RVxF motif completely abolished this regulation. Immunoprecipitation assays of tagged GEXP15 or PP1 in P. berghei followed by immunoblot or mass spectrometry analyses confirmed their interaction and showed that they are present both in schizont and gametocyte stages in shared protein complexes involved in the spliceosome and proteasome pathways and known to play essential role in parasite development. Phenotypic analysis of viable GEXP15 deficient P. berghei blood parasites showed that they were unable to develop lethal infection in BALB/c mice or to establish experimental cerebral malaria in C57BL/6 mice. Further, although deficient parasites produced gametocytes they did not produce any oocysts/sporozoites indicating a high fitness cost in the mosquito. Global proteomic and phosphoproteomic analyses of GEXP15 deficient schizonts revealed a profound defect with a significant decrease in the abundance and an impact on phosphorylation status of proteins involved in regulation of gene expression or invasion. Moreover, depletion of GEXP15 seemed to impact mainly the abundance of some specific proteins of female gametocytes. Our study provides the first insight into the contribution of a PP1 regulator to Plasmodium virulence and suggests that GEXP15 affects both the asexual and sexual life cycle., Competing Interests: The authors have declared that no competing interests exist.

Germ cell immortality, or transgenerational maintenance of the germ line, could be promoted by mechanisms that could occur in either mitotic or meiotic germ cells. Here we report for the first time that the GSP-2 PP1/Glc7 phosphatase promotes germ cell immortality. Small RNA-induced genome silencing is known to promote germ cell immortality, and we identified a separation-of-function allele of C. elegans gsp-2 that is compromised for germ cell immortality and is also defective for small RNA-induced genome silencing and meiotic but not mitotic chromosome segregation. Previous work has shown that GSP-2 is recruited to meiotic chromosomes by LAB-1, which also promoted germ cell immortality. At the generation of sterility, gsp-2 and lab-1 mutant adults displayed germline degeneration, univalents, histone methylation and histone phosphorylation defects in oocytes, phenotypes that mirror those observed in sterile small RNA-mediated genome silencing mutants. Our data suggest that a meiosis-specific function of GSP-2 ties small RNA-mediated silencing of the epigenome to germ cell immortality. We also show that transgenerational epigenomic silencing at hemizygous genetic elements requires the GSP-2 phosphatase, suggesting a functional link to small RNAs. Given that LAB-1 localizes to the interface between homologous chromosomes during pachytene, we hypothesize that small localized discontinuities at this interface could promote genomic silencing in a manner that depends on small RNAs and the GSP-2 phosphatase., Competing Interests: The authors have declared that no competing interests exist.

The posttranslational modification of histones is crucial in spermatogenesis, as in other tissues; however, during spermiogenesis, histones are replaced with protamines, which are critical for the tight packaging of the DNA in sperm cells. Protamines are also posttranslationally modified by phosphorylation and dephosphorylation, which prompted our investigation of the underlying mechanisms and biological consequences of their regulation. On the basis of a screen that implicated the heat shock protein Hspa4l in spermatogenesis, we generated mice deficient in Hspa4l ( Hspa4l -null mice), which showed male infertility and the malformation of sperm heads. These phenotypes are similar to those of Ppp1cc -deficient mice, and we found that the amount of a testis- and sperm-specific isoform of the Ppp1cc phosphatase (Ppp1cc2) in the chromatin-binding fraction was substantially less in Hspa4l -null spermatozoa than that in those of wild-type mice. We further showed that Ppp1cc2 was a substrate of the chaperones Hsc70 and Hsp70 and that Hspa4l enhanced the release of Ppp1cc2 from these complexes, enabling the freed Ppp1cc2 to localize to chromatin. Pull-down and in vitro phosphatase assays suggested the dephosphorylation of protamine 2 at serine 56 (Prm2 Ser 56 ) by Ppp1cc2. To confirm the biological importance of Prm2 Ser 56 dephosphorylation, we mutated Ser 56 to alanine in Prm2 (Prm2 S56A). Introduction of this mutation to Hspa4l -null mice ( Hspa4l -/- ; Prm2 S56A/S56A ) restored the malformation of sperm heads and the infertility of Hspa4l -/- mice. The dephosphorylation signal to eliminate phosphate was crucial, and these results unveiled the mechanism and biological relevance of the dephosphorylation of Prm2 for sperm maturation in vivo., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Protein phosphatase 1 isoforms α, β, and γ (PP1α, PP1β, and PP1γ) are highly homologous in the catalytic domains but have distinct subcellular localizations. In this study, we utilized both primary cell culture and knockout mice to investigate the isoform-specific roles of PP1s in the heart. In both neonatal and adult cardiac myocytes, PP1β was mainly localized in the nucleus, compared to the predominant presence of PP1α and PP1γ in the cytoplasm. Adenovirus-mediated overexpression of PP1α led to decreased phosphorylation of phospholamban, which was not influenced by overexpression of either PP1β or PP1γ. Interestingly, only cardiac-specific knockout of PP1β resulted in increased HDAC7 phosphorylation, consistent with the predominant nuclear localization of PP1β. Functionally, deletion of either PP1 isoform resulted in reduced fractional shortening in aging mice, however only PP1β deletion resulted in interstitial fibrosis in mice as early as 3 weeks of age. Deletion of neither PP1 isoform had any effect on pathological cardiac hypertrophy induced by 2 weeks of pressure overload stimulation. Together, our data suggest that PP1 isoforms have differential localizations to regulate the phosphorylation of their specific substrates for the physiological function in the heart., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences.)

