Overexpression Of Protein Phosphatase 2A Research Articles

LncRNA ENST00000440246.1 Promotes Alzheimer's Disease Progression by Targeting PP2A.

Alzheimer's disease (AD) is an extremely prevalent neurodegenerative disease. Long noncoding RNAs (lncRNAs) play pivotal roles in the regulation of AD. However, the function of most lncRNAs in AD remains to be elucidated. In this study, the effects of lncRNA ENST00000440246.1 on the biological characteristics of AD were explored. Differentially expressed lncRNAs in AD were identified through bioinformatics analysis and peripheral blood from thirty AD patients was collected to verify the expression of these lncRNAs by quantitative real-time polymerase chain reaction (RT-qPCR). The correlations between lncRNAs and the Mini-Mental State Examination (MMSE) or the Montreal Cognitive Assessment (MoCA) were assessed by Pearson's correlation analysis. Immunofluorescence (IF), Cell Counting Kit-8 (CCK-8) and flow cytometry assays were conducted to evaluate the biological effect of ENST00000440246.1 and protein phosphatase 2A (PP2A) in SK-N-SH cells. Gene expression at the protein and mRNA levels was analyzed by Western blotting and RT-qPCR. The interaction between PP2A and ENST00000440246.1 was confirmed by IntaRNA and RNA pulldown assays. ENST00000440246.1 was upregulated and significantly negatively correlated with the MMSE and MoCA scores and the overexpression of ENST00000440246.1 inhibited cell proliferation and facilitated apoptosis and Aβ expression in SK-N-SH cells. Mechanistically, ENST00000440246.1 targeted PP2A and regulated AD-related gene expression. The silencing of ENST00000440246.1 had the opposite effect. Furthermore, PP2A overexpression reversed the influence of ENST00000440246.1 overexpression in SK-N-SH cells. In conclusion, ENST00000440246.1 could promote AD progression by targeting PP2A, which indicates that ENST00000440246.1 has the potential to be a diagnostic target in AD.

Sodium Tanshinone IIA Sulfonate Ameliorates Oxygen-glucose Deprivation/Reoxygenation-induced Neuronal Injury via Protection of Mitochondria and Promotion of Autophagy.

Sodium tanshinone IIA sulfonate (STS) has shown significant clinical therapeutic effects in cerebral ischemic stroke (CIS), but the molecular mechanisms of neuroprotection remain partially known. The purpose of this study was to explore whether STS plays a protective role in oxygen-glucose deprivation/reoxygenation (OGD/R)-induced neuronal injury by regulating microglia autophagy and inflammatory activity. Co-cultured microglia and neurons were subjected to OGD/R injury, an in vitro model of ischemia/reperfusion (I/R) injury with or without STS treatment. Expression of protein phosphatase 2A (PP2A) and autophagy-associated proteins Beclin 1, autophagy related 5 (ATG5), and p62 in microglia was determined by Western blotting. Autophagic flux in microglia was observed with confocal laser scanning microscopy. Neuronal apoptosis was measured by flow cytometric and TUNEL assays. Neuronal mitochondrial function was determined via assessments of reactive oxygen species generation and mitochondrial membrane potential integrity. STS treatment markedly induced PP2A expression in microglia. Forced overexpression of PP2A increased levels of Beclin 1 and ATG5, decreased the p62 protein level, and induced autophagic flux. Silencing of PP2A or administration of 3-methyladenine inhibited autophagy and decreased the production of anti-inflammatory factors (IL-10, TGF-β and BDNF) and induced the release of proinflammatory cytokines (IL-1β, IL-2 and TNF-α) by STS-treated microglia, thereby inducing mitochondrial dysfunction and apoptosis of STS-treated neurons. STS exerts protection against neuron injury, and the PP2A gene plays a crucial role in improving mitochondrial function and inhibiting neuronal apoptosis by regulating autophagy and inflammation in microglia.

Magnesium isoglycyrrhizinate prevents cadmium-induced activation of JNK and apoptotic hepatocyte death by reversing ROS-inactivated PP2A.

