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Biased holoenzyme assembly of protein phosphatase 2A (PP2A): From cancer to small molecules

Open AccessPublished:October 31, 2022DOI:https://doi.org/10.1016/j.jbc.2022.102656
      Protein phosphatase 2A (PP2A) is a family of serine threonine phosphatases responsible for regulating protein phosphorylation, thus opposing the activity of cellular kinases. PP2A is composed of a catalytic subunit (PP2A Cα/β) and scaffolding subunit (PP2A Aα/β) and various substrate-directing B regulatory subunits. PP2A biogenesis is regulated at multiple levels. For example, the sequestration of the free catalytic subunit during the process of biogenesis avoids promiscuous phosphatase activity. Posttranslational modifications of PP2A C direct PP2A heterotrimeric formation. Additionally, PP2A functions as a haploinsufficient tumor suppressor, where attenuated PP2A enzymatic activity creates a permissive environment for oncogenic transformation. Recent work studying PP2A in cancer showed that its role in tumorigenesis is more nuanced, with some holoenzymes being tumor suppressive, while others are required for oncogenic transformation. In cancer biology, PP2A function is modulated through various mechanisms including the displacement of specific B regulatory subunits by DNA tumor viral antigens, by recurrent mutations, and through loss of carboxymethyl-sensitive heterotrimeric complexes. In aggregate, these alterations bias PP2A activity away from its tumor suppressive functions and toward oncogenic ones. From a therapeutic perspective, molecular glues and disruptors present opportunities for both the selective stabilization of tumor-suppressive holoenzymes and disruption of holoenzymes that are pro-oncogenic. Collectively, these approaches represent an attractive cancer therapy for a wide range of tumor types. This review will discuss the mechanisms by which PP2A holoenzyme formation is dysregulated in cancer and the current therapies that are aimed at biasing heterotrimer formation of PP2A for the treatment of cancer.

      Keywords

      Abbreviations:

      HCC (hepatocellular carcinoma), PTM (posttranslational modification)
      Reversible protein phosphorylation is a critical posttranslational modification (PTM), regulated by the opposing action of two classes of proteins: kinases and phosphatases. Kinases catalyze the addition of a phosphoryl group onto a target protein while phosphatases catalyze the removal of these same phosphoryl groups. Protein phosphorylation can occur on serine, threonine, and tyrosine residues but the vast majority (98%) occurs on serine and threonine residues (
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      ). The addition or removal of these phosphoryl groups is a central mechanism for the regulation of diverse cellular processes including, but not limited to, the activation state of enzymes, protein subcellular localization, membrane transport, protein–protein interactions, and protein degradation. Importantly, while phosphatases have been viewed simply as the erasers of protein phosphorylation, in reality, they serve as critical regulators of the duration and timing of a phosphorylation signal (
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      ). Proteins often harbor multiple phosphorylation sites, allowing for additional complexity and layering of signal transduction. Within a cell, protein phosphorylation is highly dynamic and regulated in both time and space, allowing for a cell to rapidly respond to a plethora of microenvironmental cues and signals. Overall, balanced and regulated protein phosphorylation is key to cellular homeostasis, and its dysregulation is hallmark to the pathogenesis of many diseases, including cancer.
      While there are roughly 500 identified cellular kinases, paradoxically, there have been only approximately 60 identified phosphatases (
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      ). This imbalance led to an early misconception that phosphatases had relatively nonspecific enzymatic activity. Many protein phosphatases, however, are composed of multimeric protein complexes, allowing for the assembly of a repertoire of structurally distinct holoenzymes, explaining the observed difference in the number of kinases versus phosphatases present in our proteome. The serine/threonine protein phosphatase 2A (PP2A) is a perfect example of how the structural diversity of protein phosphatases establish both the broad repertoire of target proteins while allowing for substrate specificity for a given heterotrimeric complex. PP2A is serine/threonine-directed phosphatase family, where the active holoenzyme is composed of a scaffolding subunit (PP2A Aα/β), a catalytic subunit (PP2A Cα/β), and one substrate-directing B regulatory subunit. Both the A and C subunits have two isoforms, α and β, with the α isoform being the more highly expressed isoform for both subunits (
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      ). The active site of the catalytic subunit (PP2A C) has two manganese atoms that when bound assists in substrate catalysis by binding to the phosphoryl group allowing for the hydrolysis of serine or threonine phosphate esters (
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      ). The various regulatory B subunits have been divided broadly into four structurally distinct groups: B55/PR55/B, B56/PR61/B′, PR48/PR72/PR130/B′′, and Striatin/PR93/PR110/B′′′. Within this review, we will refer to these subunit families as the first family name listed. Each structural family contains multiple isoforms and splice variants resulting in the ability to form over 60 different PP2A heterotrimers to regulate the activity, subcellular localization, and substrate specificity of PP2A (Fig. 1) (
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      Figure thumbnail gr1
      Figure 1PP2A is a family of heterotrimeric complexes that regulates many different cellular processes that are crucial for oncogenic transformation. PP2A is composed of a catalytic subunit (PP2A C) and a scaffolding subunit (PP2A A) with two isoforms (α and β) and various substrate-directing B regulatory subunits. The different families of B regulatory subunits, which can be further subsetted by their respective isoforms (
      • Brautigan D.L.
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      ,
      • Smoly I.
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      ,
      • Cho U.S.
      • Xu W.
      Crystal structure of a protein phosphatase 2A heterotrimeric holoenzyme.
      ,
      • Wlodarchak N.
      • Xing Y.
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      ,
      • Zolnierowicz S.
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      ).
      In aggregate, PP2A activity, accounted by all members of the heterotrimeric family, establishes this protein superfamily as a master regulator of a plethora of cellular processes including, but not limited to, cell cycle progression and mitotic exit, the DNA damage response, extracellular mitogenic signals, and cell metabolism (
      • Wlodarchak N.
      • Xing Y.
      PP2A as a master regulator of the cell cycle.
      ). Given the critical role PP2A plays in cellular homeostasis, its biogenesis is highly regulated at multiple levels. This regulation starts with unbound PP2A C (
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      Alpha4 is an essential regulator of PP2A phosphatase activity.
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      The structural basis for tight control of PP2A methylation and function by LCMT-1.
      ). Free PP2A C binds to alpha 4 (α4) leading to the inactivation of the PP2A C catalytic site and protecting it from degradation thereby preventing any damaging promiscuous unregulated catalytic activity while maintaining an ample reserve of PP2A C that can be reactivated by the PP2A Phosphatase activator (PTPA) to rapidly respond to changes in cellular homeostasis (Fig. 2A) (
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      Structural basis of PP2A activation by PTPA, an ATP-dependent activation chaperone.
      ). PTPA reactivates PP2A C by stabilizing its active site, thus promoting binding of ATP phosphoryl groups, which in turn changes the binding preferences of manganese ions ultimately priming the active site for ATP hydrolysis (
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      Structural basis of PP2A activation by PTPA, an ATP-dependent activation chaperone.
      ). PP2A activity is also regulated through PTMs. The reversible carboxymethylation of L309 on the C-terminal tail of PP2A C results in a molecular signal, which guides PP2A heterotrimeric formation (
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      ). Protein methylation at this site occurs at the carboxy terminus of the very terminal amino acid of the protein. This is an extremely unique PTM, found in PP2A and closely related phosphatases. The presence of methylation on L309, catalyzed by Leucine Carboxyl Methyltransferase 1 (LCMT-1), blocks the negative charge on the terminal carboxylic acid, allowing for binding of both B55 and B56 family members to the PP2A A/C dimer (Fig. 2B). Conversely, Protein Phosphatase Methylesterase 1 (PME-1) mediated removal of this PTM keeps the negatively charged terminal carboxylic acid, resulting in steric hindrance preventing B55 and B56 family member binding. However, some PP2A regulatory subunit families, including the Striatin and PR72/130 families can bind regardless of the carboxymethylation status of PP2A AC heterodimer (Fig. 2C).
      Figure thumbnail gr2
      Figure 2PP2A biogenesis is highly complex and regulated at multiple levels. A, free PP2A Cα/β is sequestered and inactivated by α4 preventing any promiscuous phosphatase activity and generating a reserve of catalytic subunit. In response to cellular stimuli, PTPA will reactivate PP2A Cα/β allowing PP2A biogenesis to begin by binding to PP2A Aα/β to form the PP2A A/C dimer (
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      Alpha4 is an essential regulator of PP2A phosphatase activity.
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      Structural basis of PP2A activation by PTPA, an ATP-dependent activation chaperone.
      ). B, PP2A biogenesis is further focused through the carboxymethylation of the C-terminal tail of PP2A Cα/β catalyzed by LCMT-1 and ultimately generating a bias in heterotrimeric formation toward B regulatory subunits that show enhanced affinity. The B56 family is shown, but it is not the only carboxymethyl-sensitive B subunit family (
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      Recurrent PPP2R1A mutations in uterine cancer act through a dominant-negative mechanism to promote malignant cell growth.
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      Expression of protein phosphatase 2A mutants and silencing of the regulatory B alpha subunit induce a selective loss of acetylated and detyrosinated microtubules.
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      Mutation of Tyr307 and Leu309 in the protein phosphatase 2A catalytic subunit favors association with the alpha 4 subunit which promotes dephosphorylation of elongation factor-2.
      ). C, conversely, PME-1 acts to remove carboxymethylation shifting the balance of PP2A heterotrimeric formation toward carboxymethylation-insensitive heterotrimers like PR70-PP2A (
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      PME-1 modulates protein phosphatase 2A activity to promote the malignant phenotype of endometrial cancer cells.
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      PP2A inhibitor PME-1 drives kinase inhibitor resistance in glioma cells.
      ).
      There have been a number of reviews that have provided comprehensive summaries of PP2A’s role in cancer and how this phosphatase family can be targeted therapeutically (
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      Mathematical models of protein kinase signal transduction.
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      ). Here, we will review and discuss the mechanisms that bias PP2A holoenzyme assembly in human cancer and how recent research has shown that small molecules can be used to stabilize or inhibit specific PP2A holoenzymes as a potential therapeutic strategy for the treatment of a diverse range of cancers.

      PP2A heterotrimer dysregulation in cancer

      PP2A was established as a tumor suppressor through the study of okadaic acid and the Simian Virus 40 small T antigen (SV40 ST). These studies demonstrated that loss of PP2A function through either inhibition of its catalytic activity or through the displacement of regulatory B subunits was a key step in driving cellular transformation and tumorigenesis (
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      • Sontag E.
      Simian virus 40 small tumor antigen induces deregulation of the actin cytoskeleton and tight junctions in kidney epithelial cells.
      ). Multiple cellular and mouse models, now supported by patient genetic data, have established PP2A as a haploinsufficient tumor suppressor (
      • Sablina A.A.
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      The role of PP2A A subunits in tumor suppression.
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      • Chen W.
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      Cancer-associated PP2A Aalpha subunits induce functional haploinsufficiency and tumorigenicity.
      ,
      • Zhao Z.
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      • et al.
      PPP2R2A prostate cancer haploinsufficiency is associated with worse prognosis and a high vulnerability to B55alpha/PP2A reconstitution that triggers centrosome destabilization.
      ,
      • Jackson J.B.
      • Pallas D.C.
      Circumventing cellular control of PP2A by methylation promotes transformation in an Akt-dependent manner.
      ). Interestingly, partial or complete knockdown of only certain PP2A subunits were sufficient to induce oncogenic transformation, suggesting that specific regulatory subunits are responsible for PP2A’s tumor suppressive activities. Furthermore, the Cancer Dependency Map (DepMap.org) identified PPP2CA and PPP2R1A, which encode the α isoforms of the catalytic and scaffolding subunits of PP2A, respectively, as common essential genes for cellular survival (
      • Fan J.L.
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      [Phenotype and mechanism of inducible ppp2r1a knockout mouse model].
      ,
      • Lange L.
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      Patterning and gastrulation defects caused by the t(w18) lethal are due to loss of Ppp2r1a.
      ). Collectively, this suggests that tumor cells select a window of PP2A activity, maintaining activity essential for cellular function, while selectively inactivating PP2A’s tumor-suppressive functions, allowing for uncontrolled cancer cell growth. The concept of PP2A inactivation in human cancer has been extensively reviewed (
      • Zhao Z.
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      • Wasserman J.S.
      • Fowle H.
      • et al.
      PPP2R2A prostate cancer haploinsufficiency is associated with worse prognosis and a high vulnerability to B55alpha/PP2A reconstitution that triggers centrosome destabilization.
      ,
      • Kim J.W.
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      STRIPAK directs PP2A activity toward MAP4K4 to promote oncogenic transformation of human cells.
      ,
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      Identification of PP2A complexes and pathways involved in cell transformation.