Myosin phosphatase (MP) holoenzyme is a Ser/Thr specific enzyme, which is the member of protein phosphatase type 1 (PP1) family and composed of a PP1 catalytic subunit (PP1c/PPP1CB) and a myosin phosphatase targeting subunit (MYPT1/PPP1R12A). PP1c is required for the catalytic activity of the holoenzyme, while MYPT1 regulates MP through targeting the holoenzyme to its substrates. Above the well-characterized function of MP, as the major regulator of smooth muscle contractility mediating the dephosphorylation of 20 kDa myosin light chain, accumulating data support its role in other, non-contractile functions. In this review, we summarize the scaffold function of MP holoenzyme and its roles in processes such as cell cycle, development, gene expression regulation and neurotransmitter release. In particular, we highlight novel interacting proteins of MYPT1 and pathophysiological functions of MP relevant to tumorigenesis, insulin resistance and neurodegenerative disorders. This article is part of a Special Issue entitled: Protein Phosphatases as Critical Regulators for Cellular Homeostasis edited by Prof. Peter Ruvolo and Dr. Veerle Janssens., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Protein synthesis is one of the most complex and energy-consuming processes in eukaryotic cells and therefore is tightly regulated. One of the main mechanisms of translational control is post-translational modifications of the components of translational apparatus. Phosphorylation status of translation factors depends on the balanced action of kinases and phosphatases. While many kinase-dependent events are well defined, phosphatases that counteract phosphorylation are rarely determined. This mini-review focuses on the regulation of activity of translational initiation factors by serine/threonine phosphatases., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Normal functioning of the brain is dependent upon a complex web of communication between numerous cell types. Within neuronal networks, the faithful transmission of information between neurons relies on an equally complex organization of inter- and intra-cellular signaling systems that act to modulate protein activity. In particular, post-translational modifications (PTMs) are responsible for regulating protein activity in response to neurochemical signaling. The key second messenger, cyclic adenosine 3',5'-monophosphate (cAMP), regulates one of the most ubiquitous and influential PTMs, phosphorylation. While cAMP is canonically viewed as regulating the addition of phosphate groups through its activation of cAMP-dependent protein kinases, it plays an equally critical role in regulating removal of phosphate through indirect control of protein phosphatase activity. This dichotomy of regulation by cAMP places it as one of the key regulators of protein activity in response to neuronal signal transduction throughout the brain. In this review we focus on the role of cAMP in regulation of the serine/threonine phosphatases protein phosphatase 1 (PP1) and protein phosphatase 2A (PP2A) and the relevance of control of PP1 and PP2A to regulation of brain function and behavior., (Copyright © 2018. Published by Elsevier B.V.)

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., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Integrated stress response (ISR) is a signaling system in which phosphorylation of eukaryotic translation initiation factor 2α (eIF2α) by stress-specific kinases and subsequent activation of activation transcription factor (ATF) 4 help restore cellular homeostasis following exposure to environmental stresses. ISR activation has been observed in metabolic diseases, including hepatic steatosis (HS), steatohepatitis (SH), and insulin resistance (IR), but it remains unclear whether ISR contributes to disease pathogenesis or represents an innate defense mechanism against metabolic stresses. Constitutive repressor of eIF2α phosphorylation (CReP) is a critical regulatory subunit of the eIF2α phosphatase complex. Here, we show that CReP ablation causes constitutive eIF2α phosphorylation in the liver, which leads to activation of the ATF4 transcriptional program including increased fibroblast growth factor 21 (FGF21) production. Liver-specific CReP knockout (CReP LKO ) mice exhibited marked browning of white adipose tissue (WAT) and increased energy expenditure and insulin sensitivity in an FGF21-dependent manner. Furthermore, CReP LKO mice were protected from high-fat diet (HFD)-induced obesity, HS, and IR. Acute CReP ablation in liver of HFD-induced obese mice also reduced adiposity and improved glucose homeostasis. Conclusion: These data suggest that CReP abundance is a critical determinant for eIF2α phosphorylation and ensuing ISR activation in the liver. Constitutive ISR activation in the liver induces FGF21 and confers protection from HFD-induced adiposity, IR, and HS in mice. Augmenting hepatic ISR may represent a therapeutic approach to treat metabolic disorders., (© 2018 by the American Association for the Study of Liver Diseases.)

This study was designed to investigate the effects of astragalosides on cardiac diastolic function, and an emphasis was placed on the variation of the upstream molecular regulators of phospholamban. Chronic heart failure (CHF) rats were induced by ligaturing the left anterior coronary artery, and rats in the therapeutic groups were treated with either a 50 mg/kg dose of captopril, 10 mg/kg dose of astragalosides or 20 mg/kg dose of astragalosides. Four weeks after treatment, the ratio of the early and atrial peak filling velocities (E/A) and maximal slope diastolic pressure decrement (-dp/dt) both decreased in CHF rats (by 30.3% and 25.5%, respectively) and significantly increased in 20 mg/kg astragalosides and captopril-treated rats. The protein phosphatase-1 activity was lower in the 20 mg/kg astragalosides group than in the CHF group (0.22 vs 0.44, P < 0.01), and the inhibitor-1 levels in the astragalosides and captopril-treated groups were increased. Chronic heart failure increased expression of protein kinase C-α and calcium-sensing receptor, and these changes were attenuated by astragalosides therapy. Astragalosides restored the diastolic dysfunction of chronic heart failure rats, possibly by downregulation of calcium-sensing receptor and protein kinase C-α, which in turn augmented inhibitor-1 expression, reduced protein phosphatase-1 activity and increased phospholamban phosphorylation., (Copyright © 2018 Elsevier Masson SAS. All rights reserved.)

Background & Aims: In colorectal tumors, hypoxia causes resistance to therapy and promotes metastasis. Loss of the tumor suppressor p53 (encoded by TP53) provides cancer cells with a selective advantage under conditions of hypoxia, but little is known about the mediators of this effect., Methods: Isogenic colorectal cancer (CRC) cell lines with different TP53 genotypes were placed under conditions of hypoxia. We examined the effects on levels and activity of microRNA-34a (MIR34A) in CRC cells. We determined the expression and localization of protein phosphatase 1 regulatory inhibitor subunit 11 (PPP1R11, also called INH3, HCGV, IPP3, HCGV, TCTE5, TCTEX5, or CFAP255) in 82 human colon cancers. We analyzed data on human colorectal carcinomas from the Cancer Genome Atlas collection to determine whether expression of PPP1R11 was affected by altered level or activity of p53, markers of epithelial-to-mesenchymal transition (EMT), or MIR34A or was associated with metastasis. We determined the effects of disruption Mir34a, Mir34b, and Mir34c in ApcMin/+ mice. DLD-1 cells were transfected with small inhibitor RNAs against PPP1R1, injected into the tail veins of immune-compromised mice, and followed by noninvasive bioluminescence imaging., Results: The hypoxia inducible factor 1 alpha subunit (HIF1A) directly repressed the MIR34A gene in p53-defective CRC cells, whereas expression of MIR34A was induced in p53-proficient CRC cells exposed to hypoxia. Down-regulation of MIR34A was required for hypoxia-induced EMT, invasion and migration, and activation of STAT3 in CRC cells. We identified PPP1R11, whose product inhibits PP1, as a target of MIR34A. PPP1R11 mediates phosphorylation (activation) of STAT3, so expression of MIR34A reduced activation of STAT3 in p53-deficient CRC cells. Ectopic expression of PPP1R11 in CRC cells induced EMT, invasion, and migration, which all required STAT3. Increased expression of PPP1R11 in p53-deficient CRC cells was required for hypoxia-induced EMT, invasion, migration, and resistance to 5-fluorouracil, as well as metastasis of xenograft tumors to lungs of mice. Adenomas and derived tumoroids of ApcMin/+ mice with disruption of Mir34a, Mir34b, and Mir34c had increased levels of PPP1R11. Colorectal tumors from patients had increased levels of PPP1R11 at areas of invasion, compared with other areas of the tumor; increased level PPP1R11 associated with TP53 mutations and metastasis to the liver., Conclusions: HIF1A represses, whereas p53 increases, expression of MIR34A in CRC cells. MIR34A reduces expression of PPP1R11 to prevent activation of STAT3 and inhibit the EMT and metastasis. Strategies to target this pathway might be developed to inhibit CRC metastasis and overcome resistance to therapy associated with hypoxia., (Copyright © 2017 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Using a high-throughput approach, we identified lemur tyrosine kinase 2 (LMTK2) as a novel determinant of cell sensitivity to TRAIL. LMTK2 is a poorly characterized serine/threonine kinase believed to play a role in endosomal membrane trafficking and neuronal physiology, and recently found to be mutated in diverse tumor types. We show that LMTK2 silencing sensitizes immortalized epithelial cells and cancer cells to TRAIL, and this phenomenon is accompanied by changes in the expression of BCL2 family members. In epithelial cells, LMTK2 targeting causes the down-regulation of the BCL2 and BCL-xL anti-apoptotic proteins and the reciprocal up-regulation of the pro-apoptotic protein BIM, while, in cancer cells, LMTK2 knock-down reduces BCL2 without increasing BIM levels. We provide evidence that both BIM and BCL2 proteins are regulated by LMTK2 in a GSK3β- and PP1A-dependent manner and that their perturbation, together with BCL-xL reduction, determines an increased sensitivity not only to TRAIL, but also to other compounds. Overall, our findings suggest a broad function of LMTK2 in the regulation of the apoptotic pathway and highlight LMTK2 as a novel candidate target to increase the cytotoxic activity of chemotherapeutic compounds., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