Cadmium (Cd) induces reactive oxygen species (ROS)-mediated hepatocyte apoptosis and consequential liver disorders. This study aimed to investigate the effect of magnesium isoglycyrrhizinate (MgIG) on Cd-induced hepatotoxicity. L02 and AML-12 cells were used to study MgIG hepatoprotective effects. Cd-evoked apoptosis, ROS and protein phosphatase 2A (PP2A)/c-Jun N-terminal kinase (JNK) cascade disruption were analysed by cell viability assay, 6-diamidino-2-phenylindole (DAPI) and TdT-mediated dUTP Nick-End Labeling (TUNEL) staining, ROS imaging and Western blotting. Pharmacological and genetic approaches were used to explore the mechanisms. We show that MgIG attenuated Cd-evoked hepatocyte apoptosis by blocking JNK pathway. Pre-treatment with SP600125 or ectopic expression of dominant-negative c-Jun enhanced MgIG's anti-apoptotic effects. Further investigation found that MgIG rescued Cd-inactivated PP2A. Inhibition of PP2A activity by okadaic acid attenuated the MgIG's inhibition of the Cd-stimulated JNK pathway and apoptosis; in contrast, overexpression of PP2A strengthened the MgIG effects. In addition, MgIG blocked Cd-induced ROS generation. Eliminating ROS by N-acetyl-l-cysteine abrogated Cd-induced PP2A-JNK pathway disruption and concurrently reinforced MgIG-conferred protective effects, which could be further slightly strengthened by PP2A overexpression. Our findings indicate that MgIG is a promising hepatoprotective agent for the prevention of Cd-induced hepatic injury by mitigating ROS-inactivated PP2A, thus preventing JNK activation and hepatocyte apoptosis.

PP2A protects podocytes against Adriamycin-induced injury and epithelial-to-mesenchymal transition via suppressing JIP4/p38-MAPK pathway.

Protein phosphatase 2A (PP2A) is one of the major protein serine/threonine phosphatases (PPPs) with regulatory effects on several cellular processes, but its role and function in Adriamycin (ADR)-treated podocytes injury needs to be further explored. Mice podocytes were treated with ADR and PP2A inhibitor (okadaic acid, OA). After transfection, cell apoptosis was detected by flow cytometry. Expressions of podocytes injury-, apoptosis- and epithelial-to-mesenchymal transition (EMT)- and JNK-interacting protein 4/p38-Mitogen-Activated Protein Kinase (JIP4/p38-MAPK) pathway-related factors were measured using quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot as needed. Interaction between PP2A and JIP4/MAPK pathway was confirmed using co-immunoprecipitation (Co-Ip) assay. In podocytes, ADR inhibited PP2A, Nephrin and Wilms' tumor (WT) 1 expressions yet upregulated apoptosis and Desmin expression, and suppressing PP2A expressionenhanced the effects. PP2A overexpression reversed the effects of ADR on PP2A and podocyte injury-related factors expressions and apoptosis of podocytes. JIP4 was the candidate gene interacting with both PP2A and p38-MAPK pathway, and PP2A overexpression alleviated the effects of ADR on p38-MAPK pathway-related factors expressions. Additionally, in ADR-treated podocytes, PP2A suppression enhanced the effects of ADR, yet silencing of JIP4 reversed the effects of PP2A suppression on regulating p38-MAPK pathway-, apoptosis- and EMT-related factors expressions and apoptosis, with upregulations of B-cell lymphoma-2 (Bcl-2) and E-cadherin and down-regulations of Bcl-2 associated protein X (Bax), cleaved (C)-casapse-3, N-cadherin, Vimentin and Snail. PP2A protects ADR-treated podocytes against injury and EMT by suppressing JIP4/p38-MAPK pathway, showing their interaction in podocytes.

