Regulation Of Protein Phosphatase 2A Activity Research Articles

Abstract 425: Protein Kinase Activity of Pi3kγ Regulates Insulin-βArs Cross-talk

β adrenergic receptor (βAR) function is regulated by G-protein coupled receptor (GPCR) kinase (GRK) driven desensitization and protein phosphatase 2A (PP2A) mediated resensitization to its classical agonist. Surprisingly, Insulin modulates (βARs) function thus regulating cardiac function. Although insulin is known to modulate βAR function through GRKs, less is known about insulin mediated resensitization mechanisms. PI3Kγ is activated by GPCRs regulates resensitization by inhibiting PP2A activity. Therefore, we tested whether insulin could mediate βAR dysfunction through inhibition of resensitization. Co-immunoprecipitation in cardiac lysates and surface plasmon resonance (SPR) with purified proteins show that PI3Kγ interacts with GRK-2 resulting in recruitment of GRK2-PI3Kγ to the β2AR complex upon insulin. Furthermore, use of PI3K inhibitors significantly reduced Insulin-stimulated β2ARs phosphorylation in HEK293 cells. The role of PI3Kγ was further validated by the loss of insulin stimulated β 2 AR phosphorylation in PI3Kγ-knockout (KO) mouse embryonic fibroblasts (MEFs), shRNA and CRIPSR knockdown of PI3Kγ. Data shows that PI3Kγ inhibits PP2A activity at the βAR complex upon insulin while loss of PI3Kγ unravels this inhibition resulting in increased PP2A activity leading to β2AR dephosphorylation and resensitization. Mechanistically, PI3Kγ inhibits PP2A activity at the β2AR complex by phosphorylating an endogenous inhibitor of PP2A (I2PP2A). CRISPR knockout and siRNA knockdown of endogenous I2PP2A in HEK293 cells restored PP2A activity resulting in β2AR dephosphorylation despite PI3Kγ. Furthermore, β blocker (propranolol) pretreatment did not affect β2AR phosphorylation and there was no β-arrestin recruitment to the βAR complex with Insulin. Together these studies show that Insulin mediates β2AR desensitization through β-agonist and β-arrestin independent mechanisms wherein, PI3Kγ-mediated regulation of PP2A activity plays a pivotal role in cardiac βAR function in hyperinsulemic conditions like diabetes and obesity.

UBE3A-mediated PTPA ubiquitination and degradation regulate PP2A activity and dendritic spine morphology

Deficiency in the E3 ubiquitin ligase UBE3A leads to the neurodevelopmental disorder Angelman syndrome (AS), while additional dosage of UBE3A is linked to autism spectrum disorder. The mechanisms underlying the downstream effects of UBE3A gain or loss of function in these neurodevelopmental disorders are still not well understood, and effective treatments are lacking. Here, using stable-isotope labeling of amino acids in mammals and ubiquitination assays, we identify PTPA, an activator of protein phosphatase 2A (PP2A), as a bona fide ubiquitin ligase substrate of UBE3A. Maternal loss of Ube3a (Ube3am-/p+) increased PTPA level, promoted PP2A holoenzyme assembly, and elevated PP2A activity, while maternal 15q11-13 duplication containing Ube3a down-regulated PTPA level and lowered PP2A activity. Reducing PTPA level in vivo restored the defects in dendritic spine maturation in Ube3am-/p+ mice. Moreover, pharmacological inhibition of PP2A activity with the small molecule LB-100 alleviated both reduction in excitatory synaptic transmission and motor impairment in Ube3am-/p+ mice. Together, our results implicate a critical role of UBE3A-PTPA-PP2A signaling in the pathogenesis of UBE3A-related disorders and suggest that PP2A-based drugs could be potential therapeutic candidates for treatment of UBE3A-related disorders.

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.

