Protein Phosphatase 2A Phosphatase Activity Research Articles

Endothelial PP2AC prevents endotoxemia‐induced acute lung injury by suppressing STAT3 activity

Acute lung injury (ALI) is a critical hypoxemic respiratory failure characterized by alveolar‐capillary damage, unchecked inflammation, vascular leak, and pulmonary edema secondary to endothelial barrier dysfunction. Identifying the candidate molecules targeting mechanisms for excessive production of proinflammatory cytokines and endothelial dysfunction are warranted for therapeutic intervention. Recent studies show that the signal transducers and activators of transcription 3 (STAT3) phosphorylation in critical for inflammatory cytokines production and systemic inflammation. Protein phosphatase 2A (PP2A) is a hetero‐trimeric, ubiquitously expressed Ser/Thr phosphatase. The phosphatase activity of PP2A is regulated by its catalytic C subunit (PP2AC). PP2A plays an essential role in modulating tissue inflammation by maintaining the phosphorylation/de‐phosphorylation balance of multiple proteins. So far, however, no studies have deciphered PP2AC isoform‐a function in regulating pulmonary vascular endothelial permeability and inflammatory signaling. We hypothesize that PP2AC expression in vascular endothelial cell (EC) protects the lung against sepsis‐induced lung injury. Wild‐type (WT) and endothelial specific PP2AC‐deficient mice (PP2ACCKO) were challenged with lipopolysaccharide. We found that lipopolysaccharide‐induced inflammatory response, characterized by up‐regulated level of IL‐1b, IL‐6, MPO activity and pulmonary vascular hyper‐permeability in lung tissue of PP2ACCKO mice was significantly elevated as compared to WT littermate control. Mechanistically, we showed that PP2AC deletion in mouse lung‐EC and in human lung‐EC markedly augmented Ser727‐phosphorylation‐mediated STAT3 activation and MMP9 level in response to lipopolysaccharide. Intriguingly, administration of a STAT3 inhibitor suppressed lipopolysaccharide‐induced production of MMP9 and proinflammatory cytokines IL‐1b, IL‐6, MPO activity and pulmonary vascular hyper‐permeability and thus rescued the augmented inflammatory phenotype in PP2ACCKO mice lung. In conclusion, these data showed the protective role of endothelial‐PP2AC in limiting endothelial barrier dysfunction and inflammation, and thereby it attenuates sepsis‐induced lung injury by preventing hyper‐activated STAT3 signaling.

Phosphatase inhibition by sodium orthovanadate displays anti-inflammatory action by suppressing AKT-IKKβ signaling in RAW264.7 cells

Sodium orthovanadate (Na3VO4) is an inhibitor of phosphatases that acts as a phosphate analog and is being developed as an anti-diabetes drug. Phosphatases play important roles in inflammatory signal pathways by modulating the removal of phosphate moieties of key signaling proteins. However, the role of protein phosphatases on the inflammatory response has not been fully established. In this study, we investigated how phosphatases can control the inflammatory response using Na3VO4 in LPS-stimulated RAW264.7 cells and explored the molecular mechanisms by NO assay, mRNA analysis, immunoblotting analysis, kinase assay, luciferase reporter gene assay, and mutation strategy. Na3VO4 decreased the release of nitric oxide (NO) and suppressed the expression of pro-inflammatory genes at the transcriptional level, without cytotoxicity. The translocation of nuclear factor (NF)-κB subunits into the nucleus and the level of p-IκBα were reduced by Na3VO4, as was IKKβ activity. Na3VO4 inhibited NF-κB-Luc activity under AKT1/2 and IKKβ overexpression. However, the inhibitory effect of Na3VO4 against NF-κB-Luc was not observed in the group overexpressing both AKT2 and IKKβ-M10, a mutant in which the 10 serine residues in the autophosphorylated region of the C-terminal were replaced with alanine. Na3VO4 directly decreased the activity of protein phosphatase 1α (PP1α) and protein phosphatase 2 A (PP2A) by 95%. Phosphatase inhibition by Na3VO4 also selectively suppressed AKT-IKKβ signaling by directly blocking the phosphatase activity of PP1 and PP2A, consequently down-regulating NF-κB and inflammatory gene expression. Therefore, these results suggest that vanadium compounds including Na3VO4 can be developed as anti-inflammatory drugs.