Reduction of protein phosphatase 2A (PP2A) complexity reveals cellular functions and dephosphorylation motifs of the PP2A/B′δ holoenzyme

Protein phosphatase 2A (PP2A) is a large enzyme family responsible for most cellular Ser/Thr dephosphorylation events. PP2A substrate specificity, localization, and regulation by second messengers rely on more than a dozen regulatory subunits (including B/R2, B'/R5, and B″/R3), which form the PP2A heterotrimeric holoenzyme by associating with a dimer comprising scaffolding (A) and catalytic (C) subunits. Because of partial redundancy and high endogenous expression of PP2A holoenzymes, traditional approaches of overexpressing, knocking down, or knocking out PP2A regulatory subunits have yielded only limited insights into their biological roles and substrates. To this end, here we sought to reduce the complexity of cellular PP2A holoenzymes. We used tetracycline-inducible expression of pairs of scaffolding and regulatory subunits with complementary charge-reversal substitutions in their interaction interfaces. For each of the three regulatory subunit families, we engineered A/B charge-swap variants that could bind to one another, but not to endogenous A and B subunits. Because endogenous Aα was targeted by a co-induced shRNA, endogenous B subunits were rapidly degraded, resulting in expression of predominantly a single PP2A heterotrimer composed of the A/B charge-swap pair and the endogenous catalytic subunit. Using B'δ/PPP2R5D, we show that PP2A complexity reduction, but not PP2A overexpression, reveals a role of this holoenzyme in suppression of extracellular signal-regulated kinase signaling and protein kinase A substrate dephosphorylation. When combined with global phosphoproteomics, the PP2A/B'δ reduction approach identified consensus dephosphorylation motifs in its substrates and suggested that residues surrounding the phosphorylation site play roles in PP2A substrate specificity.

Reduced Local Response to Corticosteroids in Eosinophilic Chronic Rhinosinusitis with Asthma.

Eosinophilic chronic rhinosinusitis (ECRS), a subgroup of chronic rhinosinusitis with nasal polyps, is recognized as a refractory eosinophilic disorder characterized by both upper and lower airway inflammation. In some severe cases, disease control is poor, likely due to local steroid insensitivity. In this study, we focused on protein phosphatase 2A (PP2A), a key factor regulating glucocorticoid receptor (GR) nuclear translocation, and examined its association with local responses to corticosteroids in eosinophilic airway inflammation. Our results indicated reduced responses to corticosteroids in nasal epithelial cells from ECRS patients with asthma, which were also associated with decreased PP2A mRNA expression. Eosinophil peroxidase stimulates elevated PP2A phosphorylation levels, reducing PP2A protein expression and activity. In addition, mRNA levels of inflammatory mediators (TSLP, IL-25, IL-33, CCL4, CCL5, CCL11, and CCL26) associated with eosinophilic airway inflammation in epithelial cells were increased in nasal polyps (eosinophil-rich areas) compared with those in uncinate process tissues (eosinophil-poor areas) from the same patients. PP2A reduction by siRNA reduced GR nuclear translocation, whereas PP2A overexpression by plasmid transfection, or PP2A activation by formoterol, enhanced GR nuclear translocation. Collectively, our findings indicate that PP2A may represent a promising therapeutic target in refractory eosinophilic airway inflammation characterized by local steroid insensitivity.

Roles of I2PP2A in the downregulation of eNOS Ser1177 phosphorylation by angiotensin II-activated PP2A

The chronic elevation of angiotensin II (Ang II) is an important cause of endothelial dysfunction (ED). The Ang II/type 1 receptor (AT1R) signaling pathway can cause endothelial nitric oxide synthase (eNOS)/nitric oxide (NO) dysfunction through various mechanisms leading to ED. The modulation of eNOS phosphorylated at Ser1177 is an important mechanism upregulating eNOS activity. Protein phosphatase 2 A (PP2A) has been reported to dephosphorylate eNOS at Ser1177. The PP2A inhibitor 2 protein (I2PP2A) is a specific endogenous inhibitor that binds the catalytic subunit of PP2A and directly inhibits PP2A activity. Therefore, we hypothesized that Ang II might attenuate I2PP2A expression to activate PP2A, which downregulates eNOS Ser 1177 phosphorylation, leading to eNOS dysfunction. In our study, we used Ang II-treated human umbilical vein endothelial cells (HUVECs) and, found that the eNOS Ser1177 phosphorylation levels were downregulated, the activity of PP2A was increased, and I2PP2A expression was decreased. Furthermore, these effects were blocked by candesartan (CAN). The phosphorylation levels of eNOS Ser1177 were decreased after I2PP2A was knocked down by specific siRNA but increased after I2PP2A overexpression. We also found that the Ang II treatment decreased the association of I2PP2A with PP2A but increased the association between PP2A and eNOS. Taken together, our results suggest that Ang II activates PP2A by downregulating the I2PP2A expression through the AT1R signaling pathway leading to the loss of eNOS Ser1177 phosphorylation and ED.

