Imeglimin Ameliorates ß-Cell Apoptosis by Modulating the Endoplasmic Reticulum Homeostasis Pathway.

The effects of imeglimin, a novel antidiabetes agent, on ß-cell function remain unclear. Here, we unveiled the impact of imeglimin on ß-cell survival. Treatment with imeglimin augmented mitochondrial function, enhanced insulin secretion, promoted ß-cell proliferation, and improved ß-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 ß-cells under ER stress. Imeglimin failed to protect against ER stress-induced ß-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 ß-cells and increased ß-cell mass in Akita mice. Imeglimin also protected against ß-cell apoptosis in both human islets and human pluripotent stem cell-derived ß-like cells. Taken together, imeglimin modulates the ER homeostasis pathway, which results in the prevention of ß-cell apoptosis both in vitro and in vivo.

Sensitization of the UPR by loss of PPP1R15A promotes fibrosis and senescence in IPF.

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-ß-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.

Regulation of Synaptic Transmission and Plasticity by Protein Phosphatase 1.

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, ß, 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.

LACTB suppresses melanoma progression by attenuating PP1A and YAP interaction.

Very limited progress has been made in the management of advanced melanoma, especially melanoma of uveal origin. Lactamase ß (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.

The doxorubicin-induced cell motility network is under the control of the ceramide-activated protein phosphatase 1 alpha.

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.

Protein phosphatase 1 alpha enhances glucocorticoid receptor activity by a mechanism involving phosphorylation of serine-211.

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.

Protein phosphatase 1 activity controls a balance between collective and single cell modes of migration.

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.

Essential role of GEXP15, a specific Protein Phosphatase type 1 partner, in Plasmodium berghei in asexual erythrocytic proliferation and transmission.

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.

Dephosphorylation of protamine 2 at serine 56 is crucial for murine sperm maturation in vivo.

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 Ser56) by Ppp1cc2. To confirm the biological importance of Prm2 Ser56 dephosphorylation, we mutated Ser56 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.

The meiotic phosphatase GSP-2/PP1 promotes germline immortality and small RNA-mediated genome silencing.

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.

Differential localizations of protein phosphatase 1 isoforms determine their physiological function in the heart.

Protein phosphatase 1 isoforms α, ß, and γ (PP1α, PP1ß, 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, PP1ß 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 PP1ß or PP1γ. Interestingly, only cardiac-specific knockout of PP1ß resulted in increased HDAC7 phosphorylation, consistent with the predominant nuclear localization of PP1ß. Functionally, deletion of either PP1 isoform resulted in reduced fractional shortening in aging mice, however only PP1ß 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.

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

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.

Myosin phosphatase: Unexpected functions of a long-known enzyme.

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.

cAMP regulation of protein phosphatases PP1 and PP2A in brain.

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.

Serine-threonine protein phosphatases: Lost in translation.

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.

Astragalosides increase the cardiac diastolic function and regulate the "Calcium sensing receptor-protein kinase C-protein phosphatase 1" pathway in rats with heart failure.

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.

Preemptive Activation of the Integrated Stress Response Protects Mice From Diet-Induced Obesity and Insulin Resistance by Fibroblast Growth Factor 21 Induction.

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 (CRePLKO ) mice exhibited marked browning of white adipose tissue (WAT) and increased energy expenditure and insulin sensitivity in an FGF21-dependent manner. Furthermore, CRePLKO 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.

Antagonistic Effects of p53 and HIF1A on microRNA-34a Regulation of PPP1R11 and STAT3 and Hypoxia-induced Epithelial to Mesenchymal Transition in Colorectal Cancer Cells.

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.

Lemur tyrosine kinase 2 (LMTK2) is a determinant of cell sensitivity to apoptosis by regulating the levels of the BCL2 family members.

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 GSK3ß- 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.

GADD34 Keeps the mTOR Pathway Inactivated in Endoplasmic Reticulum Stress Related Autophagy.

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.