Protein Phosphatase 2A Phosphorylation Research Articles

A new paradigm for regulation of protein phosphatase 2A function via Src and Fyn kinase–mediated tyrosine phosphorylation

Protein phosphatase 2A (PP2A) is a major phospho-Ser/Thr phosphatase and a key regulator of cellular signal transduction pathways. While PP2A dysfunction has been linked to human cancer and neurodegenerative disorders such as Alzheimer’s disease (AD), PP2A regulation remains relatively poorly understood. It has been reported that the PP2A catalytic subunit (PP2Ac) is inactivated by a single phosphorylation at the Tyr307 residue by tyrosine kinases such as v-Src. However, multiple mass spectrometry studies have revealed the existence of other putative PP2Ac phosphorylation sites in response to activation of Src and Fyn, two major Src family kinases (SFKs). Here, using PP2Ac phosphomutants and novel phosphosite-specific PP2Ac antibodies, we show that cellular pools of PP2Ac are instead phosphorylated on both Tyr127 and Tyr284 upon Src activation, and on Tyr284 following Fyn activation. We found these phosphorylation events enhanced the interaction of PP2Ac with SFKs. In addition, we reveal SFK-mediated phosphorylation of PP2Ac at Y284 promotes dissociation of the regulatory Bα subunit, altering PP2A substrate specificity; the phosphodeficient Y127/284F and Y284F PP2Ac mutants prevented SFK-mediated phosphorylation of Tau at the CP13 (pSer202) epitope, a pathological hallmark of AD, and SFK-dependent activation of ERK, a major growth regulatory kinase upregulated in many cancers. Our findings demonstrate a novel PP2A regulatory mechanism that challenges the existing dogma on the inhibition of PP2A catalytic activity by Tyr307 phosphorylation. We propose dysregulation of SFK signaling in cancer and AD can lead to alterations in PP2A phosphorylation and subsequent deregulation of key PP2A substrates, including ERK and Tau.

Role of PP2A in Racial Differences in Endothelial Dysfunction

BackgroundAfrican Americans (AA) exhibit a higher prevalence of endothelial dysfunction and an exaggerated response to inflammatory cytokines compared to Caucasian Americans (CA). Endothelial dysfunction is characterized by a reduction in endothelial nitric oxide synthase (eNOS) activity and ultimately a lower nitric oxide production. Data obtained from bovine endothelial cells show that a reduction in eNOS activity in response to an inflammatory stimulus is mediated by protein phosphatase 2A (PP2A). Additionally, higher PP2A activity is associated with a decrease in the phosphorylation of PP2A at the Tyrosine 307 residue (p‐PP2AY307). Therefore, the purpose of this study was to investigate the role of PP2A in racial differences seen in endothelial dysfunction in response to an inflammatory stimulus.MethodsHuman umbilical vein endothelial cells (HUVECs) (n=6 cell lines; 3 CA & 3 AA) were cultured under two conditions: control or tumor necrosis factor‐α (TNF‐α) (10 ng/mL, 4 hours). Western blotting was used to measure protein expression of p‐PP2AY307, PP2A, p‐eNOSS1177 and eNOS. A two‐way ANOVA was used to assess the effects of race and conditions.ResultsNo racial differences were found in the protein expression of PP2A (Fig. 1B) and eNOS (Fig. 2B) at baseline or with TNF‐α stimulation. However, AA had a lower p‐PP2AY307 expression (p=0.021; Fig. 1A) compared to CA HUVECs under both conditions. Additionally, AA had a lower p‐eNOSS1177 expression (p=0.008; Fig. 2A) compared to CA HUVECs under both condition.ConclusionLower p‐PP2AY307 and p‐eNOSS1177 levels in AA endothelial cells could be attributed to an increase in PP2A activity that can ultimately inhibit eNOS phosphorylation and decrease nitic oxide production. Therefore, PP2A activity can explain, in part, racial differences observed in endothelial dysfunction and consequently cardiovascular diseases.Support or Funding InformationRacial differences in p‐PP2AY307 and PP2A protein expression in HUVECs after 4 hours of incubation with TNF‐α (10ng/ml).A) Densiometric quantification and representative Western blot of p‐PP2AY307 and B) Densiometric quantification and representative Western blot of PP2A. (# p= .02 in AA compared to CA HUVECs, n= 4 independent experiments. Data are shown as mean ± SEM).Figure 1Racial differences in p‐eNOSS1177 and eNOS protein expression in HUVECs after 4 hours of incubation with TNF‐α (10ng/ml).A) Densiometric quantification and representative Western blot of p‐eNOSS1177 and B) Densiometric quantification and representative Western blot of eNOS. (# p= .008 in AA compared to CA HUVECs, n= 4 independent experiments. Data are shown as mean ± SEM).Figure 2AcknowledgementsFunded partially by AU PGOP Fellowship.