Berberine attenuates pulmonary arterial hypertension via protein phosphatase 2A signaling pathway both in vivo and in vitro.

Excessive proliferation, migration, and antiapoptosis of pulmonary artery (PA) smooth muscle cells (PASMCs) underlies the development of pulmonary vascular remodeling. The innervation of the PA is predominantly sympathetic, and increased levels of circulating catecholamines have been detected in pulmonary arterial hypertension (PAH), suggesting that neurotransmitters released by sympathetic overactivation may play an essential role in PAH. However, the responsible mechanism remains unclear. Here, to investigate the effects of norepinephrine (NE) on PASMCs and the related mechanism, we used 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide, the proliferating cell nuclear antigen and the cell counting kit-8 assay to evaluate the proliferation of PASMCs, Boyden chamber migration, and wound-healing assays to assess migration and western blot analysis to investigate protein expression. We demonstrated that the phosphorylation level of the protein phosphatase 2A (PP2A) catalytic subunit (Y307) was higher in PAH patients and PAH models than in controls, both in vivo and in vitro. In addition, NE induced the proliferation and migration of PASMCs, which was attenuated by berberine (BBR), a Chinese herbal medicine, and/or PP2A overexpression. PP2A inhibition worsened NE-induced PAH and could not be reversed by BBR. Thus, PP2A is critical in driving PAH, and BBR may alleviate PAH via PP2A signaling pathways, thereby offering a potential therapeutic option for PAH.

Forskolin Induces Hyperphosphorylation of Tau Accompanied by Cell Cycle Reactivation in Primary Hippocampal Neurons

Abnormally hyperphosphorylated tau is the major protein constituent of neurofibrillary tangles (NFTs) in the brain of Alzheimer disease (AD) patients. Cell cycle reactivation is considered an important neuropathological feature of AD, and re-expression and activation of cell cycle regulators are known to occur in neurons containing NFTs. The aim of the present study was to investigate cell cycle reactivation during tau hyperphosphorylation in primary hippocampal neurons. We used forskolin, a specific activator of PKA, to induce tau hyperphosphorylation in cultured primary hippocampal neurons, and then measured levels of cyclin D1 and cyclin B1. We found that forskolin induced hyperphosphorylation of tau at Ser214, Ser396, and Ser202/Thr205 sites, attaining peak levels at 6, 12, and 12h, respectively, while returning to normal levels at 24h. Forskolin also induced a sustained cAMP elevation and PKA activation, which peaked at 6h, in association with activation and overexpression of protein phosphatase-2A (PP-2A) at 24h. The tau hyperphosphorylation was accompanied by increases in cyclin D1 and cyclin B1 levels; immunostaining showed overlapping distribution of hyperphosphorylated tau and cyclin D1 and cyclin B1 in primary hippocampal neurons. Forskolin induced hyperphosphorylation of tau and increased cyclin D1 and cyclin B1 protein levels in HEK293/tau441 cells, but not in the HEK293/vector cells, whereas the PKA inhibitor H89 inhibited the effects of forskolin on tau hyperphosphorylation and cyclin D1 and cyclin B1 protein levels. These findings suggest that forskolin induces tau hyperphosphorylation, which is itself necessary for the subsequent increases of cyclin D1 and cyclin B1 levels.

Fusarochromanone-induced reactive oxygen species results in activation of JNK cascade and cell death by inhibiting protein phosphatases 2A and 5.

Recent studies have shown that fusarochromanone (FC101), a mycotoxin, is cytotoxic in a variety of cell lines. However, the molecular mechanism underlying its cytotoxicity remains elusive. Here we found that FC101 induced cell death in COS7 and HEK293 cells in part by activating JNK pathway. This is evidenced by the findings that inhibition of JNK with SP600125 or expression of dominant negative c-Jun partially prevented FC101-induced cell death. Furthermore, we observed that FC101-activated JNK pathway was attributed to induction of reactive oxygen species (ROS). Pretreatment with N-acetyl-L-cysteine (NAC), a ROS scavenger and antioxidant, suppressed FC101-induced activation of JNK and cell death. Moreover, we noticed that FC101 inhibited the serine/threonine protein phosphatases 2A (PP2A) and 5 (PP5) in the cells, which was abrogated by NAC. Overexpression of PP2A or PP5 partially prevented FC101-induced activation of JNK and cell death. The results indicate that FC101-induced ROS inhibits PP2A and PP5, leading to activation of JNK pathway and consequently resulting in cell death.

