Protein Phosphatase 2A Activity Research Articles

H2 O2 induces PP2A demethylation to downregulate mTORC1 signaling in HEK293 cells.

Mammalian target of rapamycin (mTOR) is a Ser/Thr protein kinase that functions as an ATP and amino acid sensor to govern cell growth and proliferation by mediating mitogen- and nutrient-dependent signal transduction. Protein phosphatase 2A (PP2A), a ubiquitously expressed serine/threonine phosphatase, negatively regulates mTOR signaling. Methylation of PP2A is catalyzed by leucine carboxyl methyltransferase-1 (LCMT1) and reversed by protein phosphatase methylesterase 1 (PME-1), which regulates PP2A activity and substrate specificity. However, whether PP2A methylation is related to mTOR signaling is still unknown. In this study, we examined the effect of PP2A methylation on mTOR signaling in HEK293 cells under oxidative stress. Our results show that oxidative stress induces PP2A demethylation and inhibits the mTORC1 signaling pathway. Next, we examined two strategies to block PP2A demethylation under oxidative stress. One strategy was to prevent PP2A demethylation using a PME-1 inhibitor; the other strategy was to activate PP2A methylation via overexpression of LCMT1. The results show that both the PME-1 inhibitor and LCMT1 overexpression prevent the mTORC1 signaling suppression induced by oxidative stress. Additionally, LCMT1 overexpression rescued cell viability and the mitochondrial membrane potential decrease in response to oxidative stress. These results demonstrate that H2 O2 induces PP2A demethylation to downregulate mTORC1 signaling. These findings provide a novel mechanism for the regulation of PP2A demethylation and mTORC1 signaling under oxidative stress.

Leucine Carboxyl Methyltransferase Downregulation and Protein Phosphatase Methylesterase Upregulation Contribute Toward the Inhibition of Protein Phosphatase 2A by α-Synuclein.

The pathology of Parkinson’s disease (PD) is characterized by intracellular neurofibrillary tangles of phosphorylated α-synuclein (α-syn). Protein phosphatase 2A (PP2A) is responsible for α-syn dephosphorylation. Previous work has demonstrated that α-syn can regulate PP2A activity. However, the mechanisms underlying α-syn regulation of PP2A activity are not well understood. In this study, we found that α-syn overexpression induced increased α-syn phosphorylation at serine 129 (Ser129), and PP2A inhibition, in vitro and in vivo. α-syn overexpression resulted in PP2A demethylation. This demethylation was mediated via downregulated leucine carboxyl methyltransferase (LCMT-1) expression, and upregulated protein phosphatase methylesterase (PME-1) expression. Furthermore, LCMT-1 overexpression, or PME-1 inhibition, reversed α-syn-induced increases in α-syn phosphorylation and apoptosis. In addition to post-translational modifications of the catalytic subunit, regulatory subunits are involved in the regulation of PP2A activity. We found that the levels of regulatory subunits which belong to the PPP2R2 subfamily, not the PPP2R5 subfamily, were downregulated in the examined brain regions of transgenic mice. Our work identifies a novel mechanism to explain how α-syn regulates PP2A activity, and provides the optimization of PP2A methylation as a new target for PD treatment.

Se-Methylselenocysteine Ameliorates Neuropathology and Cognitive Deficits by Attenuating Oxidative Stress and Metal Dyshomeostasis in Alzheimer Model Mice.