Cornel Iridoid Glycoside Suppresses Tau Hyperphosphorylation and Aggregation in a Mouse Model of Tauopathy through Increasing Activity of PP2A

rTg4510 mice are transgenic mice expressing P301L mutant tau and have been developed as an animal model of tauopathy including Alzheimer's Disease (AD). Cornel Iridoid Glycoside (CIG) is an active ingredient extracted from Cornus officinalis, a traditional Chinese herb. The purpose of the present study was to investigate the effects of CIG on tau pathology and underlying mechanisms using rTg4510 mice. The cognitive functions were detected by Morris water maze and objective recognition tests. Western blotting and immunofluorescence were conducted to measure the levels of phosphorylated tau and related proteins. Serine/threonine phosphatase assay was applied to detect the activity of protein phosphatase 2A (PP2A). Intragastric administration of CIG for 3 months improved learning and memory abilities, prevented neuronal and synapse loss, halted brain atrophy, elevated levels of synaptic proteins, protected cytoskeleton, reduced tau hyperphosphorylation and aggregation in the brain of rTg4510 mice. In the mechanism studies, CIG increased the activity of PP2A, elevated the methylation of PP2A catalytic C (PP2Ac) at leucine 309, decreased the phosphorylation of PP2Ac at tyrosine 307, and increased protein expression of leucine carboxyl methyltransferase 1 (LCMT-1), protein tyrosine phosphatase 1B (PTP1B), and protein phosphatase 2A phosphatase activator (PTPA) in the brain of rTg4510 mice. CIG might have the potential to treat tauopathy such as AD via activating PP2A.

Precision Therapy for Aggressive Endometrial Cancer by Reactivation of Protein Phosphatase 2A.

Critically important to reducing uterine cancer mortality is the development of more effective therapy for aggressive endometrial cancers, including uterine serous cancer and uterine carcinosarcoma, which together account for over half of deaths due to endometrial cancer. About one-third of these aggressive endometrial cancers harbor mutations in the protein phosphatase 2A (PP2A) Aα scaffold subunit encoded by PPP2R1A In this issue, the study by Taylor and colleagues elucidates the role of a highly recurrent PP2A-Aα-subunit mutation PPP2R1A P179R as a biological driver of aggressive endometrial cancer. Compelling data demonstrate that the P179R mutation alters PP2A-Aα protein conformation, impairing holoenzyme formation and reducing PP2A phosphatase activity to promote endometrial cancer progression. Restoration of wild-type PPP2R1A in P179R-mutant endometrial cancer cells increases phosphatase activity and inhibits tumor growth in vivo Furthermore, a small-molecule activator of PP2A (SMAP) phenocopies restoration of wild-type PPP2R1A to suppress tumor growth. These promising results are an important advance toward effective precision therapy for aggressive endometrial cancer.See related article by Taylor et al., p. 4242.

PS1000 POST‐TRANSLATIONAL MODIFICATIONS OF PROTEIN PHOSPHATASE 2A (PP2A) IN MIXED LINEAGE LEUKEMIA (MLL)

Background:Mixed lineage leukemia (MLL) is the most common leukemia in infant patients. This leukemia is due to chromosomal translocations at locus 11q23 and is associated with poor clinical outcome. Novel, targeted therapeutic approaches are needed. Protein phosphatase 2A (PP2A) is a serine threonine phosphatase which regulates the phosphorylation of several kinases, including Erk, Akt, GSK3 which are fundamental for MLL cells’ survival. PP2A is a trimeric protein complex in which a core dimer formed between the scaffold subunit and the catalytic subunit is associated with one of the many regulatory subunits that facilitate and direct the interaction of the trimer with substrate proteins. PP2A activity is regulated by interaction with PP2A inhibitor proteins (PIPs), as well as by post‐translational modifications of PP2A complex components.Aims:In this context, our aim is to understand how PP2A post‐translational modifications affect the activity of PP2A on its downstream targets and to investigate whether PP2A re‐activation, by preventing phosphorylation and activation of collateral pathways, might represent a valid therapeutic strategy for MLL.Methods:12 different leukemic cell lines and 8 primary samples from MLL patients were included in this study. We measured the PP2A phosphatase activity by a colorimetric assay using a synthetic phospho‐peptide and malachite green reagent. The protein levels of PP2A, phospho‐PP2A, demethyl‐PP2A, LCMT‐1, PME‐1, Akt, phospho‐Akt, Erk, and phospho‐Erk were determined by western blot using specific antibodies.Results:Compared to healthy bone marrow, we found an increase in the phosphorylation at Tyrosine 307 (Tyr307) and a decrease in the methylation at Leucine 309 (Leu309) of the catalytic subunit of PP2A in MLL samples. These changes in the post‐translational modifications of the catalytic subunit correlated with a decrease in the phosphatase activity of PP2A. Methylation at Leu309 has been described as an absolute requirement for the binding of the regulatory subunit B55α to PP2A core. B55α was found up‐regulated in MLL cell lines and primary samples compared to healthy bone marrow. The methylation of the catalytic subunit of PP2A is catalyzed by leucine carboxyl methyltransferase (LCMT‐1), which we found down‐regulated in MLL samples, whereas demethylation is catalyzed by the phosphatase methylesterase (PME‐1) that was found up‐regulated in MLL. In line with the inactivation of PP2A in the MLL samples, PP2A downstream targets such as Akt and Erk were found hyper‐phosphorylated in MLL samples.Summary/Conclusion:Our results suggest that PP2A phosphatase activity inhibition might sustain in MLL samples a persistent serine/threonine phosphorylation of PP2A substrates, such as Akt and Erk that mediate pro‐survival and anti‐apoptotic signals. This inhibition might be explained, at least in part, by phosphorylation and demethylation of PP2A catalytic subunit. A better understanding of the mechanisms that regulate PP2A activity could provide new strategies to rescue PP2A phosphatase activity and target fundamental mechanisms of leukemic survival and chemotherapy resistance.

