Phosphatase 2A Holoenzyme Research Articles

Emerging Roles of B56 Phosphorylation and Binding Motif in PP2A-B56 Holoenzyme Biological Function.

Protein serine/threonine phosphatase 2A (PP2A) regulates diverse cellular processes via the formation of ~100 heterotrimeric holoenzymes. However, a scarcity of knowledge on substrate recognition by various PP2A holoenzymes has greatly prevented the deciphering of PP2A function in phosphorylation-mediated signaling in eukaryotes. The review summarized the contribution of B56 phosphorylation to PP2A-B56 function and proposed strategies for intervening B56 phosphorylation to treat diseases associated with PP2A-B56 dysfunction; it especially analyzed recent advancements in LxxIxEx B56-binding motifs that provide the molecular details of PP2A-B56 binding specificity and, on this basis, explored the emerging role of PP2A-B56 in the mitosis process, virus attack, and cancer development through LxxIxE motif-mediated PP2A-B56 targeting. This review provides theoretical support for discriminatingly targeting specific PP2A holoenzymes to guide PP2A activity against specific pathogenic drivers.

Prediction of PP2A Holoenzymes Guided By the Structural Characterization of Methylation Independent PP2A Assembly

Introduction: The dysregulation of phosphorylation-dependent signaling is a hallmark of leukemogenesis. Kinase inhibitors have revolutionized the outcomes of patients with chronic myeloid leukemia. An orthogonal, but less explored way to target phosphorylation pathways is to activate phosphatases, such as the ubiquitously expressed protein- phosphatase 2A (PP2A). PP2A is an essential serine/threonine phosphatase that regulates various cellular processes, including cell growth, division, and differentiation. Dysregulation of PP2A has been linked to leukemia. PP2A is a modular multi-subunit enzyme: One scaffold subunit (A) binds to a catalytic subunit (C) to form a core AC heterodimer, which together with one of many regulatory (B) subunits forms the active enzyme. The regulatory (B) subunits are classified into 4 distinct subfamilies without significant homology. The combinatorial assembly of these subunits generates a wide range of PP2A complexes, resulting in diverse substrate specificity and subcellular localization. The detailed mechanism of PP2A regulation remains elusive and reports about a governing role of methylation of the carboxy-terminal Leucin of PP2A C are conflicting. Understanding the underlying molecular regulation of PP2A activity is of significant interest for future therapeutic development for acute myeloid leukemia. Methods: Recombinant PP2A was expressed in insect cells, and purified by affinity purification, anion exchange chromatography and size exclusion chromatography (SEC). Extensive quality control, including liquid chromatography-mass spectrometry, SDS-PAGE gel and fast protein liquid chromatography was performed. The degree of methylation of PP2A C Leucine 309 was confirmed by mass spectrometry. We validated protein stability with differential scanning fluorimetry. Pull-down assays and size exclusion chromatography characterized complex formation. A calorimetric assay using p-nitrophenyl phosphate as a substrate functionally assessed PP2A in vitro. Structural information was obtained by X-ray crystallography and AlphaFold Multimer. Results: Using in vitro biochemistry, including pull down and size exclusion chromatography, we demonstrate that methylation of the carboxy terminus of the PP2A C subunit is dispensable for PP2A assembly in vitro. Absence of methylation of Leucine 309 was confirmed by mass-spectrometry. We show by SEC that instead PP2A complex formation is dependent on B subunit concentration. Carboxy-terminal methylation of the PP2A C subunit was also not required for functional activity in a calorimetric assay using p-nitrophenyl phosphate as a substrate. PP2A formed spontaneously in vitro and was enzymatically active independent of methylation of Leucine 309. To corroborate these findings, we determined the X-ray crystal structure of the PP2A:PPP2B56E:PPP2C complex to 2.7 Å resolution with the unmethylated Leucine 309. Interestingly our experimental structure superimposed with an AlphaFold Multimer model of the PP2A trimer. Building on this finding, we predicted the models of all 64 possible PP2A complexes providing a framework for further study and structural analysis of PP2A complexes and their activation. These studies provide key information for the structural design and development of novel drugs for hematologic malignancies by targeting of selected PP2A complexes and clarified the role of Leucine 309 methylation in PP2A activation. Conclusion: Methylation of the carboxy-terminal Leucine 309 of the PP2A C subunitis dispensable for PP2A activity in vitro. Integrative structural biology studies, combining biochemistry, X-ray crystallography and AlphaFold Multimer provide a comprehensive map of PP2A complexes. These studies have the potential to accelerate the drug discovery process and unlock PP2A activation as a new therapeutic modality for leukemia.

Abstract B064: The role of PP2A-B56α in KRAS-mediated pancreatic tumorigenesis

Abstract Protein phosphatase 2A (PP2A) is a major serine-threonine phosphatase that regulates many cellular pathways including KRAS, whose oncogenic mutation is prevalent in 95% of patients with Pancreatic Ductal Adenocarcinoma (PDAC). Previous research has identified a decrease in global PP2A activity and an increase in the expression of PP2A inhibitors in PDAC cell lines, suggesting that suppression of PP2A activity may be pertinent in PDAC maintenance. Importantly, PP2A has low mutation rates in PDAC, making it a viable target for therapeutic reactivation. While PP2A has been shown to have global tumor suppressive capabilities, the regulation of specific pathways by PP2A can be altered based on PP2A holoenzyme composition. Therefore, there is a critical need to understand the mechanisms by which oncogenic KRAS can affect PP2A function and differential substrate targeting in PDAC. The PP2A holoenzyme consists of 3 subunits: the scaffolding subunit (A), the catalytic subunit (C), and the regulatory subunit (B). There are 16 different B subunits that can be incorporated into the PP2A holoenzyme that are responsible for substrate specificity. The B56α subunit of PP2A has been shown to negatively regulate cellular transformation. Our research aims to investigate the mechanisms by which PP2A-B56α is regulated through oncogenic KRAS and how suppression of B56α impacts the initiation and progression of PDAC. To determine how oncogenic KRAS alters the dynamics of PP2A-B56α and overall PP2A activity we utilized tet-inducible KRASG12D cell lines to allow direct manipulation of KRAS mutational activation. Using this system, we have identified time dependent alterations in cancerous inhibitor of PP2A (CIP2A) following induction of KRASG12D expression, indicating that PP2A suppression may be an early event in PDAC initiation. Consistent with this hypothesis, we characterized changes in the acceleration of PDAC formation in vivo using the Ptf1a-Cre; LSL- KRASG12D (KC) genetic mouse model combined with a B56α hypomorph model (KCBhm/hm). Our data show that the loss of B56α accelerates PDAC initiation, with an increase in pancreatic precursor lesion (PanIN) number and a decrease in healthy acinar area. In response to B56α loss, similar acceleration of acinar to ductal metaplasia (ADM) kinetics were observed in a 3D-cultured ADM Assay. Furthermore, when 3D-cultured acinar cells were treated with a small molecule activator of PP2A (SMAP), SMAP treatment resulted in smaller and fewer ductal structures, preventing the ADM process. Collectively, these data suggest that PP2A-B56α plays a regulatory role in cellular plasticity and loss contributes to PDAC initiation. Future studies will investigate how mutant KRAS-mediated CIP2A expression effects overall PP2A phosphatase activity and how subsequent sequestration of B56α contributes to development of PDAC. Together, these studies identify PP2A as a critical regulator of KRAS-induced cellular plasticity and support reactivation of PP2A as a novel therapeutic strategy in PDAC patients. Citation Format: Samantha L Tinsley, Rebecca A. Shelley, Gagan K. Mall, Ella Rose D. Chianis, Mary C. Thoma, Marina Pasca di Magliano, Goutham Narla, Rosalie C. Sears, Brittany L. Allen-Petersen. The role of PP2A-B56α in KRAS-mediated pancreatic tumorigenesis [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr B064.

