Regulation Of Protein Phosphatase 2A Research Articles

The PP2A regulator IER5L supports prostate cancer progression

Prostate cancer exhibits high prevalence and accounts for a high number of cancer-related deaths. The discovery and characterization of molecular determinants of aggressive prostate cancer represents an active area of research. The Immediate Early Response (IER) family of genes, which regulate Protein Phosphatase 2A (PP2A) activity, has emerged among the factors that influence cancer biology. Here, we show that the less studied member of this family, Immediate Early Response 5 like (IER5L), is upregulated in aggressive prostate cancer. Interestingly, the upregulation of IER5L expression exhibits a robust association with metastatic disease in prostate and is recapitulated in other cancer types. In line with this observation, IER5L silencing reduces foci formation, migration and invasion ability in a variety of human and murine prostate cancer cell lines. In vivo, using zebrafish and immunocompromised mouse models, we demonstrate that IER5L-silencing reduces prostate cancer tumor growth, dissemination, and metastasis. Mechanistically, we characterize the transcriptomic and proteomic landscapes of IER5L-silenced cells. This approach allowed us to identify DNA replication and monomeric G protein regulators as downstream programs of IER5L through a pathway that is consistent with the regulation of PP2A. In sum, we report the alteration of IER5L in prostate cancer and beyond and provide biological and molecular evidence of its contribution to tumor aggressiveness.

Synergistic suppression of BDNF via epigenetic mechanism deteriorating learning and memory impairment caused by Mn and Pb co-exposure

Microglia, the resident immune cells of the central nervous system (CNS), play a dual role in neurotoxicity by releasing the NLR Family Pyrin Domain Containing 3 (NLRP3) inflammasome and brain-derived neurotrophic factor (BDNF) in response to environmental stress. Suppression of BDNF is implicated in learning and memory impairment induced by exposure to manganese (Mn) or lead (Pb) individually. Methyl CpG Binding Protein 2 (MeCp2) and its phosphorylation status are related to BDNF suppression. Protein phosphatase2A (PP2A), a member of the serine/threonine phosphatases family, dephosphorylates substrates based on the methylation state of its catalytic C subunit (PP2Ac). However, the specific impairment patterns and molecular mechanisms resulting from co-exposure to Mn and Pb remain unclear. Therefore, the purpose of this study was to explore the effects of Mn and Pb exposure, alone and in combination, on inducing neurotoxicity in the hippocampus of mice and BV2 cells, and to determine whether simultaneous exposure to both metals exacerbate their toxicity. Our findings reveal that co-exposure to Mn and Pb leads to severe learning and memory impairment in mice, which correlates with the accumulation of metals in the hippocampus and synergistic suppression of BDNF. This suppression is accompanied by up-regulation of the epigenetic repressor MeCp2 and its phosphorylation status, as well as demethylation of PP2Ac. Furthermore, inhibition of PP2Ac demethylation using ABL127, an inhibitor for its protein phosphatase methylesterase1 (PME1), or knockdown of MeCp2 via siRNA transfection in vitro effectively increases BDNF expression and mitigates BV2 cell damage induced by Mn and Pb co-exposure. We also observe abnormal activation of microglia characterized by enhanced release of the NLRP3 inflammasome, Casepase-1 and pro-inflammatory cytokines IL-1β, in the hippocampus of mice and BV2 cells. In summary, our experiments demonstrate that simultaneous exposure to Mn and Pb results in more severe hippocampus-dependent learning and memory impairment, which is attributed to epigenetic suppression of BDNF mediated by PP2A regulation.

Protein phosphatase 2A modulation and connection with miRNAs and natural products.