Proteomics and phosphoproteomics study of LCMT1 overexpression and oxidative stress: overexpression of LCMT1 arrests H2O2-induced lose of cells viability

ABSTRACTObjectives: Protein phosphatase 2A (PP2A), a major serine/threonine phosphatase, is also known to be a target of ROS. The methylation of PP2A can be catalyzed by leucine carboxyl methyltransferase-1 (LCMT1), which regulates PP2A activity and substrate specificity.Methods: In the previous study, we have showed that LCMT1-dependent PP2Ac methylation arrests H2O2-induced cell oxidative stress damage. To explore the possible protective mechanism, we performed iTRAQ-based comparative quantitative proteomics and phosphoproteomics studies of H2O2-treated vector control and LCMT1-overexpressing cells.Results: A total of 4480 non-redundant proteins and 3801 unique phosphopeptides were identified by this means. By comparing the H2O2-regulated proteins in LCMT1-overexpressing and vector control cells, we found that these differences were mainly related to protein phosphorylation, gene expression, protein maturation, the cytoskeleton and cell division. Further investigation of LCMT1 overexpression-specific regulated proteins under H2O2 treatment supported the idea that LCMT1 overexpression induced ageneral dephosphorylation of proteins and indicated increased expression of non-erythrocytic hemoglobin, inactivation of MAPK3 and regulation of proteins related to Rho signal transduction, which were known to be linked to the regulation of the cytoskeleton.Discussion: These data provide proteomics and phosphoproteomics insights into the association of LCMT1-dependent PP2Ac methylation and oxidative stress and indirectly indicate that the methylation of PP2A plays an important role against oxidative stress.

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.

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.

Regulation of protein phosphatase 2A (PP2A) tumor suppressor function by PME-1.

Protein phosphatase 2A (PP2A) plays a major role in maintaining cellular signaling homeostasis by dephosphorylation of a variety of signaling proteins and acts as a tumor suppressor. Protein phosphatase methylesterase-1 (PME-1) negatively regulates PP2A activity by highly complex mechanisms that are reviewed here. Importantly, recent studies have shown that PME-1 promotes oncogenic MAPK/ERK and AKT pathway activities in various cancer types. In human glioma, high PME-1 expression correlates with tumor progression and kinase inhibitor resistance. We discuss the emerging cancer-associated function of PME-1 and its potential clinical relevance.

CaTip41 regulates protein phosphatase 2A activity, CaRad53 deactivation and the recovery of DNA damage-induced filamentation to yeast form in Candida albicans.

Phosphorylation and dephosphorylation of the checkpoint kinase CaRad53 is crucial for fungal cells in response to genotoxic stresses. The protein phosphatase 2A (PP2A) CaPph3/CaPsy2 phosphatase complex is involved in CaRad53 dephosphorylation in Candida albicans. In view of the role of ScTip41/ScTap42 in regulating PP2A phosphatases in Saccharomyces cerevisiae, we have explored the function of CaTip41 in C. albicans. Here, we show that CaTIP41 is a functional ortholog of ScTIP41 in the sensitivity of S. cerevisiae cells to rapamycin. Deletion of CaTIP41 causes C. albicans cells to be sensitive to DNA damaging agents, methylmethane sulfonate (MMS) and cisplatin, and resistant to both rapamycin and caffeine. Accordingly, expression of CaTip41 increases in response to MMS and cisplatin. In addition, C. albicans cells lacking CaTIP41 show a delay in the recovery from MMS-induced filamentation to yeast form, decreased PP2A activity and a defect in deactivation of CaRad53 during recovery from DNA damage. Through yeast two-hybrid assay we show that CaTip41 interacts with either CaPph3, CaPsy2 or CaTap42. Therefore, CaTip41 plays regulatory roles in both the CaRad53 deactivation during recovery from DNA damage and the target of rapamycin signaling pathway.

Immediate-early response 5 (IER5) interacts with protein phosphatase 2A and regulates the phosphorylation of ribosomal protein S6 kinase and heat shock factor 1

Immediate-early response 5 (IER5) is a growth factor-inducible protein with homology to the N-terminus of IER2. Deletion analysis shows that a large region of IER5, including the N-terminal region, is involved in cell growth and stress resistance. The N-terminal region mediates IER5 oligomerization and binding to the B55 regulatory subunit of protein phosphatase 2A (PP2A). IER5 physically interacts with the PP2A target proteins ribosomal protein S6 kinase (S6K) and heat shock factor 1 (HSF1), and the interactions are essential for the reduced phosphorylation of S6K and HSF1. Our data indicate that oligomeric IER5 regulates PP2A activity and cell growth.