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.

Novel Regulation of PKC-induced Inflammation by Akt and Protein Phosphatase 2A in Ovarian Granulosa Cells

PKC-mediated inflammation is important in ovarian physiology. The roles of Akt and protein phosphatase 2A (PP2A) in PKC-mediated inflammation in ovarian granulosa cells (GCs) remain mostly unclear. PKC activator phorbol 12-myristate 13-acetate induced the Akt phosphorylation in rat primary GCs but reduced the Akt phosphorylation in KGN human GCs. In rat GCs, an inhibitory effect of PI3K inhibitor wortmannin and a stimulatory effect of Akt activator SC79 on PKC-induced cyclooxygenase-2 (COX-2)/PGE2production were noted; wortmannin and SC79 acted oppositely in human GCs. In rat GCs, PP2A inhibitor okadaic acid further enhanced the PKC-mediated promoter activation and elevation of mRNA and protein levels of the COX-2 gene, whereas PP2A activator sodium selenate attenuated the PKC-mediated COX-2 expression and promoter activation. PKC activation did not affect PP2A phosphorylation, but okadaic acid indeed augmented the PKC-induced NF-κB nuclear translocation. Thus, PP2A appears to act as a negative modulator in PKC-mediated cellular inflammation in rat GCs, at least in part due to its attenuating effect on the PKC-induced NF-κB activation.

Endoplasmic reticulum stress induces liver cells apoptosis after brain death by suppressing the phosphorylation of protein phosphatase 2A.

The present study aimed to investigate whether brain death (BD) induces the activation of endoplasmic reticulum stress (ERS) and protein phosphatase 2A (PP2A), and reveal the possible association with BD-induced liver cell apoptosis. A total of 30 healthy adult male Sprague-Dawley rats were randomized into three groups: Sham-operated group (S), BD group and 4-phenylbutyric acid group (BD + 4-PBA), with 10 rats in each group. All rats were anesthetized. The model of BD was established by inflating a balloon catheter that was placed into the extradural space after anesthesia. 4-PBA was administered via an intraperitoneal injection when the BD model was established. Anesthesia of the S group of rats was maintained for 6 h. Liver tissues were harvested after 6 h of BD. HE staining was used to evaluate the damage of liver. Terminal deoxynucleotidyl transferase-mediated 2′-deoxyuridine 5′-triphosphate nick-end labeling staining was used to observe the apoptosis of liver cells. Activation of ERS and PP2A was examined by western blotting and immunohistochemical staining. We reported that the apoptosis of liver cells after BD was significantly promoted than in the S group. Activation of ERS and PP2A was induced in the BD group when compared with S group. Phosphorylation of PP2A was suppressed in BD group. Application of 4-PBA decreased the activation of ERS and apoptosis rate compared with the BD group. In addition, activation of PP2A in the BD + 4-PBA group was decreased due to the reduction of PP2A phosphorylation compared with the BD group, but the levels were higher than in the S group. (P<0.05). In summary, our results indicated that BD induced ERS, then activated PP2A by suppressing the phosphorylation of PP2A, resulting in the apoptosis of liver cells.