Protein phosphatase 2A (PP2A), a heterotrimeric holoenzyme (scaffolding, catalytic, and regulatory subunits), regulates dephosphorylation for more than half of serine/threonine phosphosites and exhibits diverse cellular functions. Although several studies on natural products and miRNAs have emphasized their impacts on PP2A regulation, their connections lack systemic organization. Moreover, only part of the PP2A family has been investigated. This review focuses on the PP2A-modulating effects of natural products and miRNAs' interactions with potential PP2A targets in cancer and non-cancer cells. PP2A-modulating natural products and miRNAs were retrieved through a literature search. Utilizing the miRDB database, potential PP2A targets of these PP2A-modulating miRNAs for the whole set (17 members) of the PP2A family were retrieved. Finally, PP2A-modulating natural products and miRNAs were linked via a literature search. This review provides systemic directions for assessing natural products and miRNAs relating to the PP2A-modulating functions in cancer and disease treatments.

B56δ long-disordered arms form a dynamic PP2A regulation interface coupled with global allostery and Jordan's syndrome mutations.

Intrinsically disordered regions (IDR) and short linear motifs (SLiMs) play pivotal roles in the intricate signaling networks governed by phosphatases and kinases. B56δ (encoded by PPP2R5D) is a regulatory subunit of protein phosphatase 2A (PP2A) with long IDRs that harbor a substrate-mimicking SLiM and multiple phosphorylation sites. De novo missense mutations in PPP2R5D cause intellectual disabilities (ID), macrocephaly, Parkinsonism, and a broad range of neurological symptoms. Our single-particle cryo-EM structures of the PP2A-B56δ holoenzyme reveal that the long, disordered arms at the B56δ termini fold against each other and the holoenzyme core. This architecture suppresses both the phosphatase active site and the substrate-binding protein groove, thereby stabilizing the enzyme in a closed latent form with dual autoinhibition. The resulting interface spans over 190 Å and harbors unfavorable contacts, activation phosphorylation sites, and nearly all residues with ID-associated mutations. Our studies suggest that this dynamic interface is coupled to an allosteric network responsive to phosphorylation and altered globally by mutations. Furthermore, we found that ID mutations increase the holoenzyme activity and perturb the phosphorylation rates, and the severe variants significantly increase the mitotic duration and error rates compared to the normal variant.

EB1C forms dimer and interacts with protein phosphatase 2A (PP2A) to regulate fiber elongation in upland cotton (Gossypium hirsutum)

Cotton is the most economically important natural fiber crop grown in more than sixty-five countries of the world. Fiber length is the main factor affecting fiber quality, but the existing main varieties are short in length and cannot suit the higher demands of the textile industry. It is necessary to discover functional genes that enable fiber length improvement in cotton through molecular breeding.In this study, overexpression of GhEB1C in Arabidopsis thaliana significantly promotes trichomes, tap roots, and root hairs elongation. The molecular regulation of GhEB1C involves its interactions with itself and GhB'ETA, and the function of GhEB1C regulation mainly depends on the two cysteine residues located at the C-terminal. In particular, the function activity of GhEB1C protein triggered with the regulation of protein phosphatase 2A, while silencing of GhEB1C in cotton significantly influenced the fiber protrusions and elongation mechanisms., Further, influenced the expression of MYB-bHLH-WD40 complex, brassinosteroids, and jasmonic acid-related genes, which showed that transcriptional regulation of GhEB1C is indispensable for cotton fiber formation and elongation processes.Our study analyzed the brief molecular mechanism of GhEB1C regulation. Further elucidated that GhEB1C can be a potential target gene to improve cotton fiber length through transgenic breeding.