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      3D clusters of somatic mutations in cancer reveal numerous rare mutations as functional targets.
      ,
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      Recurrent PPP2R1A mutations in uterine cancer act through a dominant-negative mechanism to promote malignant cell growth.
      ,
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      The structure of the protein phosphatase 2A PR65/A subunit reveals the conformation of its 15 tandemly repeated HEAT motifs.
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      ). However, in the sections later, we will focus specifically on how PP2A alterations in cancer lead to biased holoenzyme assembly, allowing tumor cells to capitalize on the progrowth and survival functions of PP2A.

      SV40 small T antigen

      As stated previously, one of the key findings supporting PP2A’s role as a tumor suppressor was through the study of the DNA tumor virus SV40. SV40 expresses two key viral antigens, the small and large T antigen. Expression of these viral antigens facilitates human cellular transformation. The large T antigen (SV40 LT) binds and inactivates the tumor suppressor proteins p53 and RB (
      • Sullivan C.S.
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      T antigens of simian virus 40: molecular chaperones for viral replication and tumorigenesis.
      ,
      • Cho U.S.
      • Morrone S.
      • Sablina A.A.
      • Arroyo J.D.
      • Hahn W.C.
      • Xu W.
      Structural basis of PP2A inhibition by small t antigen.
      ,
      • Ruediger R.
      • Hentz M.
      • Fait J.
      • Mumby M.
      • Walter G.
      Molecular model of the A subunit of protein phosphatase 2A: interaction with other subunits and tumor antigens.
      ,
      • Boehm J.S.
      • Hession M.T.
      • Bulmer S.E.
      • Hahn W.C.
      Transformation of human and murine fibroblasts without viral oncoproteins.
      ). The SV40 small T antigen (SV40 ST) was identified to bind to the PP2A Aα subunit, which remains its only known cellular target (
      • Cho U.S.
      • Morrone S.
      • Sablina A.A.
      • Arroyo J.D.
      • Hahn W.C.
      • Xu W.
      Structural basis of PP2A inhibition by small t antigen.
      ,
      • Chen W.
      • Hahn W.C.
      SV40 early region oncoproteins and human cell transformation.
      ). Structural data suggested that SV40 ST bound to PP2A Aα and blocked B regulatory subunit binding (
      • Cho U.S.
      • Morrone S.
      • Sablina A.A.
      • Arroyo J.D.
      • Hahn W.C.
      • Xu W.
      Structural basis of PP2A inhibition by small t antigen.
      ,
      • Ruediger R.
      • Hentz M.
      • Fait J.
      • Mumby M.
      • Walter G.
      Molecular model of the A subunit of protein phosphatase 2A: interaction with other subunits and tumor antigens.
      ,
      • Sablina A.A.
      • Hector M.
      • Colpaert N.
      • Hahn W.C.
      Identification of PP2A complexes and pathways involved in cell transformation.
      ). Depletion of B56γ partially phenocopied SV40 ST expression in the HEK-TER (nontransformed cellular model that expresses SV40 LT, human telomerase catalytic subunit, and oncogenic HRAS allele) model, suggesting that SV40 ST may displace more than one B regulatory subunit (
      • Moreno C.S.
      • Ramachandran S.
      • Ashby D.G.
      • Laycock N.
      • Plattner C.A.
      • Chen W.
      • et al.
      Signaling and transcriptional changes critical for transformation of human cells by simian virus 40 small tumor antigen or protein phosphatase 2A B56gamma knockdown.
      ). Additional studies have shown that depletion of B56α, PR130/PR72, and PTPA in addition to B56γ is sufficient to phenocopy SV40 ST expression in the HEK-TER model, yet depletion of B56β, B56δ, B56ε, PR48, and Striatin 3 is not (
      • Sablina A.A.
      • Hector M.
      • Colpaert N.
      • Hahn W.C.
      Identification of PP2A complexes and pathways involved in cell transformation.
      ). Supporting the structural data, it was shown that the expression of SV40 ST led to the degradation of B55α, a subunit subject to proteasomal degradation when not bound to A/C (
      • Kwun H.J.
      • Shuda M.
      • Camacho C.J.
      • Gamper A.M.
      • Thant M.
      • Chang Y.
      • et al.
      Restricted protein phosphatase 2A targeting by Merkel cell polyomavirus small T antigen.
      ). Interestingly, SV40 ST was found to associate with PP2A independent of carboxymethylation of the C subunit at L309 (
      • Yu X.X.
      • Du X.
      • Moreno C.S.
      • Green R.E.
      • Ogris E.
      • Feng Q.
      • et al.
      Methylation of the protein phosphatase 2A catalytic subunit is essential for association of Balpha regulatory subunit but not SG2NA, striatin, or polyomavirus middle tumor antigen.
      ). Recently, further mechanistic insight into SV40 ST–mediated transformation has been elucidated. The expression of SV40 ST was shown to promote interaction of the PP2A A/C dimer with Striatin 4, a methylation independent regulatory subunit (
      • Kim J.W.
      • Berrios C.
      • Kim M.
      • Schade A.E.
      • Adelmant G.
      • Yeerna H.
      • et al.
      STRIPAK directs PP2A activity toward MAP4K4 to promote oncogenic transformation of human cells.
      ). This bias in PP2A heterotrimer formation was shown to facilitate the recruitment of the Striatin Interacting Phosphatase and Kinase (STRIPAK) complex, which redirected PP2A activity to dephosphorylate MAP4K4 and induce cellular transformation through the activation of the Hippo pathway effector, YAP1, a known cellular oncogene (
      • Chen C.
      • Shi Z.
      • Zhang W.
      • Chen M.
      • He F.
      • Zhang Z.
      • et al.
      Striatins contain a noncanonical coiled coil that binds protein phosphatase 2A A subunit to form a 2:2 heterotetrameric core of striatin-interacting phosphatase and kinase (STRIPAK) complex.
      ,
      • Seo G.
      • Han H.
      • Vargas R.E.
      • Yang B.
      • Li X.
      • Wang W.
      MAP4K interactome reveals STRN4 as a key STRIPAK complex component in hippo pathway regulation.
      ). Importantly, it was shown that Striatin 4 was required for SV40 ST–mediated cellular transformation (
      • Kim J.W.
      • Berrios C.
      • Kim M.
      • Schade A.E.
      • Adelmant G.
      • Yeerna H.
      • et al.
      STRIPAK directs PP2A activity toward MAP4K4 to promote oncogenic transformation of human cells.
      ). Combined, the expression of SV40 ST results in a redirection of PP2A activity, away from the formation of tumor-suppressive holoenzymes and toward a tumor promoting PP2A-STRN4 complex.

      PP2A Aα alterations

      Loss of PP2A function through decreased expression or heterozygous somatic mutations of Aα induce tumorigenesis. Complete loss of Aα, however, has been shown to be nonviable in mouse, rat, and drosophila models and homozygous mutations in Aα have not been documented in human cancer (
      • Fan J.L.
      • Wang F.P.
      • Wang S.
      • Liu X.L.
      • Wu X.N.
      • Chen W.
      • et al.
      [Phenotype and mechanism of inducible ppp2r1a knockout mouse model].
      • Hu M.W.
      • Wang Z.B.
      • Jiang Z.Z.
      • Qi S.T.
      • Huang L.
      • Liang Q.X.
      • et al.
      Scaffold subunit Aalpha of PP2A is essential for female meiosis and fertility in mice.
      ,
      • Panicker N.
      • Coutman M.
      • Lawlor-O'Neill C.
      • Kahl R.G.S.
      • Roselli S.
      • Verrills N.M.
      Ppp2r2a knockout mice reveal that protein phosphatase 2A regulatory subunit, PP2A-B55alpha, is an essential regulator of neuronal and epidermal embryonic development.
      ). Furthermore, hemizygous loss of Aα occurs recurrently (42%) in prostate adenocarcinomas, and hemizygous loss of Aα occurs more frequently (greater than 75%) in metastatic disease (
      • Zhao Z.
      • Kurimchak A.
      • Nikonova A.S.
      • Feiser F.
      • Wasserman J.S.
      • Fowle H.
      • et al.
      PPP2R2A prostate cancer haploinsufficiency is associated with worse prognosis and a high vulnerability to B55alpha/PP2A reconstitution that triggers centrosome destabilization.
      ). The loss of Aα scaffold subunit expression has been shown to lead to the decreased expression of B55α in multiple cell lines, and prostate cancer cells with Aα loss were markedly sensitive to B55α reconstitution (
      • Zhao Z.
      • Kurimchak A.
      • Nikonova A.S.
      • Feiser F.
      • Wasserman J.S.
      • Fowle H.
      • et al.
      PPP2R2A prostate cancer haploinsufficiency is associated with worse prognosis and a high vulnerability to B55alpha/PP2A reconstitution that triggers centrosome destabilization.
      ,
      • O'Connor C.M.
      • Hoffa M.T.
      • Taylor S.E.
      • Avelar R.A.
      • Narla G.
      Protein phosphatase 2A Aalpha regulates Abeta protein expression and stability.
      ).
      While Aα has been shown to have decreased expression in cancer, an additional mechanism by which the Aα subunit is inactivated is through mutation of the scaffolding subunit of PP2A. The scaffolding subunit is a highly flexible protein made up of 15 tandem Hunting Elongation A subunit Tor (HEAT) repeats (
      • Groves M.R.
      • Hanlon N.
      • Turowski P.
      • Hemmings B.A.
      • Barford D.
      The structure of the protein phosphatase 2A PR65/A subunit reveals the conformation of its 15 tandemly repeated HEAT motifs.
      ). Based on the crystal structure, it was hypothesized that binding of the various B regulatory subunits and the catalytic subunit was facilitated by highly conserved amino acids located within the intrarepeat loops and that these residues may present a vulnerability that could be exploited by cancer to selectively inactivate the tumor-suppressive functions of PP2A (
      • Ruediger R.
      • Hentz M.
      • Fait J.
      • Mumby M.
      • Walter G.
      Molecular model of the A subunit of protein phosphatase 2A: interaction with other subunits and tumor antigens.
      ). In fact, through both targeted and large-scale genomic sequencing efforts, two mutational hotspots were identified, located within HEAT repeats 5 and 7 of the PP2A scaffolding subunit (
      • Gao J.
      • Chang M.T.
      • Johnsen H.C.
      • Gao S.P.
      • Sylvester B.E.
      • Sumer S.O.
      • et al.
      3D clusters of somatic mutations in cancer reveal numerous rare mutations as functional targets.
      ,
      • Gao J.
      • Aksoy B.A.
      • Dogrusoz U.
      • Dresdner G.
      • Gross B.
      • Sumer S.O.
      • et al.
      Integrative analysis of complex cancer genomics and clinical profiles using the cBioPortal.
      ,
      • Kamburov A.
      • Lawrence M.S.
      • Polak P.
      • Leshchiner I.
      • Lage K.
      • Golub T.R.
      • et al.
      Comprehensive assessment of cancer missense mutation clustering in protein structures.
      ). These mutational hotspots cluster in the region that directly contacts different B regulatory subunits (
      • Cho U.S.
      • Xu W.
      Crystal structure of a protein phosphatase 2A heterotrimeric holoenzyme.
      ,
      • Ruediger R.
      • Hentz M.
      • Fait J.
      • Mumby M.
      • Walter G.
      Molecular model of the A subunit of protein phosphatase 2A: interaction with other subunits and tumor antigens.
      ,
      • Kamburov A.
      • Lawrence M.S.
      • Polak P.
      • Leshchiner I.
      • Lage K.
      • Golub T.R.
      • et al.
      Comprehensive assessment of cancer missense mutation clustering in protein structures.
      ,
      • Stanevich V.
      • Zheng A.
      • Guo F.
      • Jiang L.
      • Wlodarchak N.
      • Xing Y.
      Mechanisms of the scaffold subunit in facilitating protein phosphatase 2A methylation.
      ,
      • Brautigan D.L.
      • Farrington C.
      • Narla G.
      Targeting protein phosphatase PP2A for cancer therapy: development of allosteric pharmaceutical agents.
      ). It is important to note that these mutations are heterozygous, leaving the possibility that they might not only result in a loss of PP2A tumor-suppressive function but might also promote gain of function properties.
      Further research has been conducted to better understand how and if mutations to Aα alter holoenzyme binding, and a comprehensive summary of all the mutations studied can be found in Figure 3, highlighting that different mutations display varying degrees of impaired subunit binding (
      • Chen W.
      • Arroyo J.D.
      • Timmons J.C.
      • Possemato R.
      • Hahn W.C.
      Cancer-associated PP2A Aalpha subunits induce functional haploinsufficiency and tumorigenicity.
      ,
      • Gao J.
      • Chang M.T.
      • Johnsen H.C.
      • Gao S.P.