The balance of protein synthesis and proteolysis (i.e. proteostasis) is maintained by a complex regulatory network in which mTOR (mechanistic target of rapamycin serine/threonine kinase) pathway and unfolded protein response are prominent positive and negative actors. The interplay between the two systems has been revealed; however the mechanistic details of this crosstalk are largely unknown. The aim of the present study was to investigate the elements of crosstalk during endoplasmic reticulum stress and to verify the key role of GADD34 in the connection with the mTOR pathway. Here, we demonstrate that a transient activation of autophagy is present in endoplasmic reticulum stress provoked by thapsigargin or tunicamycin, which is turned into apoptotic cell death. The transient phase can be characterized by the elevation of the autophagic marker LC3II/I, by mTOR inactivation, AMP-activated protein kinase activation and increased GADD34 level. The switch from autophagy to apoptosis is accompanied with the appearance of apoptotic markers, mTOR reactivation, AMP-activated protein kinase inactivation and a decrease in GADD34. Inhibition of autophagy by 3-methyladenine shortens the transient phase, while inhibition of mTOR by rapamycin or resveratrol prolongs it. Inhibition of GADD34 by guanabenz or transfection of the cells with siGADD34 results in down-regulation of autophagy-dependent survival and a quick activation of mTOR, followed by apoptotic cell death. The negative effect of GADD34 inhibition is diminished when guanabenz or siGADD34 treatment is combined with rapamycin or resveratrol addition. These data confirm that GADD34 constitutes a mechanistic link between endoplasmic reticulum stress and mTOR inactivation, therefore promotes cell survival during endoplasmic reticulum stress., Competing Interests: The authors have declared that no competing interests exist.

Phosphoprotein phosphatase 1 (PPP1) catalytic subunit gamma 2 (PPP1CC2), a PPP1 isoform, is largely restricted to testicular germ cells and spermatozoa. The key to understanding PPP1 regulation in male germ cells lies in the identification and characterisation of its interacting partners. This study was undertaken to determine the expression patterns of the several ankyrin repeat protein variant 2 (SARP2), a PPP1-interacting protein, in testis and spermatozoa. SARP2 was found to be highly expressed in testis and spermatozoa, and its interaction with human spermatozoa endogenous PPP1CC2 was confirmed by immunoprecipitation. Expression analysis by RT-qPCR revealed that SARP2 and PPP1CC2 mRNA levels were significantly higher in the spermatocyte fraction. However, microscopy revealed that SARP2 protein was only present in the nucleus of elongating and mature spermatids and in spermatozoa. In spermatozoa, SARP2 was prominently expressed in the connecting piece and flagellum, as well as, to a lesser extent, in the acrosome. A yeast two-hybrid approach was used to detect SARP2-interacting proteins and a relevant interaction with a novel sperm-associated antigen 9 (SPAG9) variant, a testis and spermatozoa-specific c-Jun N-terminal kinase-binding protein, was validated in human spermatozoa. Given the expression pattern of SARP2 and its association with PPP1CC2 and SPAG9, it may play a role in spermiogenesis and sperm function, namely in sperm motility and the acrosome reaction.

Protein phosphatase 1γ (PP1γ), a member of mammalian protein phosphatases, serine/threonine phosphatases, catalyzes the majority of protein dephosphorylation events and regulates diverse cellular processes, such as neuronal signaling, muscle contraction, glycogen synthesis, and cell proliferation. However, its expression and potential functions in human glioma is unclear. In this study, we detected the high expression of PP1γ and phosphorylated p65 (p-p65) in human glioma tissues. Besides, we demonstrated that upregulation of PP1γ was tightly related to poor 5-year survival via systemic statistical analysis. Employing serum-starved and re-feeding models of U251 and U87MG, we observed the increasing expression of PP1γ and p-p65 were accompanied by the cell proliferation markers cyclin D1 and proliferating cell nuclear antigen (PCNA). Employing depletion-PP1γ models, we found downregulated PP1γ and p-p65 compared with upregulated IκBα, which indicates the inhibition of NF-κB pathway, and flow cytometry analysis confirmed the weakened cell proliferation. Moreover, we found that the translocation of p65 into the nucleus was impaired. Collectively, we identified the positive correlation between upregulation of PP1γ and human glioma cell proliferation and that knock-down of PP1γ alleviated the glioma proliferation by reducing p65 transportation into the nucleus. The results showed that PP1γ could accelerate human glioma proliferation via the NF-κB pathway.