324-LB: A Novel Link between Mig6 and PP2a in Beta-Cell Apoptosis

Hyperglycemia in both type 1 and 2 diabetes is toxic to pancreatic beta cells and exacerbates the decline in functional beta cell mass in both diseases. Thus, identifying the molecular mechanism for how this glucotoxicity negatively impacts beta cells is essential for identifying new therapeutic targets to treat both forms of diabetes. We identified that EGF receptor feedback inhibitor Mig6 is induced in islets cultured in glucotoxic conditions and that Mig6 promotes apoptosis in beta cells. To define how the adaptor protein Mig6 promotes apoptosis, we sought to identify its interacting partners. Following immunoprecipitation of flag-tagged Mig6 in Ins1-derived 832/13 cells cultured in normal and toxic conditions, mass spectrometric analysis revealed protein phosphatase 2A (PP2A). PP2A controls many cellular functions ranging from metabolism, cell cycle, DNA replication, growth and apoptosis, and PP2A can be exploited by tumor cells to promote cell survival. Thus, we hypothesized Mig6 interacts with PP2A to regulate beta cell apoptosis. We cultured 832/13 cells and mouse islets in low (5 mM) or high glucose (HG, 20 mM) for up to 48h and validated that glucotoxicity increased methylation and decreased phosphorylation of PP2A (indicating activation). HG also increased expression of Mig6, CHOP (ER stress), γH2AX (DNA damage response), and cleaved caspase 3 (CC3; apoptosis). Compared to controls, siRNA-mediated suppression of Mig6 in 832/13 cells increased methylated PP2A and decreased γH2AX and CC3, suggesting Mig6 antagonizes beta cell survival. We generated mice lacking Mig6 in beta cells (Mig6βKO) by crossing Mig6-floxed mice (Mig6fl/fl) with RIP-Cre mice. Beta cell mass and glucose homeostasis were similar between Mig6βKO and control Mig6fl/fl littermates. Islets from Mig6βKO mice had increased methylated PP2A during HG compared to controls. We speculate the Mig6-PP2A interaction limits PP2A activation; hence, disrupting Mig6-PP2A or inhibiting Mig6 could promote compensatory beta cell survival by activating PP2A. Disclosure A. Rezaeizadeh: None. B.M. Bauer: None. P.T. Fueger: None.

The Post-Ischemic Increase in GluA2 Ser880 Phosphorylation Involves NADPH Oxidase

Most 2-amino-3-(3-hydroxy-5-methyl-isoxazol-4-yl) propanoic acid receptors (AMPARs) expressed on adult hippocampal pyramidal neurons contain the edited form of GluA2 (Q607R) and are thus impermeable to Ca2+/Zn2+ entry. Following ischemic injury, these receptors undergo a subunit composition change, switching from a GluA2-containing Ca2+/Zn2+-impermeable AMPAR to a GluA2-lacking Ca2+/Zn2+-permeable AMPAR. Recent studies indicate that an oxidative stress signaling pathway is responsible for the I/R-induced changes in AMPAR subunit composition. Studies suggest that nicotinamide adenine dinucleotide phosphate-oxidase (NADPH oxidase), a superoxide generator, is the source that initiates the oxidative stress-signaling cascade during post-ischemic reperfusion. The objective of the present study was to determine if suppression of NADPH oxidase activity prevents the increase in phosphorylation and subsequent internalization of the GluA2 AMPAR subunit during reperfusion of post-ischemic hippocampal slices. In this study, we demonstrated that exposure of adult rat hippocampal slices to oxygen glucose deprivation/reperfusion (OGD/R) results in an increase in Ser880 phosphorylation of the GluA2 subunit. The increase in Ser880 phosphorylation resulted in the dissociation of GluA2 from the scaffolding proteins Glutamate receptor-interacting protein 1 (GRIP1) and AMPAR binding protein (ABP), thus enabling the association of GluA2 with protein interacting with C kinase 1 (PICK1). OGD/R also resulted in an increase in the association of activated protein kinase C ? (PKC?) with PICK1. We have found that pharmacological inhibition of NADPH oxidase with apocynin diminishes the OGD/R-induced increase in activated PKC? association with PICK1 and subsequent Ser880 phosphorylation of GluA2. Suppression of NADPH oxidase activity also blunted OGD/R-induced decreased association of GluA2 with the scaffolding proteins GRIP1 and ABP. Protein phosphatase 2A (PP2A), which regulates PKC? activity by dephosphorylating the kinase, was inactivated by OGD/R-induced increase in tyrosine phosphorylation of the phosphatase (Y307). Inhibition of NADPH oxidase activity ameliorated OGD/R-induced PP2A phosphorylation and inactivation. Our findings are consistent with a model of OGD/R-induced Ser880 phosphorylation of GluA2 that implicates NADPH oxidase mediated inactivation of PP2A and sustained PKC? phosphorylation of GluA2.

Ketamine, a Clinically Used Anesthetic, Inhibits Vascular Smooth Muscle Cell Proliferation via PP2A-Activated PI3K/Akt/ERK Inhibition.