      • Sylvester B.E.
      • Sumer S.O.
      • et al.
      3D clusters of somatic mutations in cancer reveal numerous rare mutations as functional targets.
      ,
      • Haesen D.
      • Abbasi Asbagh L.
      • Derua R.
      • Hubert A.
      • Schrauwen S.
      • Hoorne Y.
      • et al.
      Recurrent PPP2R1A mutations in uterine cancer act through a dominant-negative mechanism to promote malignant cell growth.
      ,
      • Yu X.X.
      • Du X.
      • Moreno C.S.
      • Green R.E.
      • Ogris E.
      • Feng Q.
      • et al.
      Methylation of the protein phosphatase 2A catalytic subunit is essential for association of Balpha regulatory subunit but not SG2NA, striatin, or polyomavirus middle tumor antigen.
      ,
      • O'Connor C.M.
      • Leonard D.
      • Wiredja D.
      • Avelar R.A.
      • Wang Z.
      • Schlatzer D.
      • et al.
      Inactivation of PP2A by a recurrent mutation drives resistance to MEK inhibitors.
      ,
      • Taylor S.E.
      • O'Connor C.M.
      • Wang Z.
      • Shen G.
      • Song H.
      • Leonard D.
      • et al.
      The highly recurrent PP2A Aalpha-subunit mutation P179R alters protein structure and impairs PP2A enzyme function to promote endometrial tumorigenesis.
      ,
      • Lenaerts L.
      • Reynhout S.
      • Verbinnen I.
      • Laumonnier F.
      • Toutain A.
      • Bonnet-Brilhault F.
      • et al.
      The broad phenotypic spectrum of PPP2R1A-related neurodevelopmental disorders correlates with the degree of biochemical dysfunction.
      ,
      • Antao N.V.
      • Marcet-Ortega M.
      • Cifani P.
      • Kentsis A.
      • Foley E.A.
      A cancer-associated missense mutation in PP2A-Aalpha increases centrosome clustering during mitosis.
      ,
      • Jeong A.L.
      • Han S.
      • Lee S.
      • Su Park J.
      • Lu Y.
      • Yu S.
      • et al.
      Patient derived mutation W257G of PPP2R1A enhances cancer cell migration through SRC-JNK-c-Jun pathway.
      ,
      • Ruediger R.
      • Ruiz J.
      • Walter G.
      Human cancer-associated mutations in the Aalpha subunit of protein phosphatase 2A increase lung cancer incidence in Aalpha knock-in and knockout mice.
      ,
      • Nguyen B.
      • Fong C.
      • Luthra A.
      • Smith S.A.
      • DiNatale R.G.
      • Nandakumar S.
      • et al.
      Genomic characterization of metastatic patterns from prospective clinical sequencing of 25,000 patients.
      ). The mutations studied include those in the mutational hotspots but also those outside of these regions. As illustrated in Figure 3, different mutations to Aα display different degrees of impaired B regulatory subunit binding. Several of the recurrent mutations (P179R, R182W, R183G/Q/W, and S256F) display impaired binding to B regulatory subunits of the B55 and B56 families and display increased binding to Striatin family members (
      • Chen W.
      • Arroyo J.D.
      • Timmons J.C.
      • Possemato R.
      • Hahn W.C.
      Cancer-associated PP2A Aalpha subunits induce functional haploinsufficiency and tumorigenicity.
      ,
      • Gao J.
      • Chang M.T.
      • Johnsen H.C.
      • Gao S.P.
      • Sylvester B.E.
      • Sumer S.O.
      • et al.
      3D clusters of somatic mutations in cancer reveal numerous rare mutations as functional targets.
      ,
      • Haesen D.
      • Abbasi Asbagh L.
      • Derua R.
      • Hubert A.
      • Schrauwen S.
      • Hoorne Y.
      • et al.
      Recurrent PPP2R1A mutations in uterine cancer act through a dominant-negative mechanism to promote malignant cell growth.
      ,
      • Yu X.X.
      • Du X.
      • Moreno C.S.
      • Green R.E.
      • Ogris E.
      • Feng Q.
      • et al.
      Methylation of the protein phosphatase 2A catalytic subunit is essential for association of Balpha regulatory subunit but not SG2NA, striatin, or polyomavirus middle tumor antigen.
      ,
      • O'Connor C.M.
      • Leonard D.
      • Wiredja D.
      • Avelar R.A.
      • Wang Z.
      • Schlatzer D.
      • et al.
      Inactivation of PP2A by a recurrent mutation drives resistance to MEK inhibitors.
      ,
      • Taylor S.E.
      • O'Connor C.M.
      • Wang Z.
      • Shen G.
      • Song H.
      • Leonard D.
      • et al.
      The highly recurrent PP2A Aalpha-subunit mutation P179R alters protein structure and impairs PP2A enzyme function to promote endometrial tumorigenesis.
      ,
      • Lenaerts L.
      • Reynhout S.
      • Verbinnen I.
      • Laumonnier F.
      • Toutain A.
      • Bonnet-Brilhault F.
      • et al.
      The broad phenotypic spectrum of PPP2R1A-related neurodevelopmental disorders correlates with the degree of biochemical dysfunction.
      ,
      • Antao N.V.
      • Marcet-Ortega M.
      • Cifani P.
      • Kentsis A.
      • Foley E.A.
      A cancer-associated missense mutation in PP2A-Aalpha increases centrosome clustering during mitosis.
      ,
      • Jeong A.L.
      • Han S.
      • Lee S.
      • Su Park J.
      • Lu Y.
      • Yu S.
      • et al.
      Patient derived mutation W257G of PPP2R1A enhances cancer cell migration through SRC-JNK-c-Jun pathway.
      ,
      • Ruediger R.
      • Ruiz J.
      • Walter G.
      Human cancer-associated mutations in the Aalpha subunit of protein phosphatase 2A increase lung cancer incidence in Aalpha knock-in and knockout mice.
      ,
      • Nguyen B.
      • Fong C.
      • Luthra A.
      • Smith S.A.
      • DiNatale R.G.
      • Nandakumar S.
      • et al.
      Genomic characterization of metastatic patterns from prospective clinical sequencing of 25,000 patients.
      ) (Fig. 3A). Interestingly, this pattern of heterotrimer bias mirrors that of SV40 ST, but the dependence of the transformative potential of these Aα mutants on Striatin 4 binding, for example, has not been explored. Additionally, coimmunoprecipitated complexes from both Aα-P179R and Aα-R183W mutant expressing cells displayed reduced PP2A enzymatic activity, even when this activity was normalized for C subunit binding in the complex (
      • O'Connor C.M.
      • Leonard D.
      • Wiredja D.
      • Avelar R.A.
      • Wang Z.
      • Schlatzer D.
      • et al.
      Inactivation of PP2A by a recurrent mutation drives resistance to MEK inhibitors.
      ,
      • Taylor S.E.
      • O'Connor C.M.
      • Wang Z.
      • Shen G.
      • Song H.
      • Leonard D.
      • et al.
      The highly recurrent PP2A Aalpha-subunit mutation P179R alters protein structure and impairs PP2A enzyme function to promote endometrial tumorigenesis.
      ). This suggests that there may be other proteins or mechanisms responsible for their tumor-promoting activity beyond their selective B regulatory subunit loss. In support of this concept, the Aα-S256F mutant demonstrated increased binding to Tip41-like protein (TIPRL), which has been shown to inhibit PP2A activity by binding to the demethylated tail of the C subunit effectively sequestering the catalytic subunit (
      • Haesen D.
      • Abbasi Asbagh L.
      • Derua R.
      • Hubert A.
      • Schrauwen S.
      • Hoorne Y.
      • et al.
      Recurrent PPP2R1A mutations in uterine cancer act through a dominant-negative mechanism to promote malignant cell growth.
      • Scorsato V.
      • Lima T.B.
      • Righetto G.L.
      • Zanchin N.I.
      • Brandao-Neto J.
      • Sandy J.
      • et al.
      Crystal structure of the human Tip41 orthologue, TIPRL, reveals a novel fold and a binding site for the PP2Ac C-terminus.
      ). While the hotspot mutations have similar patterns of disruption of B subunit binding, there are important differences for C subunit binding. For example, the Aα-P179R mutation impairs binding to the catalytic subunit disrupting PP2A A/C dimer formation, resulting in catalytic subunit degradation, which is not seen for other mutants (Fig. 3B) (
      • Haesen D.
      • Abbasi Asbagh L.
      • Derua R.
      • Hubert A.
      • Schrauwen S.
      • Hoorne Y.
      • et al.
      Recurrent PPP2R1A mutations in uterine cancer act through a dominant-negative mechanism to promote malignant cell growth.
      • Taylor S.E.
      • O'Connor C.M.
      • Wang Z.
      • Shen G.
      • Song H.
      • Leonard D.
      • et al.
      The highly recurrent PP2A Aalpha-subunit mutation P179R alters protein structure and impairs PP2A enzyme function to promote endometrial tumorigenesis.
      ).
      Figure thumbnail gr3
      Figure 3PP2A Aα mutants effect the PP2A interactome in a graded manner. A, the different cancer-associated PP2A Aα mutants can be broken down by the degree to which they lose binding to PP2A Cα/β. B, similarly, the mutants can be broken down by the degree to which they lose binding to the B55 and B56 family members. Collectively, this illustrates that the scaffold mutants that demonstrate loss of catalytic subunit often demonstrate coordinate loss of association with the more tumor-suppressive B regulatory subunits (
      • Chen W.
      • Arroyo J.D.
      • Timmons J.C.
      • Possemato R.
      • Hahn W.C.
      Cancer-associated PP2A Aalpha subunits induce functional haploinsufficiency and tumorigenicity.
      ,
      • Gao J.
      • Chang M.T.
      • Johnsen H.C.
      • Gao S.P.
      • Sylvester B.E.
      • Sumer S.O.
      • et al.
      3D clusters of somatic mutations in cancer reveal numerous rare mutations as functional targets.
      ,
      • Haesen D.
      • Abbasi Asbagh L.
      • Derua R.
      • Hubert A.
      • Schrauwen S.
      • Hoorne Y.
      • et al.
      Recurrent PPP2R1A mutations in uterine cancer act through a dominant-negative mechanism to promote malignant cell growth.
      ,
      • Yu X.X.
      • Du X.
      • Moreno C.S.
      • Green R.E.
      • Ogris E.
      • Feng Q.
      • et al.
      Methylation of the protein phosphatase 2A catalytic subunit is essential for association of Balpha regulatory subunit but not SG2NA, striatin, or polyomavirus middle tumor antigen.
      ,
      • O'Connor C.M.
      • Leonard D.
      • Wiredja D.
      • Avelar R.A.
      • Wang Z.
      • Schlatzer D.
      • et al.
      Inactivation of PP2A by a recurrent mutation drives resistance to MEK inhibitors.
      ,
      • Taylor S.E.
      • O'Connor C.M.
      • Wang Z.
      • Shen G.
      • Song H.
      • Leonard D.
      • et al.
      The highly recurrent PP2A Aalpha-subunit mutation P179R alters protein structure and impairs PP2A enzyme function to promote endometrial tumorigenesis.
      ,
      • Lenaerts L.
      • Reynhout S.
      • Verbinnen I.
      • Laumonnier F.
      • Toutain A.
      • Bonnet-Brilhault F.
      • et al.
      The broad phenotypic spectrum of PPP2R1A-related neurodevelopmental disorders correlates with the degree of biochemical dysfunction.
      ,
      • Antao N.V.
      • Marcet-Ortega M.
      • Cifani P.
      • Kentsis A.
      • Foley E.A.
      A cancer-associated missense mutation in PP2A-Aalpha increases centrosome clustering during mitosis.
      ,
      • Jeong A.L.
      • Han S.
      • Lee S.
      • Su Park J.
      • Lu Y.
      • Yu S.
      • et al.
      Patient derived mutation W257G of PPP2R1A enhances cancer cell migration through SRC-JNK-c-Jun pathway.
      ). Recurrent PPP2R1A hotspot mutations with greater than ten independent observations in cancer were bolded to demonstrate the effect on PP2A heterotrimer formation (
      • Nguyen B.
      • Fong C.
      • Luthra A.
      • Smith S.A.
      • DiNatale R.G.
      • Nandakumar S.
      • et al.
      Genomic characterization of metastatic patterns from prospective clinical sequencing of 25,000 patients.
      ).
      The cumulative effect of Aα mutations is a bias in PP2A heterotrimer formation that results in the activation of cellular signaling, which promotes the growth and survival of cancer cells. Studies of the downstream signaling of these mutants suggest that the cellular signaling changes upon mutation of PP2A may occur in both a cell type–dependent and context-dependent manner. For example, in Kirsten Rat Sarcoma Virus (KRAS) mutated cancer cells, it was shown that the Aα-R183W mutation potentiates mitogen-activated protein kinase (MAPK) pathway signaling through increased phosphorylation of Extracellular Signal-regulated Kinase ½ (ERK1/2) (
      • O'Connor C.M.