Disturbances in PP2A methylation and one-carbon metabolism compromise Fyn distribution, neuritogenesis, and APP regulation

The nonreceptor protein tyrosine kinase Fyn and protein Ser/Thr phosphatase 2A (PP2A) are major multifunctional signaling molecules. Deregulation of Fyn and altered PP2A methylation are implicated in cancer and Alzheimer's disease (AD). Here, we tested the hypothesis that the methylation state of PP2A catalytic subunit, which influences PP2A subunit composition and substrate specificity, can affect Fyn regulation and function. Using Neuro-2a (N2a) neuroblastoma cell models, we first show that methylated PP2A holoenzymes containing the Bα subunit coimmunoprecipitate and copurify with Fyn in membrane rafts. PP2A methylation status regulates Fyn distribution and Fyn-dependent neuritogenesis, likely in part by affecting actin dynamics. A methylation-incompetent PP2A mutant fails to interact with Fyn. It perturbs the normal partitioning of Fyn and amyloid precursor protein (APP) in membrane microdomains, which governs Fyn function and APP processing. This correlates with enhanced amyloidogenic cleavage of APP, a hallmark of AD pathogenesis. Conversely, enhanced PP2A methylation promotes the nonamyloidogenic cleavage of APP in a Fyn-dependent manner. Disturbances in one-carbon metabolic pathways that control cellular methylation are associated with AD and cancer. Notably, they induce a parallel loss of membrane-associated methylated PP2A and Fyn enzymes in N2a cells and acute mouse brain slices. One-carbon metabolism also modulates Fyn-dependent process outgrowth in N2a cells. Thus, our findings identify a novel methylation-dependent PP2A/Fyn signaling module. They highlight the underestimated importance of cross talks between essential metabolic pathways and signaling scaffolds that are involved in normal cell homeostasis and currently being targeted for cancer and AD treatment.

Abstract PR-008: The role of PP2A-B56α in acinar-to-ductal metaplasia and initiation of pancreatic ductal adenocarcinoma

Abstract Protein phosphatase 2A (PP2A) is a major serine-threonine phosphatase that regulates many cellular pathways including KRAS, whose oncogenic mutation is prevalent in 95% of patients with Pancreatic Ductal Adenocarcinoma (PDAC). Previous research has identified a decrease in global PP2A activity and an increase in the expression PP2A inhibitors in PDAC cell lines, suggesting that suppression of PP2A activity may be pertinent in PDAC maintenance. Importantly, PP2A has low mutation rates in PDAC, making it a viable target for therapeutic reactivation. While PP2A has been shown to have global tumor suppressive capabilities, the regulation of specific pathways by PP2A can be altered based on PP2A holoenzyme composition. Therefore, there is a critical need to understand the mechanisms by which oncogenic KRAS can affect PP2A function and differential substrate targeting in PDAC. The PP2A holoenzyme consists of 3 subunits: the scaffolding subunit (A), the catalytic subunit (C), and the regulatory subunit (B). There are 16 different B subunits that can be incorporated into the PP2A holoenzyme that are responsible for substrate specificity. The B56α subunit of PP2A has been shown to negatively regulate cellular transformation and has decreased expression in aggressive subtypes of PDAC, indicating that suppression of this subunit may aid in PDAC tumorgenicity. Our research aims to investigate the mechanisms by which PP2A-B56α is regulated downstream of oncogenic KRAS and how suppression of B56α impacts the initiation and progression of PDAC. In order to determine how oncogenic KRAS alters the dynamics of PP2A-B56α and overall PP2A activity we utilized tet-inducible KRASG12D cell lines to allow direct manipulation of KRAS mutational activation. Using this system, we have identified time dependent alterations in cancerous inhibitor of PP2A (CIP2A) following induction of KRASG12D expression, indicating that PP2A suppression may be an early event in PDAC initiation. Consistent with this hypothesis, we characterized changes in the acceleration of PDAC formation in vivo using the Ptf1a-Cre; LSL-KRASG12D (KC) genetic mouse model combined with a B56α hypomorph model (KCBhm/hm). Our data show that the loss of B56α accelerates PDAC initiation, with an increase in pancreatic precursor lesion (PanIN) number and a decrease in healthy acinar area. Similar results were seen in an ex vivo acinar-to-ductal metaplasia assay, suggesting that B56α alters pancreatic cellular plasticity in a cell-autonomous manner. Future studies will investigate how mutant KRAS expression effects overall PP2A phosphatase activity, holoenzyme formation, and sequestration of B56α by endogenous inhibitors to further understand how suppression of PP2A-B56α contributes to development of PDAC. Together, these studies identify PP2A as a critical regulator of KRAS-induced cellular plasticity and support therapeutic reactivation of PP2A as a novel therapeutic strategy in PDAC patients. Citation Format: Samantha L. Tinsley, Rebecca A. Shelley, Mary C. Thoma, Goutham Narla, Rosalie C. Sears, Brittany A. Allen-Petersen. The role of PP2A-B56α in acinar-to-ductal metaplasia and initiation of pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2020 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2020;80(22 Suppl):Abstract nr PR-008.