Abstract P1067: IRS Mediated Recruitment Of PI3Kgamma By Insulin Inhibits Beta-adrenergic Receptor Function

Insulin (INS) treatment results in impaired response to β-agonist isoproterenol (ISO) associated with phosphorylated β2-adrenergic receptor (β2AR) and impaired cAMP response. Although INS does not mediate dissociation of G-protein Gβγ subunits, yet surprisingly recruits phosphoinositide 3-kinase γ (PI3Kγ) to the plasma membrane. Correspondingly, knock-down of PI3Kγ (PI3Kγ KD) in HEK 293 or ablation in primary adult cardiomyocytes and fibroblasts (isolated from PI3Kγ knockout mice) abrogated β2AR phosphorylation reflecting a key role for PI3Kγ in retaining β2AR phosphorylation. Also, adult cardiomyocytes isolated from C57Bl6 mice showed significant loss of in vitro ISO-stimulated cardiomyocyte contraction upon INS pre-treatment which was remarkably preserved in the PI3Kγ knockout cardiomyocytes despite INS. As PI3Kγ is traditionally recruited to the βAR complex by the Gβγ subunits, we hypothesized that INS-mediates Gβγ-independent recruitment of PI3Kγ to the βAR complex underlying its dysfunction. INS stimulation is known to recruit insulin receptor substrate (IRS 1 & 2) to the receptor complex, and therefore, we tested whether PI3Kγ interacts with IRS 1/2 leading to Gβγ-independent recruitment of PI3Kγ to the β2AR complex. Immunoprecipitation PI3Kγ following INS showed that PI3Kγ interacts with IRS2 recruiting PI3Kγ to the β2AR complex bypassing the traditional Gβγ-dependent pathway. Since PI3Kγ KD resulted in loss of INS-mediated β2AR phosphorylation, we tested whether PI3Kγ inhibits protein phosphatase 2A (PP2A) function impairing β2AR de-phosphorylation. INS treatment resulted in significant loss of β2AR-associated PP2A activity which was rescued in PI3Kγ KD cells showing that PI3Kγ impairs PP2A function. Furthermore, our studies show that PI3Kγ phosphorylates the endogenous inhibitor of PP2A, I2PP2A which then robustly binds and inhibits PP2A activity impairing β2AR dephosphorylation. Consistently, CRISPR ablation of I2PP2A relieves this inhibition on PP2A leading to increased PP2A activity. This reduces accumulation of phosphorylated βARs despite the presence of PI3Kγ showing an underappreciated regulation of PP2A by insulin through non-canonical recruitment of PI3Kγ that impairs β2AR function.

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

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

Epigallocatechine-3-gallate Inhibits the Adipogenesis of Human Mesenchymal Stem Cells via the Regulation of Protein Phosphatase-2A and Myosin Phosphatase.

Epigallocatechin-3-gallate (EGCG) has widespread effects on adipocyte development. However, the molecular mechanisms of EGCG are not fully understood. We investigate the adipogenic differentiation of human-derived mesenchymal stem cells, including lipid deposition and changes in the expression and phosphorylation of key transcription factors, myosin, protein phosphatase-2A (PP2A), and myosin phosphatase (MP). On day 6 of adipogenic differentiation, EGCG (1–20 µM) suppressed lipid droplet formation, which was counteracted by an EGCG-binding peptide for the 67 kDa laminin receptor (67LR), suggesting that EGCG acts via 67LR. EGCG decreased the phosphorylation of CCAAT-enhancer-binding protein beta via the activation of PP2A in a protein kinase A (PKA)-dependent manner, leading to the partial suppression of peroxisome proliferator-activated receptor gamma (PPARγ) and adiponectin expression. Differentiated cells exhibited a rounded shape, cortical actin filaments, and lipid accumulation. The EGCG treatment induced cell elongation, stress fiber formation, and less lipid accumulation. These effects were accompanied by the degradation of the MP target subunit-1 and increased the phosphorylation of the 20 kDa myosin light chain. Our results suggest that EGCG acts as an agonist of 67LR to inhibit adipogenesis via the activation of PP2A and suppression of MP. These events are coupled with the decreased phosphorylation and expression levels of adipogenic transcription factors and changes in cell shape, culminating in curtailed adipogenesis.

PP2A and cancer epigenetics: a therapeutic opportunity waiting to happen.