Resveratrol prevents cadmium activation of Erk1/2 and JNK pathways from neuronal cell death via protein phosphatases 2A and 5.

Cadmium (Cd), a toxic environmental contaminant, induces neurodegenerative disorders. Resveratrol, a natural product, has been found to exert neuroprotective effects. However, little is known regarding the effect of resveratrol on Cd-evoked neurotoxicity. Here, we show that resveratrol effectively reversed Cd-elicited cell viability reduction, morphological change, nuclear fragmentation and condensation, as well as activation of caspase-3 in neuronal cells, implying neuroprotection against Cd-poisoning by resveratrol. Further research revealed that both c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinases 1/2 (Erk1/2) were involved in the inhibitory effect of resveratrol on Cd-induced cell death, as selective inhibitors of Erk1/2 (U0126) and JNK (SP600125), or over-expression of dominant negative mitogen-activated protein kinase kinase 1 (MKK1) or dominant negative c-Jun potentiated resveratrol's prevention of Cd-induced phosphorylation of JNK and Erk1/2, as well as cell death in neuronal cells. Interestingly, resveratrol potently rescued the cells from Cd-induced suppression of protein phosphatases 2A (PP2A) and 5 (PP5) activity. Over-expression of PP2A or PP5 strengthened the inhibitory effects of resveratrol on Cd-induced activation of Erk1/2 and/or JNK, as well as cell death. The results indicate that resveratrol prevents Cd-induced activation of Erk1/2 and JNK pathways and neuronal cell death in part via activating PP2A and PP5. Our findings strongly support the notion that resveratrol may serve as a potential therapeutic agent in the prevention of Cd-induced neurodegenerative diseases.

Overexpression of protein phosphatase 2A in a murine model of chronic myocardial infarction leads to increased adverse remodeling but restores the regulation of β-catenin by glycogen synthase kinase 3β

Background/ObjectivesIncreased activity of cardiac protein phosphatases is an important feature in human heart failure. Several different protein phosphatases (PP) are involved in the regulation of excitation–contraction-coupling of the myocardium. Protein phosphatase 2A (PP2A) is a serine/threonine phosphatase consisting of a dimeric core enzyme and tissue-specific subunits. In this study we used transgenic mice overexpressing PP2A to further investigate the role of PP2A in cardiac remodeling after myocardial infarction. Methods and resultsAdult male CD-1 mice overexpressing the catalytic subunit α of PP2A (αMHC-PP2A; TG) underwent chronic LAD-ligation or sham surgery, respectively; wildtype littermates (WT) were used as controls. Cardiac function was determined by echocardiography before and 28days after LAD-ligation. 28days after MI, the animals were sacrificed and cardiac remodeling was analyzed in histological sections and by Western blots. PP2A overexpression leads to dilated cardiomyopathy in mice, and increased cardiomyocyte hypertrophy and fibrosis of the remote myocardium can be seen after myocardial infarction. However, we found an improved survival of TG in the subacute phase after MI in comparison to WT. On the molecular level, TG shows reduced expression of SERCA and CaMKII alpha both under basal condition as well 28days after MI. Additionally, the regulation of the Akt/GSK3β/β-catenin pathway is severely disturbed in TG at baseline where a significant activation of Akt is found that coincides with the typical phosphorylation of GSK3β. However, this does not lead to the accumulation of β-catenin — on the contrary: phosphorylation-induced degradation of β-catenin is significantly enhanced. ConclusionTransgenic overexpression of myocardial PP2A causes adverse remodeling which coincides with a disruption of the classical Akt/GSK3/β-catenin pathway under baseline conditions that is restored to normal values in chronic myocardial infarction. Even so overall survival of TG after myocardial infarction was not constrained and survival after day 2 post MI was improved.