Chronic Cigarette Smoke Exposure Subdues PP2A Activity by Enhancing Expression of the Oncogene CIP2A.

Phosphatase activity of the major serine threonine phosphatase, protein phosphatase 2A (PP2A), is blunted in the airways of individuals with chronic obstructive pulmonary disease (COPD), which results in heightened inflammation and proteolytic responses. The objective of this study was to investigate how PP2A activity is modulated in COPD airways. PP2A activity and endogenous inhibitors of PP2A were investigated in animal and cell models of COPD. In primary human bronchial epithelial (HBE) cells isolated from smokers and donors with COPD, we observed enhanced expression of cancerous inhibitor of PP2A (CIP2A), an oncoprotein encoded by the KIAA1524 gene, compared with cells from nonsmokers. CIP2A expression was induced by chronic cigarette smoke exposure in mice that coincided with a reduction in PP2A activity, airspace enlargements, and loss of lung function, as determined by PP2A phosphatase activity, mean linear intercept analysis, and forced expiratory volume in 0.05 second/forced vital capacity. Modulating CIP2A expression in HBE cells by silencing RNA or chemically with erlotinib enhanced PP2A activity, reduced extracellular-signal-regulated kinase phosphorylation, and reduced the responses of matrix metalloproteinases 1 and 9 in HBE cells isolated from subjects with COPD. Enhanced epithelial growth factor receptor responses in cells from subjects with COPD were observed to modulate CIP2A expression levels. Our study indicates that chronic cigarette smoke induction of epithelial growth factor receptor signaling and CIP2A expression can impair PP2A responses that are associated with loss of lung function and enhancement of proteolytic responses. Augmenting PP2A activity by manipulating CIP2A expression may represent a feasible therapeutic approach to counter smoke-induced lung disease.

KIAA1199 promotes metastasis of colorectal cancer cells via microtubule destabilization regulated by a PP2A/stathmin pathway.

Tumor metastasis is the main cause of death in advanced colorectal cancer. Our previous research showed that upregulation of KIAA1199 predicted poorer outcomes, and promoted cell motility and tumor metastasis in colorectal cancer, with the mechanisms not being fully elucidated. Here, we demonstrate that silencing of KIAA1199 results in reduced tumor metastasis in the orthotopic transplantation tumor model of colorectal cancer. Importantly, we find that KIAA1199 interacts with protein phosphatase 2A (PP2A) through the C-terminal domain and increases phosphatase activity of PP2A, which is essential for KIAA1199-mediated cell motility. Moreover, we identify stathmin, a microtubule-destabilizing protein, as a downstream of KIAA1199-PP2A complex. KIAA1199-induced dephosphorylation of stathmin results in microtubule destabilization and leads to enhanced cell motility. Furthermore, a microtubule-stabilizing drug paclitaxel could prevent KIAA1199-induced microtubule destabilization, and inhibit cell migration and invasion in vitro and tumor metastasis in vivo in colorectal cancer. Collectively, our study reveals that KIAA1199 promotes metastasis of colorectal cancer cells via microtubule destabilization regulated by a PP2A/stathmin pathway, and suggests that KIAA1199 may be a promising target for preventing metastasis in colorectal cancer.

133 - Tumor Pro-oxidant Enviroment Stabilizes Onco-protein c-Myc via Sustained Phosphorylation at Serine 62 that Promotes its Oncogenic Activity

c-Myc is arguably one of the most notorious oncoproteins known. c-Myc activity and oncogenic potential, are in turn, intricately regulated by its stabilizing phosphorylation at the Serine 62 (S62) residue. Phosphorylation at the S62 site is mediated by RAS activated RAF/MEK/ERK signaling cascade, while its dephosphorylation is under the governance of the protein phosphatase 2A (PP2A). Our recent work highlighted the critical involvement of cellular redox status, in particular superoxide anion, in inhibiting PP2A-mediated dephosphorylation of Bcl-2 at serine 70, thus stabilizing its anti-apoptotic activity1. Here we report that the stabilization and activity of the onco-protein c-myc is significantly amplified by the 'pro-oxidant' tumor microenvironment, which profoundly impacted PP2A phosphatase activity via nitrative inhibition of the B56α regulatory subunit. The B56α regulatory arm is responsible for delivering PP2A substrate phosphoprotein-c-Myc to the catalytic C subunit within the AC core dimer. Tyrosine nitration of the B56α isoform renders it incapable of binding the core dimer causing c-Myc to remain hyperphosphorylated at the serine 62 site. Sustained serine 62 phosphorylated c-Myc inhibits its ubiquitination and degradation by the 26S proteasome. Hitherto driven c-Myc stability is further complemented with a moderate increase in its activity and gain of chemoresistance in osteosarcoma cells. This paradigm shift in our understanding of c-Myc stability as a function of tumor redox microenvironment could give us valuable cues in providing a pathway for therapeutic intervention targeting post-translational regulation of c-Myc in human cancer.