Se-methylselenocysteine (SMC) is a major selenocompound in selenium (Se)-enriched plants. Se is vital for proper brain function, and Se-deficient is considered to be related with cognitive impairment and Alzheimer's disease (AD). The potential of SMC in intervening cognitive deficits and neuropathology of triple transgenic AD (3 × Tg-AD) mice is evaluated for the first time. AD mice are treated with SMC (0.75 mg kg-1 BW per day) in their drinking water for 10 months. Results reveal that SMC 1) reduces oxidative stress and neuro-inflammation; 2) modulates the distribution and levels of several metal ions; 3) decreases amyloid-β peptide (Aβ) generation by inhibiting the expression of its precursor protein APP and β-secretase (BACE1); and 4) attenuates tau hyperphosphorylation and neurofibrillary tangles (NFT) formation via promoting protein phosphatase 2A (PP2A) activity, thereby preserving synaptic proteins and neuron activities and finally improving spatial learning and memory deficits in AD model mice. The authors suggest that the inhibitory effect of SMC on MEK/ERK activation may play a critical role in intervening AD progression. These results reveal that SMC is powerful in ameliorating AD-related neuropathology and cognitive deficits via modulating oxidative stress, metal homeostasis, and extracellular signal-regulated kinase (ERK) activation.

Aspirin restores ABT-737-mediated apoptosis in human renal carcinoma cells

Aspirin is a novel chemopreventive agent against malignancy. However, outcomes of aspirin monotherapy of renal cell carcinoma (RCC) are inconsistent across studies. ABT-737, an BH3 mimetic inhibitor, is also a promising antitumor drug. Cancer cells including those from RCC, that have high levels of Mcl-1, are refractory to ABT-737-induced apoptosis. We here investigated how aspirin treatment modulates the ABT-737-induced apoptosis. Using the in vitro model of human 786-O cells, we showed that aspirin had sensitized cells to ABT-737 induced apoptosis. Such aspirin-induced changes of ABT-737 resistance was accompanied by a host of biochemical events like protein phosphatase 2A (PP2A) activation, AKT dephosphorylation, Mcl-1/FLICE inhibiting protein (FLIP)/XIAP downregulation, and Bax mitochondrial redistribution. The PP2A inhibitor, okadaic acid, was able to reverse the apirin-induced apoptotic changes. Apart from the aspirin treatment, Mcl-1 silencing also rendered cells vulnerable to ABT-737 induced apoptosis. Since PP2A, Akt, and Mcl-1 play critical roles in RCC malignancy and treatment resistance, our present study showed that aspirin, an alternative adjuvant agent, had recalled ABT-737 sensitivity in the RCC cells through processes involving the PP2A/Akt/Mcl-1 axis.

Analysis of Potential Alterations Affecting SETBP1 as a Novel Contributing Mechanism to Inhibit PP2A in Colorectal Cancer Patients.

The functional loss of the tumor suppressor protein phosphatase 2A (PP2A) occurs in a wide variety of human cancers including colorectal cancer (CRC), and SET overexpression has been reported as a key contributing mechanism to inhibit PP2A. Although SET binding protein 1 (SETBP1) overexpression and gain of function mutations have been described in several hematological malignancies as common events that increase the expression levels of the PP2A inhibitor SET, thereby leading to PP2A inactivation, the potential existence of SETBP1 alterations in CRC still remains unexplored. We studied the expression profile of SETBP1 by Western blot in a set of CRC cell lines and patient samples. Moreover, we performed co-immunoprecipitation assays to analyze the formation of the previously reported SETBP1-SET-PP2A inhibitory complex. Furthermore, we evaluated the mutational status of SETBP1 by pyrosequencing assays in a cohort of 55 CRC patients with metastatic disease after the immunohistochemical characterization of SET and p-PP2A expression in this cohort. We found high SETBP1 expression in several CRC lines but only in two of the patients analyzed. In addition, we demonstrated the formation of the SETBP1-SET-PP2A heterotrimeric complex in CRC cells. However, we failed to detect SETBP1 mutations in any of the CRC patient samples included in the study. Our results suggest that SETBP1 expression is mainly similar o lower in colorectal cancer tissue compared to normal colonic mucosa. However, its overexpression is a low prevalent alteration which could contribute to inhibit PP2A in CRC through the formation of a SETBP1-SET-PP2A complex in some CRC patients. Moreover, SETBP1 mutations are, if exist, rare events in CRC patients.