Abstract 3403: Drugging the undruggable: Lessons learned from protein phosphatase 2A

Abstract Protein phosphatase 2A (PP2A) is a key tumor suppressor responsible for the dephosphorylation of many oncogenic signaling pathways. The PP2A holoenzyme is comprised of a scaffolding subunit (A), which serves as the structural platform for the catalytic subunit (C) and for an array of regulatory subunits (B) to assemble. Impairment of PP2A is essential for the pathogenesis of many diseases including cancer. In cancer, PP2A is inactivated through a variety of mechanisms including somatic mutation of the Aαsubunit. Our studies show that the most recurrent Aαmutation, P179R, results in an altered protein conformation which prevents the catalytic subunit from binding. Additionally, correcting this mutation, by expressing wild type PP2A Aαin cell lines harboring the P179R mutation, causes a reduction in tumor growth and metastasis. Given its central role in human disease pathogenesis, many strategies have been developed to therapeutically target PP2A.Our lab developed a series of small molecules activators of protein phosphatase 2A. One of our more advanced analogs in this series, DT-061, drives dephosphorylation and degradation of select pathogenic substrates of PP2A such as c-MYC in cellular and in vivo systems. Additionally, we have demonstrated the phosphomimetics of MYC that prevent PP2A mediated dephosphorylation and degradation markedly reduce the anti-tumorigenic activity of this series of PP2A activators further validating the target-substrate specificity of this approach. Specific mutations in the site of drug interaction or overexpression of the DNA tumor virus small T antigen which has been shown to specifically bind to and inactivate PP2A abrogate the in vivo activity of this small molecule series further validating the PP2A specificity of this approach. Importantly, treatment with DT-061 results in tumor growth inhibition in an array of in vivocancer models and marked regressions in combination with MEKi and PARPi.To further define the mechanism of action of this small molecule series, we have used cryo-electron microscopy (cryo-EM) to visualize directly theinteraction between DT-061 and a PP2A heterotrimeric complex. We have identified molecular interactions between DT-061 and all three PP2A subunits that prevent dissociation of the active enzyme through the marked prolongation of the kOFF of the native complex. Furthermore, we demonstrate that DT-061 specifically stabilizes the B56α-PP2A holoenzyme in a fully assembled, active state to dephosphorylate oncogenic targets such as c-MYC in both cellular and in vivo systems. This 3.6 Å structure identifies dynamic molecular interactions between the three distinct PP2A subunits and highlight the inherent mechanisms of PP2A complex assembly and disassembly in both cell free and cellular systems. Thus, our findings provide fundamental insights into PP2A complex assembly and regulation, identify a unique interfacial stabilizing mode of action for the therapeutic targeting of previously undruggable proteins, and aid in the development of phosphatase-based therapeutics tailored against disease specific phosphor-protein targets. The marriage of multidisciplinary scientific practices has allowed us to present here a previously unrecognized therapeutic strategy of complex stabilization for the activation of endogenous disease combating enzymes. Citation Format: Derek Taylor, Goutham Narla. Drugging the undruggable: Lessons learned from protein phosphatase 2A [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 3403.

PPP2R5D-Related Intellectual Disability and Neurodevelopmental Delay: A Review of the Current Understanding of the Genetics and Biochemical Basis of the Disorder.

Protein Phosphatase 2 Regulatory Subunit B′ Delta (PPP2R5D)-related intellectual disability (ID) and neurodevelopmental delay results from germline de novo mutations in the PPP2R5D gene. This gene encodes the protein PPP2R5D (also known as the B56 delta subunit), which is an isoform of the subunit family B56 of the enzyme serine/threonine-protein phosphatase 2A (PP2A). Clinical signs include intellectual disability (ID); autism spectrum disorder (ASD); epilepsy; speech problems; behavioral challenges; and ophthalmologic, skeletal, endocrine, cardiac, and genital malformations. The association of defective PP2A activity in the brain with a wide range of severity of ID, along with its role in ASD, Alzheimer’s disease, and Parkinson’s-like symptoms, have recently generated the impetus for further research into mutations within this gene. PP2A, together with protein phosphatase 1 (PP1), accounts for more than 90% of all phospho-serine/threonine dephosphorylations in different tissues. The specificity for a wide variety of substrates is determined through nearly 100 different PP2A holoenzymes that are formed by at least 23 types of regulatory B subunits, and two isoforms each of the catalytic subunit C and the structural subunit A. In the mammalian brain, PP2A-mediated protein dephosphorylation plays an important role in learning and memory. The PPP2R5D subunit is highly expressed in the brain and the PPP2A–PPP2R5D holoenzyme plays an important role in maintaining neurons and regulating neuronal signaling. From 2015 to 2017, 25 individuals with PPP2R5D-related developmental disorder were diagnosed. Since then, Whole-Exome Sequencing (WES) has helped to identify more unrelated individuals clinically diagnosed with a neurodevelopmental disorder with pathological variants of PPP2R5D. In this review, we discuss the current understanding of the clinical and genetic aspects of the disorder in the context of the known functions of the PP2A–PPP2R5D holoenzyme in the brain, as well as the pathogenic mutations in PPP2R5D that lead to deficient PP2A–PPP2R5D dephosphorylation and their implications during development and in the etiology of autism, Parkinson’s disease, Alzheimer’s disease, and so forth. In the future, tools such as transgenic animals carrying pathogenic PPP2R5D mutations, and patient-derived induced pluripotent stem cell lines need to be developed in order to fully understand the effects of these mutations on different neural cell types.