Abnormal proliferation of vascular smooth muscle cells (VSMCs) gives rise to major pathological processes involved in the development of cardiovascular diseases. The use of anti-proliferative agents for VSMCs offers potential for the treatment of vascular disorders. Intravenous anesthetics are firmly established to have direct effects on VSMCs, resulting in modulation of blood pressure. Ketamine has been used for many years in the intensive care unit (ICU) for sedation, and has recently been considered for adjunctive therapy. In the present study, we investigated the effects of ketamine on platelet-derived growth factor BB (PDGF-BB)-induced VSMC proliferation and the associated mechanism. Ketamine concentration-dependently inhibited PDGF-BB-induced VSMC proliferation without cytotoxicity, and phosphatidylinositol 3-kinase (PI3K) and extracellular signal-regulated protein kinase (ERK) inhibitors, LY294002 and PD98059, respectively, have similar inhibitory effects. Ketamine was shown to attenuate PI3K, Akt, and ERK1/2 phosphorylation induced by PDGF-BB. Okadaic acid, a selective protein phosphatase 2A (PP2A) inhibitor, significantly reversed ketamine-mediated PDGF-BB-induced PI3K, Akt, and ERK1/2 phosphorylation; a transfected protein phosphatse 2a (pp2a) siRNA reversed Akt and ERK1/2 phosphorylation; and 3-O-Methyl-sphingomyeline (3-OME), an inhibitor of sphingomyelinase, also significantly reversed ERK1/2 phosphorylation. Moreover, ketamine alone significantly inhibited tyrosine phosphorylation and demethylation of PP2A in a concentration-dependent manner. In addition, the pp2a siRNA potently reversed the ketamine-activated catalytic subunit (PP2A-C) of PP2A. These results provide evidence of an anti-proliferating effect of ketamine in VSMCs, showing activation of PP2A blocks PI3K, Akt, and ERK phosphorylation that subsequently inhibits the proliferation of VSMCs. Thus, ketamine may be considered a potential effective therapeutic agent for reducing atherosclerotic process by blocking the proliferation of VSMCs.

Phosphorylation of protein phosphatase 2A facilitated an early stage of chemical carcinogenesis

Protein phosphatase 2A (PP2A) is a serine-threonine phosphatase that regulates cell signaling pathways. Its inactivation is correlated with tumor malignancy, possibly due to the effects on cell differentiation and malignant cell transformation. Therefore, it has been noted that PP2A could be a promising target for cancer therapy. In our previous study of the hepatocarcinogen estragole (ES), cell proliferation may be required to convert ES-specific DNA adducts to mutations. To explore the trigger for cell proliferation, gpt delta rats were administered ES by gavage at doses of 3, 30 and 300mg/kg/day for 4weeks. ES-induced cell proliferation and gene mutations were observed at only the high dose whereas ES-specific DNA adducts were detected in a dose-dependent manner. Western blot analyses revealed activation of the Akt and ERK pathways without activation of upstream regulators, such as c-Raf, PKC and, PI3K. Phosphorylation of the PP2A C subunit at Tyr307 was found along with phosphorylation of Src. The overall data might imply that PP2A inactivation is responsible for cell cycle progression through activation of the Akt and ERK pathways at high doses of ES. Based on γ-H2AX immunohistochemistry and Western blot analysis for Rad51 protein, the resultant mutation spectra showed large deletion mutations that might result from double strand breaks of DNA. Thus, it is likely that inactivation of PP2A resulted in acceleration and exacerbation of gene mutations. We conclude that PP2A might contribute to an early stage of chemical carcinogenesis, suggesting that PP2A could be a molecular target of primary cancer prevention.