Subthalamic nucleus deep brain stimulation protects neurons by activating autophagy via PP2A inactivation in a rat model of Parkinson's disease

Deep brain stimulation (DBS) of the subthalamic nucleus (STN) is an effective therapeutic strategy for alleviating disability in patients with moderate to severe Parkinson's disease (PD). Preclinical studies have shown that stimulation of the rat STN can protect against nigral dopaminergic neuron loss. However, the underlying mechanism is unclear. To investigate the molecular basis of the neuroprotective effects of STN stimulation, a rat model of PD was established by unilaterally injecting 6-hydroxydopamine (6-OHDA) into the striatum. PD rats were subjected to DBS of the STN (STN-DBS) and the effects on motor symptoms and number of nigral tyrosine hydroxylase-positive (TH+) neurons was examined. We found that STN-DBS improved movement disorder and mitigated the loss of TH+ neurons induced by 6-OHDA. Furthermore, STN-DBS blocked protein phosphatase (PP)2A activation induced by 6-OHDA and led to the phosphorylation of B cell lymphoma (Bcl)-2, thereby increasing its activity. This induced its disassociation from Beclin1, a positive regulator of autophagy, leading to autophagy and inhibition of apoptosis. These findings demonstrate for the first time that STN-DBS could exert neuroprotective effects against 6-OHDA-induced cell injury in PD by inducing autophagy via PP2A inactivation and dissociation of the Bcl-2/Beclin1 complex, thereby providing a molecular basis of STN-DBS neuroprotection for PD.

Membrane-Initiated Estrogen Receptor Signaling Mediates Metabolic Homeostasis via Central Activation of Protein Phosphatase 2A.

Women gain weight and their diabetes risk increases as they transition through menopause; these changes can be partly reversed by hormone therapy. However, the underlying molecular mechanisms mediating these effects are unknown. A novel knock-in mouse line with the selective blockade of the membrane-initiated estrogen receptor (ER) pathway was used, and we found that the lack of this pathway precipitated excessive weight gain and glucose intolerance independent of food intake and that this was accompanied by impaired adaptive thermogenesis and reduced physical activity. Notably, the central activation of protein phosphatase (PP) 2A improved metabolic disorders induced by the lack of membrane-initiated ER signaling. Furthermore, the antiobesity effect of estrogen replacement in a murine menopause model was abolished by central PP2A inactivation. These findings define a critical role for membrane-initiated ER signaling in metabolic homeostasis via the central action of PP2A.

Antiphospholipid antibodies induce thrombosis by PP2A activation via apoER2-Dab2-SHC1 complex formation in endothelium

AbstractIn the antiphospholipid syndrome (APS), antiphospholipid antibody (aPL) recognition of β2 glycoprotein I promotes thrombosis, and preclinical studies indicate that this is due to endothelial nitric oxide synthase (eNOS) antagonism via apolipoprotein E receptor 2 (apoER2)-dependent processes. How apoER2 molecularly links these events is unknown. Here, we show that, in endothelial cells, the apoER2 cytoplasmic tail serves as a scaffold for aPL-induced assembly and activation of the heterotrimeric protein phosphatase 2A (PP2A). Disabled-2 (Dab2) recruitment to the apoER2 NPXY motif promotes the activating L309 methylation of the PP2A catalytic subunit by leucine methyl transferase-1. Concurrently, Src homology domain-containing transforming protein 1 (SHC1) recruits the PP2A scaffolding subunit to the proline-rich apoER2 C terminus along with 2 distinct regulatory PP2A subunits that mediate inhibitory dephosphorylation of Akt and eNOS. In mice, the coupling of these processes in endothelium is demonstrated to underlie aPL-invoked thrombosis. By elucidating these intricacies in the pathogenesis of APS-related thrombosis, numerous potential new therapeutic targets have been identified.