      • Leonard D.
      • Wiredja D.
      • Avelar R.A.
      • Wang Z.
      • Schlatzer D.
      • et al.
      Inactivation of PP2A by a recurrent mutation drives resistance to MEK inhibitors.
      ). However, in endometrial cancer cells, without KRAS mutations, Aα-R183W and other Aα mutants were shown to reduce ERK1/2 phosphorylation and increase AKT phosphorylation (
      • Haesen D.
      • Abbasi Asbagh L.
      • Derua R.
      • Hubert A.
      • Schrauwen S.
      • Hoorne Y.
      • et al.
      Recurrent PPP2R1A mutations in uterine cancer act through a dominant-negative mechanism to promote malignant cell growth.
      ,
      • O'Connor C.M.
      • Leonard D.
      • Wiredja D.
      • Avelar R.A.
      • Wang Z.
      • Schlatzer D.
      • et al.
      Inactivation of PP2A by a recurrent mutation drives resistance to MEK inhibitors.
      ,
      • Wu C.G.
      • Zheng A.
      • Jiang L.
      • Rowse M.
      • Stanevich V.
      • Chen H.
      • et al.
      Methylation-regulated decommissioning of multimeric PP2A complexes.
      ). Other PP2A scaffold mutants have also been shown to activate AKT signaling as well, including Aα-E64D, resulting in increased lung cancer progression and decreased survival in mice crossed with constitutively active RAS (
      • Ruediger R.
      • Ruiz J.
      • Walter G.
      Human cancer-associated mutations in the Aalpha subunit of protein phosphatase 2A increase lung cancer incidence in Aalpha knock-in and knockout mice.
      ,
      • Walter G.
      • Ruediger R.
      Mouse model for probing tumor suppressor activity of protein phosphatase 2A in diverse signaling pathways.
      ,
      • Ruediger R.
      • Pham H.T.
      • Walter G.
      Alterations in protein phosphatase 2A subunit interaction in human carcinomas of the lung and colon with mutations in the A beta subunit gene.
      ,
      • De Baere I.
      • Derua R.
      • Janssens V.
      • Van Hoof C.
      • Waelkens E.
      • Merlevede W.
      • et al.
      Purification of porcine brain protein phosphatase 2A leucine carboxyl methyltransferase and cloning of the human homologue.
      ). Furthermore, the Aα-R183Q/G, S256F, R182W, and P179R mutants demonstrated enhanced activation of the ribosomal protein S6 kinase, PI3K/AKT/Mammalian Target of Rapamycin (mTOR), and WNT/β-catenin pathways, all of which stimulate protein translation, cellular proliferation and differentiation, and survival (
      • Haesen D.
      • Abbasi Asbagh L.
      • Derua R.
      • Hubert A.
      • Schrauwen S.
      • Hoorne Y.
      • et al.
      Recurrent PPP2R1A mutations in uterine cancer act through a dominant-negative mechanism to promote malignant cell growth.
      ). Further research investigating the effects of different amino acid changes at one position, for example, R183W versus R183Q, will provide key insight into the nuanced differences in downstream signaling driven by specific PP2A scaffold mutations. Additionally, elucidating how the cell signaling in PP2A mutant cells is altered in the presence of different oncogenic drivers may begin to explain some of the tissue specificity of certain PP2A mutations.

      Methylation of the PP2A C subunit

      Protein methylation is a PTM that most commonly occurs on arginine and lysine residues; however, in the case of PP2A, methylation of the C-terminal tail of the catalytic subunit occurs at the terminal leucine (L309) residue, a unique reversible methylation event resulting in the formation of a carboxymethyl ester. This modification is regulated by the activity of LCMT-1, the methyltransferase, and PME-1, the methylesterase (
      • Xing Y.
      • Li Z.
      • Chen Y.
      • Stock J.B.
      • Jeffrey P.D.
      • Shi Y.
      Structural mechanism of demethylation and inactivation of protein phosphatase 2A.
      ,
      • De Baere I.
      • Derua R.
      • Janssens V.
      • Van Hoof C.
      • Waelkens E.
      • Merlevede W.
      • et al.
      Purification of porcine brain protein phosphatase 2A leucine carboxyl methyltransferase and cloning of the human homologue.
      ,
      • Ogris E.
      • Du X.
      • Nelson K.C.
      • Mak E.K.
      • Yu X.X.
      • Lane W.S.
      • et al.
      A protein phosphatase methylesterase (PME-1) is one of several novel proteins stably associating with two inactive mutants of protein phosphatase 2A.
      ,
      • Lee J.
      • Stock J.
      Protein phosphatase 2A catalytic subunit is methyl-esterified at its carboxyl terminus by a novel methyltransferase.
      ,
      • Xie H.
      • Clarke S.
      Protein phosphatase 2A is reversibly modified by methyl esterification at its C-terminal leucine residue in bovine brain.
      ,
      • Kloeker S.
      • Bryant J.C.
      • Strack S.
      • Colbran R.J.
      • Wadzinski B.E.
      Carboxymethylation of nuclear protein serine/threonine phosphatase X.
      ,
      • Ye C.
      • Sutter B.M.
      • Wang Y.
      • Kuang Z.
      • Zhao X.
      • Yu Y.
      • et al.
      Demethylation of the protein phosphatase PP2A promotes demethylation of histones to enable their function as a methyl group sink.
      ). The presence of carboxymethylation of PP2A C has been reported to have a modest to no effects on PP2A enzymatic activity in vitro, initiating inquiries into mechanisms through which this PTM regulates PP2A activity and function (
      • De Baere I.
      • Derua R.
      • Janssens V.
      • Van Hoof C.
      • Waelkens E.
      • Merlevede W.
      • et al.
      Purification of porcine brain protein phosphatase 2A leucine carboxyl methyltransferase and cloning of the human homologue.
      ,
      • Bryant J.C.
      • Westphal R.S.
      • Wadzinski B.E.
      Methylated C-terminal leucine residue of PP2A catalytic subunit is important for binding of regulatory Balpha subunit.
      ). Interestingly, it was determined that carboxymethylation of the catalytic subunit is an evolutionarily conserved mechanism to promote the formation of specific heterotrimeric complexes (
      • De Baere I.
      • Derua R.
      • Janssens V.
      • Van Hoof C.
      • Waelkens E.
      • Merlevede W.
      • et al.
      Purification of porcine brain protein phosphatase 2A leucine carboxyl methyltransferase and cloning of the human homologue.
      ,
      • Xie H.
      • Clarke S.
      Protein phosphatase 2A is reversibly modified by methyl esterification at its C-terminal leucine residue in bovine brain.
      ,
      • Wei H.
      • Ashby D.G.
      • Moreno C.S.
      • Ogris E.
      • Yeong F.M.
      • Corbett A.H.
      • et al.
      Carboxymethylation of the PP2A catalytic subunit in Saccharomyces cerevisiae is required for efficient interaction with the B-type subunits Cdc55p and Rts1p.
      ). Modification of carboxymethylation, through expression changes in PME-1 or LCMT-1, is another method by which PP2A function is pathologically subverted in cancer (
      • Perrotti D.
      • Neviani P.
      Protein phosphatase 2A: a target for anticancer therapy.
      ). Importantly, knockdown of LCMT-1 or overexpression of PME-1 was shown enhance cellular transformation in the HEK TER/B56γ model, resulting in activation of the AKT/S6K pathway (
      • Jackson J.B.
      • Pallas D.C.
      Circumventing cellular control of PP2A by methylation promotes transformation in an Akt-dependent manner.
      ).
      As stated previously, the carboxymethylation status of the catalytic subunit has been shown to alter the binding of specific B regulatory subunits to the PP2A A/C dimer. Specifically, the B55 family, B56α/ε, have been shown to be methylation-sensitive regulatory subunits, indicating that they preferentially bind to the methylated A/C dimer (
      • Jackson J.B.
      • Pallas D.C.
      Circumventing cellular control of PP2A by methylation promotes transformation in an Akt-dependent manner.
      ,
      • Haesen D.
      • Abbasi Asbagh L.
      • Derua R.
      • Hubert A.
      • Schrauwen S.
      • Hoorne Y.
      • et al.
      Recurrent PPP2R1A mutations in uterine cancer act through a dominant-negative mechanism to promote malignant cell growth.
      ,
      • Yu X.X.
      • Du X.
      • Moreno C.S.
      • Green R.E.
      • Ogris E.
      • Feng Q.
      • et al.
      Methylation of the protein phosphatase 2A catalytic subunit is essential for association of Balpha regulatory subunit but not SG2NA, striatin, or polyomavirus middle tumor antigen.
      ,
      • Wu C.G.
      • Zheng A.
      • Jiang L.
      • Rowse M.
      • Stanevich V.
      • Chen H.
      • et al.
      Methylation-regulated decommissioning of multimeric PP2A complexes.
      ,
      • De Baere I.
      • Derua R.
      • Janssens V.
      • Van Hoof C.
      • Waelkens E.
      • Merlevede W.
      • et al.
      Purification of porcine brain protein phosphatase 2A leucine carboxyl methyltransferase and cloning of the human homologue.
      ,
      • Wei H.
      • Ashby D.G.
      • Moreno C.S.
      • Ogris E.
      • Yeong F.M.
      • Corbett A.H.
      • et al.
      Carboxymethylation of the PP2A catalytic subunit in Saccharomyces cerevisiae is required for efficient interaction with the B-type subunits Cdc55p and Rts1p.
      ,
      • Lyons S.P.
      • Greiner E.C.
      • Cressey L.E.
      • Adamo M.E.
      • Kettenbach A.N.
      Regulation of PP2A, PP4, and PP6 holoenzyme assembly by carboxyl-terminal methylation.
      ,
      • Yabe R.
      • Tsuji S.
      • Mochida S.
      • Ikehara T.
      • Usui T.
      • Ohama T.
      • et al.
      A stable association with PME-1 may be dispensable for PP2A demethylation - implications for the detection of PP2A methylation and immunoprecipitation.
      ,
      • Tolstykh T.
      • Lee J.
      • Vafai S.
      • Stock J.B.
      Carboxyl methylation regulates phosphoprotein phosphatase 2A by controlling the association of regulatory B subunits.
      ,
      • Ogris E.
      • Gibson D.M.
      • Pallas D.C.
      Protein phosphatase 2A subunit assembly: the catalytic subunit carboxy terminus is important for binding cellular B subunit but not polyomavirus middle tumor antigen.
      ,
      • Ikehara T.
      • Ikehara S.
      • Imamura S.
      • Shinjo F.
      • Yasumoto T.
      Methylation of the C-terminal leucine residue of the PP2A catalytic subunit is unnecessary for the catalytic activity and the binding of regulatory subunit (PR55/B).
      ,
      • Sontag J.M.
      • Nunbhakdi-Craig V.
      • Mitterhuber M.
      • Ogris E.
      • Sontag E.
      Regulation of protein phosphatase 2A methylation by LCMT1 and PME-1 plays a critical role in differentiation of neuroblastoma cells.
      ,
      • Nunbhakdi-Craig V.
      • Schuechner S.
      • Sontag J.M.
      • Montgomery L.
      • Pallas D.C.
      • Juno C.
      • et al.
      Expression of protein phosphatase 2A mutants and silencing of the regulatory B alpha subunit induce a selective loss of acetylated and detyrosinated microtubules.
      ,
      • Chung H.
      • Nairn A.C.
      • Murata K.
      • Brautigan D.L.
      Mutation of Tyr307 and Leu309 in the protein phosphatase 2A catalytic subunit favors association with the alpha 4 subunit which promotes dephosphorylation of elongation factor-2.
      ) (Table 1). A recent study analyzed the effects of catalytic subunit carboxymethylation of various protein phosphatases on holoenzyme assembly using a proteomics-based approach (
      • Lyons S.P.
      • Greiner E.C.
      • Cressey L.E.
      • Adamo M.E.
      • Kettenbach A.N.
      Regulation of PP2A, PP4, and PP6 holoenzyme assembly by carboxyl-terminal methylation.
      ). This study supported the findings that specific members of the B55 and B56 family of regulatory subunits are sensitive to the carboxymethylation status of the catalytic subunit. Furthermore, this study showed within the B56 family, specific regulatory subunits demonstrated differential sensitivity to changes in carboxymethylation of the catalytic subunit. Specifically, B56α and B56ε were more dependent on methylation for binding compared to the B56γ and B56δ regulatory subunits. The authors also found that the PR72 family showed some sensitivity to the C subunit methylation status while Striatin family member binding was unaffected. Interestingly, the Striatin methyl independence was lost in nontransformed cell lines. This change in Striatin carboxymethyl sensitivity requires further investigation, as the authors only used one immortalized nontransformed cell line (
      • Lyons S.P.