Background and Purpose: Vasodilatation may contribute to the neuroprotective and vascular anti-remodelling effect of the tissue transglutaminase 2 (TG2) inhibitor cystamine. Here, we hypothesized that inhibition of TG2 followed by blockade of smooth muscle calcium entry and/or inhibition of Rho kinase underlies cystamine vasodilatation., Experimental Approach: We used rat mesenteric small arteries and RT-PCR, immunoblotting, and measurements of isometric wall tension, intracellular Ca(2+) ([Ca(2+)]i ), K(+) currents (patch clamp), and phosphorylation of myosin phosphatase targeting subunit 1 (MYPT1) and myosin regulatory light chain, in our experiments., Key Results: RT-PCR and immunoblotting revealed expression of TG2 in mesenteric small arteries. Cystamine concentration-dependently inhibited responses to phenylephrine, 5-HT and U46619 and for extracellular potassium. Selective inhibitors of TG2, LDN 27129 and T101, also inhibited phenylephrine contraction. An inhibitor of PLC suppressed cystamine relaxation. Cystamine relaxed and reduced [Ca(2+)]i in phenylephrine-contracted arteries. In potassium-contracted arteries, cystamine induced less relaxation without changing [Ca(2+)]i , and these relaxations were blocked by mitochondrial complex inhibitors. Blockers of Kv 7 channels, XE991 and linopirdine, inhibited cystamine relaxation and increases in voltage-dependent smooth muscle currents. Cystamine and the Rho kinase inhibitor Y27632 reduced basal MYPT1-Thr(855) phosphorylation, but only Y27632 reduced phenylephrine-induced increases in MYPT1-Thr(855) and myosin regulatory light chain phosphorylation., Conclusions and Implications: Cystamine induced vasodilatation by inhibition of receptor-coupled TG2, leading to opening of Kv channels and reduction of intracellular calcium, and by activation of a pathway sensitive to inhibitors of the mitochondrial complexes I and III. Both pathways may contribute to the antihypertensive and neuroprotective effect of cystamine., (© 2015 The British Pharmacological Society.)

TIMAP (TGF-β inhibited membrane associated protein) is a protein phosphatase 1 (PP1) regulatory subunit highly abundant in endothelial cells and it is involved in the maintenance of pulmonary endothelial barrier function. It localizes mainly in the plasma membrane, but it is also present in the nuclei and cytoplasm. Direct interaction of TIMAP with the eukaryotic elongation factor 1 A1 (eEF1A1) is shown by pull-down, LC-MS/MS, Far-Western and immunoprecipitations. In connection with the so called moonlighting functions of the elongation factor, eEF1A is thought to establish protein-protein interactions through a transcription-dependent nuclear export motif, TD-NEM, and to aid nuclear export of TD-NEM containing proteins. We found that a TD-NEM-like motif of TIMAP has a critical role in its specific binding to eEF1A1. However, eEF1A1 is not or not exclusively responsible for the nuclear export of TIMAP. On the contrary, TIMAP seems to regulate membrane localization of eEF1A1 as the elongation factor co-localized with TIMAP in the plasma membrane fraction of control endothelial cells, but it has disappeared from the membrane in TIMAP depleted cells. It is demonstrated that membrane localization of eEF1A1 depends on the phosphorylation state of its Thr residue(s); and ROCK phosphorylated eEF1A1 is a novel substrate for TIMAP-PP1 underlining the complex regulatory role of TIMAP in the endothelium. The elongation factor seems to be involved in the regulation of endothelial cell attachment and spreading as silencing of eEF1A1 positively affected these processes which were monitored by transendothelial resistance measurements., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Reversible protein phosphorylation at serine (Ser), threonine (Thr) and tyrosine (Tyr) residues is among the major regulatory mechanism in eukaryotic cells. The eukaryotic genome encodes many protein kinases and protein phosphatases. However, the localization, activity and specificity towards phosphatase substrates are dictated by a large array of phosphatase binding and regulatory subunits. For protein phosphatase 1 (PP1) more than 200 binding subunits have been described. The various PP1 isoforms and the binding subunits can be located throughout the cell, including in the nucleus. It follows that several nuclear specific PP1 binding proteins (PIPs) have been described and these will be discussed. Among them are PNUTS (phosphatase 1 nuclear targeting subunit), NIPP1 (nuclear inhibitor of PP1) and CREB (cAMP-responsive element-binding protein), which have all been associated with transcription. In fact PP1 can associate with transcription factors fulfilling an important regulatory function, in this respect it can bind to Hox11, human factor C1 (HCF1) and myocyte enhancer factor-2 (MEF2). PP1 also regulates cell cycle progression and centrosome maturation and splitting, again by binding to specific regulatory proteins. Moreover, PP1 together with other protein phosphatases control the entry into mitosis by regulating the activity of mitotic kinases. Thus, PP1, its binding proteins and/or the phosphorylation states of both, directly control a vast array of cell nucleus associated functions, many of which are starting to be unraveled., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Reversible phosphorylation, a fundamental regulatory mechanism required for many biological processes including memory formation, is coordinated by the opposing actions of protein kinases and phosphatases. Type I protein phosphatase (PP1), in particular, has been shown to constrain learning and memory formation. However, how PP1 might be regulated in memory is still not clear. Our previous work has elucidated that PP1 inhibitor-2 (I-2) is an endogenous regulator of PP1 in hippocampal and cortical neurons (Hou et al., 2013). Contrary to expectation, our studies of contextual fear conditioning and novel object recognition in I-2 heterozygous mice suggest that I-2 is a memory suppressor. In addition, lentiviral knock-down of I-2 in the rat dorsal hippocampus facilitated memory for tasks dependent on the hippocampus. Our data indicate that I-2 suppresses memory formation, probably via negatively regulating the phosphorylation of cAMP/calcium response element-binding protein (CREB) at serine 133 and CREB-mediated gene expression in dorsal hippocampus. Surprisingly, the data from both biochemical and behavioral studies suggest that I-2, despite its assumed action as a PP1 inhibitor, is a positive regulator of PP1 function in memory formation., Significance Statement: We found that inhibitor-2 acts as a memory suppressor through its positive functional influence on type I protein phosphatase (PP1), likely resulting in negative regulation of cAMP/calcium response element-binding protein (CREB) and CREB-activated gene expression. Our studies thus provide an interesting example of a molecule with an in vivo function that is opposite to its in vitro function. PP1 plays critical roles in many essential physiological functions such as cell mitosis and glucose metabolism in addition to its known role in memory formation. PP1 pharmacological inhibitors would thus not be able to serve as good therapeutic reagents because of its many targets. However, identification of PP1 inhibitor-2 as a critical contributor to suppression of memory formation by PP1 may provide a novel therapeutic target for memory-related diseases., (Copyright © 2015 the authors 0270-6474/15/3515082-06$15.00/0.)

Environmental enrichment has been reported to delay or restore age-related cognitive deficits, however, a mechanism to account for the cause and progression of normal cognitive decline and its preservation by environmental enrichment is lacking. Using genome-wide SAGE-Seq, we provide a global assessment of differentially expressed genes altered with age and environmental enrichment in the hippocampus. Qualitative and quantitative proteomics in naïve young and aged mice was used to further identify phosphorylated proteins differentially expressed with age. We found that increased expression of endogenous protein phosphatase-1 inhibitors in aged mice may be characteristic of long-term environmental enrichment and improved cognitive status. As such, hippocampus-dependent performances in spatial, recognition, and associative memories, which are sensitive to aging, were preserved by environmental enrichment and accompanied by decreased protein phosphatase activity. Age-associated phosphorylated proteins were also found to correspond to the functional categories of age-associated genes identified through transcriptome analysis. Together, this study provides a comprehensive map of the transcriptome and proteome in the aging brain, and elucidates endogenous protein phosphatase-1 inhibition as a potential means through which environmental enrichment may ameliorate age-related cognitive deficits.