Protein phosphatase 2A mediates JS-K-induced apoptosis by affecting Bcl-2 family proteins in human hepatocellular carcinoma HepG2 cells.

Protein phosphatase 2A (PP2A) is an important enzyme within various signal transduction pathways. The present study was investigated PP2A mediates JS-K-induced apoptosis by affecting Bcl-2 family protein. JS-K showed diverse inhibitory effects in five HCC cell lines, especially HepG2 cells. JS-K caused a dose- and time-dependent reduction in cell viability and increased in levels of LDH release. Meanwhile, JS-K- induced apoptosis was characterized by mitochondrial membrane potential reduction, Hoechst 33342+ /PI+ dual staining, release of cytochrome c (Cyt c), and activation of cleaved caspase-9/3. Moreover, JS-K-treatment could lead to the activation of protein phosphatase 2A-C (PP2A-C), decrease of anti-apoptotic Bcl-2 family-protein expression including p-Bcl-2 (Ser70), Bcl-2, Bcl-xL, and Mcl-1 as well as the increase of pro-apoptosis Bcl-2 family-protein including Bim, Bad, Bax, and Bak. Furthermore, JS-K caused a marked increase of intracellular NO levels while pre-treatment with Carboxy-PTIO (a NO scavenger) reduced the cytotoxicity effects and the apoptosis rate. Meanwhile, pre-treatment with Carboxy-PTIO attenuated the JS-K-induced up-regulation of PP2A, Cyt c, and cleaved-caspase-9/3 activation. The silencing PP2A-C by siRNA could abolish the activation of PP2A-C, down-regulation of anti-apoptotic Bcl-2 family-protein (p-Bcl-2, Bcl-2, Bcl-xL, and Mcl-1), increase of pro-apoptosis Bcl-2 family-protein (Bim, Bad, Bax, and Bak) and apoptotic-related protein (Cyt c, cleaved caspase-9/3) that were caused by JS-K in HepG2 cells. In addition, pre-treatment with OA (a PP2A inhibitor) also attenuated the above effects induced by JS-K. In summary, NO release from JS-K induces apoptosis through PP2A activation, which contributed to the regulation of Bcl-2 family proteins.

Small-Molecule Activators of Protein Phosphatase 2A for the Treatment of Castration-Resistant Prostate Cancer.

Primary prostate cancer is generally treatable by androgen deprivation therapy, however, later recurrences of castrate-resistant prostate cancer (CRPC) that are more difficult to treat nearly always occur due to aberrant reactivation of the androgen receptor (AR). In this study, we report that CRPC cells are particularly sensitive to the growth-inhibitory effects of reengineered tricyclic sulfonamides, a class of molecules that activate the protein phosphatase PP2A, which inhibits multiple oncogenic signaling pathways. Treatment of CRPC cells with small-molecule activators of PP2A (SMAP) in vitro decreased cellular viability and clonogenicity and induced apoptosis. SMAP treatment also induced an array of significant changes in the phosphoproteome, including most notably dephosphorylation of full-length and truncated isoforms of the AR and downregulation of its regulatory kinases in a dose-dependent and time-dependent manner. In murine xenograft models of human CRPC, the potent compound SMAP-2 exhibited efficacy comparable with enzalutamide in inhibiting tumor formation. Overall, our results provide a preclinical proof of concept for the efficacy of SMAP in AR degradation and CRPC treatment.Significance: A novel class of small-molecule activators of the tumor suppressor PP2A, a serine/threonine phosphatase that inhibits many oncogenic signaling pathways, is shown to deregulate the phosphoproteome and to destabilize the androgen receptor in advanced prostate cancer. Cancer Res; 78(8); 2065-80. ©2018 AACR.