Downregulation of protein phosphatase 2A by apolipoprotein E: Implications for Alzheimer's disease.

The apolipoprotein E ε4 allele is the single most important genetic risk factor associated with Alzheimer's disease (AD). Tau phosphorylation and hyperphosphorylation is an underlying feature of AD and is regulated by specific kinases and phosphatases. Among phosphatases, protein phosphatase 2A (PP2A) is the principal tau dephosphorylating enzyme in the brain. Several abnormalities of PP2A have been reported in AD, including among others decreased protein levels of PP2A, decreased mRNA and protein levels of the catalytic subunit PP2AC and variable regulatory B subunits and reduced methylation of the catalytic subunit, all of which results in disruption of the PP2A phosphatase activity. In earlier studies we described a novel mechanism for ApoE as a transcription factor that binds regions of double-stranded DNA with high affinity, including the promoter regions of ~3000 different genes. The list of genes also included PPP2R5E (B56ε), a regulatory B' subunit of protein phosphatase 2A. Using a combination of A172 human glioblastoma cells, ApoE3/4 and ApoE-/- NSC and human postmortem tissue, we now demonstrate that ApoE not only binds to the PPP2R5E promoter but also triggers a significant reduction in PP2A activity by two mechanisms: 1) ApoE transcriptionally represses PPP2R5E and reduces protein expression, and 2) ApoE triggers demethylation of the catalytic subunit (PP2AC) of PP2A, resulting in the disruption of the PPP2R5E-PP2AC complex. Our results indicated a significant down-regulation of PPP2R5E gene expression and reduction in PP2A activity by ApoE4 compared with ApoE3. This may also explain an elevated Tau phosphorylation in AD human brains that featured at least one ApoE4 allele. Thus, our present work links ApoE and PPP2R5E expression to a reduction in the PP2A catalytic activity that has implications for Alzheimer's disease.

High CFTR expression in Philadelphia chromosome-positive acute leukemia protects and maintains continuous activation of BCR-ABL and related signaling pathways in combination with PP2A.

Cystic fibrosis transmembrane conductance regulator (CFTR) is classified as an anion channel transporter of Cl− and HCO3−. Through interactions with its PDZ domain, CFTR is capable of regulating other proteins, such as protein phosphatase 2A (PP2A). The aberrant expression and mutation of CFTR have been observed in several tumor, but not in philadelphia chromosome–positive(Ph+) acute leukemia, including Ph+ B cell acute lymphoblastic leukemia(Ph+ B-ALL) and chronic myelogenous leukemia blast crisis phases (CML-BC). In this study, we demonstrated the mean expression level of CFTR in Ph+ acute leukemia cells was markedly higher than that in Ph- B-ALL and CML-chronic phase cells. CFTRinh-172, a classic CFTR inhibitor, down-regulated the expression of CFTR, p-BCR-ABL and classical Wnt/β-catenin signaling in Ph+ acute leukemia cells, while imatinib had no effect on CFTR. Importantly, reduced efficacy of CFTRinh-172 was closely associated with elevated PP2A phosphatase activity. Furthermore, we confirmed an interaction between CFTR and the PP2AA subunit in K562 cells. In addition, we demonstrated CFTR and PP2AA interact in the cytosol, resulting in PP2A complex inactivation and increased degradation of PP2A substrates via the lysosomal/proteasome pathway. In conclusion, our results showed CFTR was highly expressed in Ph+ acute leukemia, which protected and maintained the continuous activation of BCR-ABL and the canonical Wnt/β-catenin signaling pathway by decreasing PP2A phosphatase activity. According to this working model of the CFTR-PP2A-BCR-ABL axis, targeting the CFTR protein will activate PP2A and may offer a new treatment strategy for Ph+ acute leukemia, especially for patients exhibiting high levels of CFTR expression.