Unlabelled: The liver has robust regenerative potential in response to damage, but hepatic steatosis (HS) weakens this potential. We found that the enhanced integrated stress response (ISR) mediated by phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF2α) impairs regeneration in HS and that growth arrest and DNA damage-inducible 34 (Gadd34)-dependent suppression of ISR plays a crucial role in fatty liver regeneration. Although mice fed a high-fat diet for 2 weeks developed moderate fatty liver with no increase in eIF2α phosphorylation before 70% hepatectomy, they showed impaired liver regeneration as a result of reduced proliferation and increased death of hepatocytes with increased phosphorylation of eIF2α and ISR. An increased ISR through Gadd34 knockdown induced C/EBP homologous protein (CHOP)-dependent apoptosis and receptor-interacting protein kinase 3-dependent necrosis, resulting in increased hepatocyte death during fatty liver regeneration. Furthermore, Gadd34 knockdown and increased phosphorylation of eIF2α decreased cyclin D1 protein and reduced hepatocyte proliferation. In contrast, enhancement of Gadd34 suppressed phosphorylation of eIF2α and reduced CHOP expression and hepatocyte apoptosis without affecting hepatocyte proliferation, clearly improving fatty liver regeneration. In more severe fatty liver of leptin receptor-deficient db/db mice, forced expression of hepatic Gadd34 also promoted hepatic regeneration after hepatectomy., Conclusion: Gadd34-mediated regulation of ISR acts as a physiological defense mechanism against impaired liver regeneration resulting from steatosis and is thus a possible therapeutic target for impaired regeneration in HS., (© 2014 by the American Association for the Study of Liver Diseases.)

The regulation of Plasmodium falciparum protein phosphatase type 1 (PfPP1) activity remains to be deciphered. Data from homologous eukaryotic type 1 protein phosphatases (PP1) suggest that several protein regulators should be involved in this essential process. One such regulator, named PfI2 based on its primary sequence homology with eukaryotic inhibitor 2 (I2), was recently shown to be able to interact with PfPP1 and to inhibit its phosphatase activity, mainly through the canonical 'RVxF' binding motif. The details of the structural and functional characteristics of this interaction are investigated here. Using NMR spectroscopy, a second site of interaction is suggested to reside between residues D94 and T117 and contains the 'FxxR/KxR/K' binding motif present in other I2 proteins. This site seems to play in concert/synergy with the 'RVxF' motif to bind PP1, because only mutations in both motifs were able to abolish this interaction completely. However, regarding the structure/function relationship, mutation of either the 'RVxF' or 'FxxR/KxR/K' motif is more drastic, because each mutation prevents the capacity of PfI2 to trigger germinal vesicle breakdown in microinjected Xenopus oocytes. This indicates that the tight association of the PfI2 regulator to PP1, mediated by a two-site interaction, is necessary to exert its function. Based on these results, the use of a peptide derived from the 'FxxR/KxR/K' PfI2 motif was investigated for its potential effect on Plasmodium growth. This peptide, fused at its N-terminus to a penetrating sequence, was shown to accumulate specifically in infected erythrocytes and to have an antiplasmodial effect., (© 2014 FEBS.)

The regulation of transcription factor function in response to neuronal activity is important for development and function of the nervous system. The transcription factor Sp4 regulates the developmental patterning of dendrites, contributes to complex processes including learning and memory, and has been linked to psychiatric disorders such as schizophrenia and bipolar disorder. Despite its many roles in the nervous system, the molecular mechanisms regulating Sp4 activity are poorly understood. Here, we report a site of phosphorylation on Sp4 at serine 770 that is decreased in response to membrane depolarization. Inhibition of the voltage-dependent NMDA receptor increased Sp4 phosphorylation. Conversely, stimulation with NMDA reduced the levels of Sp4 phosphorylation, and this was dependent on the protein phosphatase 1/2A. A phosphomimetic substitution at S770 impaired the Sp4-dependent maturation of cerebellar granule neuron primary dendrites, whereas a non-phosphorylatable Sp4 mutant behaved like wild type. These data reveal that transcription factor Sp4 is regulated by NMDA receptor-dependent activation of a protein phosphatase 1/2A signaling pathway. Our findings also suggest that the regulated control of Sp4 activity is an important mechanism governing the developmental patterning of dendrites., (© 2014 International Society for Neurochemistry.)

Innate immune sensors such as Toll-like receptors (TLRs) differentially utilize adaptor proteins and additional molecular mediators to ensure robust and precise immune responses to pathogen challenge. Through a gain-of-function genetic screen, we identified the gamma catalytic subunit of protein phosphatase 1 (PP1-γ) as a positive regulator of MyD88-dependent proinflammatory innate immune activation. PP1-γ physically interacts with the E3 ubiquitin ligase TRAF6, and enhances the activity of TRAF6 towards itself and substrates such as IKKγ, whereas enzymatically inactive PP1-γ represses these events. Importantly, these activities were found to be critical for cellular innate responses to pathogen challenge and microbial clearance in both mouse macrophages and human monocyte lines. These data indicate that PP1-γ phosphatase activity regulates overall TRAF6 E3 ubiquitin ligase function and promotes NF-κB-mediated innate signaling responses.

At the 3' ends of protein-coding genes, RNA polymerase (Pol) II is dephosphorylated at tyrosine residues (Tyr1) of its C-terminal domain (CTD). In addition, the associated cleavage-and-polyadenylation factor (CPF) cleaves the transcript and adds a poly(a) tail. Whether these events are coordinated and how they lead to transcription termination remains poorly understood. Here we show that CPF from Saccharomyces cerevisiae is a Pol II-CTD phosphatase and that the CPF subunit Glc7 dephosphorylates Tyr1 in vitro. In vivo, the activity of Glc7 is required for normal Tyr1 dephosphorylation at the polyadenylation site, for recruitment of termination factors Pcf11 and Rtt103 and for normal Pol II termination. These results show that transcription termination involves Tyr1 dephosphorylation of the CTD and indicate that pre-mRNA processing by CPF and transcription termination are coupled via Glc7-dependent Pol II-Tyr1 dephosphorylation.