Neuroprotective effects of silk fibroin hydrolysate against Aβ25–35 induced cytotoxicity in SH-SY5Y cells and primary hippocampal neurons by regulating ROS inactivation of PP2A

Silk fibroin (SF) has been widely used as biomedical materials and the hydrolysate form of SF (SFH) has shown many activities. The purpose of the current study was to explore the neuroprotective effects of SFH against Aβ25–35-induced cytotoxicity in SH-SY5Y cells and primary hippocampal neurons and the possible mechanism. The results showed that SFH could improve the viabilities of SH-SY5Y and primary hippocampal neurons injured by Aβ25–35. The lowered mitochondrial membrane potential and increased cell apoptosis induced by Aβ25–35 were recovered after SFH treatment. Further study indicated that the beneficial effects of SFH were likely due to its interference with reactive oxygen species (ROS). SFH inhibited the phosphorylation of Ser/Thr protein phosphatase 2A (PP2A) by suppressing Aβ25–35-induced ROS, and then suppressed the activation of MAPKs and tau hyperphosphorylation, and protected SH-SY5Y cells and primary neurons from Aβ25–35-induced damages. SFH has the potential to be a nutraceutical for AD intervention.

Role of cantharidin in the activation of IKKα/IκBα/NF‑κB pathway by inhibiting PP2A activity in cholangiocarcinoma cell lines.

Cantharidin (CAN), a potent inhibitor of serine/threonine-protein phosphatase 2A (PP2A), is widely used in clinical practice, particularly in the treatment of advanced cancer due to its specific action on these types of cancer. In the present study, the inhibitory effect of CAN was examined in two cholangiocarcinoma cell lines (QBC939 and Hucc-t1). Following treatment with CAN, cell viability was effectively reduced in QBC939 and Hucc-t1 cells and normal human intrahepatic biliary epithelial cells. However, a slight increase in reactive oxygen species levels in QBC939 cells treated with CAN was observed post-treatment. CAN significantly inhibited cell migration and invasion in a dose-dependent manner. Western blot analysis demonstrated that the nuclear factor-κB (NF-κB) pathway was stimulated by CAN, which was confirmed by the upregulated phosphorylation levels of inhibitor of NF-κB kinase subunit α (IKKα) and NF-κB inhibitor α (IκBα) in cells, and an increased NF-κB p65 subunit level in the nucleus. The expression levels of 72 kDa type IV collagenase (MMP2) and matrix metalloproteinase 9 (MMP9) were downregulated by CAN. Notably, there was a negative association between MMP2 and MMP9 expression levels, and NF-κB p65, although NF-κB p65 regulates the expression of MMP2 and MMP9 and has a positive association with these proteins in various types of cancer. Notably, it was observed that CAN exerted specific inhibition on PP2A activity and thereby resulted in the activation of the IKKα/IκBα/NF-κB pathway. Therefore, CAN-induced cell inhibition maybe partially dependent on the activation of the IKKα/IκBα/NF-κB pathway. In conclusion, it was demonstrated that CAN selectively and effectively inhibited cholangiocarcinoma cell migration and invasion. The present study may provide a novel insight into the use of CAN as a therapeutic candidate in the treatment of cholangiocarcinoma.

Ethoxysanguinarine inhibits viability and induces apoptosis of colorectal cancer cells by inhibiting CIP2A.

Cancerous inhibitor of protein phosphatase2A (CIP2A) an endogenous inhibitor of protein phosphatase2A (PP2A), which can promote proliferation and transformation of several cancer types, has been shown to be a target for tumor therapy. The present study investigated the effects and underlying mechanisms of action of a novel natural compound, ethoxysanguinarine (Eth), on colorectal cancer(CRC) cells. MTT assay and flow cytometric assay found that Eth inhibited the viability and induced the apoptosis of the CRC cells. The inhibition of viability and activation of apoptosis was mediated through the Eth-induced decrease in CIP2A expression. Knockdown of CIP2A by RNA interference sensitized, whereas overexpression of CIP2A antagonized, Eth-induced viability inhibition and apoptosis. Furthermore, western blot analysis suggested that Eth inhibited phosphorylation of CIP2A downstream molecule protein kinaseB via the activation of PP2A. CRC xenograft tests also confirmed the antitumor effect of Eth invivo. These results advance our understanding of Eth-induced viability inhibition and apoptosis, implying the requirement for further investigation of Eth as a CIP2A inhibitor for cancer therapies.