PP2A Balances Glucose Metabolism and Foxo Activation to Maintain Cellular Redox Homeostasis in Acute Lymphoblastic Leukemia

Introduction: Protein phosphatase 2A (PP2A) is a Ser/Thr phosphatase negatively regulates a diverse set of signaling pathways promoting tumor growth. While PP2A functions as tumor suppressor in multiple types of cancer, here, we demonstrate an unexpected pro-survival role of PP2A in pre-B acute lymphoblastic leukemia (ALL) cells. By calibrating PI3K-AKT-mTOR signaling strength, PP2A regulates glycolysis rate and thereby balances energy demands against anti-oxidant protection of pre-B ALL cells. In addition, PP2A reinstates activity of FOXO factors by dephosphorylation and thereby enables the anti-oxidant function of FOXO1 and FOXO3. Consistent with previous studies, we find PP2A is dispensable for the survival of myeloid leukemia cells which indicates a lineage-specific role of PP2A. Combined with genetic study and small molecule inhibitor, we verify that regulation in patient-derived xenografts and highlight PP2A as a therapeutic target in pre-B ALL.Results: Consistent with a divergent role of PP2A in pre-B ALL (compared to CML), we found that high mRNA levels of PP2A subunits at the time of diagnosis predict poor outcome of children (COG P9906; n=207) and adults (ECOG 2993; n=215) with ALL. Consistent with these findings, mutations in PP2A subunits are extremely rare in B cell malignancies but relatively common in solid tumors and myeloid malignancies (COSMIC). We therefore, studied the function of PP2A in a genetic mouse model for Cre-induced deletion of Ppp2r1a in BCR-ABL1 (Ph+) ALL. Inducible activation of Cre reduced protein expression of the targeted PP2A subunit A and the catalytic subunit C, which leads to near-complete loss of PP2A phosphatase activity. Conversely, Cre-mediated deletion increased phosphorylation levels of FoxO1, FoxO3a, p70S6K and S6 ribosomal protein, which indicated elevated PI3K-Akt-mTOR signaling. Acute deletion of Ppp2r1afl/fl in B cell-lineage ALL cells dramatically affected survival and colony formation, both of which could be rescued by overexpression of wildtype PP2A. PI3K and mTOR inhibitors also have rescue effect on PP2A deficient ALL cells in growth-competition assay. Luciferase-labeled PP2A-deleted ALL cells showed reduced cell growth and leukemia progression after being transplanted into recipient mice. However, Cre-mediated deletion had no deleterious effects in a Ppp2r1afl/fl CML model. This lineage-specific role of PP2A was verified by inducible CEBPα expression to reprogram B cell lineage ALL cells into myeloid cells.Interestingly, inducible deletion of PP2A caused profound imbalances of glucose metabolism in Ph+ ALL but not in CML cells. Upon PP2A-deletion, ALL cells showed higher glycolytic flux shunted into lactate rather than NADPH production. By employing glucose flux metabolic profiling assay using [1,2-13C2]-D-glucose tracer, we found elevated glycolysis and repressed pentose phosphate pathway (PPP) flux in PP2A-deleted pre-B ALL cells. Lower NADPH/NADP ratio and higher reactive oxygen species level in PP2A-deleted pre-B ALL cells, together with decreased anti-oxidant gene expression, increased DNA damage, including H2AX phosphorylation and p53 expression. The mechanistic role of ROS downstream of PP2A was supported by a strong rescue effect of overexpression of the antioxidant catalase in PP2A-deleted cells. The unexpected role of PP2A in Ph+ ALL was further validated by CRISPR-Cas9 mediated disruption of PPP2R1A in ALL xenografts derived from three patients. In addition, a PP2A specific inhibitor LB-100 (in clinical trial for solid tumors) was employed to pharmacologically inhibit PP2A activity. Low micromolar concentrations of LB-100 induced cell death in patient-derived ALL xenografts in parallel with ROS-accumulation and increased S6 and H2AX phosphorylation.Conclusion: Here we revealed an unexpected role of PP2A in maintaining redox homeostasis in pre-B ALL cells. By regulating AKT-mTOR signaling, PP2A keeps the balance of glycolysis and PPP to meet the energy demands of pre-B ALL cells and avoids extreme levels of oxidative stress. We confirmed this pro-survival role of PP2A in both genetic mouse ALL model and human Ph+ ALL-patients derived leukemia cells. These findings highlight PP2A as a therapeutic target and suggest that agents like the PP2A inhibitor LB-100 may be of interest for pre-clinical development and testing. DisclosuresNo relevant conflicts of interest to declare.

Glutaminolysis Was Induced by TGF-β1 through PP2Ac Regulated Raf-MEK-ERK Signaling in Endothelial Cells.