The epigenetic state of chromatin is altered by regulators which influence gene expression in response to environmental stimuli. While several post-translational modifications contribute to chromatin accessibility and transcriptional programs, our understanding of the role that specific phosphorylation sites play is limited. In cancer, kinases and phosphatases are commonly deregulated resulting in increased oncogenic signaling and loss of epigenetic regulation. Aberrant epigenetic states are known to promote cellular plasticity and the development of therapeutic resistance in many cancer types, highlighting the importance of these mechanisms to cancer cell phenotypes. Protein Phosphatase 2A (PP2A) is a heterotrimeric holoenzyme that targets a diverse array of cellular proteins. The composition of the PP2A complex influences its cellular targets and activity. For this reason, PP2A can be tumor suppressive or oncogenic depending on cellular context. Understanding the nuances of PP2A regulation and its effect on epigenetic alterations can lead to new therapeutic avenues that afford more specificity and contribute to the growth of personalized medicine in the oncology field. In this review, we summarize the known PP2A-regulated substrates and potential phosphorylation sites that contribute to cancer cell epigenetics and possible strategies to therapeutically leverage this phosphatase to suppress tumor growth.

Abstract P427: Inhibition Of PP2A By Insulin Impairs Beta-Adrenergic Receptor Function

Insulin impairs β2-adrenergic receptor (β2AR) function via trans-phosphorylation through G protein-coupled receptor kinase 2 (GRK2). However, less is known about dephosphorylation mechanisms mediated by protein phosphatase 2A (PP2A) during this insulin-β2AR cross-talk. Pharmacologic or genetic inhibition of phosphoinositide 3-kinase γ (PI3Kγ) unexpectedly resulted in significant reduction of insulin-mediated β2AR phosphorylation. Interestingly, β2AR-associated phosphatase activity was inhibited by insulin but was reversed by knock-down of PI3Kγ showing negative regulation of PP2A by PI3Kγ. Co-immunoprecipitation and surface plasmon resonance studies using purified proteins showed that GRK2 and PI3Kγ form a complex and could be recruited to β2ARs as GRK2 interacts with insulin receptor substrate (IRS) following insulin treatment. Further, co-immunoprecipitation studies showed that PI3Kγ directly interacted with both IRS-1 and IRS-2 but only IRS-2 interaction with PI3Kγ significantly increased following insulin stimulation. These results indicated that PI3Kγ could also be directly recruited to the receptor complex by IRS-2. Consistently, β-blocker pretreatment did not reduce insulin-mediated β2AR phosphorylation indicating agonist- and Gβγ-independent non-canonical regulation of receptor function. Mechanistically, PI3Kγ inhibits PP2A activity at the βAR complex by phosphorylating an intracellular inhibitor of PP2A (I2PP2A). Knock-down or CRISPR ablation of endogenous I2PP2A unlocked PP2A inhibition mediating β2AR dephosphorylation showing an unappreciated acute regulation of PP2A in mediating insulin-β2AR cross-talk.

Deletion or inhibition of prolyl oligopeptidase blocks lithium-induced phosphorylation of GSK3b and Akt by activation of protein phosphatase 2A.

Alterations in prolyl oligopeptidase (PREP) activity have been connected, for example, with bipolar and major depressive disorder, and several studies have reported that lack or inhibition of PREP blocks the effects of lithium on inositol 1,4,5-triphosphate (IP3 ) levels. However, the impact of PREP modulation on other intracellular targets of lithium, such as glycogen synthase kinase 3 beta (GSK3b) or protein kinase B (Akt), has not been studied. We recently found that PREP regulates protein phosphatase 2A (PP2A), and because GSK3b and Akt are PP2A substrates, we studied if PREP-related lithium insensitivity is dependent on PP2A. To assess this, HEK-293 and SH-SY5Y cells with PREP deletion or PREP inhibition (KYP-2047) were exposed to lithium, and thereafter, the phosphorylation levels of GSK3b and Akt were measured by Western blot. As expected, PREP deletion and inhibition blocked the lithium-induced phosphorylation on GSK3b and Akt in both cell lines. When lithium exposure was combined with okadaic acid, a PP2A inhibitor, KYP-2047 did not have effect on lithium-induced GSK3b and Akt phosphorylation. Therefore, we conclude that PREP deletion or inhibition blocks the intracellular effects of lithium on GSK3b and Akt via PP2A activation.