Microcystin-Leucine-Arginine Induces Tau Pathology Through Bα Degradation via Protein Phosphatase 2A Demethylation and Associated Glycogen Synthase Kinase-3β Phosphorylation

Microcystin-leucine-arginine (MC-LR) has been implicated as a potential environmental factor in Alzheimer's disease because of its potent inhibition of protein phosphatase 2A (PP2A) activity, but experimental evidence to support its detailed neurotoxic effects and their underlying mechanisms has been lacking. The present study investigated the role of PP2A catalytic subunit (PP2Ac) demethylation and its link with glycogen synthase kinase-3β (GSK)-3β in tau hyperphosphorylation induced by MC-LR. The results showed that MC-LR treatment significantly increased demethylation of PP2Ac, with a concomitant increase in GSK-3β phosphorylation at Ser9 resulting in elevated tau hyperphosphorylation at PP2A-favorable sites in SH-SY5Y cells and rat hippocampus. Coimmunoprecipitation experiments showed that MC-LR treatment dissociated PP2Ac from Bα, making it incompetent in binding tau, thus causing tau hyperphosphorylation. Moreover, we found that inhibition of PP2A resulted in an increase in phosphorylation of GSK-3β at Ser9 and a decrease in GSK-3β activity, which further promoted demethylation of PP2Ac induced by MC-LR. These findings suggest a scenario in which MC-LR-mediated demethylation of PP2Ac is associated with GSK-3β phosphorylation at Ser9 and contributes to dissociation of Bα from PP2Ac, which would result in Bα degradation and disruption of PP2A/Bα-tau interactions, thus promoting tau hyperphosphorylation and paired helical filaments-tau accumulation and, consequently, axonal degeneration and cell death.

Stemness Is Enhanced in Gastric Cancer by a SET/PP2A/E2F1 Axis.

Gastric cancer is the fifth most common malignancy and the third leading cause of cancer-related deaths worldwide. Chemotherapies against gastric cancer often fail, with cancer recurrence due potentially to the persistence of cancer stem cells. This unique subpopulation of cells in tumors possesses the ability to self-renew and dedifferentiate. These cancer stem cells are critical for initiation, maintenance, metastasis, and relapse of cancers; however, the molecular mechanisms supporting cancer stemness remain largely unknown. Increased kinase and decreased phosphatase activity are hallmarks of oncogenic signaling. Protein phosphatase 2A (PP2A) functions as a tumor-suppressor enzyme, and elevated levels of SET/I2PP2A, an endogenous PP2A protein inhibitor, are correlated with poor prognosis of several human cancers. Here, it was determined that SET expression was elevated in tumor tissue in a gastric cancer mouse model system, and SET expression was positively correlated with poor survival of human gastric cancer patients. Mechanistically, SET knockdown decreased E2F1 levels and suppressed the stemness of cancer cell lines. Immunoprecipitations show SET associated with the PP2A-B56 complex, and the B56 subunit interacted with the E2F1 transcription factor. Treatment of gastric cancer cells with the SET-targeting drug OP449 increased PP2A activity, decreased E2F1 protein levels, and suppressed stemness of cancer cells. These data indicate that a SET/PP2A/E2F1 axis regulates cancer cell stemness and is a potential target for gastric cancer therapy.Implications: This study highlights the oncogenic role of SET/I2PP2A in gastric cancer and suggests that SET maintains cancer cell stemness by suppressing PP2A activity and stabilizing E2F1. Mol Cancer Res; 16(3); 554-63. ©2018 AACR.