Vascular endothelial cells can survive under hypoxic and inflammatory conditions by alterations of the cellular energy metabolism. In addition to high rates of glycolysis, glutaminolysis is another important way of providing the required energy to support cellular sprouting in such situations. However, the exact mechanism in which endothelial cells upregulate glutaminolysis remains unclear. Here we demonstrated that protein phosphatase 2A (PP2A)-mediated Raf-MEK-ERK signaling was involved in glutaminolysis in endothelial cells. Using models of human umbilical vein endothelial cells (HUVECs) treated with transforming growth factor-β1 (TGF-β1), we observed a dramatic induction in cellular glutamate levels accompanied by Raf-MEK-ERK activation. By addition of U0126, the specific inhibitor of MEK1/2, the expression of kidney-type glutaminase (KGA, a critical glutaminase in glutaminolysis) was significantly decreased. Moreover, inhibition of PP2A by okadaic acid (OA), a specific inhibitor of PP2A phosphatase activity or by depletion of its catalytic subunit (PP2Ac), led to a significant inactivation of Raf-MEK-ERK signaling and reduced glutaminolysis in endothelial cells. Taken together, these results indicated that PP2A-dependent Raf-MEK-ERK activation was involved in glutaminolysis and inhibition of PP2A signals was sufficient to block Raf-MEK-ERK pathway and reduced glutamine metabolism in endothelial cells.

ROTUNDA3 function in plant development by phosphatase 2A-mediated regulation of auxin transporter recycling

The shaping of organs in plants depends on the intercellular flow of the phytohormone auxin, of which the directional signaling is determined by the polar subcellular localization of PIN-FORMED (PIN) auxin transport proteins. Phosphorylation dynamics of PIN proteins are affected by the protein phosphatase 2A (PP2A) and the PINOID kinase, which act antagonistically to mediate their apical-basal polar delivery. Here, we identified the ROTUNDA3 (RON3) protein as a regulator of the PP2A phosphatase activity in Arabidopsis thaliana. The RON3 gene was map-based cloned starting from the ron3-1 leaf mutant and found to be a unique, plant-specific gene coding for a protein with high and dispersed proline content. The ron3-1 and ron3-2 mutant phenotypes [i.e., reduced apical dominance, primary root length, lateral root emergence, and growth; increased ectopic stages II, IV, and V lateral root primordia; decreased auxin maxima in indole-3-acetic acid (IAA)-treated root apical meristems; hypergravitropic root growth and response; increased IAA levels in shoot apices; and reduced auxin accumulation in root meristems] support a role for RON3 in auxin biology. The affinity-purified PP2A complex with RON3 as bait suggested that RON3 might act in PIN transporter trafficking. Indeed, pharmacological interference with vesicle trafficking processes revealed that single ron3-2 and double ron3-2 rcn1 mutants have altered PIN polarity and endocytosis in specific cells. Our data indicate that RON3 contributes to auxin-mediated development by playing a role in PIN recycling and polarity establishment through regulation of the PP2A complex activity.

Protein phosphatase 2A plays an important role in migration of bone marrow stroma cells.

Administration of bone marrow stroma cells (BMSCs) has the potential to ameliorate degenerative disorders and to repair injured sites. The homing of transplanted BMSCs to damaged tissues is a critical property of engraftment. Therefore, it is important to understand signal molecules controlling migration of BMSCs. Here, we demonstrate that serine-threonine protein phosphatase 2A (PP2A) is responsive to migration of BMSCs. Pharmacological Inhibition of PP2A, using okadaic acid (OA), leads to attenuated cell migration in rat primary BMSCs both in the absence or presence of stromal cell-derived factor-1 (SDF-1). Consistent with the above findings, knockdown of the main catalytic subunit PP2Acα using small interfering RNA also attenuates chemotaxis of BMSCs. On the other hand, cell viability of BMSCs remains unchanged with OA treatment or knockdown of PP2Acα subunit. Moreover, we observed an upregulation of PP2A-B55β in transcription level after SDF-1 treatment, indicating their potential role as the functioning regulatory subunit of PP2A phosphatase in BMSCs migration model. Collectively, these data provide first insight into the modulation of BMSCs migration by PP2A phosphatase activity and lay a foundation for exploring PP2A signaling as a modulating target for BMSCs transplantation.

PP2A Is Required for B Cell Survival and Represents a Therapeutic Target in Acute Lymphoblastic Leukemia