PP2A regulates SCF-induced cardiac stem cell migration through interaction with p38 MAPK

AimsPrevious studies have shown that stem cell factor (SCF) induces the migration of cardiac stem cells (CSCs) and helps to repair myocardial infarctions. Earlier studies on the migration mechanism only focused on the activation of kinases; here, we aimed to explore the functional role of protein phosphatase 2A (PP2A) in SCF-induced CSC migration. Main methodsCSCs were treated with SCF, PP2A enzymatic activity was measured, the phosphorylation levels of PP2A, p38 MAPK and cofilin were evaluated using western blot. Transwell assay was used to determine the migratory ability of CSCs. Key findingsIn vitro, SCF induced the phosphorylation of p38 MAPK and cofilin, leading to the migration of CSCs. Cofilin acted as a downstream signal of p38 MAPK. PP2A was involved in this process. Further studies revealed that PP2A was inactivated via phosphorylation at Tyr307 by SCF and the inactivation/phosphorylation was mediated by activated p38 MAPK, as p38 MAPK inhibitor SB203580 or siRNA prevented SCF-induced inactivation and phosphorylation of PP2A. When CSCs were pretreated with PP2A inhibitor (okadaic acid, OA), SCF-induced CSC migration and the downstream signals were enhanced, and the enhancement was reversed when p38 MAPK was blocked. Additionally, co-immunoprecipitation showed a direct interaction of PP2A with p38 MAPK. SignificanceOur results indicated that PP2A regulated the SCF-induced activation of p38 MAPK/cofilin signaling pathway and subsequent migration of CSCs by interaction with p38 MAPK.

Abstract 2383: Nicotinamide N-methyltransferase inhibits Protein Phosphatase 2A activity by promoting Y307 phosphorylation

Abstract Recent evidence indicates that suppression of protein phosphatase 2A (PP2A) activity plays an essential role in malignant transformation and cancer cell maintenance. Despite the importance of PP2A as a tumor suppressor in cancer, the mechanisms by which other enzymes regulate PP2A remain largely unknown. We recently identified Nicotinamide N-methyltransferase (NNMT) as a novel regulator of PP2A activity in glioblastoma (GBM). NNMT is a cytosolic N-methyl transferase which utilizes methyl groups from S-Adenosyl methionine (SAM), forming the by-product S-Adenosyl Homocysteine (SAH). PP2A becomes activated following the trimer formation of the three PP2A subunits, which requires the methylation of the catalytic subunit of PP2A (PP2A C) at L309.In cancer, NNMT has been reported to decrease the cellular methylation potential (SAM:SAH) and correspond with decreased PP2A activity. It was hypothesized that the decrease in methylation potential accounts for the decrease in PP2A activity by kinetically disfavoring the methylation of PP2A C. Interestingly, when we overexpressed NNMT in GBM cell lines and used LC-MS/MS (LC-MS Quadrupole Time-of-Flight and LC-MS Triple Quad) to monitor the methylation potential, we discovered that overexpression of NNMT increased methylation potential, however, still lead to a decrease in PP2A activity. As a result, we hypothesized that NNMT suppresses PP2A activity in a methylation-independent manner. In an effort to identify an alternative mechanism by which NNMT decreases PP2A activity, we found that NNMT expression was required for the phosphorylation of PP2A C at residue T307, which inhibits PP2A trimer formation and activation. Further, we found that NNMT expression leads to the constitutive activation of the PP2A target serine/threonine kinases (STKs): Akt, MAPK, and SAPK/JNK. NNMT silencing resulted in the deactivation of these STKs and radiation-induced cellular stress was not sufficient to rescue their activation. This suggests that NNMT-induced PP2A suppression is essential for the activation of these kinases. Overall, this study demonstrates a novel mechanism independent of methylation by which NNMT represses the activity of tumor suppressor PP2A. Citation Format: John R. Jacob, Arnab Chakravarti, Kamalakannan Palanichamy. Nicotinamide N-methyltransferase inhibits Protein Phosphatase 2A activity by promoting Y307 phosphorylation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2383. doi:10.1158/1538-7445.AM2017-2383

PP2A deactivation is a common event in oral cancer and reactivation by FTY720 shows promising therapeutic potential.

Protein phosphatase 2A (PP2A) is a tumor suppressor gene, that has been frequently deactivated in many types of cancer. However, its molecular and clinical relevance in oral squamous cell carcinoma (OSCC) remain unclear. Here we show that, PP2A deactivation is a common event in oral cancer cells and hyperphosphorylation in its tyrosine-307 (Y307) residue contributes to PP2A deactivation. PP2A restoration by FTY720 treatment reduced cell growth and decreased GSK-3β phosphorylation without significantly altering other PP2A targets. We further detected PP2A phosphorylation in 262 OSCC tissues. Increased expression of p-PP2A in the tumor tissues was significantly correlated with higher N2/N3-stage (aOR = 2.1, 95%CI: 1.2-3.8). Patients with high p-PP2A expression had lower overall survival rates than those with low expression. Hazard ratio analysis showed that, high p-PP2A expression was significantly associated with mortality density (aOR = 2.2, 95%CI: 1.2-4.0) and lower 10-year overall survival (p = 0.027) in lymph node metastasis. However, no interaction was observed between p-PP2A expression and lymph node metastasis. All our results suggest that PP2A is frequently deactivated in oral cancer and determines poor outcome, restoring its expression by FTY720 can be an alternative therapeutic approach in OSCC.