Abstract 3325: A protein phosphatase 2A-ATM complex regulates the DNA damage response and pro-survival signaling

Abstract Background: Ataxia telangiectasia mutated (ATM), a S/T protein kinase, is a major regulator of the DNA damage response (DDR) and is activated by DNA double-strand breaks (DSB) caused by ionizing radiation (IR) and radiomimetic drugs. ATM controls cell cycle checkpoints and DSB repair leading to cell cycle arrest and recovery, or apoptosis. ATM also regulates pro-survival AKT signaling. We and other groups have previously reported that an ATM-dependent phosphatase negatively controls AKT phosphorylation in response to insulin and IR. Many DDR proteins, phosphorylated after DNA damage are subsequently dephosphorylated by phosphatases such as protein phosphatase 2A (PP2A) to reverse the DDR and reset the cell to normalcy. In addition to dephophorylating AKT, PP2A controls the G2/M checkpoint and dephosphorylates γ-H2AX after IR. Recently, it was reported that ATM negatively regulates PP2A activity by phosphorylating its scaffolding subunit Aα. Objective: We previously reported that a small molecule inhibitor of the ATM kinase results in AKT dephosphorylation. The present study was aimed at determining how ATM and PP2A are co-regulated during the DDR and pro-survival signaling. We hypothesized that ATM phosphorylates PP2A-Aα thereby inhibiting phosphatase activity to increase AKT signaling. Results: We generated specific PP2A-Aα mutations at S401; a S401A mutant which cannot be phosphorylated and a phospho-mimetic S401D mutant. Co-immunoprecipitation studies in HEK293 cells over-expressing PP2A-Aα WT, S401A or S401D showed that ATM, AKT and the PP2A catalytic subunit (PP2A-C) bind to the WT and S401A mutant but not the S401D mutant. We then generated PP2A-Aα conditional knockout mouse embryonic fibroblasts (MEFs) expressing PP2A-Aα-WT, S401A or S401D transgenes by floxing out the endogenous PP2A-Aα alleles with Cre. Treatment with insulin increased pAKT (S473) levels in cells expressing the S401D mutant relative to WT and S401A. Furthermore, H2AX phosphorylation after low dose of IR showed prolonged phosphorylation whereas AKT showed decreased phosphorylation, suggesting that there was a delay in re-establishing the pre-IR, non-phosphorylated state. Additionally, the S401D mutant was retained in the cytoplasm after IR and was less efficient in DSB repair. S401D mutant cells were also more radiosensitive relative to the WT and S401A. Conclusion: Taken together, our findings suggest that ATM-mediated phosphorylation of PP2A-Aα at S401 modulates the DDR and AKT pro-survival signaling and perhaps other proteins modulating DSB repair. These studies are very important as they will help elucidate the intimate regulatory relationship between PP2A and ATM and establish a more detailed mechanisms how a single, critical amino acid (S401) in PP2A-Aα affects the DDR, DSB repair and survival. Citation Format: Amrita D. Sule, Mary Tokarz, Mostafa Ahmed, Glen E. Kellogg, Kristoffer C. Valerie. A protein phosphatase 2A-ATM complex regulates the DNA damage response and pro-survival signaling. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3325. doi:10.1158/1538-7445.AM2015-3325

Midline 1 directs lytic granule exocytosis and cytotoxicity of mouse killer T cells.

Midline 1 (MID1) is a microtubule-associated ubiquitin ligase that regulates protein phosphatase 2A activity. Loss-of-function mutations in MID1 lead to the X-linked Opitz G/BBB syndrome characterized by defective midline development during embryogenesis. Here, we show that MID1 is strongly upregulated in murine cytotoxic lymphocytes (CTLs), and that it controls TCR signaling, centrosome trafficking, and exocytosis of lytic granules. In accordance, we find that the killing capacity of MID1(-/-) CTLs is impaired. Transfection of MID1 into MID1(-/-) CTLs completely rescued lytic granule exocytosis, and vice versa, knockdown of MID1 inhibited exocytosis of lytic granules in WT CTLs, cementing a central role for MID1 in the regulation of granule exocytosis. Thus, MID1 orchestrates multiple events in CTL responses, adding a novel level of regulation to CTL activation and cytotoxicity.