Targeting T-ALL Cells with Potent Activators of the PP2A Protein Phosphatase Tumor Suppressor

Background Protein phosphatase 2A (PP2A) represents a family of potent tumor suppressors that are often suppressed in human cancers by upregulation of proteins that inhibit subunit assembly into active enzyme complexes. Thus, restoration of PP2A has assumed increasing importance for cancer treatment. In earlier work, we found that perphenazine (PPZ) kills leukemic T cells by activating PP2A (Gutierrez et al. 2014. J Clin Invest.). PPZ also acts to inhibit dopamine D2 receptor (DRD2) in the basal ganglia, which causes movement disorders at dosages less than those needed to kill T-ALL cells, effectively precluding "repurposing" of PPZ for the therapy of T-ALL. Hypothesis We sought to identify PPZ analogues that activate PP2A and induce apoptosis in T-ALL cells but lack the ability to bind and inhibit DRD2. Such analogues would not induce the movement disorders that have limited the usefulness of PPZ as an anti-leukemic drug. Methods PP2A is a heterotrimeric phosphatase that is assembled from three classes of subunits, encoded by two possible "A" genes, 15 "B" genes and 2 "C" genes, providing 60 distinct PP2A holoenzymes that can potentially form in the cell. To identify the key subunits of PP2A required for the antitumor activity of PPZ in T-ALL cells, we knocked out each subunit in human T-ALL cell lines and treated these cells with PPZ. Biochemical reporter assays were established to identify a phenothiazine derivative, iHAP1, that efficiently reactivates PP2A but does not inhibit dopamine signaling. iHAP1 preclinical studies included in vivo efficacy and toxicity in T-ALL xenograft models and in vitro efficacy in 248 cancer cell lines from 35 distinct tumor types. Results Using this approach, we found that knockout of each of three specific subunit genes of PP2A - PPP2R1A, PPP2CA and PPP2R5E - uniquely conferred resistance to PPZ treatment in T-ALL cell lines,. An independent Immunoprecipitation followed by western blotting indicated that all three subunits form a functional PP2A heterotrimeric holoenzyme complex in response to PPZ treatment. Narla and coworkers have published extensively about a series of compounds called "small molecule PP2 activators" or SMAPs (Sangodkar et al. 2017 J Clin Invest.). Intriguingly, we showed that activities of SMAP compounds depend on a different "B subunit" - PPP2R2A - and target different signal transduction pathways. Based on this finding, we sought to identify analogues of PPZ that more potently activate PP2A through this mechanism and kill T-ALL cells but lack inhibitory activity against DRD2. Testing more than 80 analogues of PPZ revealed a highly potent PP2A activator, iHAP1 (improvedHeterocyclic Activators of PP2A 1). iHAP1 is ten times more potent than PPZ in its ability to activate PP2A and kill tumor cells, but does not measurably inhibit dopamine signalling. iHAP1 is highly active as an antitumor drug human T-ALL xenograft models, without causing untoward movement disorders or other toxicity in vivo. Phosphoproteomics analysis followed by detailed biochemical assays revealed that the potent antitumor activity of PPZ and iHAP1 is mediated by dephosphorylation of MYBL2, a transcription factor that is essential for expression of genes whose products mediate prometaphase, and thus for cancer cell growth and survival. SMAP compounds do not dephosphorylate the same transcription factor and rather target other phosphoproteins. Thus, the potent PP2A activator iHAP1 drives three specific PP2A subunits into an active trimeric phosphatase and this drug is highly active against T-ALL and other hematologic malignancies. iHAP1 did not exhibit measurable toxicity up to 80 mg/kg/day PO or IM for 30 days in preclinical studies of human leukemia xenografts growing in immunosuppressed mice, providing a therapeutic index for this lead compound in preclinical studies. Conclusions Our findings show that small molecules promote the assembly of unique PP2A complexes with different regulatory subunits and substrates, allowing detailed structure and function studies of PP2A family members. A goal is to identify small molecules that assemble PP2A enzymes containing each of the remaining 13 regulatory PP2A subunits, thereby targeting a diverse array of substrates crucial to the pathogenesis of cancer and other diseases. Disclosures Gray: Gatekeeper: Equity Ownership; Syros: Equity Ownership; Petra: Equity Ownership; C4: Equity Ownership; B2S: Equity Ownership; Soltego: Equity Ownership; Novartis: Research Funding; Takeda: Research Funding; Astellas: Research Funding; Taiho: Research Funding; Janssen: Research Funding; Kinogen: Research Funding; Voronoi: Research Funding; Her2llc: Research Funding; Deerfield: Research Funding; Sanofi: Research Funding. Fischer:C4 Therapeutics: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Research Funding; Deerfield: Consultancy, Research Funding; Astellas: Research Funding.

Abstract 368: B-lymphoid transcriptional repression of the pentose phosphate pathway reveals a unique therapeutic vulnerability of B cell malignancies

Abstract Introduction Activation during normal immune responses and oncogenic transformation impose increased metabolic demands on B-cells and their ability to retain redox homeostasis. While the serine/threonine-protein phosphatase 2A (PP2A) was identified as tumor suppressor in multiple types of cancer, our genetic studies revealed an unexpected lineage-specific dependency on PP2A in a broad range of B-cell malignancies. Results PP2A regulates glycolysis rate and balances energy supply against anti-oxidant protection of B-cells through the pentose-phosphate pathway (PPP). In contrast to robust PPP-activity in other hematopoietic lineages, B-lymphoid transcription factors (PAX5, IKZF1) restrict PPP-activity by transcriptional repression of G6PD and other rate-limiting PPP enzymes. Constitutively low PPP-activity and limited capacity to balance redox fluctuations cause a unique dependency on PP2A in B-cell tumors. Pharmacological ablation of PP2A and PPP-activity identify this pathway as a novel lineage-specific therapeutic target in a broad range of B-cell malignancies. Highlights • Conditional deletion of Ppp2r1a, the central scaffold assembling the PP2A holoenzyme, induces acute cell death of early and mature B-cells but does not affect other hematopoietic lineages. • PP2A redirects glucose carbon utilization from glycolysis to the PPP to mitigate oxidative stress. • B-cell malignancies exhibit constitutively low PPP activity, owing to B-cell-specific transcriptional repression of G6PD and other rate-limiting PPP enzymes. • Transcriptional repression of PPP activity in B-cells represents the mechanistic basis for the unique dependency of B-cell malignancies on PP2A. • Small molecule inhibitors of PP2A and G6PD act synergistically and overcome conventional drug-resistance in B-cell tumors. Summary Genetic lesions of PP2A are frequent in solid tumors and myeloid leukemia, but not in B-cell malignancies. Unlike other types of cancer, our genetic studies revealed an essential role of PP2A in B-cell tumors. Thereby, PP2A redirects glucose carbon utilization from glycolysis to PPP to salvage oxidative stress. This unique vulnerability reflects constitutively low PPP activity in B-cells and transcriptional repression of G6PD and other key PPP enzymes by the B-cell transcription factors PAX5 and IKZF1. Reflecting B-cell-specific transcriptional repression of PPP activity, glucose carbon utilization in B-cells is heavily skewed in favor of glycolysis resulting in lack of PPP-dependent antioxidant protection. These findings reveal a novel gatekeeper function of the PPP in a broad range of B-cell malignancies that can be efficiently targeted by small molecule inhibition of PP2A and G6PD. Citation Format: Gang Xiao, Zhengshan Chen, Lai N. Chan, Daniel Braas, Thomas G. Graeber, Huimin Geng, Hassan Jumaa, Xiaoyan Jiang, Markus Müschen. B-lymphoid transcriptional repression of the pentose phosphate pathway reveals a unique therapeutic vulnerability of B cell malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 368.