Role of GSK3&amp;lt;i&amp;gt;β&amp;lt;/i&amp;gt; and PP2A on Regulation of Tau Phosphorylation in Hippocampus and Memory Impairment in ICV-STZ Animal Model of Alzheimer’s Disease

Intracerebroventricular administration (ICV) of streptozotocin (STZ) in rats has been associated to desensitization of the insulin receptor (IR) and biochemical changes similar to those occurring in Alzheimer’s disease (AD) or older brains, so it has been proposed as a suitable model for studying some of the pathological features of AD sporadic type (SAD). In this study, we investigated the role of glycogen synthase kinase 3β (GSK3β) and protein phosphatase 2A (PP2A) in the regulation of the phosphorylation of tau (p-tau). Results showed that ICV-STZ treated rats had deficits in short- (1.5-h) and long-term (24- and 48-h) memory after one month of ICV-STZ treatment and six months relative to control rats. The memory deficit was associated to increasing [F(3, 12) = 31.48, p < 0.0001] p-tau in the hippocampus but not in prefrontal cortex (PFC). Likewise, STZ reduced phosphorylation of GSK3β (p-GSK3β) and PP2A in hippocampus and PFC, indicating that GSK3β and PP2A contributed to regulation of p-tau. These data supporting the model with ICV-STZ in rat are adequate to study the progressive memory impairment associated to hyperphosphorylation of tau and the cascade of insulin receptor signaling; confirm that phosphatidyl-inositol-3 kinase-protein kinase B (PI3K-PKB/Akt-GSK3β) and PP2A are involved in the modulation of proteins responsible for the regulation of neurodegeneration in AD.

The impact of tau hyperphosphorylation at Ser262 on memory and learning after global brain ischaemia in a rat model of reversible cardiac arrest

An increase in phosphorylated tau (p-tau) is associated with Alzheimer's disease (AD), and brain hypoxia. Investigation of the association of residue-specific tau hyperphosphorylation and changes in cognition, leads to greater understanding of its potential role in the pathology of memory impairment. The aims of this study are to investigate the involvement of the main metabolic kinases, Liver Kinase B1 (LKB1) and Adenosine Monophosphate Kinase Protein Kinase (AMPK), in tau phosphorylation-derived memory impairment, and to study the potential contribution of the other tau kinases and phosphatases including Glycogen Synthase Kinase (GSK-3β), Protein kinase A (PKA) and Protein Phosphatase 2A (PP2A). Spatial memory and learning were tested in a rat global brain ischemic model of reversible cardiac arrest (CA). The phosphorylation levels of LKB1, AMPK, GSK-3β, PP2A, PKA and tau-specific phosphorylation were assessed in rats, subjected to ischaemia/reperfusion and in clinically diagnosed AD and normal human brains. LKB1 and AMPK phosphorylation increased 4 weeks after CA as did AMPK related p-tau (Ser262). The animals showed unchanged levels of GSK-3β specific p-tau (Ser202/Thr205), phospho-PP2A (Tyr307), total GSK-3β, PP2A, phospho-cAMP response element-binding protein (CREB) which is an indicator of PKA activity, and no memory deficits. AD brains had hyperphosphorylated tau in all the residues of Ser262, Ser202 and Thr205, with increased phosphorylation of both AMPK (Thr172) and GSK-3β (Ser9), and reduced PP2A levels. Our data suggests a crucial role for a combined activation of tau kinases and phosphatases in adversely affecting memory and that hyperphosphorylation of tau in more than one specific site may be required to create memory deficits.