Cucurbitacin B induces inhibitory effects via CIP2A/PP2A/Akt pathway in glioblastoma multiforme.

Cancerous inhibitor of protein phosphatase 2A (CIP2A) is a human oncoprotein that is overexpressed in multiple types of tumors and promotes the proliferation and transformation of cancer cells. However, whether CIP2A can be a new drug target for human glioblastoma multiforme (GBM) is largely unclear. In the present study, we demonstrated that the overexpression of CIP2A promotes invasive behavior in GBM, and a natural compound, cucurbitacin B (CuB), shows an anti-proliferative and anti-invasion effect in GBM cell lines. CuB effectively induces apoptosis, downregulates CIP2A expression and its downstream signaling molecule, phospho-Akt, and upregulates protein phosphatase 2A (PP2A) activity. Overexpression of CIP2A reduced CuB-inhibited growth and invasion in GBM cells. Silencing CIP2A enhanced CuB-induced invasion inhibition and apoptosis in GBM. CuB combined with cisplatin synergistically inhibited GBM cells. CuB also inhibited tumor growth in murine models. Western blot results further revealed that CuB downregulates CIP2A, and phospho-Akt in vivo. In summary, inhibition of CIP2A determines the effects of CuB-induced invasive behavior inhibition and apoptosis in GBM cells. These characteristics render CuB as a promising candidate drug for further development and for designing new effective CIP2A inhibitors.

PP2A Inhibits Cervical Cancer Cell Migration by Dephosphorylation of p-JNK, p-p38 and the p-ERK/MAPK Signaling Pathway.

Protein phosphatase 2A (PP2A) was reported to play an important role in cancer development; however, the relationship between PP2A and cervical cancer development has yet to be fully understood. The present study aimed to explore the role of PP2A in the development of cervical cancer. Serum levels of PP2A were detected by ELISA in 23 patients with cervical cancer and 30 patients with benign cervical lesions. Furthermore, the PP2A activities and the mRNA and protein levels of PP2A were measured in cervical cancer (n=8) and chronic cervicitis (n=10) tissues. The results showed that the serum levels of PP2A were significantly reduced in patients with cervical cancer. Further studies showed that not only the activities of PP2A but also the mRNA and protein levels of PP2A were significantly decreased in cervical cancer tissues. Wound healing and Transwell assays demonstrated that pharmacological and genetic upregulation of PP2A could inhibit the migration of HeLa cells, but the downregulation of PP2A promoted cellular migration. The activation of PP2A also inhibited the remodeling of actin and the activity of mitogen-activated protein kinases (MAPKs) including p-JNK, p-p38 and p-ERK. Meanwhile, the activation of PP2A was found to downregulate MMP-9 levels, which further inhibited the migration and invasion of HeLa cells. In conclusion, our data suggest that the activity and expression of PP2A are significantly reduced in cervical cancer tissues, and the activation of PP2A may inhibit the migration of cervical cancer cells by inhibiting the phosphorylation of p-JNK, p-p38 and the p-ERK/MAPK signaling pathway as well as by downregulating MMP-9, implying that PP2A plays an important role in cervical cancer development.

Non-genomic mechanisms of protein phosphatase 2A (PP2A) regulation in cancer.