Knockdown of phosphotyrosyl phosphatase activator induces apoptosis via mitochondrial pathway and the attenuation by simultaneous tau hyperphosphorylation.

Phosphotyrosyl phosphatase activator (PTPA) is decreased in the brains of Alzheimer's disease (AD) and the AD transgenic mouse models. Here, we investigated whether down-regulation of PTPA affects cell viability and the underlying mechanisms. We found that PTPA was located in the integral membrane of mitochondria, and knockdown of PTPA induced cell apoptosis in HEK293 and N2a cell lines. PTPA knockdown decreased mitochondrial membrane potential and induced Bax translocation into the mitochondria with a simultaneous release of Cyt C, activation of caspase-3, cleavage of poly (DNA ribose) polymerase (PARP), and decrease in Bcl-xl and Bcl-2 protein levels. Over-expression of Protein phosphatase 2A (PP2A) catalytic subunit (PP2AC ) did not rescue the apoptosis induced by PTPA knockdown, and PTPA knockdown did not affect the level of and their phosphorylation of mitogen-activated protein kinases (MAPKs), indicating that PP2A and MAPKs were not involved in the apoptosis induced by PTPA knockdown. In the cells with over-expression of tau, PTPA knockdown induced PP2A inhibition and tau hyperphosphorylation but did not cause significant cell death. These data suggest that PTPA deficit causes apoptotic cell death through mitochondrial pathway and simultaneous tau hyperphosphorylation attenuates the PTPA-induced cell death. Phosphotyrosyl phosphatase activator (PTPA) is decreased in the brains of Alzheimer's disease (AD) and AD transgenic mouse models. Here, we investigated whether down-regulation of PTPA affects cell viability. We found that PTPA located in the integral membrane of mitochondria, and knockdown of PTPA induced cell apoptosis in HEK293 and N2a cell lines by decreasing mitochondrial membrane potential, which leads to translocation of Bax and a simultaneous release of Cyt C. In the cells with tau over-expression, PTPA knockdown inactivated PP2A to phosphorylate tau to avoid cell apoptosis which induced by PTPA knockdown.

Hepatitis C virus dysregulates glucose homeostasis by a dual mechanism involving induction ofPGC1αand dephosphorylation of FoxO1

The maintenance of glucose homeostasis is a complex process in which the insulin signalling pathway plays a major role. Disruption of insulin-regulated glucose homeostasis is frequently observed in chronic hepatitis C (CHC) infection and might potentially contribute to type 2 diabetes mellitus (T2DM) development. Presently, the mechanism that links HCV infection to insulin resistance remains unclear. Previously, we have reported that HCV protein expression in HCV transgenic mice (B6HCV) leads to an overexpression of protein phosphatase 2A (PP2A) through an ER stress response. In the present work, we describe an association of FoxO1 hypophosphorylation and upregulation of both PGC-1α and G6Pase to phenotypic hyperglycaemia and insulin resistance in B6HCV mice. In vitro, we observed that PGC1α is concomitantly induced with PP2A. Moreover, we show that the enhanced PP2A expression is sufficient to inhibit insulin-induced FoxO1 phosphorylation via blockade of insulin-mediated Akt activation or/and through direct association and dephosphorylation of pS-FoxO1. Consequently, we found that the gluconeogenic gene glucose-6-phosphatase is upregulated. These observations were confirmed in liver biopsies obtained from CHC patients. In summary, our results show that HCV-mediated upregulation of PP2A catalytic subunit alters signalling pathways that control hepatic glucose homeostasis by inhibiting Akt and dephosphorylation of FoxO1.