Abstract 13927: Allosteric Activation of PP2A Inhibits Experimental Abdominal Aortic Aneurysm

Introduction: In abdominal aortic aneurysms (AAA), recent studies strongly implicate the kinase, Mitogen-activated protein kinase (MAPK) as an essential contributor to its pathogenesis. Upregulation of Protein phosphatase 2A (PP2A) activity has been shown to be of significant therapeutic value, however pharmaceutically tractable approaches to directly activate PP2A remain elusive in AAA disease. Hypothesis: In light of the potential phosphatase effects PP2A can exert on MAPK protein targets, we hypothesized that PP2A activation by small molecule activators of PP2A (SMAPs) might mitigate AAA progression. Methods: Oral administration of the small molecule activator of PP2A, termed DT-1154, was performed for 4 weeks in apolipoprotein E-null (ApoE-/-) mice concomitant with subcutaneous Angiotensin II infusion. Human abdominal artery tissue as well as abdominal artery tissue from Ang II-infused ApoE-/- mice (+/- DT-1154) were examined by PCR, Western blotting and IHC. Results: To examine its activation state, Phosphorylated-PP2A (Y307) was stained in human and murine AAA tissues, with an increased phospho-PP2A signal indicating less cellular phosphatase activity. Examination of these tissues indeed revealed increased phosphorylation of PP2A compared to control non-AAA tissues indicating a marked reduction of PP2A activation. This strongly suggests that compromised PP2A activation can drive AAA formation. Oral administration of DT-1154 also resulted in significantly reduced AAA incidence and aortic dilation in these pro-AAA ApoE-/- mice. Finally, mechanistic studies indicated that blockade of ERK1/2 and NF-kB signaling by DT-1154 contributed to its effects in ameliorating AAA progression. Conclusions: These studies demonstrate a role of PP2A in AAA etiology and implicate that activation of PP2A may serve as a novel strategy to limit human AAA progression.

Morroniside-Induced PP2A Activation Antagonizes Tau Hyperphosphorylation in a Cellular Model of Neurodegeneration.

An accumulation of hyperphosphorylated tau in the brain is a hallmark of Alzheimer's disease (AD). Deficits in protein phosphatase 2A (PP2A) are associated with tau hyperphosphorylation in AD. To investigate the effects of morroniside (MOR), isolated from Cornus officinalis, on tau hyperphosphorylation and its underlying mechanisms related to PP2A. SK-N-SH cells were pretreated with 50-200 μM MOR for 24 h followed by 20 nM okadaic acid (OA) for 6 h. PP2Ac siRNA was transfected into HEK293 cells to determine the direct interaction of MOR with PP2A. Western blotting was used to measure the expression of proteins and enzymes. PP2A activity was measured by molybdenum blue spectrophotometry. Pretreatment with MOR improved the cellular morphological damage and inhibited tau hyperphosphorylation in SK-N-SH cells induced by OA, a PP2A inhibitor. Moreover, MOR increased PP2A activity, concurrent with a decrease in the expression of demethylated PP2A at Leu309 and phosphorylated PP2A at Tyr307. MOR decreased protein phosphatase methylesterase 1 (PME-1) expression and the ratio of PME-1/leucine carboxyl methyltransferase 1 (LCMT-1). Furthermore, MOR treatment decreased the phosphorylation of Src at Tyr416, which regulates the phosphorylation of PP2A. MOR had no effect on PP2Ac expression and tau hyperphosphorylation in PP2Ac siRNA-transfected cells. MOR attenuated OA-induced tau hyperphosphorylation via PP2A activation, and its mechanism might be related to the regulation of PP2Ac post-translational modification and upstream enzymes such as Src and PME-1.

Resistin, a fat-derived secretory factor, promotes metastasis of MDA-MB-231 human breast cancer cells through ERM activation.

Resistin, an adipocyte-secreted factor, is known to be elevated in breast cancer patients. However, the molecular mechanism by which resistin acts is not fully understood. The aim of this study was to investigate whether resistin could stimulate invasion and migration of breast cancer cells. Here, we report that resistin stimulated invasion and migration of breast cancer cells as well as phosphorylation of c-Src. Inhibition of c-Src blocked resistin-induced breast cancer cell invasion. Resistin increased intracellular calcium concentration, and chelation of intracellular calcium blocked resistin-mediated activation of Src. Resistin also induced phosphorylation of protein phosphatase 2A (PP2A). Inhibition of c-Src blocked resistin-mediated PP2A phosphorylation. In addition, resistin increased phosphorylation of PKCα. Inhibition of PP2A enhanced resistin-induced PKCα phosphorylation, demonstrating that PP2A activity is critical for PKCα phosphorylation. Resistin also increased phosphorylation of ezrin, radixin, and moesin (ERM). Additionally, ezrin interacted with PKCα, and resistin promoted co-localization of ezrin and PKCα. Either inhibition of c-Src and PKCα or knock-down of ezrin blocked resistin-induced breast cancer cells invasion. Moreover, resistin increased expression of vimentin, a key molecule for cancer cell invasion. Knock-down of ezrin abrogated resistin-induced vimentin expression. These results suggest that resistin play as a critical regulator of breast cancer metastasis.