PP2A Inactivation Mediated by PPP2R4 Haploinsufficiency Promotes Cancer Development.

Protein phosphatase 2A (PP2A) complexes counteract many oncogenic kinase pathways. In cancer cells, PP2A function can be compromised by several mechanisms, including sporadic mutations in its scaffolding A and regulatory B subunits or more frequently through overexpression of cellular PP2A inhibitors. Here, we identify a novel genetic mechanism by which PP2A function is recurrently affected in human cancer, involving haploinsufficiency of PPP2R4, a gene encoding the cellular PP2A activator PTPA. Notably, up to 70% of cancer patients showed a heterozygous deletion or missense mutations in PPP2R4 Cancer-associated PTPA mutants exhibited decreased abilities to bind the PP2A-C subunit or activate PP2A and failed to reverse the tumorigenic phenotype induced by PTPA suppression, indicating they function as null alleles. In Ppp2r4 gene-trapped (gt) mice showing residual PTPA expression, total PP2A activity and methylation were reduced, selectively affecting specific PP2A holoenzymes. Both PTPAgt/gt and PTPA+/gt mice showed higher rates of spontaneous tumors, mainly hematologic malignancies and hepatocellular adenomas and carcinomas. These tumors exhibited increased c-Myc phosphorylation and increased Wnt or Hedgehog signaling. We observed a significant reduction in lifespan in PTPA+/gt mice compared with wild-type mice. In addition, chemical-induced skin carcinogenesis was accelerated in PTPA+/gt compared with wild-type mice. Our results provide evidence for PPP2R4 as a haploinsufficient tumor suppressor gene, defining a high-penetrance genetic mechanism for PP2A inhibition in human cancer. Cancer Res; 77(24); 6825-37. ©2017 AACR.

Overexpression of the PP2A-C5 gene confers increased salt tolerance in Arabidopsis thaliana

ABSTRACTProtein phosphatase 2A (PP2A) was shown to play important roles in biotic and abiotic stress signaling pathways in plants. PP2A is made of 3 subunits: a scaffolding subunit A, a regulatory subunit B, and a catalytic subunit C. It is believed that the B subunit recognizes specific substrates and the C subunit directly acts on the selected substrates, whereas the A subunit brings a B subunit and a C subunit together to form a specific PP2A holoenzyme. Because there are multiple isoforms for each PP2A subunit, there could be hundreds of novel PP2A holoenzymes in plants. For an example, there are 3 A subunits, 17 B subunits, and 5 C subunits in Arabidopsis, which could form 255 different PP2A holoenzymes. Understanding the roles of these PP2A holoenzymes in various signaling pathways is a challenging task. In a recent study,1 we discovered that PP2A-C5, the catalytic subunit 5 of PP2A, plays an important role in salt tolerance in Arabidopsis. We found that a knockout mutant of PP2A-C5 (i.e. pp2a-c5–1) was very sensitive to salt treatments, whereas PP2A-C5-overexpressing plants were more tolerant to salt stresses. Genetic analyses between pp2a-c5–1 and Salt-Overly-Sensitive (SOS) mutants indicated that PP2A-C5 does not function in the same pathway as SOS genes. Using yeast 2-hybrid analysis, we found that PP2A-C5 interacts with several vacuolar membrane bound chloride channel proteins. We hypothesize that these vacuolar chloride channel proteins might be PP2A-C5's substrates in vivo, and the action of PP2A-C5 on these channel proteins could increase or activate their activities, thereby result in accumulation of the chloride and sodium contents in vacuoles, leading to increased salt tolerance in plants.

Abstract 3180: Regulation of stem cells by the tumor suppressor PP2A-B56α

Abstract Protein phosphatase 2 A (PP2A) is one of the major cellular Serine-Threonine phosphatases and regulates numerous signaling pathways, including stem cell self-renewal, proliferation, differentiation, and cell survival. The PP2A holoenzyme has 3 subunits: a catalytic (C) subunit, a structural (A) subunit, and a variable regulatory (B) subunit, which directs PP2A to specific targets. PP2A inhibition is required to completely transform human cells and siRNA screens have shown that loss of B56α, B56γ, or PR72/PR130 B subunits results in cell transformation, which are involved in negatively regulating oncogenic pathways including Wnt, Akt, and Myc. Moreover, PP2A plays an important role in regulating human embryonic stem cells, where both the A and C subunit levels and PP2A activity gradually increase during the course of differentiation. Our preliminary data also demonstrates in breast cancer cell lines that B56α overexpression decreases the number of mammospheres whereas inhibiting B56α increases them. Further research is needed to address the function of specific PP2A complexes in regulating stem cells. Despite the fact that B56α depletion is important for human cell transformation, no one has reported on its role in vivo and no mouse model has ever been generated to our knowledge. Therefore, to study the role of B56α in stem cells and cell transformation in vivo, we have generated B56α knockout mice. We generated B56α knockout mice using commercially available gene trap ES cells. The gene trap sits in the first intron of the B56α gene and, through a splice acceptor site, splices out the first exon to the gene trap and therefore stops whole gene transcription in the whole body. The primary phenotypic effect we observed with B56α loss was spontaneous skin lesion formation. Thus far 40% of mice have developed skin lesions between 11 to 21 months and showed hyper-proliferation of the epidermis, hair follicles, and dermis. Increased number of hair follicles prompted us to perform stem cell assays using BrDU to label long term label-retaining cells and the colony assays for the fast adhering keratinocyte stem cells. We found an increase in skin stem cell numbers. In addition to skin lesions, we found increased inflammation both in skin lesions, liver, and spleen and some enlarged lymph nodes. Next we asked whether loss of B56α also increased the number of hematopoietic stem cell. Although the number of hematopoietic stem cells in the periphery was unchanged under normal condition, treating mice with granulocyte colony-stimulating factor increased the number of circulating stem cells. Our model suggests that loss of B56α increases the number of stem cells and that B56α is important for cells to maintain homeostasis; B56α loss can lead to increased activity of important oncogenes, such as cMyc, leading to aberrant cell growth and defects in stem cell maintenance. More experiments need to be done to understand the primary effect of B56α loss in skin and the interplay between skin lesions and inflammation. Citation Format: Mahnaz Janghorban, Derek Zachman, Xiaoyan Wang, Anupriya Agarwal, William Fleming, Rosalie Sears. Regulation of stem cells by the tumor suppressor PP2A-B56α. [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 3180. doi:10.1158/1538-7445.AM2015-3180