Identification and development of a KASP functional marker of TaTAP46‐5A associated with kernel weight in wheat (Triticum aestivum)

AbstractTAP46 (Type 2A‐phosphatase‐Associated Protein 46 kDa) is homologous to yeast TAP42 or the animal α4 gene and is a crucial gene in the target of rapamycin regulation of protein phosphatase 2A. It has been proven to influence the thousand‐kernel weight (TKW) and kernel size in transgenic wheat. To rapidly identify high‐yield wheat germplasms of TaTAP46, four interlinked single nucleotide polymorphisms were identified by resequencing the TaTAP46‐5A gene in 32 varieties, forming two haplotypes: Hap1 and Hap2. A KASP marker, −1462A/C, was developed to identify Hap1/Hap2. A 157 Chinese wheat mini core collection and 348 Chinese modern collection were constructed for association analysis of the two haplotypes and their phenotypes. The results showed that the Hap1/Hap2 of TaTAP46‐5A was significantly associated with the TKW and kernel size. Hap1 was a favourable haplotype with higher TKW and larger kernel size compared to those of Hap2. Hap1 is a stable high yield type of the TaTAP46 gene that has been continuously selected in high‐yield wheat breeding worldwide. The germplasm of Hap1 can be selected quickly using the KASP primer −1462A/C, to improve breeding efficiency.

Direct-Acting Small Molecule Activators of PP2A (SMAPs) in Myeloid Malignancies: Understanding Mechanisms of Cytotoxicity to Inform Rational Combinatorial Therapeutic Strategies