TAP46 Plays a Positive Role in the ABSCISIC ACID INSENSITIVE5-Regulated Gene Expression in Arabidopsis

TAP46 is a protein phosphatase2A (PP2A)-associated protein that regulates PP2A activity in Arabidopsis (Arabidopsis thaliana). To study how PP2A is involved in abscisic acid (ABA) signaling in plants, we studied the function of TAP46 in ABA-regulated seed maturation and seedling development. Expression of TAP46 coincides with the action of ABA in developing seeds and during seed germination, and the TAP46 transcript reaches to the highest level in mature seeds. Real-time polymerase chain reaction analysis indicates that external ABA can increase TAP46 transcript level transiently during seed germination. Overexpression of TAP46 increases plant sensitivity to ABA, while tap46 knockdown mutants are less sensitive to ABA during seed germination, suggesting that TAP46 functions positively in ABA signaling. Overexpression of TAP46 also leads to lower PP2A activity, while tap46-1 knockdown mutant displays higher PP2A activity, suggesting that TAP46 negatively regulates PP2A activity in Arabidopsis. Both TAP46 and PP2A interact with the ABA-regulated transcription factor ABA INSENSITIVE5 (ABI5) in vivo, and TAP46's binding to ABI5 can stabilize ABI5. Furthermore, TAP46's binding to the phosphorylated ABI5 may prevent PP2A or PP2A-like protein phosphatases from removing the phosphate from ABI5, thereby maintaining ABI5 in its active form. Overexpression of TAP46 and inhibition of activities of PP2A or PP2A-like protein phosphatases can increase transcript levels of several ABI5-regulated genes, suggesting that TAP46 is a positive factor in the ABA-regulated gene expression in Arabidopsis.

Phosphatidic Acid (PA) Binds PP2AA1 to Regulate PP2A Activity and PIN1 Polar Localization

Phospholipase D (PLD) exerts broad biological functions in eukaryotes through regulating downstream effectors by its product, phosphatidic acid (PA). Protein kinases and phosphatases, such as mammalian target of rapamycin (mTOR), Protein Phosphatase 1 (PP1) and Protein Phosphatase 2C (PP2C), are PA-binding proteins that execute crucial regulatory functions in both animals and plants. PA participates in many signaling pathways by modulating the enzymatic activity and/or subcellular localization of bound proteins. In this study, we demonstrated that PLD-derived PA interacts with the scaffolding A1 subunit of Protein Phosphatase 2A (PP2A) and regulates PP2A-mediated PIN1 dephosphorylation in Arabidopsis. Genetic and pharmacological studies showed that both PA and PP2A participate in the regulation of auxin distribution. In addition, both the phosphorylation status and polar localization of PIN1 protein were affected by PLD inhibitors. Exogenous PA triggered the membrane accumulation of PP2AA1 and enhanced the PP2A activity at membrane, while PLD inhibition resulted in the reduced endosomal localization and perinuclear aggregation of PP2AA1. These results demonstrate the important role of PLD-derived PA in normal PP2A-mediated PIN dephosphorylation and reveal a novel mechanism, in which PA recruits PP2AA1 to the membrane system and regulates PP2A function on membrane-targeted proteins. As PA and PP2A are conserved among eukaryotes, other organisms might use similar mechanisms to mediate multiple biological processes.

Phosphorylation of SET Protein at Ser171 by Protein Kinase D2 Diminishes Its Inhibitory Effect on Protein Phosphatase 2A