Abstract 5287: Identification of metadherin in a complex with RACK1/PP2A in mammary epithelial cells

Abstract Protein Phosphatase 2A (PP2A) is a major serine/threonine phosphatase in cells. It consists of a catalytic (C), structural (A) and regulatory/variable B-type subunits. PP2A has a critical role to play in homeostasis where it functions as a tumour suppressor. Changes in the activity of PP2A have a direct role in maintaining the transformed phenotype in cancer. However, the mechanism of action of PP2A during this process is poorly understood. Our understanding is limited because of the complex nature of PP2A holoenzyme assembly, and because it acts through a wide variety of signalling pathways. We have identified the scaffolding protein RACK1 as a PP2A interacting protein and we showed that RACK1 regulates PP2A activity to mediate crosstalk between growth factor and adhesion signalling by an IGF-I-independent mechanism(1, 2) . Our hypothesis is that RACK1 plays a critical role in determining how PP2A functions in signalling pathways. Using a series of truncation mutations and peptide array technology, we have identified and mapped the binding sites between RACK1 and the catalytic subunit of PP2A. Using a series of specific point mutations of these PP2A sites, we show that the RACK1/PP2A interaction is critical to maintain PP2A activity. Disruption of the RACK1/PP2A interaction has dramatic consequences for cell adhesion, proliferation and migration in MCF-7 cells. Our hypothesis is that RACK1 regulates the cellular distribution of PP2A and scaffolds PP2A to a specific subset of cellular targets. Using 3D in vitro breast cancer models and mass spectroscopy approaches, we have identified several proteins that interact with the RACK1/PP2A complex. One of these proteins, Metadherin has been shown to play a role in drug resistance(3). It has also been linked to the progression of many cancers(4). Using a series of novel approaches we have mapped the Metadherin interaction site on RACK1 and PP2A and confirmed that RACK1 and Metadherin colocalise at the cell membrane. Overexpression of Metadherin promotes IGF-I-mediated cell migration and invasion. Taken together, our data suggest that RACK1 scaffolds PP2A to Metadherin where it regulates Metadherin activity and cell migration in breast cancer. 1. Kiely, PA., et al. “Tyrosine 302 in RACK1 is essential for insulin-like growth factor-I-mediated competitive binding of PP2A and β1 integrin and for tumor cell proliferation and migration.” Journal of Biological Chemistry 283.34 (2008): 22952-22961. 2. Kiely, PA., et al. “Insulin-like growth factor I controls a mutually exclusive association of RACK1 with protein phosphatase 2A and β1 integrin to promote cell migration.” Molecular and cellular biology 26.11 (2006): 4041-4051. 3. Wei, Yong, Guohong Hu, and Yibin Kang. “Metadherin as a link between metastasis and chemoresistance.” Cell Cycle 8.14 (2009): 2131-2137. 4. Brown, DM., and Erkki R. “Metadherin, a cell surface protein in breast tumors that mediates lung metastasis.” Cancer cell 5.4 (2004): 365-374. Citation Format: Maeve L. Kiely, Rosemary O'Connor, Patrick A. Kiely. Identification of metadherin in a complex with RACK1/PP2A in mammary epithelial cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5287. doi:10.1158/1538-7445.AM2014-5287

Abstract 2325: Identification of RACK1 as a novel regulatory subunit of PP2A.

Abstract Protein Phosphatase 2A (PP2A) is a major serine/threonine phosphatase in cells. It consists of a catalytic (C), structural (A) and regulatory/variable B-type subunits. PP2A plays a critical role as a tumour suppressor. Changes in the activity of PP2A have a direct role in maintaining the transformed phenotype in cancer. However, the mechanism of action of PP2A during this process is poorly understood. Understanding is limited by the complex nature of PP2A holoenzyme assembly, and because it acts through a wide variety of signalling pathways. The regulatory B-type subunits have a critical role in targeting PP2A to specific areas of the cell and also in regulating the overall activity and substrate specificity of PP2A. To date, at least 15 known PP2A B-type subunits have been identified but their detection is difficult because of the transient nature of their interaction with the PP2A holoenzyme. The scaffolding protein RACK1 is a PP2A interacting protein and we have published a mechanism by which PP2A regulates IGF-IR and adhesion signalling through RACK1 [1, 2]. Our hypothesis is that RACK1 plays a critical role in determining how PP2A functions in signalling pathways and may function as a regulatory subunit of PP2A. We predict it does this by modulating the activity of PP2A and regulating its distribution to a specific subset of targets. We examined how the PP2A holoenzyme is assembled in MCF-7 cells. We have found that both the catalytic subunit and structural subunit of PP2A (PP2A C and PP2A A respectively) are assembled on the RACK1 scaffold in response to IGF-I. Our findings indicate that both the C subunit and the A subunit of PP2A are released from RACK1 in response to IGF-I and reassembled after 30 minutes IGF-I stimulation. We used immobilized peptide arrays representing the entire PP2A-catalytic protein to identify amino acids on the catalytic subunit of PP2A that are required for the binding of RACK1. Analysis of these residues revealed the binding sites for RACK1 on PP2A and supports their role in mediating the interaction with RACK1. When cells are transfected with specific mutations of PP2A C that disrupt the association with RACK1, cells show decreased PP2A activity and deregulated cell adhesion and migration suggesting that RACK1 has an essential role to play in the overall activity and substrate specificity of PP2A. This data supports our hypothesis that RACK1 functions as a B-type subunit of PP2A which is required for the regulation of PP2A activity. Our data also indicates that RACK1 targets PP2A to specific locations to promote IGF-I-mediated cell migration and proliferation in tumour cells. 1. Kiely, P.A., et al. Molecular and cellular biology, 2006. 26(11): p. 4041-4051. 2. Kiely, P.A., et al. Journal of Biological Chemistry, 2008. 283(34): p. 22952-22961. Citation Format: Maeve L. Kiely, Rosemary O'Connor, Patrick A. Kiely. Identification of RACK1 as a novel regulatory subunit of PP2A. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2325. doi:10.1158/1538-7445.AM2013-2325