The protein phosphatase 2A (PP2A) functions as a tumor suppressor by counteracting oncogenic kinase signaling. Suppression of PP2A activity represents a critical process in the development and maintenance of many neoplasms, including myeloid malignancies; PP2A inhibition de-represses numerous proliferative and survival signaling pathways. However, complete loss of PP2A is lethal to cells. As such, indirect reactivation of PP2A has emerged as a promising therapeutic strategy for myeloid leukemias. Yet the broad applicability of such a strategy remains uncertain, as it relies on targeting endogenous PP2A inhibitors. In the present work we investigated the effects of direct acting small molecule activators of PP2A (SMAPs) (Sandokar et al. JCI 2017) on myeloid leukemia cells and uncovered a novel link between PP2A reactivation and NOXA-mediated apoptosis. This mechanism explains our observation of cooperativity between SMAPs and the BH3 mimetic ABT-199 (venetoclax). Additionally, we found that SMAPs elicit a protective stress response that can be overcome by inhibition ofprotein kinase R (PKR)-like endoplasmic reticulum kinase (PERK).Given the known role of PP2A inactivation in both acute and chronic myeloid leukemia (AML and CML), we first tested the activity of SMAPs against cell lines derived from both diseases. Across all cell lines tested (CML- K562, Meg-01; AML- MOLM-14, HL-60, OCI-AML3, THP-1) SMAPs caused a dose-dependent decrease in cell viability. It is known that reactivation of PP2A induces apoptosis in myeloid leukemia cells, though this area has yet to be thoroughly investigated. Therefore, we first sought to clarify the mode of cell death in response to PP2A reactivation. Both by biochemical and flow cytometric analyses, SMAPs showed activation of the intrinsic apoptotic cascade. Furthermore, SMAP-mediated cell death was blocked by co-treatment with the caspase inhibitor z-VAD-FMK (50 μM). We previously demonstrated the target specificity of SMAPs for PP2A with methods including expression of SV40 small T antigen and PP2A alpha subunit drug-binding mutants (Sandokar et al. JCI 2017). Here, we confirmed that pre-treatment with the PP2A inhibitor okadaic acid at a dose that retains selectivity for PP2A (10 nM) attenuated SMAP-induced cell death and cleavage of PARP and caspase-3.Next, we investigated the effectors of SMAP-mediated apoptosis. To this end, we performed Western blots to detect changes in abundance and/or phosphorylation of a panel of Bcl-2 family member proteins. Interestingly, a strong upregulation of the pro-apoptotic protein NOXA occurred consistently across AML and CML cell lines. Further RT-PCR studies showed this increase to occur at the transcript level. Knockdown studies confirmed the importance of NOXA for SMAP-mediated apoptosis. These data represent the first reported link between PP2A and NOXA regulation. Since SMAPs target the NOXA-MCL1 axis of the Bcl-2 family, we next tested their efficacy in combination with the Bcl-2-selective BH3 mimetic ABT-199. We found co-treatment with SMAPs and ABT-199 to exhibit enhanced cell death compared to either agent alone.Concurrent with NOXA elevation, SMAP treatment modulated protein expression and phosphorylation levels for several known pathways downstream of PP2A. Interestingly, we additionally observed increased phosphorylation of eukaryotic translation initiation factor 2 subunit alpha (eIF2α) in SMAP-treated cells which correlated with upregulation of mRNA and protein for the downstream transcription factor ATF4. Blockade of eIF2α phosphorylation with PERK inhibitor GSK2656157 (350 nM) prevented ATF4 elevation and enhanced SMAP-induced apoptosis. This result supports the idea that the integrated stress response (ISR) via PERK-eIF2α-ATF4 acts to counter apoptosis in the context of PP2A reactivation in myeloid leukemia cells. Thus, the efficacy of SMAPs and other PP2A-activating therapeutics may be enhanced by combination with both BH3 mimetics and inhibitors of the ISR. DisclosuresNarla:Mount Sinai School of Medicine: Patents & Royalties: patents on the small molecule PP2A activators (GN).

CDC25B partners with PP2A to induce AMPK activation and tumor suppression in triple negative breast cancer

Cell division cycle 25 (CDC25) dual specificity phosphatases positively regulate the cell cycle by activating cyclin-dependent kinase/cyclin complexes. Here, we demonstrate that in addition to its role in cell cycle regulation, CDC25B functions as a regulator of protein phosphatase 2A (PP2A), a major cellular Ser/Thr phosphatase, through its direct interaction with PP2A catalytic subunit. Importantly, CDC25B alters the regulation of AMP-activated protein kinase signaling (AMPK) by PP2A, increasing AMPK activity by inhibiting PP2A to dephosphorylate AMPK. CDC25B depletion leads to metformin resistance by inhibiting metformin-induced AMPK activation. Furthermore, dual inhibition of CDC25B and PP2A further inhibits growth of 3D organoids isolated from patient derived xenograft model of breast cancer compared to CDC25B inhibition alone. Our study identifies CDC25B as a regulator of PP2A, and uncovers a mechanism of controlling the activity of a key energy metabolism marker, AMPK.

Sphingosine Analogs and Protein Phosphatase 2A as a Molecular Targeted Cancer Therapy: A Mini Systematic Review