Background: The Ser/Thr Protein phosphatase 2A (PP2A) attenuates activity of RAS-ERK and PI3K-AKT signaling pathways and functions as important tumor suppressor in chronic myeloid leukemia (CML) by downregulating output of oncogenic BCR-ABL1 signaling. Restoration of PP2A activity has been proposed for the treatment of CML and eradication of TKI-resistant leukemia CML-stem cells (Neviani et al., 2013). While the tumor suppressor function of PP2A was independently confirmed by multiple groups, our analyses of PP2A function during early B cell development suggested a role for PP2A to contribute to B cell survival. Therefore, we studied whether PP2A has different roles in B-lineage ALL and CML and whether these mechanistic differences are relevant for patients with B-lineage and B-lymphoid blast crisis CML.Results: Consistent with a divergent role of PP2A in B-lineage ALL (compared to CML), we found that high mRNA levels of PP2A subunits at the time of diagnosis predict poor outcome of children (COG P9906; n=207) and adults (ECOG 2993; n=215) with ALL. We therefore, studied the function of PP2A in a genetic mouse model for Cre-induced deletion of Ppp2r1a in BCR-ABL1 (Ph+) ALL. Inducible activation of Cre reduced protein expression of the targeted PP2A subunit A but also the catalytic subunit C and was paralleled by near-complete loss of PP2A phosphatase activity. Conversely, Cre-mediated deletion increased phosphorylation levels of p70S6K and S6 ribosomal protein, which indicated elevated mTOR signaling. Acute deletion of Ppp2r1afl/fl in B cell-lineage ALL cells dramatically affected survival and colony formation, both of which could be rescued by overexpression of wildtype PP2A. In agreement with previous studies, however, Cre-mediated deletion had no deleterious effects in a Ppp2r1afl/fl CML model. To verify that the observed difference between B cell and myeloid disease was indeed lineage-dependent, we reprogrammed B cell lineage ALL cells into myeloid CML using inducible overexpression of CEBPα. As expected, CEBPα-driven myeloid lineage conversion prevented cell death upon deletion of PP2A, demonstrating that PP2A-function represents a B cell-specific vulnerability.We next transplanted luciferase-labeled Ppp2r1afl/fl ALL cells into recipient mice and monitored cell growth and leukemia progression. Cre-mediated deletion significantly prolonged overall survival of recipient mice that were transplanted with Ppp2r1afl/fl ALL cells. Although mice died eventually from leukemia, the ALL cells isolated from the mice retained Ppp2r1a floxed alleles in all cases studied demonstrating that fatal leukemia arose from few subclones that had escaped Cre-mediated deletion of PP2A.Interestingly, inducible deletion of PP2A caused profound imbalances of glucose metabolism in B cell lineage Ph+ ALL but not CML-like cells. Upon PP2A-deletion, ALL cells showed higher glycolytic flux shunted into lactate rather than NADPH production. Lower NADPH/NADP ratio and higher reactive oxygen species level in PP2A-deleted ALL cells, together with decreased anti-oxidant gene expression, increased H2AX phosphorylation and p53 expression indicated impaired balance of glycolytic flux may account for increased death of those cells. This notion was supported by a strong rescue effect of overexpression of the antioxidant catalase in PP2A-deleted cells.The unexpected role of PP2A in Ph+ ALL was further validated by CRISPR-Cas9 mediated disruption of PPP2R1A in ALL xenografts derived from three patients. In addition, a PP2A specific inhibitor LB-100 (in clinical trial for solid tumors) was employed to pharmacologically inhibit PP2A activity. Low micromolar concentrations of LB-100 induced cell death in patient-derived ALL xenografts in parallel with ROS-accumulation and increased S6 and H2AX phosphorylation.Conclusion: Cre-mediated ablation of PP2A in mouse Ph+ ALL cells induced rapid cell death through excessively oxidative stress but not in myeloid CML cells. We confirmed this pro-survival role of PP2A in human Ph+ ALL-patients derived leukemia cells through both CRISPR-Cas9 mediated genetic study and PP2A inhibition by a novel small molecule LB-100 (Jie Lu et al., 2009). These findings highlight PP2A as a therapeutic target with potential relevance in Ph+ ALL and B-lymphoid blast crisis progression of CML. DisclosuresNo relevant conflicts of interest to declare.

Disruption and inactivation of the PP2A complex promotes the proliferation and angiogenesis of hemangioma endothelial cells through activating AKT and ERK.

Hemangioma is a benign vascular neoplasm of unknown etiology. In this study, we generated an endothelial-specific PyMT gene-expressing transgenic mouse model that spontaneously develops hemangioma. Based on this transgenic model, a specific binding between PyMT and the core AC dimer of protein phosphatase 2A (PP2A) was verified in hemangioma vascular endothelial cells. The binding between PyMT and the PP2A AC dimer resulted in dissociation of the B subunit from the PP2A complex and inactivation of PP2A phosphatases, which in turn activated AKT and ERK signaling and promoted cell proliferation, migration and angiogenesis in vitro and tumorigenesis in vivo. Consistent with the in vitro findings, decreased PP2A phosphatase activity and disruption of the PP2A heterotrimeric complex were also observed in both primary transgene-positive TG(+) mouse hemangioma endothelial cells (TG(+) HEC cells) and human proliferating phase hemangioma endothelial (human HEC-P) cells, but not in transgene-negative TG(-) mouse normal vascular endothelial cells (TG(-) NEC cells) and human involuting phase hemangioma endothelial (human HEC-I) cells. Further, it was observed that in human hemangioma cells, endoglin could compete with the PP2A/A, C subunits for binding to the PP2A/B subunit, thereby resulting in dissociation of the B subunit from the PP2A complex. Treatment of Tie2/PyMT transgenic mice with the PP2A activator FTY720 significantly delayed the occurrence of hemangioma. Our data provide evidence of a previously unreported anti-proliferation and anti-angiogenesis effect of PP2A in vascular endothelial cells, and show the therapeutic value of PP2A activators in hemangioma.