Protein phosphatase 1 (PP1) and Ca2+/calmodulin-dependent protein kinase δ (CaMKIIδ) are upregulated in heart disorders. Alternative splicing factor (ASF), a major splice factor for CaMKIIδ splicing, can be regulated by both protein kinase and phosphatase. Here we determine the role of PP1 isoforms in ASF-mediated splicing of CaMKIIδ in cells. We found that 1) PP1γ, but not α or β isoform, enhanced the splicing of CaMKIIδ in HEK293T cells; 2) PP1γ promoted the function of ASF, evidenced by the existence of ASF-PP1γ association as well as the PP1γ overexpression- or silencing-mediated change in CaMKIIδ splicing in ASF-transfected HEK293T cells; 3) CaMKIIδ splicing was promoted by overexpression of PP1γ and impaired by application of PP1 inhibitor 1 (I1PP1) or pharmacological inhibitor tautomycetin in primary cardiomyocytes; 4) CaMKIIδ splicing and enhancement of ASF-PP1γ association induced by oxygen-glucose deprivation followed by reperfusion (OGD/R) were potentiated by overexpression of PP1γ and suppressed by inhibition of PP1γ with I1PP1 or tautomycetin in primary cardiomyocytes; 5) functionally, overexpression and inhibition of PP1γ, respectively, potentiated or suppressed the apoptosis and Bax/Bcl-2 ratio, which were associated with the enhanced activity of CaMKII in OGD/R-stimulated cardiomyocytes; and 6) CaMKII was required for the OGD/R induced- and PP1γ exacerbated-apoptosis of cardiomyocytes, evidenced by a specific inhibitor of CaMKII KN93, but not its structural analog KN92, attenuating the apoptosis and Bax/Bcl-2 ratio in OGD/R and PP1γ-treated cells. In conclusion, our results show that PP1γ promotes the alternative splicing of CaMKIIδ through its interacting with ASF, exacerbating OGD/R-triggered apoptosis in primary cardiomyocytes.

Background and Purpose: Lysosome-associated membrane protein 3 (LAMP3) is induced by the PKR-like ER kinase (PERK)/activating transcription factor 4 (ATF4)-arm of the unfolded protein response (UPR) during hypoxia. LAMP3 has prognostic value in breast cancer patients treated with radiotherapy. Here, we specifically investigated the role of the PERK/ATF4/LAMP3-arm in the radiation response of breast cancer cells., Material and Methods: Radiosensitivity of breast cancer cells was examined after siRNA-mediated knockdown of PERK, ATF4 and LAMP3. Activation of DNA damage repair proteins was evaluated by Western blotting and immunocytochemistry., Results: Knockdown of the PERK/ATF4/LAMP3-arm and chemical inhibition of PERK could radiosensitise MDA-MB-231 cells significantly. Western blot analysis of several DNA damage repair proteins showed that LAMP3 knockdowns had an attenuated DNA damage response after radiation compared to controls. γ-H2AX foci analysis revealed that LAMP3 knockdowns had a reduced number of positive cells after irradiation, indicating that their DNA damage repair signalling response is decreased. In addition, the effect of autophagy inhibition was examined and revealed a radiosensitising effect and the presence of residual γ-H2AX foci., Conclusions: The PERK/ATF4/LAMP3-arm causes radioresistance of breast cancer cells by increasing DNA damage repair signalling. Inhibition of PERK and/or autophagy may sensitise tumours to radiotherapy., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

The metabotropic glutamate receptor type 7 (mGluR7) is the predominant group III mGluR in the presynaptic active zone, where it serves as an autoreceptor to inhibit neurotransmitter release. Our previous studies show that PKC phosphorylation of mGluR7 on Ser-862 is a key mechanism controlling constitutive and activity-dependent surface expression of mGluR7 by regulating a competitive interaction of calmodulin and protein interacting with C kinase (PICK1). As receptor phosphorylation and dephosphorylation are tightly coordinated through the precise action of protein kinases and phosphatases, dephosphorylation by phosphatases is likely to play an active role in governing the activity-dependent or agonist-induced changes in mGluR7 receptor surface expression. In the present study, we find that the serine/threonine protein phosphatase 1 (PP1) has a crucial role in the constitutive and agonist-induced dephosphorylation of Ser-862 on mGluR7. Treatment of neurons with PP1 inhibitors leads to a robust increase in Ser-862 phosphorylation and increased surface expression of mGluR7. In addition, Ser-862 phosphorylation of both mGluR7a and mGluR7b is a target of PP1. Interestingly, agonist-induced dephosphorylation of mGluR7 is regulated by PP1, whereas NMDA-mediated activity-induced dephosphorylation is not, illustrating there are multiple signaling pathways that affect receptor phosphorylation and trafficking. Importantly, PP1γ1 regulates agonist-dependent Ser-862 dephosphorylation and surface expression of mGluR7.

Potential mechanisms of the reversible temperature-dependent immobilization of fowl sperm were investigated. At 30 °C, motility of demembranated fowl sperm was inhibited by adding 2 mM ethylene glycol-bis (2-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA), but restored immediately after the subsequent addition of 2 mM CaCl(2), whereas at 40 °C, such additions did not appreciably affect motility (which remained almost negligible). With intact sperm, 10(-9) to 10(-3) M Ca(2+) had no effect on motility at 30 °C, which remained high. In contrast, intact sperm at 40 °C were almost immotile below 10(-5) M Ca(2+), and then gradually recovered motility at higher Ca(2+) concentrations. The negligible motility of demembranated sperm at 40 °C, and at 30 °C in the presence of EGTA, was stimulated by addition of 100 nM of the protein phosphatase inhibitor calyculin A. Dynein-ATPase activities of sperm at 40 °C in the presence of 2 mM EGTA, 2 μM CaCl(2), 2 mM CaCl(2,) or 100 nM calyculin A were higher than those at 30 °C. Therefore, stimulation of fowl sperm motility by temperature, Ca(2+), and phosphatase inhibition was not simply associated with an increase of flagellar dynein-ATPase activity. Furthermore, Ca(2+) was essential, at the axonemal level, for initiation of the 'intrinsic' motility of fowl sperm at 30 °C, but this Ca(2+)-dependent mechanism might be different from that involved in restoration of motility of intact sperm at 40 °C. In addition, perhaps inhibition of protein phosphatase activity was involved in initiation of sperm motility, but acting at a location different from Ca(2+) on the axoneme., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Hyperosmotic stress induces the contractile response of vascular smooth muscle cells (VSMCs). Previous studies have demonstrated that cytoskeleton reorganization and Rho/Rho-kinase-mediated inactivation of myosin light chain phosphatase (MLCP) play an important role in hyperosmotic vasoconstriction, but the precise mechanism is unknown. This study aimed to investigate the contractile response of endothelium-denuded rings of rat aortas to hyperosmolar sucrose (160 mM) in the presence or absence of inhibitors for various protein kinases. We found that the hyperosmotic constriction of aortic rings was attenuated not only by ML-7 or hydroxyfasudil, specific inhibitor for myosin light chain kinase (MLCK) or Rho-kinase, respectively, but also by SB203580, a specific inhibitor for p38 mitogen-activated kinase (p38 MAPK). Hyperosmolar sucrose evoked a transient increase in cytosolic free Ca(2+) in rat VSMCs, and this response was not affected by SB203580. Western blot analysis of proteins extracted from rings showed that the hyperosmolar sucrose stimulated phosphorylation of the Rho-kinase-mediated myosin phosphatase target subunit 1, myosin light chain (MLC), and p38 MAPK. The experiments performed using a combination of the kinase inhibitors showed that hyperosmolarity-induced MLC phosphorylation is partially mediated via the SB203580-sensitive pathway and is independent of both MLCK and Rho-kinase-mediated inactivation of MLCP. Furthermore, the hyperosmolarity-induced increase in the F-actin/G-actin ratio in rings was attenuated not only by hydroxyfasudil but also by SB203580. These results suggest that p38 MAPK is involved in hyperosmotic vasoconstriction via stimulation of MLC phosphorylation and cytoskeleton reorganization through pathways independent of activation of MLCK and/or Rho-kinase-mediated mechanisms.