Background: Regulation of protein phosphatase 2A (PP2A) plays an important role in hematologic and solid neoplasms. Therefore, the use of sphingosine analogs as anti-neoplastic drugs has shown potential due to their role as PP2A activators. Objective: Investigation of whether sphingosine analogs bind to endogenous inhibitor proteins of PP2A, such as I2 PP2A/SET and/or CIP2A, and whether this binding reactivates PP2A, allowing it to resume its role as a tumor suppressor. Methods: Literature from the PubMed database was searched and those articles related to PP2A and sphingosine analogs were reviewed. Results: Utilization of sphingosine analogs in hematologic and solid neoplasms revealed numerous mechanisms of inducing cell death. Regulation of PP2A through modulation of I2 PP2A/SET and/or CIP2A was demonstrated in a variety of neoplastic processes; however, unique mechanisms such as cell necrosis via the production of reactive oxygen species was also appreciated. Conclusion: Only certain malignancies expressed endogenous inhibitor proteins, yet sphingosine analogs were able to induce cell death in neoplasms that did not express these proteins. This suggests that sphingosine analogs may be utilized for anti-neoplastic therapy via reactivation of PP2A however, it is not the exclusive mechanism for inducing cell death. Further investigation of sphingosine analogs as a novel or adjunctive chemotherapeutic treatment is warranted.

Abstract 2981: Biased activation of PP2A through selective heterotrimer binding and stabilization

Abstract Development of molecularly targeted therapies have shifted our clinical approach to cancer allowing for an integrated treatment approach where tumor genotype and molecular profiles are paired in a clinically actionable manner with specific targeted therapies. This approach maximizes efficacy and minimizes toxicity for each patient. While the vast majority of molecularly targeted therapies have focused on the development of kinase inhibition, an equally effective yet underexplored strategy exists in the activation or regulation of phosphatases. The tumor suppressor Protein Phosphatase 2A (PP2A) negatively regulates multiple oncogenic drivers common to a broad range of human malignancies including MYC, AKT, and ERK; and has been demonstrated as a critical tumor suppressor. In cancer, PP2A is inactivated through several mechanisms including increased expression of endogenous inhibitors, post-translational modifications of the catalytic subunit, and somatic mutation in its catalytic, regulatory or scaffold subunits. The vast mechanisms employed to dysregulate and inhibit PP2A in cancer, highlights it's potential as an endogenous molecular target to combat malignant growth. Our laboratory has been involved in the development of a novel series of small molecule activators of PP2A (SMAPs). Using structural, biochemical, cellular, and in vivo model systems we reveal how the lead SMAP, DT-061, specifically stabilizes a B56 containing PP2A holoenzyme, in its fully assembled, active state, to direct enzymatic activity towards specific oncogenic targets, including its well-known substrate c-MYC. Our structure identifies a unique interfacial small molecule binding pocket that involves all three PP2A subunits to prevent dissociation of select B56 subunit containing holoenzymes. Elucidating the mechanism of SMAPs allowed us to explore appropriate clinical placement for this targeting strategy and investigate potential mechanisms of resistance. For example, in vivo xenograft studies of cancer-associated Aα mutants, most notably R183W, demonstrates that these mutations render malignant cells resistant to these SMAPs and further support the mechanism of SMAPs described. Thus, our findings provide fundamental insights into PP2A complex assembly and regulation, identify a unique interfacial stabilizing mode of action for therapeutic targeting, and aid in the development of phosphatase-based therapeutics tailored against disease specific phospho-protein targets. Citation Format: Daniel Leonard, Wei Huang, Sudeh Izadmehr, Caitlin M. O'Connor, Danica D. Wiredja, Zhizhi Wang, Nilesh Zaware, Daniela M. Schlatzer, Stefan Schuchner, Janna Kiselar, Yinghua Chen, Nikhil Vasireddi, Abbey L. Perl, Matthew D. Glasky, Wenqing Xu, David L. Brautigan, Egon Ogris, Derek J. Taylor, Goutham Narla. Biased activation of PP2A through selective heterotrimer binding and stabilization [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2981.

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

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

A dynamic charge-charge interaction modulates PP2A:B56 substrate recruitment.