PP2A regulates autophagy in two alternative ways in Drosophila

Protein phosphatase 2A (PP2A) holoenzyme is a heterotrimeric complex, consisting of A, B and C subunits. The catalytic subunit PP2A-C (microtubule star/mts) binds to the C-terminal part of the scaffold protein PP2A-A (PP2A-29B). In Drosophila, there are three different forms of B subunits (widerborst/wdb, twins/tws and PP2A-B'), which determine the subcellular localization and substrate specificity of the holoenzyme. Previous studies demonstrated that PP2A is involved in the control of TOR-dependent autophagy both in yeast and mammals. Furthermore, in Drosophila, wdb genetically interacts with the PtdIns3K/PTEN/Akt signaling cascade, which is a main upstream regulatory system of dTOR. Here we demonstrate that in Drosophila, two different PP2A complexes (containing B' or wdb subunit) play essential roles in the regulation of starvation-induced autophagy. The PP2A-A/wdb/C complex acts upstream of dTOR, whereas the PP2A-A/B'/C complex functions as a target of dTOR and may regulate the elongation of autophagosomes and their subsequent fusion with lysosomes. We also identified three Drosophila Atg orthologs (Atg14, Atg17 and Atg101), which represent potential targets of the PP2A-A/B'/C complex during autophagy.

HEAT Repeat 1 Motif Is Required for B56γ-containing Protein Phosphatase 2A (B56γ-PP2A) Holoenzyme Assembly and Tumor-suppressive Function

Protein phosphatase 2A (PP2A) enzyme consists of a heterodimeric core (AC core) comprising a scaffolding subunit (A), a catalytic subunit (C), and a variable regulatory subunit (B). Earlier studies suggest that upon DNA damage, a specific B subunit, B56γ, bridges the PP2A AC core to p53, leading to dephosphorylation of p53 at Thr-55, induction of the p53 transcriptional target p21, and the inhibition of cell proliferation and transformation. In addition to dephosphorylation of p53, B56γ-PP2A also inhibits cell proliferation and transformation by an unknown mechanism. B56γ contains 18 α-helices that are organized into eight HEAT (Huntington-elongation-A subunit-TOR) repeat motifs. Although previous crystal structure study has revealed the residues of B56γ that directly contact the A and C subunits, the contribution of HEAT repeats to holoenzyme assembly and to B56γ-PP2A tumor-suppressive function remains to be elucidated. Here, we show that HEAT repeat 1 is required for the interaction of B56γ with the PP2A AC core and, more importantly, for B56γ-PP2A tumor-suppressive function. Within this region, we identified a tumor-associated mutation, C39R, which disrupts the interaction of B56γ with the AC core and thus was unable to mediate dephosphorylation of p53 by PP2A. Furthermore, due to its lack of AC interaction, C39R was also unable to promote the p53-independent tumor-suppressive function of B56γ-PP2A. This study provides structural insight into the PP2A holoenzyme assembly and emphasizes the importance of HEAT repeat 1 in B56γ-PP2A tumor-suppressive function.

Abstract 924: Identification of somatic mutations of the PPP2R1A gene in subtypes of endometrial and ovarian carcinomas

Abstract Somatic mutations in PPP2R1A have been found at low frequencies in lung, breast cancer and melanoma and most recently through whole-exome sequencing in ovarian clear cell carcinoma. We examined whole-transcriptome sequencing data from ovarian clear cell carcinomas (OCCC) and identified a cluster of mutations in exon 5 of PPP2R1A. The protein phosphatase 2, regulatory subunit A, alpha (PPP2R1A, or PR65a) gene is the scaffolding subunit of the serine-threonine protein phosphatase 2A (PP2A) holoenzyme. PP2A is involved in growth and survival cellular pathways, and has been suggested to play a role as a tumor suppressor gene in some human cancers. The PP2A holoenzyme is made up of the A scaffolding subunit (isoforms α and β), the C catalytic subunit (isoforms α and β and multiple regulatory B subunits also with many isoforms. PPP2R1A is made up of 15 HEAT domains forming a horseshoe-like multi-helical structure that interacts with both the C and B subunits of PP2A. We have examined the frequency of PPP2R1A mutations across the spectrum of both ovarian and endometrial carcinomas. DNA from endometrial and ovarian subtypes was sequenced across exons 5 and 6 of PPP2R1A using PCR based Sanger sequencing, and somatic mutation status was verified by sequencing matched normal DNA. Targeted sequencing of PPP2R1A revealed somatic missense mutations in 36.7% (18 of 49) of endometrial high-grade serous primary tumors. This high frequency of mutations has not previously been reported and suggests a strong link to the pathology of this disease subtype. Mutations were also identified at a lower frequency of 4.9% (3 of 61) in endometrial endometrioid carcinomas, and ovarian primary tumors at 11.6% (5 of 43) in endometrioid, 4% (2 of 50) in clear cell, 0% (0 of 50) in high-grade serous and 0% (0 of 12) of low-grade serous carcinomas. These nucleotide missense mutations result in the modification of amino acid residues that are highly conserved within the intrarepeat helix of the HEAT domains of PPP2R1A. Previous mutation based biochemical studies have identified these amino acid residues as important in the interaction between the regulatory B subunit and PPP2R1A. This indicates a strong functional role of PPP2R1A in stabilization of the PP2A complex, and a probable role in cellular tumorgenesis. A closer survey of mutations found within PPP2R1A and the PP2A complex, as well as functional characterization of this pathway, may pave the way to defining biologically relevant pathways and targets within endometrial carcinomas, ultimately leading to improved patient-targeted therapies and survival. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 924. doi:10.1158/1538-7445.AM2011-924