We previously reported that protein kinase D2 (PKD2) in T cells is promptly activated after T-cell receptor (TCR) stimulation and involved in the activation of interleukin-2 promoter and T cell death, and that one of its candidate substrate is SET protein, a natural inhibitor for protein phosphatase 2A (PP2A). In this study, we investigated the target amino acid residues of SET phosphorylated by PKD2 and the effects of phosphorylation of SET on PP2A phosphatase activity. In vitro kinase assay using various recombinant SET mutants having Ser/Thr to Ala substitutions revealed that Ser171 of SET is one of the sites phosphorylated by PKD2. Recombinant SET with phosphorylation-mimic Ser171 to Glu substitution reduced its inhibitory effects on PP2A phosphatase activity compared with Ser171 to Ala substituted or wild-type SET. In addition, knockdown of PKD2 in Jurkat cells by RNAi or treatment of human CD4+ T cell clone with the PKD2 inhibitor Gö6976 resulted in reduced PP2A activity after TCR-stimulation judged from phosphorylation status of Tyr307 of the catalytic subunit of PP2A. These results suggest that PKD2 is involved in the regulation of PP2A activity in activated T cells through phosphorylation of Ser171 of SET.

Association of decreased expression of protein phosphatase 2A subunit PR55γ (PPP2R2C) with an increased risk of metastases and prostate cancer-specific mortality.

4669 Background: The protein phosphatase 2A (PP2A) holoenzyme complex negatively controls cell growth, differentiation and apoptosis. Down regulation of PP2A activity is associated with oncogenic transformation of papillomas and is a target for the small-T viral oncogene. The PR55γ (2R2C) subunit regulates PP2A target specificity, inhibiting c-Src activity in some cell lines. c-Src activity enhances growth and invasion in prostate cancer. This study evaluates whether PPP2R2C protein levels in radical prostatectomy (RP) specimens were predictive for the development of metastases or prostate-cancer specific mortality (PCSM). Methods: From 1954-1997, 1004 consecutive patients underwent RP at Virginia Mason Medical Center in Seattle. Metastases were confirmed radiographically or by tissue diagnosis. Death certificates confirmed PCSM. Patients without stored tissue were excluded. 34 patients with metastases or PCSM were matched to patients without recurrence (NR) at a 1:2 ratio. Paraffin-embedded tissue was stained with anti-PPP2R2C monoclonal antibody, clone 6D1 (Abnova, Taiwan). A pathologist evaluated the percentage and intensity of nuclei stained using a 4-point scale. Statistical analysis was performed on SAS. Results: Mean follow up was 10.26 years. PPP2R2C was detected in 13/18 (72.2%) PCSM, 18/22 (81.8%) metastases and 45/49 (91.8%) NR patients. Mean expression was lower in cancerous vs benign glands, 1.74 vs 2.04 (p = 0.001). Expression intensity from PCSM vs NR was 1.45 vs 1.80 (p = 0.041). On multivariate analysis, expression was lower in metastases or PCSM versus NR 1.79 vs 2.35 (p = 0.002), independent of pre-op PSA and Gleason grade. Conclusions: PPP2R2C loss was highly correlated with metastasis and PCSM. Patients with the lowest levels of expression were at the highest risk for PCSM. As tissue was obtained at time of surgery, this analysis reflects a truly prognostic biomarker, independent of Gleason grade and PSA. We have shown that PPP2R2C scoring is a useful metric to identify patients at high risk of developing advanced disease. With further validation may allow for more accurate individual patient treatment plans and counseling.

Cyclin G-associated kinase regulates protein phosphatase 2A by phosphorylation of its B'γ subunit

Protein phosphatase 2A (PP2A) bearing the B’γ (= B’α/B56γ1/PR61γ) subunit is recruited to dephosphorylation targets by cyclin G. We demonstrate here that cyclin G-associated kinase (GAK), a component of the GAK/B’γ/cyclin G complex, directly phosphorylates the B’γ-Thr104 residue and regulates PP2A activity. Indeed, an anti-B’γ-pT104 antibody detected immunofluorescence signals at the chromosome and centrosome during mitosis; these signals were reduced by siRNA-mediated GAK knockdown. After DNA damage by γ-irradiation, the chromosome signals formed foci that colocalized with a DNA double-strand break (DSB) marker H2AX-pS139 (γH2AX) and CHK2-pT68. Moreover, B’γ-pT104 enhanced PP2A holoenzyme assembly and PP2A activity, as shown by the results of an in vitro phosphatase assay. These results suggest a novel role for GAK as a regulator of dephosphorylation events under the control of the PP2A B’γ subunit.