      • Greiner E.C.
      • Cressey L.E.
      • Adamo M.E.
      • Kettenbach A.N.
      Regulation of PP2A, PP4, and PP6 holoenzyme assembly by carboxyl-terminal methylation.
      ). It is important to note that many of these studies use mutation to the C-terminal tail to decrease PP2A C carboxymethylation but distinguishing the effects of carboxymethylation status from the loss of a direct amino acid interaction with B regulatory subunits remain challenging. A summary of the published studies on the effects of PP2A C mutations on carboxymethylation is presented in Table 2 (
      • Jackson J.B.
      • Pallas D.C.
      Circumventing cellular control of PP2A by methylation promotes transformation in an Akt-dependent manner.
      • Haesen D.
      • Abbasi Asbagh L.
      • Derua R.
      • Hubert A.
      • Schrauwen S.
      • Hoorne Y.
      • et al.
      Recurrent PPP2R1A mutations in uterine cancer act through a dominant-negative mechanism to promote malignant cell growth.
      ,
      • Yu X.X.
      • Du X.
      • Moreno C.S.
      • Green R.E.
      • Ogris E.
      • Feng Q.
      • et al.
      Methylation of the protein phosphatase 2A catalytic subunit is essential for association of Balpha regulatory subunit but not SG2NA, striatin, or polyomavirus middle tumor antigen.
      ,
      • Wu C.G.
      • Zheng A.
      • Jiang L.
      • Rowse M.
      • Stanevich V.
      • Chen H.
      • et al.
      Methylation-regulated decommissioning of multimeric PP2A complexes.
      ,
      • De Baere I.
      • Derua R.
      • Janssens V.
      • Van Hoof C.
      • Waelkens E.
      • Merlevede W.
      • et al.
      Purification of porcine brain protein phosphatase 2A leucine carboxyl methyltransferase and cloning of the human homologue.
      ,
      • Wei H.
      • Ashby D.G.
      • Moreno C.S.
      • Ogris E.
      • Yeong F.M.
      • Corbett A.H.
      • et al.
      Carboxymethylation of the PP2A catalytic subunit in Saccharomyces cerevisiae is required for efficient interaction with the B-type subunits Cdc55p and Rts1p.
      ,
      • Lyons S.P.
      • Greiner E.C.
      • Cressey L.E.
      • Adamo M.E.
      • Kettenbach A.N.
      Regulation of PP2A, PP4, and PP6 holoenzyme assembly by carboxyl-terminal methylation.
      ,
      • Yabe R.
      • Tsuji S.
      • Mochida S.
      • Ikehara T.
      • Usui T.
      • Ohama T.
      • et al.
      A stable association with PME-1 may be dispensable for PP2A demethylation - implications for the detection of PP2A methylation and immunoprecipitation.
      ,
      • Tolstykh T.
      • Lee J.
      • Vafai S.
      • Stock J.B.
      Carboxyl methylation regulates phosphoprotein phosphatase 2A by controlling the association of regulatory B subunits.
      ,
      • Ogris E.
      • Gibson D.M.
      • Pallas D.C.
      Protein phosphatase 2A subunit assembly: the catalytic subunit carboxy terminus is important for binding cellular B subunit but not polyomavirus middle tumor antigen.
      ,
      • Ikehara T.
      • Ikehara S.
      • Imamura S.
      • Shinjo F.
      • Yasumoto T.
      Methylation of the C-terminal leucine residue of the PP2A catalytic subunit is unnecessary for the catalytic activity and the binding of regulatory subunit (PR55/B).
      ,
      • Sontag J.M.
      • Nunbhakdi-Craig V.
      • Mitterhuber M.
      • Ogris E.
      • Sontag E.
      Regulation of protein phosphatase 2A methylation by LCMT1 and PME-1 plays a critical role in differentiation of neuroblastoma cells.
      ,
      • Nunbhakdi-Craig V.
      • Schuechner S.
      • Sontag J.M.
      • Montgomery L.
      • Pallas D.C.
      • Juno C.
      • et al.
      Expression of protein phosphatase 2A mutants and silencing of the regulatory B alpha subunit induce a selective loss of acetylated and detyrosinated microtubules.
      ,
      • Chung H.
      • Nairn A.C.
      • Murata K.
      • Brautigan D.L.
      Mutation of Tyr307 and Leu309 in the protein phosphatase 2A catalytic subunit favors association with the alpha 4 subunit which promotes dephosphorylation of elongation factor-2.
      ).
      Table 1The effect of PP2A Cα mutations on the PP2A interactome
      Change to PP2A CB55 familyB56 familyPR familyStriatin familyViral antigensRef
      αδγαδεγPR130/PR72PR59PR70/PR48StriatinStriatin 3Striatin 4SV40 STPySTPyMT
      H59Q<0.151–0.700.40–0.151(
      • Yu X.X.
      • Du X.
      • Moreno C.S.
      • Green R.E.
      • Ogris E.
      • Feng Q.
      • et al.
      Methylation of the protein phosphatase 2A catalytic subunit is essential for association of Balpha regulatory subunit but not SG2NA, striatin, or polyomavirus middle tumor antigen.
      )
      D85N<0.151(
      • Yu X.X.
      • Du X.
      • Moreno C.S.
      • Green R.E.
      • Ogris E.
      • Feng Q.
      • et al.
      Methylation of the protein phosphatase 2A catalytic subunit is essential for association of Balpha regulatory subunit but not SG2NA, striatin, or polyomavirus middle tumor antigen.
      )
      <0.1011(
      • Jackson J.B.
      • Pallas D.C.
      Circumventing cellular control of PP2A by methylation promotes transformation in an Akt-dependent manner.
      )
      R89A<0.151(
      • Yu X.X.
      • Du X.
      • Moreno C.S.
      • Green R.E.
      • Ogris E.
      • Feng Q.
      • et al.
      Methylation of the protein phosphatase 2A catalytic subunit is essential for association of Balpha regulatory subunit but not SG2NA, striatin, or polyomavirus middle tumor antigen.
      )
      H118Q<0.151(
      • Yu X.X.
      • Du X.
      • Moreno C.S.
      • Green R.E.
      • Ogris E.
      • Feng Q.
      • et al.
      Methylation of the protein phosphatase 2A catalytic subunit is essential for association of Balpha regulatory subunit but not SG2NA, striatin, or polyomavirus middle tumor antigen.
      )
      T301D<0.151(
      • Yu X.X.
      • Du X.
      • Moreno C.S.
      • Green R.E.
      • Ogris E.
      • Feng Q.
      • et al.
      Methylation of the protein phosphatase 2A catalytic subunit is essential for association of Balpha regulatory subunit but not SG2NA, striatin, or polyomavirus middle tumor antigen.
      )
      0.52(
      • Sontag J.M.
      • Nunbhakdi-Craig V.
      • Mitterhuber M.
      • Ogris E.
      • Sontag E.
      Regulation of protein phosphatase 2A methylation by LCMT1 and PME-1 plays a critical role in differentiation of neuroblastoma cells.
      )
      T301Stop<0.150.70–10.70–11(
      • Yu X.X.
      • Du X.
      • Moreno C.S.
      • Green R.E.
      • Ogris E.
      • Feng Q.
      • et al.
      Methylation of the protein phosphatase 2A catalytic subunit is essential for association of Balpha regulatory subunit but not SG2NA, striatin, or polyomavirus middle tumor antigen.
      )
      01(
      • Sontag J.M.
      • Nunbhakdi-Craig V.
      • Mitterhuber M.
      • Ogris E.
      • Sontag E.
      Regulation of protein phosphatase 2A methylation by LCMT1 and PME-1 plays a critical role in differentiation of neuroblastoma cells.
      )
      T304A>2>21–0.71(
      • Yu X.X.
      • Du X.
      • Moreno C.S.
      • Green R.E.
      • Ogris E.
      • Feng Q.
      • et al.
      Methylation of the protein phosphatase 2A catalytic subunit is essential for association of Balpha regulatory subunit but not SG2NA, striatin, or polyomavirus middle tumor antigen.
      )
      1.73 ± 0.450.46 ± 0.101.13 ± 0.130.59 ± 0.09(
      • Kaur A.
      • Denisova O.V.
      • Qiao X.
      • Jumppanen M.
      • Peuhu E.
      • Ahmed S.U.
      • et al.
      PP2A inhibitor PME-1 drives kinase inhibitor resistance in glioma cells.
      )
      1.79 ± 0.120.50 ± 0.111.12 ± 0.620.60 ± 0.15(
      • Kaur A.
      • Denisova O.V.
      • Qiao X.
      • Jumppanen M.
      • Peuhu E.
      • Ahmed S.U.
      • et al.
      PP2A inhibitor PME-1 drives kinase inhibitor resistance in glioma cells.
      )
      1.50.5(
      • Sontag J.M.
      • Nunbhakdi-Craig V.
      • Mitterhuber M.
      • Ogris E.
      • Sontag E.
      Regulation of protein phosphatase 2A methylation by LCMT1 and PME-1 plays a critical role in differentiation of neuroblastoma cells.
      )
      T304D<0.151–0.701–0.70(
      • Yu X.X.
      • Du X.
      • Moreno C.S.
      • Green R.E.
      • Ogris E.
      • Feng Q.
      • et al.
      Methylation of the protein phosphatase 2A catalytic subunit is essential for association of Balpha regulatory subunit but not SG2NA, striatin, or polyomavirus middle tumor antigen.
      )
      00.22 ± 0.61.92 ± 0.881.51 ± 0.22(
      • Kaur A.
      • Denisova O.V.
      • Qiao X.
      • Jumppanen M.
      • Peuhu E.
      • Ahmed S.U.
      • et al.
      PP2A inhibitor PME-1 drives kinase inhibitor resistance in glioma cells.
      )
      00.22 ± 0.082.38 ± 0.941.90 ± 0.40(
      • Kaur A.
      • Denisova O.V.
      • Qiao X.
      • Jumppanen M.
      • Peuhu E.
      • Ahmed S.U.
      • et al.
      PP2A inhibitor PME-1 drives kinase inhibitor resistance in glioma cells.
      )
      01(
      • Sontag J.M.
      • Nunbhakdi-Craig V.
      • Mitterhuber M.
      • Ogris E.
      • Sontag E.
      Regulation of protein phosphatase 2A methylation by LCMT1 and PME-1 plays a critical role in differentiation of neuroblastoma cells.
      )
      T304K<0.103(
      • Sontag J.M.
      • Nunbhakdi-Craig V.
      • Mitterhuber M.
      • Ogris E.
      • Sontag E.
      Regulation of protein phosphatase 2A methylation by LCMT1 and PME-1 plays a critical role in differentiation of neuroblastoma cells.
      )
      <0.151(
      • Kaur A.
      • Denisova O.V.
      • Qiao X.
      • Jumppanen M.
      • Peuhu E.
      • Ahmed S.U.
      • et al.
      PP2A inhibitor PME-1 drives kinase inhibitor resistance in glioma cells.
      )
      T304N<0.151(
      • Yu X.X.
      • Du X.
      • Moreno C.S.
      • Green R.E.
      • Ogris E.
      • Feng Q.
      • et al.
      Methylation of the protein phosphatase 2A catalytic subunit is essential for association of Balpha regulatory subunit but not SG2NA, striatin, or polyomavirus middle tumor antigen.
      )
      03(
      • Sontag J.M.
      • Nunbhakdi-Craig V.
      • Mitterhuber M.
      • Ogris E.
      • Sontag E.
      Regulation of protein phosphatase 2A methylation by LCMT1 and PME-1 plays a critical role in differentiation of neuroblastoma cells.
      )
      Y307E<0.15>2>21(
      • Yu X.X.
      • Du X.
      • Moreno C.S.
      • Green R.E.
      • Ogris E.
      • Feng Q.
      • et al.
      Methylation of the protein phosphatase 2A catalytic subunit is essential for association of Balpha regulatory subunit but not SG2NA, striatin, or polyomavirus middle tumor antigen.
      )
      0.02 ± 0.040.05 ± 0.070.20 ± 0.100.69 ± 0.11(
      • Yu X.X.
      • Du X.
      • Moreno C.S.
      • Green R.E.
      • Ogris E.
      • Feng Q.
      • et al.
      Methylation of the protein phosphatase 2A catalytic subunit is essential for association of Balpha regulatory subunit but not SG2NA, striatin, or polyomavirus middle tumor antigen.
      )
      000.18 ± 0.050.57 ± 0.08(
      • Kaur A.
      • Denisova O.V.
      • Qiao X.