Activation of the Tumor Suppressor PP2A Emerges as a Potential Therapeutic Strategy for Treating Prostate Cancer.

Protein phosphatase 2A (PP2A) is a tumor suppressor complex that has recently been reported as a novel and highly relevant molecular target in prostate cancer (PCa). However, its potential therapeutic value remains to be fully clarified. We treated PC-3 and LNCaP cell lines with the PP2A activators forskolin and FTY720 alone or combined with the PP2A inhibitor okadaic acid. We examined PP2A activity, cell growth, prostasphere formation, levels of PP2A phosphorylation, CIP2A and SET expression, and AKT and ERK activation. Interestingly, both forskolin and FTY720 dephosphorylated and activated PP2A, impairing proliferation and prostasphere formation and inducing changes in AKT and ERK phosphorylation. Moreover, FTY720 led to reduced CIP2A levels. Treatment with okadaic acid impaired PP2A activation thus demonstrating the antitumoral PP2A-dependent mechanism of action of both forskolin and FTY720. Levels of PP2A phosphorylation together with SET and CIP2A protein expression were studied in 24 PCa patients and both were associated with high Gleason scores and presence of metastatic disease. Altogether, our results suggest that PP2A inhibition could be involved in PCa progression, and the use of PP2A-activating drugs might represent a novel alternative therapeutic strategy for treating PCa patients.

Zinc binds to and directly inhibits protein phosphatase 2A in vitro

Zinc induces protein phosphatase 2A (PP2A) inactivation and tau hyperphosphorylation through PP2A (tyrosine 307) phosphorylation in cells and the brain, but whether Zn(2+) has a direct inhibitory effect on PP2A is not clear. Here we explored the effect of Zn(2+) on PP2A and their direct interaction in vitro. The results showed that Zn(2+) mimicked the inhibitory effect of okadaic acid on protein phosphatase and prevented tau dephosphorylation in N2a cell lysates. PP2A activity assays indicated that a low concentration (10 μmol/L) of Zn(2+) inhibited PP2A directly. Further Zn(2+)-IDA-agarose affinity binding assays showed that Zn(2+) bound to and inhibited PP2Ac(51-270) but not PP2Ac(1-50) or PP2Ac(271-309). Taken together, Zn(2+) inhibits PP2A directly through binding to PP2Ac(51-270) in vitro.

Lipid Nanosystems Enhance the Bioavailability and the Therapeutic Efficacy of FTY720 in Acute Myeloid Leukemia.

Protein phosphatase 2A (PP2A) is a serin-threonin phosphatase that regulates many proteins critical for malignant cell behavior; therefore, PP2A is considered to be a human tumor suppressor. In this study, we assessed the pharmacokinetic profile and the antileukemic effects of the PP2A activator FTY720, free or encapsulated in lipid nanoparticles, in in vitro and in vivo models of acute myeloid leukemia. FTY720 lipid nanoparticles presented diameters around 210 nm, with an encapsulation efficiency up to 75% and significantly increased FTY720 oral bioavailability. In addition, FTY720 restores PP2A phosphatase activity and decreases phosphorylation of PP2A and its targets Akt, ERK1/2 and STAT5, all implicated in the pathogenesis of acute myeloid leukemia. Moreover, FTY720 exerts an additive anti-leukemic effect in combination with drugs used in standard induction therapy. Importantly, FTY720 lipid nanoparticles were more efficient at inducing cell growth arrest and apoptosis than FTY720 solution. Finally, oral administration of FTY720 lipid nanoparticles to mice every three days was as effective in reducing acute myeloid leukemia xenograft tumor growth as daily oral administration of FTY720. These results provide the first evidence for the potential use of FTY720 lipid nanoparticles as an oral therapeutic agent in acute myeloid leukemia.