Inhibition of Protein Phosphatase 2A Activity by PI3Kγ Regulates β-Adrenergic Receptor Function

Phosphoinositide 3-kinase γ (PI3Kγ) is activated by Gprotein-coupled receptors (GPCRs). We show here that PI3Kγ inhibits protein phosphatase 2A (PP2A) at the β-adrenergic receptor (βAR, a GPCR) complex altering G protein coupling. PI3Kγ inhibition results in significant increase of βAR-associated phosphatase activity leading to receptor dephosphorylation and resensitization preserving cardiac function. Mechanistically, PI3Kγ inhibits PP2A activity at the βAR complex by phosphorylating an intracellular inhibitor of PP2A (I2PP2A) on serine residues 9 and 93, resulting in enhanced binding to PP2A. Indeed, enhanced phosphorylation of β2ARs is observed with a phosphomimetic I2PP2A mutant that was completely reversed with a mutant mimicking dephosphorylated state. siRNA depletion of endogenous I2PP2A augments PP2A activity despite active PI3K resulting in β2AR dephosphorylation and sustained signaling. Our study provides the underpinnings of a PI3Kγ-mediated regulation of PP2A activity that has significant consequences on receptor function with broad implications in cellular signaling.

Sphingosine Interaction with Acidic Leucine-rich Nuclear Phosphoprotein-32A (ANP32A) Regulates PP2A Activity and Cyclooxygenase (COX)-2 Expression in Human Endothelial Cells

Sphingolipid metabolites regulate cell fate by acting on specific cellular targets. Although the influence of sphingolipids in cellular signaling has been well recognized, the exact molecular targets and how these targets influence cellular signaling mechanisms remain poorly understood. Toward this goal, we used affinity chromatography coupled with proteomics technology and identified acidic leucine-rich nuclear phosphoprotein-32A (ANP32A), an inhibitor of protein phosphatase 2A (PP2A) as a direct target of sphingosine, N,N'-dimethyl sphingosine (DMS) and phytosphingosine but not dihydrosphingosine or sphingosine 1-phosphate. Treatment of human umbilical vein endothelial cells (HUVEC) with DMS, which is not phosphorylated by sphingosine kinases, led to the activation of PP2A activity. Suppression of ANP32A with siRNA enhanced basal and DMS-activated PP2A activity suggesting that the sphingoid base binds to and relieves the inhibitory action of ANP32A on the PP2A complex. Indeed, DMS relieved the ANP32A-mediated inhibition of PP2A enzyme complex in vitro. Interestingly, DMS treatment induced the p38 stress-activated protein kinase (SAPK) and expression of cyclooxygenase (COX)-2 transcript and protein. Knockdown of ANP32A expression further induced p38 SAPK and COX-2. These data identify ANP32A as a novel molecular target of sphingoid bases that regulates cellular signaling events and inflammatory gene expression.

The CAG repeat in SCA12 functions as a cis element to up-regulate PPP2R2B expression

PPP2R2B, a protein widely expressed in neurons, regulates the protein phosphatase 2A (PP2A) activity for dephosphorylation of tau and other substrates. CAG repeat expansion at the 5'-end of the PPP2R2B gene causes autosomal dominant spinocerebellar ataxia type 12. In the present study, we investigated the roles of CAG repeats and flanking cis elements and the associated proteins in controlling PPP2R2B expression. Deletion/site-directed mutagenesis, in silico searches and cDNA overexpression revealed that CREB1 and SP1 bind to the conserved sequence upstream the CAG repeats to up-regulate PPP2R2B expression, whereas TFAP4 binds to the conserved sequence downstream the CAG repeats to down-regulate PPP2R2B expression. The binding of CREB1, SP1, and TFAP4 to the PPP2R2B promoter was further confirmed by DNA pull-down and ChIP-PCR assays. CAG repeats itself also function as a cis element to up-regulate PPP2R2B expression as AT repeat length has no effect on PPP2R2B expression. Together, our data provide evidence that CREB1, SP1, and TFAP4 play roles in modulating PPP2R2B expression, thus offering a mechanism of regulating PP2A activity as the treatment of neurodegenerative diseases associated with abnormal PP2A activity.