Dopamine (DA) signaling in the medial prefrontal cortex (mPFC) plays a critical role in the processing of emotional information and memory encoding. Activation of DA D4 receptors within the prelimbic (PLC) division of the mPFC bidirectionally modulates emotional memory by strongly potentiating the salience of normally nonsalient emotional memories but blocking the acquisition of suprathreshold emotionally salient fear memories. Previous in vitro studies have shown that activation of cortical DA D4 receptors can bidirectionally modulate levels of α-calcium calmodulin-dependent kinase II (α-CaMKII), a molecule essential for learning and memory. Using an olfactory fear conditioning procedure in rats combined with microinfusions into the mPFC, we examined the potential role of D4 receptor-mediated control of emotional memory salience through signaling via CaMKII, cAMP/protein kinase A (PKA), and protein phosphatase-1 (PP1) signaling. We report that CaMKII blockade prevents the ability of intra-mPFC DA D4 receptor activation to potentiate the salience of subthreshold fear memory. In contrast, blockade of either cAMP/PKA or PP1 signaling pathways rescued the blockade of suprathreshold fear memory via intra-mPFC D4 receptor activation. Our results demonstrate that modulation of emotional memory salience via intra-mPFC DA D4 receptor transmission depends upon downstream signaling via CaMKII, cAMP/PKA, and PP1 substrates.

Patients with Krabbe disease, a genetic demyelinating syndrome caused by deficiency of galactosyl-ceramidase and the resulting accumulation of galactosyl-sphingolipids, develop signs of a dying-back axonopathy compounded by a deficiency of large-caliber axons. Here, we show that axonal caliber in Twitcher mice, an animal model for Krabbe disease, is impaired in peripheral axons and is accompanied by a progressive reduction in the abundance and phosphorylation of the three neurofilament (NF) subunits. These changes correlate with an increase in the density of NFs per cross-sectional area in numerous mutant peripheral axons and abnormal increases in the activity of two serine/threonine phosphatases (PP1 and PP2A) in mutant tissue. Similarly, acutely isolated mutant cortical neurons show abnormal phosphorylation of NFs. Psychosine, the neurotoxin accumulated in Krabbe disease, was sufficient to induce abnormal dephosphorylation of NF subunits in a normal motor neuron cell line as well as in acutely isolated normal cortical neurons. This in vitro effect was mediated by PP1 and PP2A, which specifically dephosphorylated NFs. These results demonstrate that the reduced caliber observed in some axons in Krabbe disease involves abnormal dephosphorylation of NFs. We propose that a psychosine-driven pathogenic mechanism through deregulated phosphotransferase activities may be involved in this process., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Animal cells undergo dramatic actin-dependent changes in shape as they progress through mitosis; they round up upon mitotic entry and elongate during chromosome segregation before dividing into two [1-3]. Moesin, the sole Drosophila ERM-family protein [4], plays a critical role in this process, through the construction of a stiff, rounded metaphase cortex [5-7]. At mitotic exit, this rigid cortex must be dismantled to allow for anaphase elongation and cytokinesis through the loss of the active pool of phospho-Thr559moesin from cell poles. Here, in an RNA interference (RNAi) screen for phosphatases involved in the temporal and spatial control of moesin, we identify PP1-87B RNAi as having elevated p-moesin levels and reduced cortical compliance. In mitosis, RNAi-induced depletion of PP1-87B or depletion of a conserved noncatalytic PP1 phosphatase subunit Sds22 leads to defects in p-moesin clearance from cell poles at anaphase, a delay in anaphase elongation, together with defects in bipolar anaphase relaxation and cytokinesis. Importantly, similar cortical defects are seen at anaphase following the expression of a constitutively active, phosphomimetic version of moesin. These data reveal a new role for the PP1-87B/Sds22 phosphatase, an important regulator of the metaphase-anaphase transition, in coupling moesin-dependent cell shape changes to mitotic exit., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Background: Epithelial barrier defects are well known in coeliac disease, but the mechanisms are only poorly defined. It is unclear, whether barrier disturbance reflects upregulated epithelial transcytosis or paracellular leakage., Objective: To characterise the molecular structure and function of the epithelial tight junction (TJ) and mechanisms of its dysregulation., Methods: Molecular analysis of proteins involved in TJ assembly and their regulation was performed by western blotting and confocal microscopy correlated to electrophysiology., Results: A complex alteration of the composition of epithelial TJ proteins (with more pore-forming claudins like claudin-2 and a reduction in tightening claudins like claudin-3, -5 and -7) was found for protein expression and subcellular localisation, responsible for an increase in paracellular biotin-NHS uptake. In contrast, epithelial apoptosis was only moderately elevated (accounting for a minor portion of barrier defects) and epithelial gross lesions--for example, at cell extrusion zones, were absent. This TJ alteration was linked to an altered localisation/expression of proteins regulating TJ assembly, the polarity complex protein Par-3 and the serine-/threonine phosphatase PP-1., Conclusions: Changes in cell polarity proteins Par-3 and PP-1 are associated with altered expression and assembly of TJ proteins claudin-2, -3, -5 and -7 and ZO-1, causing paracellular leakage in active coeliac disease.