Study of Protein Phosphatase 2A (PP2A) Activity in LPS-Induced Tolerance Using Fluorescence-Based and Immunoprecipitation-Aided Methodology.

Protein phosphatase 2A (PP2A) is one of the most abundant intracellular serine/threonine (Ser/Thr) phosphatases accounting for 1% of the total cellular protein content. PP2A is comprised of a heterodimeric core enzyme and a substrate-specific regulatory subunit. Potentially, at least seventy different compositions of PP2A exist because of variable regulatory subunit binding that accounts for various activity modulating numerous cell functions. Due to the constitutive phosphatase activity present inside cells, a sensitive assay is required to detect the changes of PP2A activity under various experimental conditions. We optimized a fluorescence assay (DIFMU assay) by combining it with prior anti-PP2A immunoprecipitation to quantify PP2A-specific phosphatase activity. It is also known that prior exposure to lipopolysaccharides (LPS) induces “immune tolerance” of the cells to subsequent stimulation. Herein we report that PP2A activity is upregulated in tolerized peritoneal macrophages, corresponding to decreased TNF-α secretion upon second LPS stimulation. We further examined the role of PP2A in the tolerance effect by using PP2ACαlox/lox;lyM-Cre conditional knockout macrophages. We found that PP2A phosphatase activity cannot be further increased by tolerance. TNF-α secretion from tolerized PP2ACαlox/lox;lyM-Cre macrophages is higher than tolerized control macrophages. Furthermore, we showed that the increased TNF-α secretion may be due to an epigenetic transcriptionally active signature on the promoter of TNF-α gene rather than regulation of the NFκB/IκB signaling pathway. These results suggest a role for increased PP2A activity in the regulation of immune tolerance.

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.

Protein Phosphatase 2A Mediates Oxidative Stress Induced Apoptosis in Osteoblasts.

Osteoporosis is one of the most common bone diseases, which is characterized by a systemic impairment of bone mass and fragility fractures. Age-related oxidative stress is highly associated with impaired osteoblastic dysfunctions and subsequent osteoporosis. In osteoblasts (bone formation cells), reactive oxygen species (ROS) are continuously generated and further cause lipid peroxidation, protein damage, and DNA lesions, leading to osteoblastic dysfunctions, dysdifferentiations, and apoptosis. Although much progress has been made, the mechanism responsible for oxidative stress induced cellular alternations and osteoblastic toxicity is still not fully elucidated. Here, we demonstrate that protein phosphatase 2A (PP2A), a major protein phosphatase in mammalian cells, mediates oxidative stress induced apoptosis in osteoblasts. Our results showed that lipid peroxidation products (4-HNE) may induce dramatic oxidative stress, inflammatory reactions, and apoptosis in osteoblasts. These oxidative stress responses may ectopically activate PP2A phosphatase activity, which may be mediated by inactivation of AKT/mTOR pathway. Moreover, inhibition of PP2A activity by okadaic acid might partly prevent osteoblastic apoptosis under oxidative conditions. These findings may reveal a novel mechanism to clarify the role of oxidative stress for osteoblastic apoptosis and provide new possibilities for the treatment of related bone diseases, such as osteoporosis.

The role of Cullin3-mediated ubiquitination of the catalytic subunit of PP2A in TRAIL signaling

Protein phosphatase 2A (PP2A) is the major serine-threonine phosphatase that regulates a number of cell signaling pathways. PP2A activity is controlled partially through protein degradation; however, the underlying mechanism is not fully understood. Here we show that PP2A/C, a catalytic subunit of PP2A, is degraded by the Cullin3 (Cul3) ligase-mediated ubiquitin-proteasome pathway. In response to death receptor signaling by tumor-necrosis factor-related apoptosis-inducing ligand (TRAIL), PP2A/C, caspase-8 and Cul3, a subunit of the cullin family of E3 ligases, are recruited into the death-inducing signaling complex (DISC) where the Cul3 ligase targets PP2A/C for ubiquitination and subsequent degradation. Functionally, knockdown of PP2A/C expression by siRNA or pharmacological inhibition of PP2A activity increases TRAIL-induced apoptosis. In cancer cells that have developed acquired TRAIL resistance, PP2A phosphatase activity is increased, and PP2A/C protein is resistant to TRAIL-induced degradation. Thus, this work identifies a new mechanism by which PP2A/C is regulated by Cul3 ligase-mediated degradation in response to death receptor signaling and suggests that inhibition of PP2A/C degradation may contribute to resistance of cancer cells to death receptor-induced apoptosis.