      • Jumppanen M.
      • Peuhu E.
      • Ahmed S.U.
      • et al.
      PP2A inhibitor PME-1 drives kinase inhibitor resistance in glioma cells.
      )
      01(
      • Kaur A.
      • Denisova O.V.
      • Qiao X.
      • Jumppanen M.
      • Peuhu E.
      • Ahmed S.U.
      • et al.
      PP2A inhibitor PME-1 drives kinase inhibitor resistance in glioma cells.
      )
      Y307F>2>2>21(
      • Sontag J.M.
      • Nunbhakdi-Craig V.
      • Mitterhuber M.
      • Ogris E.
      • Sontag E.
      Regulation of protein phosphatase 2A methylation by LCMT1 and PME-1 plays a critical role in differentiation of neuroblastoma cells.
      )
      1.65 ± 0.280.05 ± 0.040.23 ± 0.111.17 ± 0.14(
      • Yu X.X.
      • Du X.
      • Moreno C.S.
      • Green R.E.
      • Ogris E.
      • Feng Q.
      • et al.
      Methylation of the protein phosphatase 2A catalytic subunit is essential for association of Balpha regulatory subunit but not SG2NA, striatin, or polyomavirus middle tumor antigen.
      )
      1.53 ± 0.070.09 ± 0.080.42 ± 0.161.24 ± 0.22(
      • Kaur A.
      • Denisova O.V.
      • Qiao X.
      • Jumppanen M.
      • Peuhu E.
      • Ahmed S.U.
      • et al.
      PP2A inhibitor PME-1 drives kinase inhibitor resistance in glioma cells.
      )
      12(
      • Kaur A.
      • Denisova O.V.
      • Qiao X.
      • Jumppanen M.
      • Peuhu E.
      • Ahmed S.U.
      • et al.
      PP2A inhibitor PME-1 drives kinase inhibitor resistance in glioma cells.
      )
      Y307K<0.151(
      • Sontag J.M.
      • Nunbhakdi-Craig V.
      • Mitterhuber M.
      • Ogris E.
      • Sontag E.
      Regulation of protein phosphatase 2A methylation by LCMT1 and PME-1 plays a critical role in differentiation of neuroblastoma cells.
      )
      02(
      • Kaur A.
      • Denisova O.V.
      • Qiao X.
      • Jumppanen M.
      • Peuhu E.
      • Ahmed S.U.
      • et al.
      PP2A inhibitor PME-1 drives kinase inhibitor resistance in glioma cells.
      )
      Y307Q<0.151(
      • Yu X.X.
      • Du X.
      • Moreno C.S.
      • Green R.E.
      • Ogris E.
      • Feng Q.
      • et al.
      Methylation of the protein phosphatase 2A catalytic subunit is essential for association of Balpha regulatory subunit but not SG2NA, striatin, or polyomavirus middle tumor antigen.
      )
      01(
      • Sontag J.M.
      • Nunbhakdi-Craig V.
      • Mitterhuber M.
      • Ogris E.
      • Sontag E.
      Regulation of protein phosphatase 2A methylation by LCMT1 and PME-1 plays a critical role in differentiation of neuroblastoma cells.
      )
      01(
      • Kaur A.
      • Denisova O.V.
      • Qiao X.
      • Jumppanen M.
      • Peuhu E.
      • Ahmed S.U.
      • et al.
      PP2A inhibitor PME-1 drives kinase inhibitor resistance in glioma cells.
      )
      ΔL309<0.15>2>2(
      • Yu X.X.
      • Du X.
      • Moreno C.S.
      • Green R.E.
      • Ogris E.
      • Feng Q.
      • et al.
      Methylation of the protein phosphatase 2A catalytic subunit is essential for association of Balpha regulatory subunit but not SG2NA, striatin, or polyomavirus middle tumor antigen.
      )
      0.04 ± 0.040.1 ± 0.10.21 ± 0.191.52 ± 0.40(
      • Kaur A.
      • Denisova O.V.
      • Qiao X.
      • Jumppanen M.
      • Peuhu E.
      • Ahmed S.U.
      • et al.
      PP2A inhibitor PME-1 drives kinase inhibitor resistance in glioma cells.
      )
      000.15 ± 0.111.30 ± 0.35(
      • Kaur A.
      • Denisova O.V.
      • Qiao X.
      • Jumppanen M.
      • Peuhu E.
      • Ahmed S.U.
      • et al.
      PP2A inhibitor PME-1 drives kinase inhibitor resistance in glioma cells.
      )
      <0.2011(
      • Jackson J.B.
      • Pallas D.C.
      Circumventing cellular control of PP2A by methylation promotes transformation in an Akt-dependent manner.
      )
      0<0.20<0.1000.80.30.80.60.440.90.90.9(
      • Tolstykh T.
      • Lee J.
      • Vafai S.
      • Stock J.B.
      Carboxyl methylation regulates phosphoprotein phosphatase 2A by controlling the association of regulatory B subunits.
      )
      1(
      • Nunbhakdi-Craig V.
      • Schuechner S.
      • Sontag J.M.
      • Montgomery L.
      • Pallas D.C.
      • Juno C.
      • et al.
      Expression of protein phosphatase 2A mutants and silencing of the regulatory B alpha subunit induce a selective loss of acetylated and detyrosinated microtubules.
      )
      L309A0(
      • Lee J.
      • Stock J.
      Protein phosphatase 2A catalytic subunit is methyl-esterified at its carboxyl terminus by a novel methyltransferase.
      )
      1(
      • Nunbhakdi-Craig V.
      • Schuechner S.
      • Sontag J.M.
      • Montgomery L.
      • Pallas D.C.
      • Juno C.
      • et al.
      Expression of protein phosphatase 2A mutants and silencing of the regulatory B alpha subunit induce a selective loss of acetylated and detyrosinated microtubules.
      )
      L309Q0000(
      • Liberti M.V.
      • Locasale J.W.
      The Warburg effect: how does it benefit cancer cells?.
      )
      mPP2A C111(
      • Ikehara T.
      • Ikehara S.
      • Imamura S.
      • Shinjo F.
      • Yasumoto T.
      Methylation of the C-terminal leucine residue of the PP2A catalytic subunit is unnecessary for the catalytic activity and the binding of regulatory subunit (PR55/B).
      )
      PME-1 OE1(
      • Nunbhakdi-Craig V.
      • Schuechner S.
      • Sontag J.M.
      • Montgomery L.
      • Pallas D.C.
      • Juno C.
      • et al.
      Expression of protein phosphatase 2A mutants and silencing of the regulatory B alpha subunit induce a selective loss of acetylated and detyrosinated microtubules.
      )
      LCMT1 KD<0.100.160.420.160.160.660.33<0.051.161.161.16(
      • Tolstykh T.
      • Lee J.
      • Vafai S.
      • Stock J.B.
      Carboxyl methylation regulates phosphoprotein phosphatase 2A by controlling the association of regulatory B subunits.
      )
      0.220.440.660.44<0.050.550.1111(
      • Tolstykh T.
      • Lee J.
      • Vafai S.
      • Stock J.B.
      Carboxyl methylation regulates phosphoprotein phosphatase 2A by controlling the association of regulatory B subunits.
      )
      00.250.580.330.50.360.50.080.750.750.83(
      • Tolstykh T.
      • Lee J.
      • Vafai S.
      • Stock J.B.
      Carboxyl methylation regulates phosphoprotein phosphatase 2A by controlling the association of regulatory B subunits.
      )
      ABL1278000X00250X(
      • Yabe R.
      • Tsuji S.
      • Mochida S.
      • Ikehara T.
      • Usui T.
      • Ohama T.
      • et al.
      A stable association with PME-1 may be dispensable for PP2A demethylation - implications for the detection of PP2A methylation and immunoprecipitation.
      )
      The table summarizes the results of experiments including but not limited to coimmunoprecipitation, mass spectrometry, liquid chromatography, and in vitro reconstruction of PP2A heterotrimers that highlights the effect of point mutations and deletions on PP2A Cα, overexpression of LCTM-1 or PME-1, and the inhibition of PME-1 across the different B regulatory subunit families and viral antigens that comprise the PP2A interactome. Each value indicated represents the fold change in association when compared to the association seen with the WT PP2A Cα (
      • Jackson J.B.
      • Pallas D.C.
      Circumventing cellular control of PP2A by methylation promotes transformation in an Akt-dependent manner.
      ,
      • Haesen D.
      • Abbasi Asbagh L.
      • Derua R.
      • Hubert A.
      • Schrauwen S.
      • Hoorne Y.
      • et al.
      Recurrent PPP2R1A mutations in uterine cancer act through a dominant-negative mechanism to promote malignant cell growth.
      ,
      • Yu X.X.
      • Du X.
      • Moreno C.S.
      • Green R.E.
      • Ogris E.
      • Feng Q.
      • et al.
      Methylation of the protein phosphatase 2A catalytic subunit is essential for association of Balpha regulatory subunit but not SG2NA, striatin, or polyomavirus middle tumor antigen.
      ,
      • Wu C.G.
      • Zheng A.
      • Jiang L.
      • Rowse M.
      • Stanevich V.
      • Chen H.
      • et al.
      Methylation-regulated decommissioning of multimeric PP2A complexes.
      ,
      • De Baere I.
      • Derua R.
      • Janssens V.
      • Van Hoof C.
      • Waelkens E.
      • Merlevede W.
      • et al.
      Purification of porcine brain protein phosphatase 2A leucine carboxyl methyltransferase and cloning of the human homologue.
      ,
      • Wei H.
      • Ashby D.G.
      • Moreno C.S.
      • Ogris E.
      • Yeong F.M.
      • Corbett A.H.
      • et al.
      Carboxymethylation of the PP2A catalytic subunit in Saccharomyces cerevisiae is required for efficient interaction with the B-type subunits Cdc55p and Rts1p.
      ,
      • Lyons S.P.
      • Greiner E.C.
      • Cressey L.E.
      • Adamo M.E.
      • Kettenbach A.N.
      Regulation of PP2A, PP4, and PP6 holoenzyme assembly by carboxyl-terminal methylation.
      ,
      • Yabe R.
      • Tsuji S.
      • Mochida S.
      • Ikehara T.
      • Usui T.
      • Ohama T.
      • et al.
      A stable association with PME-1 may be dispensable for PP2A demethylation - implications for the detection of PP2A methylation and immunoprecipitation.
      ,
      • Tolstykh T.
      • Lee J.
      • Vafai S.
      • Stock J.B.
      Carboxyl methylation regulates phosphoprotein phosphatase 2A by controlling the association of regulatory B subunits.
      ,
      • Ogris E.
      • Gibson D.M.
      • Pallas D.C.
      Protein phosphatase 2A subunit assembly: the catalytic subunit carboxy terminus is important for binding cellular B subunit but not polyomavirus middle tumor antigen.
      ,
      • Ikehara T.
      • Ikehara S.
      • Imamura S.
      • Shinjo F.
      • Yasumoto T.
      Methylation of the C-terminal leucine residue of the PP2A catalytic subunit is unnecessary for the catalytic activity and the binding of regulatory subunit (PR55/B).
      ,
      • Sontag J.M.
      • Nunbhakdi-Craig V.
      • Mitterhuber M.
      • Ogris E.
      • Sontag E.
      Regulation of protein phosphatase 2A methylation by LCMT1 and PME-1 plays a critical role in differentiation of neuroblastoma cells.
      ,
      • Nunbhakdi-Craig V.
      • Schuechner S.
      • Sontag J.M.
      • Montgomery L.
      • Pallas D.C.
      • Juno C.
      • et al.
      Expression of protein phosphatase 2A mutants and silencing of the regulatory B alpha subunit induce a selective loss of acetylated and detyrosinated microtubules.
      ,
      • Chung H.
      • Nairn A.C.
      • Murata K.
      • Brautigan D.L.
      Mutation of Tyr307 and Leu309 in the protein phosphatase 2A catalytic subunit favors association with the alpha 4 subunit which promotes dephosphorylation of elongation factor-2.
      ).
      Table 2The effect of PP2A Cα mutations and/or overexpression of LCMT-1 or PME-1 on carboxymethylation
      Change to PP2A CmPP2A C levelsRef
      H59Q<0.15(
      • Yu X.X.
      • Du X.
      • Moreno C.S.
      • Green R.E.
      • Ogris E.
      • Feng Q.
      • et al.
      Methylation of the protein phosphatase 2A catalytic subunit is essential for association of Balpha regulatory subunit but not SG2NA, striatin, or polyomavirus middle tumor antigen.
      )
      D85N<0.15(
      • Yu X.X.
      • Du X.
      • Moreno C.S.
      • Green R.E.
      • Ogris E.
      • Feng Q.
      • et al.