Protein Phosphatase 2A Dephosphorylates CaBP4 and Regulates CaBP4 Function

CaBP4 is a neuronal Ca(2+)-binding protein that is expressed in the retina and in the cochlea, and is essential for normal photoreceptor synaptic function. CaBP4 is phosphorylated by protein kinase C zeta (PKCζ) in the retina at serine 37, which affects its interaction with and modulation of voltage-gated Ca(v)1 Ca(2+) channels. In this study, we investigated the potential role and functional significance of protein phosphatase 2A (PP2A) in CaBP4 dephosphorylation. The effect of protein phosphatase inhibitors, light, and overexpression of PP2A subunits on CaBP4 dephosphorylation was measured in in vitro assays. Pull-down experiments using retinal or transfected HEK293 cell lysates were used to investigate the association between CaBP4 and PP2A subunits. Electrophysiologic recordings of cotransfected HEK293 cells were performed to analyze the effect of CaBP4 dephosphorylation in modulating Ca(v)1.3 currents. PP2A inhibitors, okadaic acid (OA), and fostriecin, but not PP1 selective inhibitors, NIPP-1, and inhibitor 2, block CaBP4 dephosphorylation in retinal lysates. Increased phosphatase activity in light-dependent conditions reverses phosphorylation of CaBP4 by PKCζ. In HEK293 cells, overexpression of PP2A enhances the rate of dephosphorylation of CaBP4. In addition, inhibition of protein phosphatase activity by OA increases CaBP4 phosphorylation and potentiates the modulatory effect of CaBP4 on Ca(v)1.3 Ca(2+) channels in HEK293T cells. This study provides evidence that CaBP4 is dephosphorylated by PP2A in the retina. Our findings reveal a novel role for protein phosphatases in regulating CaBP4 function in the retina, which may fine tune presynaptic Ca(2+) signals at the photoreceptor synapse.

Curcumin inhibits protein phosphatases 2A and 5, leading to activation of mitogen-activated protein kinases and death in tumor cells

Curcumin can induce p53-independent apoptosis. However, the underlying mechanism remains to be defined. Here, we show that curcumin-induced apoptosis in a panel of tumor cells with mutant p53. Curcumin rapidly induced activation of the mitogen-activated protein kinases (MAPKs) including extracellular signal-regulated kinase 1/2 (Erk1/2) and c-Jun N-terminal kinase (JNK). Inhibition of JNK (with SP600125) or Erk1/2 (with U0126) partially prevented curcumin-induced cell death in the cells. Similarly, expression of dominant negative c-Jun or downregulation of Erk1/2 in part attenuated curcumin-induced cell death. It appears that curcumin-induced activation of MAPKs and apoptosis was due to induction of reactive oxygen species (ROS), as pretreatment with N-acetyl-L-cysteine, a ROS scavenger, blocked these events. Furthermore, we found that curcumin-induced activation of MAPK pathways was related to inhibition of the serine/threonine protein phosphatases 2A (PP2A) and 5 (PP5). Overexpression of PP2A or PP5 partially prevented curcumin-induced activation of JNK and Erk1/2 phosphorylation as well as cell death. The results suggest that curcumin induction of ROS activates MAPKs, at least partially by inhibiting PP2A and PP5, thereby leading to p53-independent apoptosis in tumor cells.

Overexpression of SET is a recurrent event associated with poor outcome and contributes to protein phosphatase 2A inhibition in acute myeloid leukemia

Protein phosphatase 2A is a novel potential therapeutic target in several types of chronic and acute leukemia, and its inhibition is a common event in acute myeloid leukemia. Upregulation of SET is essential to inhibit protein phosphatase 2A in chronic myeloid leukemia, but its importance in acute myeloid leukemia has not yet been explored. We quantified SET expression by real time reverse transcriptase polymerase chain reaction in 214 acute myeloid leukemia patients at diagnosis. Western blot was performed in acute myeloid leukemia cell lines and in 16 patients' samples. We studied the effect of SET using cell viability assays. Bioinformatics analysis of the SET promoter, chromatin immunoprecipitation, and luciferase assays were performed to evaluate the transcriptional regulation of SET. SET overexpression was found in 60/214 patients, for a prevalence of 28%. Patients with SET overexpression had worse overall survival (P<0.01) and event-free survival (P<0.01). Deregulation of SET was confirmed by western blot in both cell lines and patients' samples. Functional analysis showed that SET promotes proliferation, and restores cell viability after protein phosphatase 2A overexpression. We identified EVI1 overexpression as a mechanism involved in SET deregulation in acute myeloid leukemia cells. These findings suggest that SET overexpression is a key mechanism in the inhibition of PP2A in acute myeloid leukemia, and that EVI1 overexpression contributes to the deregulation of SET. Furthermore, SET overexpression is associated with a poor outcome in acute myeloid leukemia, and it can be used to identify a subgroup of patients who could benefit from future treatments based on PP2A activators.