Abstract 3955: Identification of RACK1 as a novel regulatory subunit of PP2A

Abstract Protein Phosphatase 2A (PP2A) is a major serine/threonine phosphatase with a key role to play in the regulation of cell spreading and cell migration in tumour cells. We have identified PP2A as a RACK1 interacting protein downstream of the IGF-IR (1). In tumour cells PP2A and β1 integrin compete for binding to the C-terminus of RACK1. The IGF-I-mediated release of PP2A from RACK1 results in a decrease in PP2A activity, and promotes cell migration (2). This is coupled with the IGF-I-mediated dephosphorylation of FAK and focal adhesion turnover in MCF-7 cells (3). PP2A enzymes exist as heterotrimers comprising of the catalytic C-, the structural A- and the regulatory/variable B-type subunits. The B-type subunits have a critical role in targeting the PP2A protein to specific areas of the cell and also in regulating the overall activity and substrate specificity of PP2A. To date, at least 15 known PP2A B-type subunits have been identified but their detection has proven difficult because of the transient nature of their interaction with the PP2A holoenzyme. We used in vitro PP2A activity assays to determine that RACK1 stabilizes PP2A activity. This led us to predict that RACK1 may function as a regulatory subunit of PP2A. A comparative study between RACK1 and the well studied B56 family of PP2A regulatory subunits reveals strong homology. Both of these proteins are characterized by the presence of 7 WD repeat sequences which in the case of B56 gamma facilitates PP2A holoenzyme assembly. We used immobilized peptide arrays representing the entire PP2A-catalytic protein to identify a series of amino acids on the catalytic subunit of PP2A that are required for the binding of RACK1. Mutation analysis of these residues reveals the binding site for RACK1 on PP2A. Suppression of RACK1 expression in MCF-7 and DU145 cells results in a decrease in the activity and an altered location of PP2A-C suggesting that RACK1 has an essential role to play in the overall activity and substrate specificity of PP2A. Taken together the data supports our hypothesis that RACK1 functions as a B-type subunit of PP2A which is required for the regulation of PP2A activity. Our data also indicate that RACK1 targets PP2A to specific locations to promote IGF-I-mediated cell migration and proliferation in tumour cells. 1. Kiely, P. A., O'Gorman, D., Luong, K., Ron, D., and O'Connor, R. (2006) Mol Cell Biol 26, 4041-4051 2. Kiely, P. A., Baillie, G. S., Lynch, M. J., Houslay, M. D., and O'Connor, R. (2008) J Biol Chem 3. Kiely, P. A., Baillie, G. S., Barrett, R., Buckley, D. A., Adams, D. R., Houslay, M. D., and O'Connor, R. (2009) J Biol Chem 284, 20263-20274 Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3955.

Diversity in genomic organisation, developmental regulation and distribution of the murine PR72/B" subunits of protein phosphatase 2A

BackgroundProtein phosphatase 2A (PP2A) is a serine/threonine-specific phosphatase displaying vital functions in growth and development through its role in various signalling pathways. PP2A holoenzymes comprise a core dimer composed of a catalytic C and a structural A subunit, which can associate with a variable B-type subunit. The importance of the B-type subunits for PP2A regulation cannot be overestimated as they determine holoenzyme localisation, activity and substrate specificity. Three B-type subunit families have been identified: PR55/B, PR61/B' and PR72/B", of which the latter is currently the least characterised.ResultsWe deduced the sequences and genomic organisation of the different murine PR72/B" isoforms: three genes encode nine isoforms, five of which are abundantly expressed and give rise to genuine PP2A subunits. Thereby, one novel subunit was identified. Using Northern blotting, we examined the tissue-specific and developmental expression of these subunits. All subunits are highly expressed in heart, suggesting an important cardiac function. Immunohistochemical analysis revealed a striated expression pattern of PR72 and PR130 in heart and skeletal muscle, but not in bladder smooth muscle. The subcellular localisation and cell cycle regulatory ability of several PR72/B" isoforms were determined, demonstrating differences as well as similarities.ConclusionIn contrast to PR55/B and PR61/B', the PR72/B" family seems evolutionary more divergent, as only two of the murine genes have a human orthologue. We have integrated these results in a more consistent nomenclature of both human and murine PR72/B" genes and their transcripts/proteins. Our results provide a platform for the future generation of PR72/B" knockout mice.

Identification of a PP2A-interacting protein that functions as a negative regulator of phosphatase activity in the ATM/ATR signaling pathway

Protein serine/threonine phosphatase 2A (PP2A) activity must be tightly controlled to maintain cell homeostasis. Here, we report the identification of a previously uncharacterized mammalian protein, type 2A-interacting protein (TIP), as a novel regulatory protein of PP2A and the PP2A-like enzymes PP4 and PP6. TIP is a ubiquitously expressed protein and parallels the distribution of the PP2A catalytic subunit. Unlike its role in yeast, TIP does not interact with the mammalian homolog of type 2A-associated protein of 42 kDa (Tap42), alpha4, but instead associates with PP2A, PP4 and PP6 catalytic subunits independently of mammalian target of rapamycin kinase activity. Interestingly, the 20 kDa TIP splice variant TIP_i2, which lacks amino acids 173-272 of TIP's C-terminus, does not interact with PP2A; this finding indicates that residues 173-272 are important for the assembly of the TIP.phosphatase complex. In contrast to purified PP2A holoenzymes, TIP.PP2A complexes are devoid of phosphatase activity. Furthermore, alterations in the cellular levels of TIP influence the phosphorylation state of a specific protein substrate of ataxia-telangiectasia mutated (ATM)/ATM- and Rad3-related (ATR) kinases. Elevated levels of TIP result in an increase in the phosphorylation state of this protein substrate, whereas TIP-depleted cells exhibit a significant decrease in this protein's phosphorylation state, which is reversed by treatment with the PP2A inhibitor okadaic acid. These results indicate TIP is a novel inhibitory regulator of PP2A and implicate a role for TIP.PP2A complexes within the ATM/ATR signaling pathway controlling DNA replication and repair.