Propagation of transient signals requires coordinated suppression of antagonistic phosphatase activity. Protein phosphatase 2A (PP2A) is a broad specificity serine/threonine phosphatase that functions as an antagonist of many signaling pathways associated with growth and proliferation, and endogenous inhibitory mechanisms suppress PP2A activity in response to mitogenic stimuli. These inhibitory mechanisms, including expression and activation of endogenous inhibitor proteins and phosphoregulation of PP2A subunits, are also engaged by aberrant constitutive activation of mitogenic pathways in cancer. Inhibition of PP2A activity has been shown to promote malignant transformation and endogenous inhibitory mechanisms of PP2A have been associated with malignant progression and prognosis in a wide range of cancers. Despite existence of recurrent mutations and other genetic and gene regulatory alterationsin PP2A genes, they collectively appear at relatively low frequency, and in only some cancer types. The non-genomic inhibition of PP2A activity by increased expression of endogenous PP2A inhibitor proteins greatly exceeds the frequency of genetic mutations of PP2A genes in human cancers. This feature makes PP2A an untypical tumor suppressor, and may have influenced its recognition as one of the critical human cell transformation mechanisms. We propose that non-genetic inhibition is the dominant mechanism causing loss of PP2A tumor suppressor function in cancer cells, possibly because these mechanisms do not elicit genomic instability associated with genetic loss of function of specific PP2A subunits.

Essential roles of Akt/Snail pathway in microcystin-LR-induced tight junction toxicity in Sertoli cell

Microcystin (MC)-LR is a cyclic heptapeptide that acts as a potent reproductive system toxin. However, the underlying pathways of MCLR-induced reproductive system toxicity have not been well elucidated. The blood-testis barrier is mainly constituted by tight junctions (TJs) between adjacent Sertoli cells in the seminiferous epithelium near the basement membrane. The present study was designed to investigate changes in TJs and the underlying pathway in MC-LR-induced TJs toxicity in Sertoli cell. In our study, the transepithelial electrical resistance (TER) value was decreased in a dose dependent manner due to the markers of TJs occludin, claudin and zonula occludens-1 (ZO-1) expression decline. MC-LR is shown to induce cytotoxicity by inhibiting protein phosphatase 2A (PP2A) activity. Our results also showed that the PP2A activity presented a dose-dependent decline. Moreover, MC-LR stimulated protein expression of snail by Akt/GSK-3β activation. The activated Akt/GSK-3β and snail signaling pathway largely accounted for MC-LRinduced TJs toxicity, which could be partially reversed by snail siRNA interference or AKT chemical inhibitor in TM4 cells. These findings indicated that MC-LR inhibit the protein expression of TJs, and the activation of Akt/Snail signaling pathways due to PP2A inhibition is proposed to participate in this process.

The Molecular Mechanism of Alpha-Synuclein Dependent Regulation of Protein Phosphatase 2A Activity

Background/Aims: Alpha-synuclein (α-Syn) is a neuronal protein that is highly implicated in Parkinson’s disease (PD), and protein phosphatase 2A (PP2A) is an important serine/threonine phosphatase that is associated with neurodegenerative diseases, such as PD. α-Syn can directly upregulate PP2A activity, but the underling mechanism remains unclear. Therefore, we investigated the molecular mechanism of α-Syn regulating PP2A activity. Methods: α-Syn and its truncations were expressed in E.coli, and purified by affinity chromatography. PP2A Cα and its mutants were expressed in recombinant baculovirus, and purified by affinity chromatography combined with gel filtration chromatography. The interaction between α-Syn and PP2A Cα was detected by GST pull-down assay. PP2A activity was investigated by the colorimetric assay. Results: The hydrophobic non-amyloid component (NAC) domain of α-Syn interacted with PP2A Cα and upregulated its activity. α-Syn aggregates reduced its ability to upregulate PP2A activity, since the hydrophobic domain of α-Syn was blocked during aggregation. Furthermore, in the hydrophobic center of PP2A Cα, the residue of I123 was responsible for PP2A to interact with α-Syn, and its hydrophilic mutation blocked its interaction with α-Syn as well as its activity upregulation by α-Syn. Conclusions: α-Syn bound to PP2A Cα by the hydrophobic interaction and upregulated its activity. Blocking the hydrophobic domain of α-Syn or hydrophilic mutation on the residue I123 in PP2A Cα all reduced PP2A activity upregulation by α-Syn. Overall, we explored the mechanism of α-Syn regulating PP2A activity, which might offer much insight into the basis underlying PD pathogenesis.