The recruitment of substrates by the ser/thr protein phosphatase 2A (PP2A) is poorly understood, limiting our understanding of PP2A-regulated signaling. Recently, the first PP2A:B56 consensus binding motif, LxxIxE, was identified. However, most validated LxxIxE motifs bind PP2A:B56 with micromolar affinities, suggesting that additional motifs exist to enhance PP2A:B56 binding. Here, we report the requirement of a positively charged motif in a subset of PP2A:B56 interactors, including KIF4A, to facilitate B56 binding via dynamic, electrostatic interactions. Using molecular and cellular experiments, we show that a conserved, negatively charged groove on B56 mediates dynamic binding. We also discovered that this positively charged motif, in addition to facilitating KIF4A dephosphorylation, is essential for condensin I binding, a function distinct and exclusive from PP2A-B56 binding. Together, these results reveal how dynamic, charge-charge interactions fine-tune the interactions mediated by specific motifs, providing a new framework for understanding how PP2A regulation drives cellular signaling.

Loss of myeloid-specific protein phosphatase 2A enhances lung injury and fibrosis and results in IL-10-dependent sensitization of epithelial cell apoptosis.

Protein phosphatase 2A (PP2A), a ubiquitously expressed Ser/Thr phosphatase is an important regulator of cytokine signaling and cell function. We previously showed that myeloid-specific deletion of PP2A (LysMcrePP2A-/-) increased mortality in a murine peritoneal sepsis model. In the current study, we assessed the role of myeloid PP2A in regulation of lung injury induced by lipopolysaccharide (LPS) or bleomycin delivered intratracheally. LysMcrePP2A-/- mice experienced increased lung injury in response to both LPS and bleomycin. LysMcrePP2A-/- mice developed more exuberant fibrosis in response to bleomycin, elevated cytokine responses, and chronic myeloid inflammation. Bone marrow-derived macrophages (BMDMs) from LysMcrePP2A-/- mice showed exaggerated inflammatory cytokine release under conditions of both M1 and M2 activation. Notably, secretion of IL-10 was elevated under all stimulation conditions, including activation of BMDMs by multiple Toll-like receptor ligands. Supernatants collected from LPS-stimulated LysMcrePP2A-/- BMDMs induced epithelial cell apoptosis in vitro but this effect was mitigated when IL-10 was also depleted from the BMDMs by crossing LysMcrePP2A-/- mice with systemic IL-10-/- mice (LysMcrePP2A-/- × IL-10-/-) or when IL-10 was neutralized. Despite these findings, IL-10 did not directly induce epithelial cell apoptosis but sensitized epithelial cells to other mediators from the BMDMs. Taken together our results demonstrate that myeloid PP2A regulates production of multiple cytokines but that its effect is most pronounced on IL-10 production. Furthermore, IL-10 sensitizes epithelial cells to apoptosis in response to myeloid-derived mediators, which likely contributes to the pathogenesis of lung injury and fibrosis in this model.

Fibroblast growth factor 21 ameliorates neurodegeneration in rat and cellular models of Alzheimer’s disease

Our understanding of the mechanisms underlying process in Alzheimer's disease (AD) is far from completion and new therapeutic targets are urgently needed. Recently, the link between dementia and diabetes mellitus (DM) prompted us to search for new therapeutic strategies from glucose metabolism regulators for neurodegeneration. Previous studies have indicated that fibroblast growth factor 21 (FGF21), an attractive and potential therapeutic treatment for DM, may exert diverse effects in the central nervous system. However, the specific biological function and mechanisms of FGF21 on AD is still largely unknown. We report here a study in vivo and in vitro of the neuroprotective effects of FGF21 on cell apoptosis, tau hyperphosphorylation and oxidative stress induced by amyloid β-peptide 25–35. In the present study, the results also further provided evidence for molecular mechanisms by which FGF21 exerted its beneficial effects in neuron and suggested that the regulation of protein phosphatase 2A / mitogen-activated protein kinases / hypoxia-inducible factor-1α pathway may play a key role in mediating the neuroprotective effects of FGF21 against AD-like pathologies.