Protein phosphatase 2A facilitates IL-17 production by promoting accessibility to the IL-17 locus (47.15)

Abstract Systemic lupus erythematosus (SLE) is a complex autoimmune disease caused by the combination of several immune abnormalities. T cells from SLE patients exhibit an aberrant phenotype, caused in part by increased levels of the protein phosphatase 2A (PP2A). To study the mechanisms through which PP2A alters T cell behavior and to identify the contribution of this defect to the disease, we generated a transgenic mouse that over-expresses PP2A in T cells. The transgenic mice produced abnormally high levels of IL-17A and F in vivo and in vitro. Remarkably, naïve CD4+ TPP2A cells produced IL-17 as early as 3h after stimulation. This effect was specific for IL-17 as production of type I and type II cytokines was not affected. PP2A over-expression did not promote Th17 differentiation or significant increases in Th17-related transcription factors. Due to the specificity of the phenomenon and its kinetics, we hypothesized that PP2A may facilitate transcription in the IL-17 locus. ChIP experiments revealed increased tri-methylation of H3K4 and decreased tri-methylation of H3K9 at the Il17a and Il17f promoters as well as in local conserved non-coding sequences. These changes are associated with increased chromatin accessibility. We propose that PP2A plays a central role in the transcriptional regulation of IL-17 by promoting chromatin accessibility to the IL-17 locus. This pathway may play a role in the excessive IL-17 production observed in SLE T cells.

PP2A attenuates cardiac response to LPS‐induced sepsis

Lipopolysaccharide (LPS), a cell membrane component from Gram‐negative bacteria, is involved in sepsis‐associated cardiac dysfunction. We wanted to know if protein phosphatase 2A (PP2A) influences LPS‐signaling in the heart. Therefore, we used transgenic (TG) mice with heart‐specific overexpression of PP2A. TG and littermate wild type (WT) mice were intraperitoneally injected with 30 μg LPS per g body weight or with NaCl as control (n = 14 – 18 per group). After 3 days, successful induction of LPS‐induced sepsis was monitored by enhanced plasma levels of interleukin‐6. Contractility of WT and TG hearts was analyzed using the isolated work performing heart preparation. Left ventricular systolic pressure (LVP) and the first derivative of developed pressure (+dP/dt and −dP/dt) were decreased in hearts of LPS‐treated WT mice, whereas hearts of TG mice showed no decrease of contractility after LPS‐treatment (Table). Contractile parameters Control LPS WT TG WT TG LVP (mmHg) 74±2.2 66±4.3 50±4.3* 61±1.9# +dP/dt (mmHg/s) 2481±186 2145±254 1306±246* 1901±171# −dP/dt (mmHg/s) −1371±114 −1073±139 −720±135* −1006±115 p&lt;0.05 vs. control, p&lt;0.05 vs. WT Quantitative PCR revealed that LPS increased IL‐6 mRNA expression in TG and WT hearts but TNFα mRNA levels only were enhanced in WT. In summary, LPS‐induced myocardial dysfunction was attenuated in TG mice compared to WT. It is tempting to speculate that increased PP2A activity in TG hearts is protective against LPS‐induced sepsis probably via diminished TNFα expression. (Supported by the DFG)

Biphasic Effects of Forskolin on Tau Phosphorylation and Spatial Memory in Rats

To explore the role of protein kinase A (PKA) in regulating tau phosphorylation and spatial memory, we injected forskolin, an activator of PKA, at different concentrations into the rat brains. We found that forskolin at concentrations up to 80 microM enhanced tau phosphorylation and was associated with prominent spatial memory impairment. Higher concentrations of forskolin, up to 200 microM, were associated with reduced phosphorylation levels of tau and no memory deficits. Forskolin elevated cAMP and activated PKA in a dose-dependent manner. When infused at 200 microM, forskolin also resulted in the activation and overexpression of protein phosphatase-2A (PP-2A) and attenuated the okadaic acid-induced PP-2A inhibition. These data suggest that the upregulation of PKA by forskolin to a certain level may activate PP-2A but that the latter can ameliorate the PKA-induced tau phosphorylation and memory impairment in the rats.