Alzheimer's Disease (AD) is characterized by progressive loss of cognitive function, linked to marked neuronal loss. Pathological hallmarks of the disease are the accumulation of the amyloid-β (Aβ) peptide in the form of amyloid plaques and the intracellular formation of neurofibrillary tangles (NFTs). Accumulating evidence supports a key role for protein phosphorylation in both the normal and pathological actions of Aβ as well as the formation of NFTs. NFTs contain hyperphosphorylated forms of the microtubule-binding protein tau, and phosphorylation of tau by several different kinases leads to its aggregation. The protein kinases involved in the generation and/or actions of tau or Aβ are viable drug targets to prevent or alleviate AD pathology. However, it has also been recognized that the protein phosphatases that reverse the actions of these protein kinases are equally important. Here, we review recent advances in our understanding of serine/threonine and tyrosine protein phosphatases in the pathology of AD., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Long-term memory formation is sensitive to the pattern of training sessions. Training distributed over time (spaced training) is superior at generating long-term memories than training presented with little or no rest interval (massed training). This spacing effect was observed in a range of organisms from invertebrates to humans. In the present paper, we discuss the evidence supporting cyclic-AMP response element-binding protein 2 (CREB), a transcription factor, as being an important molecule mediating long-term memory formation after spaced training. We also review the main upstream proteins that regulate CREB in different model organisms. Those include the eukaryotic translation initiation factor (eIF2α), protein phosphatase I (PP1), mitogen-activated protein kinase (MAPK), and the protein tyrosine phosphatase corkscrew. Finally, we discuss PKC activation and protein synthesis and degradation as mechanisms by which neurons decode the spacing intervals.

The integrated stress response (ISR) is an evolutionarily conserved homeostatic program activated by specific pathological states. These include amino acid deprivation, viral infection, iron deficiency, and the misfolding of proteins within the endoplasmic reticulum (ER), the so-called ER stress. Although apparently disparate, each of these stresses induces phosphorylation of a translation initiation factor, eIF2α, to attenuate new protein translation while simultaneously triggering a transcriptional program. This is achieved by four homologous stress-sensing kinases: GCN2, PKR, HRI, and PERK. In addition to these kinases, mammals possess two specific eIF2α phosphatases, GADD34 and CReP, which play crucial roles in the recovery of protein synthesis following the initial insult. They are not only important in embryonic development but also appear to play important roles in disease, particularly cancer. In this chapter, we discuss each of the eIF2α kinases, in turn, with particular emphasis on their regulation and the new insights provided by recent structural studies. We also discuss the potential for developing novel drug therapies that target the ISR., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The drs gene is an apoptosis-inducing tumor suppressor. By using drs-knockout (KO) mouse embryonic fibroblasts (MEFs), we showed that drs is involved in the host defense against viral infection. In drs-KO MEFs infected with vesicular stomatitis virus, the viral replication and protein synthesis were markedly enhanced without the upregulation of the cellular protein synthesis. Phosphorylation of S6K, S6, 4EBP1 and TSC2 proteins was closely correlated with the enhanced viral replication in drs-KO MEFs. Drs protein could associate with stress-inducible GADD34 to form a complex with TSC1/2, which suppresses mTOR activity. These findings indicate that Drs suppresses viral replication via mTOR-dependent pathway., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

The phosphorylation status of the Snf1 activation loop threonine is determined by changes in the rate of its dephosphorylation, catalyzed by the yeast PP1 phosphatase Glc7 in complex with the Reg1 protein. Previous studies have shown that Reg1 can associate with both Snf1 and Glc7, suggesting substrate binding as a mechanism for Reg1-mediated targeting of Glc7. In this study, the association of Reg1 with the three Snf1 isoforms was measured by two-hybrid analysis and coimmunoprecipitation. We found that Reg1 association with Snf1 occurred almost exclusively with the Gal83 isoform of the Snf1 complex. Nonetheless, Reg1 plays an important role in determining the phosphorylation status of all three Snf1 isoforms. We found that the rate of dephosphorylation for isoforms of Snf1 did not correlate with the amount of associated Reg1 protein. Functional chimeric β subunits containing residues from Gal83 and Sip2 were used to map the residues needed to promote Reg1 association with the N-terminal 150 residues of Gal83. The Gal83 isoform of Snf1 is the only isoform capable of nuclear localization. A Gal83-Sip2 chimera containing the first 150 residues of Gal83 was able to associate with the Reg1 protein but did not localize to the nucleus. Therefore, nuclear localization is not required for Reg1 association. Taken together, these data indicate that the ability of Reg1 to promote the dephosphorylation of Snf1 is not directly related to the strength of its association with the Snf1 complex.

Abnormal regulation of brain glycogen metabolism is believed to underlie insulin-induced hypoglycaemia, which may be serious or fatal in diabetic patients on insulin therapy. A key regulator of glycogen levels is glycogen targeted protein phosphatase 1 (PP1), which dephosphorylates and activates glycogen synthase (GS) leading to an increase in glycogen synthesis. In this study, we show that the gene PPP1R3F expresses a glycogen-binding protein (R3F) of 82.8 kDa, present at the high levels in rodent brain. R3F binds to PP1 through a classical 'RVxF' binding motif and substitution of Phe39 for Ala in this motif abrogates PP1 binding. A hydrophobic domain at the carboxy-terminus of R3F has similarities to the putative membrane binding domain near the carboxy-terminus of striated muscle glycogen targeting subunit G(M)/R(GL), and R3F is shown to bind not only to glycogen but also to membranes. GS interacts with PP1-R3F and is hyperphosphorylated at glycogen synthase kinase-3 sites (Ser640 and Ser644) when bound to R3F(Phe39Ala). Deprivation of glucose or stimulation with adenosine or noradrenaline leads to an increased phosphorylation of PP1-R3F bound GS at Ser640 and Ser644 curtailing glycogen synthesis and facilitating glycogen degradation to provide glucose in astrocytoma cells. Adenosine stimulation also modulates phosphorylation of R3F at Ser14/Ser18., (© 2011 The Authors. Journal of Neurochemistry © 2011 International Society for Neurochemistry.)

Loss of epithelial integrity often correlates with the progression of malignant tumors. Sds22, a regulatory subunit of protein phosphatase 1 (PP1), has recently been linked to regulation of epithelial polarity in Drosophila. However, its role in tumorigenesis remains obscure. In this study, using Drosophila imaginal tissue as an in vivo model system, we show that sds22 is a new potential tumor suppressor gene in Drosophila. Without sds22, cells lose epithelial architecture, and become invasive and tumorigenic when combined with Ras overexpression; conversely, sds22 overexpression can largely suppress tumorigenic growth of Ras(V12)scrib(-/-) mutant cells. Mechanistically, we show that sds22 prevents cell invasion and metastasis by inhibiting myosin II and Jun N-terminal kinase (JNK) activity downstream of PP1. Loss of this inhibition causes cells to lose epithelial organization and promotes cell invasion. Finally, human Sds22 is focally deleted and downregulated in multiple carcinomas, and this downregulation correlates with tumor progression, suggesting that sds22 inactivation may contribute to tumorigenesis and metastatic potential in human cancers via a similar mechanism.