      Methylation of the protein phosphatase 2A catalytic subunit is essential for association of Balpha regulatory subunit but not SG2NA, striatin, or polyomavirus middle tumor antigen.
      )
      <0.10(
      • Jackson J.B.
      • Pallas D.C.
      Circumventing cellular control of PP2A by methylation promotes transformation in an Akt-dependent manner.
      )
      R89A<0.15(
      • Yu X.X.
      • Du X.
      • Moreno C.S.
      • Green R.E.
      • Ogris E.
      • Feng Q.
      • et al.
      Methylation of the protein phosphatase 2A catalytic subunit is essential for association of Balpha regulatory subunit but not SG2NA, striatin, or polyomavirus middle tumor antigen.
      )
      H118Q<0.15(
      • Yu X.X.
      • Du X.
      • Moreno C.S.
      • Green R.E.
      • Ogris E.
      • Feng Q.
      • et al.
      Methylation of the protein phosphatase 2A catalytic subunit is essential for association of Balpha regulatory subunit but not SG2NA, striatin, or polyomavirus middle tumor antigen.
      )
      T301D1–0.70(
      • Yu X.X.
      • Du X.
      • Moreno C.S.
      • Green R.E.
      • Ogris E.
      • Feng Q.
      • et al.
      Methylation of the protein phosphatase 2A catalytic subunit is essential for association of Balpha regulatory subunit but not SG2NA, striatin, or polyomavirus middle tumor antigen.
      )
      0(
      • Sontag J.M.
      • Nunbhakdi-Craig V.
      • Mitterhuber M.
      • Ogris E.
      • Sontag E.
      Regulation of protein phosphatase 2A methylation by LCMT1 and PME-1 plays a critical role in differentiation of neuroblastoma cells.
      )
      T301Stop<0.15(
      • Yu X.X.
      • Du X.
      • Moreno C.S.
      • Green R.E.
      • Ogris E.
      • Feng Q.
      • et al.
      Methylation of the protein phosphatase 2A catalytic subunit is essential for association of Balpha regulatory subunit but not SG2NA, striatin, or polyomavirus middle tumor antigen.
      )
      0(
      • Sontag J.M.
      • Nunbhakdi-Craig V.
      • Mitterhuber M.
      • Ogris E.
      • Sontag E.
      Regulation of protein phosphatase 2A methylation by LCMT1 and PME-1 plays a critical role in differentiation of neuroblastoma cells.
      )
      T304A1–0.70(
      • Yu X.X.
      • Du X.
      • Moreno C.S.
      • Green R.E.
      • Ogris E.
      • Feng Q.
      • et al.
      Methylation of the protein phosphatase 2A catalytic subunit is essential for association of Balpha regulatory subunit but not SG2NA, striatin, or polyomavirus middle tumor antigen.
      )
      1–0.71(
      • Kaur A.
      • Denisova O.V.
      • Qiao X.
      • Jumppanen M.
      • Peuhu E.
      • Ahmed S.U.
      • et al.
      PP2A inhibitor PME-1 drives kinase inhibitor resistance in glioma cells.
      )
      1(
      • Sontag J.M.
      • Nunbhakdi-Craig V.
      • Mitterhuber M.
      • Ogris E.
      • Sontag E.
      Regulation of protein phosphatase 2A methylation by LCMT1 and PME-1 plays a critical role in differentiation of neuroblastoma cells.
      )
      T304D1–0.70(
      • Yu X.X.
      • Du X.
      • Moreno C.S.
      • Green R.E.
      • Ogris E.
      • Feng Q.
      • et al.
      Methylation of the protein phosphatase 2A catalytic subunit is essential for association of Balpha regulatory subunit but not SG2NA, striatin, or polyomavirus middle tumor antigen.
      )
      1–0.71(
      • Kaur A.
      • Denisova O.V.
      • Qiao X.
      • Jumppanen M.
      • Peuhu E.
      • Ahmed S.U.
      • et al.
      PP2A inhibitor PME-1 drives kinase inhibitor resistance in glioma cells.
      )
      1(
      • Frohner I.E.
      • Mudrak I.
      • Schuchner S.
      • Anrather D.
      • Hartl M.
      • Sontag J.M.
      • et al.
      PP2AC phospho-Tyr(307) antibodies are not specific for this modification but are sensitive to other PP2AC modifications including Leu(309) methylation.
      )
      T304K1–0.70(
      • Yu X.X.
      • Du X.
      • Moreno C.S.
      • Green R.E.
      • Ogris E.
      • Feng Q.
      • et al.
      Methylation of the protein phosphatase 2A catalytic subunit is essential for association of Balpha regulatory subunit but not SG2NA, striatin, or polyomavirus middle tumor antigen.
      )
      1(
      • Sontag J.M.
      • Nunbhakdi-Craig V.
      • Mitterhuber M.
      • Ogris E.
      • Sontag E.
      Regulation of protein phosphatase 2A methylation by LCMT1 and PME-1 plays a critical role in differentiation of neuroblastoma cells.
      )
      T304N1–0.70(
      • Yu X.X.
      • Du X.
      • Moreno C.S.
      • Green R.E.
      • Ogris E.
      • Feng Q.
      • et al.
      Methylation of the protein phosphatase 2A catalytic subunit is essential for association of Balpha regulatory subunit but not SG2NA, striatin, or polyomavirus middle tumor antigen.
      )
      1(
      • Sontag J.M.
      • Nunbhakdi-Craig V.
      • Mitterhuber M.
      • Ogris E.
      • Sontag E.
      Regulation of protein phosphatase 2A methylation by LCMT1 and PME-1 plays a critical role in differentiation of neuroblastoma cells.
      )
      Y307E<0.15(
      • Yu X.X.
      • Du X.
      • Moreno C.S.
      • Green R.E.
      • Ogris E.
      • Feng Q.
      • et al.
      Methylation of the protein phosphatase 2A catalytic subunit is essential for association of Balpha regulatory subunit but not SG2NA, striatin, or polyomavirus middle tumor antigen.
      )
      <0.10(
      • Kaur A.
      • Denisova O.V.
      • Qiao X.
      • Jumppanen M.
      • Peuhu E.
      • Ahmed S.U.
      • et al.
      PP2A inhibitor PME-1 drives kinase inhibitor resistance in glioma cells.
      )
      0(
      • Sontag J.M.
      • Nunbhakdi-Craig V.
      • Mitterhuber M.
      • Ogris E.
      • Sontag E.
      Regulation of protein phosphatase 2A methylation by LCMT1 and PME-1 plays a critical role in differentiation of neuroblastoma cells.
      )
      Y307F0.40–0.15(
      • Yu X.X.
      • Du X.
      • Moreno C.S.
      • Green R.E.
      • Ogris E.
      • Feng Q.
      • et al.
      Methylation of the protein phosphatase 2A catalytic subunit is essential for association of Balpha regulatory subunit but not SG2NA, striatin, or polyomavirus middle tumor antigen.
      )
      0.70–0.41(
      • Kaur A.
      • Denisova O.V.
      • Qiao X.
      • Jumppanen M.
      • Peuhu E.
      • Ahmed S.U.
      • et al.
      PP2A inhibitor PME-1 drives kinase inhibitor resistance in glioma cells.
      )
      1(
      • Sontag J.M.
      • Nunbhakdi-Craig V.
      • Mitterhuber M.
      • Ogris E.
      • Sontag E.
      Regulation of protein phosphatase 2A methylation by LCMT1 and PME-1 plays a critical role in differentiation of neuroblastoma cells.
      )
      Y307K<0.15(
      • Yu X.X.
      • Du X.
      • Moreno C.S.
      • Green R.E.
      • Ogris E.
      • Feng Q.
      • et al.
      Methylation of the protein phosphatase 2A catalytic subunit is essential for association of Balpha regulatory subunit but not SG2NA, striatin, or polyomavirus middle tumor antigen.
      )
      0(
      • Sontag J.M.
      • Nunbhakdi-Craig V.
      • Mitterhuber M.
      • Ogris E.
      • Sontag E.
      Regulation of protein phosphatase 2A methylation by LCMT1 and PME-1 plays a critical role in differentiation of neuroblastoma cells.
      )
      Y307Q<0.15(
      • Yu X.X.
      • Du X.
      • Moreno C.S.
      • Green R.E.
      • Ogris E.
      • Feng Q.
      • et al.
      Methylation of the protein phosphatase 2A catalytic subunit is essential for association of Balpha regulatory subunit but not SG2NA, striatin, or polyomavirus middle tumor antigen.
      )
      0(
      • Sontag J.M.
      • Nunbhakdi-Craig V.
      • Mitterhuber M.
      • Ogris E.
      • Sontag E.
      Regulation of protein phosphatase 2A methylation by LCMT1 and PME-1 plays a critical role in differentiation of neuroblastoma cells.
      )
      DL309<0.15(
      • Yu X.X.
      • Du X.
      • Moreno C.S.
      • Green R.E.
      • Ogris E.
      • Feng Q.
      • et al.
      Methylation of the protein phosphatase 2A catalytic subunit is essential for association of Balpha regulatory subunit but not SG2NA, striatin, or polyomavirus middle tumor antigen.
      )
      <0.10(
      • Kaur A.
      • Denisova O.V.
      • Qiao X.
      • Jumppanen M.
      • Peuhu E.
      • Ahmed S.U.
      • et al.
      PP2A inhibitor PME-1 drives kinase inhibitor resistance in glioma cells.
      )
      <0.10(
      • Jackson J.B.
      • Pallas D.C.
      Circumventing cellular control of PP2A by methylation promotes transformation in an Akt-dependent manner.
      )
      0(
      • Tolstykh T.
      • Lee J.
      • Vafai S.
      • Stock J.B.
      Carboxyl methylation regulates phosphoprotein phosphatase 2A by controlling the association of regulatory B subunits.
      )
      0(
      • Nunbhakdi-Craig V.
      • Schuechner S.
      • Sontag J.M.
      • Montgomery L.
      • Pallas D.C.
      • Juno C.
      • et al.
      Expression of protein phosphatase 2A mutants and silencing of the regulatory B alpha subunit induce a selective loss of acetylated and detyrosinated microtubules.
      )
      0.55(
      • Chung H.
      • Nairn A.C.
      • Murata K.
      • Brautigan D.L.
      Mutation of Tyr307 and Leu309 in the protein phosphatase 2A catalytic subunit favors association with the alpha 4 subunit which promotes dephosphorylation of elongation factor-2.
      )
      L309A0(
      • Lee J.
      • Stock J.
      Protein phosphatase 2A catalytic subunit is methyl-esterified at its carboxyl terminus by a novel methyltransferase.
      )
      0(
      • Nunbhakdi-Craig V.
      • Schuechner S.
      • Sontag J.M.
      • Montgomery L.
      • Pallas D.C.
      • Juno C.
      • et al.
      Expression of protein phosphatase 2A mutants and silencing of the regulatory B alpha subunit induce a selective loss of acetylated and detyrosinated microtubules.
      )
      L309Q0(
      • Sontag J.M.
      • Nunbhakdi-Craig V.
      • Mitterhuber M.
      • Ogris E.
      • Sontag E.
      Regulation of protein phosphatase 2A methylation by LCMT1 and PME-1 plays a critical role in differentiation of neuroblastoma cells.
      )
      mPP2A C1(
      • Ikehara T.
      • Ikehara S.
      • Imamura S.
      • Shinjo F.
      • Yasumoto T.
      Methylation of the C-terminal leucine residue of the PP2A catalytic subunit is unnecessary for the catalytic activity and the binding of regulatory subunit (PR55/B).
      )
      PME-1 OE<0.10(
      • Nunbhakdi-Craig V.
      • Schuechner S.
      • Sontag J.M.
      • Montgomery L.
      • Pallas D.C.
      • Juno C.
      • et al.
      Expression of protein phosphatase 2A mutants and silencing of the regulatory B alpha subunit induce a selective loss of acetylated and detyrosinated microtubules.
      )
      0.5(
      • Chung H.
      • Nairn A.C.
      • Murata K.
      • Brautigan D.L.
      Mutation of Tyr307 and Leu309 in the protein phosphatase 2A catalytic subunit favors association with the alpha 4 subunit which promotes dephosphorylation of elongation factor-2.
      )
      LCMT1 OE1.25(
      • Chung H.
      • Nairn A.C.
      • Murata K.
      • Brautigan D.L.
      Mutation of Tyr307 and Leu309 in the protein phosphatase 2A catalytic subunit favors association with the alpha 4 subunit which promotes dephosphorylation of elongation factor-2.
      )
      LCMT1 KD0(
      • Tolstykh T.
      • Lee J.
      • Vafai S.
      • Stock J.B.
      Carboxyl methylation regulates phosphoprotein phosphatase 2A by controlling the association of regulatory B subunits.
      )
      LCMT1 OE + L309Del0.6(
      • Chung H.
      • Nairn A.C.
      • Murata K.
      • Brautigan D.L.
      Mutation of Tyr307 and Leu309 in the protein phosphatase 2A catalytic subunit favors association with the alpha 4 subunit which promotes dephosphorylation of elongation factor-2.
      )
      LCMT1 OE + PME1 OE0.25(
      • Chung H.
      • Nairn A.C.
      • Murata K.
      • Brautigan D.L.
      Mutation of Tyr307 and Leu309 in the protein phosphatase 2A catalytic subunit favors association with the alpha 4 subunit which promotes dephosphorylation of elongation factor-2.
      )
      The table summarizes the results of experiments including but not limited to coimmunoprecipitation, mass spectrometry and in vitro reconstruction of PP2A heterotrimers that highlights the effect of PP2A Cα point mutations and deletions on the levels of carboxymethylation. Each value posted represents the fold change in levels of carboxymethylation when compared to the carboxymethylation levels observed by the WT PP2A Cα (
      • Jackson J.B.
      • Pallas D.C.
      Circumventing cellular control of PP2A by methylation promotes transformation in an Akt-dependent manner.
      ,
      • Haesen D.
      • Abbasi Asbagh L.
      • Derua R.
      • Hubert A.
      • Schrauwen S.
      • Hoorne Y.
      • et al.
      Recurrent PPP2R1A mutations in uterine cancer act through a dominant-negative mechanism to promote malignant cell growth.
      ,
      • Yu X.X.
      • Du X.
      • Moreno C.S.
      • Green R.E.
      • Ogris E.
      • Feng Q.
      • et al.
      Methylation of the protein phosphatase 2A catalytic subunit is essential for association of Balpha regulatory subunit but not SG2NA, striatin, or polyomavirus middle tumor antigen.
      ,
      • Wu C.G.
      • Zheng A.
      • Jiang L.
      • Rowse M.
      • Stanevich V.
      • Chen H.
      • et al.
      Methylation-regulated decommissioning of multimeric PP2A complexes.
      ,
      • De Baere I.
      • Derua R.
      • Janssens V.
      • Van Hoof C.
      • Waelkens E.
      • Merlevede W.
      • et al.
      Purification of porcine brain protein phosphatase 2A leucine carboxyl methyltransferase and cloning of the human homologue.
      ,
      • Wei H.
      • Ashby D.G.
      • Moreno C.S.
      • Ogris E.
      • Yeong F.M.
      • Corbett A.H.
      • et al.
      Carboxymethylation of the PP2A catalytic subunit in Saccharomyces cerevisiae is required for efficient interaction with the B-type subunits Cdc55p and Rts1p.
      ,
      • Lyons S.P.
      • Greiner E.C.
      • Cressey L.E.
      • Adamo M.E.
      • Kettenbach A.N.
      Regulation of PP2A, PP4, and PP6 holoenzyme assembly by carboxyl-terminal methylation.
      ,
      • Yabe R.
      • Tsuji S.
      • Mochida S.
      • Ikehara T.
      • Usui T.
      • Ohama T.
      • et al.
      A stable association with PME-1 may be dispensable for PP2A demethylation - implications for the detection of PP2A methylation and immunoprecipitation.
      ,
      • Tolstykh T.
      • Lee J.
      • Vafai S.
      • Stock J.B.
      Carboxyl methylation regulates phosphoprotein phosphatase 2A by controlling the association of regulatory B subunits.
      ,
      • Ogris E.
      • Gibson D.M.
      • Pallas D.C.
      Protein phosphatase 2A subunit assembly: the catalytic subunit carboxy terminus is important for binding cellular B subunit but not polyomavirus middle tumor antigen.
      ,
      • Ikehara T.
      • Ikehara S.
      • Imamura S.
      • Shinjo F.
      • Yasumoto T.
      Methylation of the C-terminal leucine residue of the PP2A catalytic subunit is unnecessary for the catalytic activity and the binding of regulatory subunit (PR55/B).
      ,
      • Sontag J.M.
      • Nunbhakdi-Craig V.
      • Mitterhuber M.
      • Ogris E.
      • Sontag E.
      Regulation of protein phosphatase 2A methylation by LCMT1 and PME-1 plays a critical role in differentiation of neuroblastoma cells.
      ,
      • Nunbhakdi-Craig V.
      • Schuechner S.
      • Sontag J.M.
      • Montgomery L.
      • Pallas D.C.
      • Juno C.
      • et al.
      Expression of protein phosphatase 2A mutants and silencing of the regulatory B alpha subunit induce a selective loss of acetylated and detyrosinated microtubules.
      ,
      • Chung H.
      • Nairn A.C.
      • Murata K.
      • Brautigan D.L.
      Mutation of Tyr307 and Leu309 in the protein phosphatase 2A catalytic subunit favors association with the alpha 4 subunit which promotes dephosphorylation of elongation factor-2.
      ).
      In the context of cancer, LCMT-1 and PME-1 have been shown to act as regulators of oncogenic signaling through the modulation of PP2A carboxymethylation. Importantly, knockdown of LCMT-1 or overexpression of PME-1 was shown enhance cellular transformation in the HEK TER/B56γ model. Specifically, it was shown that LCMT-1 knockdown resulted in decreased PP2A C subunit carboxymethylation and activation of the AKT/S6K pathway and that this activation of AKT signaling was necessary for the enhanced cellular transformation observed in this model system (
      • Jackson J.B.
      • Pallas D.C.
      Circumventing cellular control of PP2A by methylation promotes transformation in an Akt-dependent manner.
      ). Conversely, overexpression of PME-1 resulted in decreased carboxymethylation and increased AKT phosphorylation (
      • Wandzioch E.
      • Pusey M.
      • Werda A.
      • Bail S.
      • Bhaskar A.
      • Nestor M.
      • et al.
      PME-1 modulates protein phosphatase 2A activity to promote the malignant phenotype of endometrial cancer cells.
      ,
      • Kaur A.
      • Denisova O.V.
      • Qiao X.
      • Jumppanen M.
      • Peuhu E.
      • Ahmed S.U.
      • et al.
      PP2A inhibitor PME-1 drives kinase inhibitor resistance in glioma cells.
      ). In another study, PME-1 overexpression in glioma cells resulted in increased MEK1/2 phosphorylation and activation of downstream signaling proteins through the increased association of PP2A with the MEK1/2 complex (
      • Kaur A.
      • Denisova O.V.
      • Qiao X.
      • Jumppanen M.
      • Peuhu E.
      • Ahmed S.U.
      • et al.
      PP2A inhibitor PME-1 drives kinase inhibitor resistance in glioma cells.
      ). Similar to the studies on PP2A scaffolding point mutations, these changes in cellular signaling may be context dependent, as the overexpression of PME-1 promoted both ERK and AKT signaling in endometrial cancer and glioma cells, but this was not observed in colorectal cancer cells (
      • Wandzioch E.
      • Pusey M.
      • Werda A.
      • Bail S.
      • Bhaskar A.
      • Nestor M.
      • et al.
      PME-1 modulates protein phosphatase 2A activity to promote the malignant phenotype of endometrial cancer cells.
      ,
      • Liberti M.V.
      • Locasale J.W.
      The Warburg effect: how does it benefit cancer cells?.
      ). Combined, the effects of LCMT-1 and PME-1 expression on cellular transformation support the central role for PP2A in regulating tumorigenesis and phenocopies results obtained with individual regulatory subunit knockdown in these same models of cellular transformation.
      The levels of carboxymethylated PP2A has been correlated with cancer progression and both progression free and overall survival in patient cohorts. Decreased PP2A carboxymethylation has been associated with increased cancer progression possibly as a result of loss of specific B subunit binding to the carboxymethylated PP2A A/C dimer (
      • Kaur A.
      • Elzagheid A.
      • Birkman E.M.
      • Avoranta T.
      • Kytola V.
      • Korkeila E.
      • et al.
      Protein phosphatase methylesterase-1 (PME-1) expression predicts a favorable clinical outcome in colorectal cancer.
      ,
      • Du B.
      • Liao H.
      • Zhang S.
      Expression pattern and prognostic utility of PME-1 in patients with hepatocellular carcinoma.
      ). Additionally, modulation of PP2A carboxymethylation has been studied in the context of cancer therapeutic response, where overexpression of PME-1 was found to drive kinase inhibitor resistance in glioma cells and knockdown of PME-1 increased sensitivity to epidermal growth factor receptor (EGFR) and Polo-Like Kinase 1 (PLK1) inhibitors in lung adenocarcinoma cells (
      • Arora D.K.
      • Machhadieh B.
      • Matti A.
      • Wadzinski B.E.
      • Ramanadham S.
      • Kowluru A.
      High glucose exposure promotes activation of protein phosphatase 2A in rodent islets and INS-1 832/13 beta-cells by increasing the posttranslational carboxylmethylation of its catalytic subunit.
      ).
      Cancer cells increase the metabolism of glucose to produce increasing amounts of lactate to generate sufficient ATP to fuel the tumorigenic cellular pathways that drive cancer growth (
      • Bose S.
      • Le A.
      Glucose metabolism in cancer.
      ).The increased consumption of glucose in cancer cells may modulate PP2A activity via regulation of carboxymethylation of the catalytic subunit (
      • Stanevich V.
      • Zheng A.
      • Guo F.
      • Jiang L.
      • Wlodarchak N.
      • Xing Y.
      Mechanisms of the scaffold subunit in facilitating protein phosphatase 2A methylation.
      ,
      • Arora D.K.
      • Machhadieh B.
      • Matti A.
      • Wadzinski B.E.
      • Ramanadham S.
      • Kowluru A.
      High glucose exposure promotes activation of protein phosphatase 2A in rodent islets and INS-1 832/13 beta-cells by increasing the posttranslational carboxylmethylation of its catalytic subunit.
      ,
      • Castermans D.
      • Somers I.
      • Kriel J.
      • Louwet W.
      • Wera S.
      • Versele M.
      • et al.
      Glucose-induced posttranslational activation of protein phosphatases PP2A and PP1 in yeast.
      ). Interestingly, the scaffolding subunit that acts to immobilize and stabilize the catalytic subunit to promote LCMT-1 interaction and drive enhanced carboxymethylation of the catalytic subunit is the most frequently mutated subunit of PP2A in cancer, suggesting that cancer selects multiple mechanisms to disrupt PP2A carboxymethylation (
      • Stanevich V.
      • Zheng A.
      • Guo F.
      • Jiang L.
      • Wlodarchak N.
      • Xing Y.
      Mechanisms of the scaffold subunit in facilitating protein phosphatase 2A methylation.
      ).
      It is important to note that there are limitations in the tools available for detecting the carboxymethylation status of the C-terminal tail of the catalytic subunit, as there have been studies that have shown commonly used antibodies are unable to discriminate between the C terminus of PP2Ac, PP4c, and PP6c as a result of the high degree of sequence homology between these phosphatases. Additionally, some of the commonly used antibodies are not specific to the carboxymethylated form of the catalytic subunit (
      • Bryant J.C.
      • Westphal R.S.
      • Wadzinski B.E.
      Methylated C-terminal leucine residue of PP2A catalytic subunit is important for binding of regulatory Balpha subunit.
      ,
      • Schmitz M.H.
      • Held M.
      • Janssens V.
      • Hutchins J.R.
      • Hudecz O.
      • Ivanova E.
      • et al.
      Live-cell imaging RNAi screen identifies PP2A-B55alpha and importin-beta1 as key mitotic exit regulators in human cells.
      ,
      • Frohner I.E.
      • Mudrak I.
      • Schuchner S.
      • Anrather D.
      • Hartl M.
      • Sontag J.M.
      • et al.
      PP2AC phospho-Tyr(307) antibodies are not specific for this modification but are sensitive to other PP2AC modifications including Leu(309) methylation.
      ,
      • Mazhar S.
      • Leonard D.
      • Sosa A.
      • Schlatzer D.
      • Thomas D.
      • Narla G.
      Challenges and reinterpretation of antibody-based research on phosphorylation of Tyr(307) on PP2Ac.
      ,
      • Shouse G.
      • de Necochea-Campion R.
      • Mirshahidi S.
      • Liu X.
      • Chen C.S.
      Novel B55alpha-PP2A mutations in AML promote AKT T308 phosphorylation and sensitivity to AKT inhibitor-induced growth arrest.
      ,
      • Hein A.L.
      • Seshacharyulu P.
      • Rachagani S.
      • Sheinin Y.M.
      • Ouellette M.M.
      • Ponnusamy M.P.
      • et al.
      PR55alpha subunit of protein phosphatase 2A supports the tumorigenic and metastatic potential of pancreatic cancer cells by sustaining hyperactive oncogenic signaling.