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Biochimica et Biophysica Acta 1812 (2011) 1498–1507

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Biochimica et Biophysica Acta

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Review PP2A targeting by viral : A widespread biological strategy from DNA/RNA tumor viruses to HIV-1

Julien Guergnon a,1, Angélique N. Godet a, Amandine Galioot a, Pierre Barthélémy Falanga a, Jean-Hervé Colle b, Xavier Cayla c, Alphonse Garcia a,⁎

a Laboratoire E3 Phosphatases-Unité Signalisation Moléculaire et Activation Cellulaire, Institut Pasteur 25, rue du Dr Roux, 75015 Paris, France b Unité de Biologie des Populations Lymphocytaires, Institut Pasteur 25, rue du Dr Roux, 75015 Paris, France c Neurobiologie intégrative de la reproduction UMR 6175 INRA-CNRS-Univ. Tours-Haras Nationaux Centre INRA de Tours, PRC F-37380 Nouzilly, France

article info abstract

Article history: phosphatase 2A (PP2A) is a large family of holoenzymes that comprises 1% of total cellular proteins Received 12 May 2011 and accounts for the majority of Ser/Thr phosphatase activity in eukaryotic cells. Although initially viewed as Received in revised form 22 June 2011 constitutive housekeeping , it is now well established that PP2A proteins represent a family of highly Accepted 5 July 2011 and sophistically regulated phosphatases. The past decade, multiple complementary studies have improved Available online 10 August 2011 our knowledge about structural and functional regulation of PP2A holoenzymes. In this regard, after

Keywords: summarizing major cellular regulation, this review will mainly focus on discussing a particulate biological Viruses strategy, used by various viruses, which is based on the targeting of PP2A enzymes by viral proteins in order to Cancer specifically deregulate, for their own benefit, cellular pathways of their hosts. The impact of such PP2A Neurodegeneration targeting for research in human diseases, and in further therapeutic developments, is also discussed. PP2A holoenzymes © 2011 Elsevier B.V. All rights reserved. PP2A and human therapy

1. Introduction PP2C [1]. Today, based on sequence and structural homology of catalytic subunits, and enzymatic mechanisms, Ser/Thr phospha- An estimated one third of the cellular proteins is reversibly tases are divided into two large families, known as PPP (Phospho- phosphorylated on specific serine/threonine (Ser/Thr) residues. Protein Phosphatases) and PPM (Protein Phosphatases Metal- Reversible protein , a dynamic process controlled by dependent). Moreover, other related PPP proteins, such as PP4, the coordinated action of phosphorylating (kinases) and depho- PP5, PP6 and PP7, that occurred at low abundance and are expressed sphorylating (phosphatases) enzymes, is a key regulatory mechanism in a tissue-and development-specific manner, have also been that regulates multiple intracellular pathways. There are many characterized [2]. different Ser/Thr-specific kinases, each of them being subjected to a Numerous viruses ranging from DNA-Tumor viruses to retrovi- specific regulation, that phosphorylate Ser/Thr residues on specific ruses have developed sophisticated strategies in order to deregulate protein substrates. In contrast, to antagonize kinase activities through cellular functions via PP2A. This review summarizes our recent specific dephosphorylation of Ser/Thr residues, cells used a very knowledge on cellular PP2A proteins and discusses the functional limited number of phosphatase catalytic subunits that rely on specific relevance of interactions between PP2A and viral-proteins and their interactions with regulatory proteins to generate a large diversity of impact on deregulation of cellular pathways. Starting from the phosphatase holoenzymes. biological consequences of these interactions and their ability to The Ser/Thr phosphatases were originally classified into type-1 specifically target the deregulation of particular cellular pathways, we (PP1) or type 2 (PP2), according to their substrate specificity and also discuss how the mimicry of PP2A binding and viral proteins may sensitivity to pharmacological inhibitors. In addition type-2 phos- also serve to evolve new therapeutic developments. phatases were subdivided into major classes, including PP2A, calcium-dependent calcineurin (PP2B) and magnesium-dependent 2. Structure of cellular PP2A holoenzymes

PP2A refers to a large family of dimeric and heterotrimeric Ser/Thr phosphatases that account for the majority of Ser/Thr phosphatase ⁎ Corresponding author. Tel.: +33140613821. E-mail address: [email protected] (A. Garcia). activity in eukaryotic cells [3]. PP2AD, the PP2A core dimeric 1 Present adress: Immunité Université Pierre et Marie Curie 91 Boulevard de l'Hôpital comprises a 36 kDa catalytic PP2Ac (also called PPP2C) and a 75013 Paris France. structural A subunit, also known as PR65 or PPP2R1 (Fig. 1). In

0925-4439/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.bbadis.2011.07.001 J. Guergnon et al. / Biochimica et Biophysica Acta 1812 (2011) 1498–1507 1499

3. Regulation of cellular PP2A holoenzymes

3.1. Regulation by B subunits

Regulation of PP2A primarily involves a combinatorial assembly of

type-B subunits with PP2AD core enzyme and, in addition, PP2A activity, substrate specificity and cellular localization depend on the

nature of a B subunit. For example, in contrast to dimeric PP2AD, the trimeric ABαC holoenzyme efficiently dephosphorylates the brain microtubule-associated protein Tau, suggesting that Tau-dephos- phorylation is critically regulated by specificBα targeting of the trimeric holoenzyme [10,11]. However, as illustrated in Fig. 1,in addition to type-B subunits both specific cellular regulators and post- translational modification also regulate PP2A activity. For example binding of α4 protein (Tap42 in yeast) to the PP2A catalytic subunit generates a specific heterodimer [12]. This dimeric Tap42/α4-PP2A phosphatase can repress apoptosis in mammalian cells through negative regulation of c-Jun and [13].

3.2. Regulation by post-translational modifications of the catalytic subunit

Methylation and phosphorylation, two distinct post-translational

modifications of the PP2Ac C-terminus 304-TPDYFL-309 sequence, regulate subunit composition of PP2A as well as catalytic activity and substrate specificity of holoenzymes. PP2A phosphorylation on both Thr and Tyr residues inactivates PP2A and its reactivation requires intramolecular autodephosphorylation catalyzed by PP2A [14,15].

Thr304 phosphorylation is dependant on an autophosphorylation- activated protein kinase [16]. Tyr307 phosphorylation was found in fibroblasts overexpressing pp60 v-src and in chronic myelogenous leukemia cells that overexpress the PP2A inhibitor called SET or I2PP2A [17]. Reversible methylation regulates the binding of the third variable

type B subunit [18]. The free carboxyl group of the C-terminal Leu309 residue is methylated by the S-adenosylmethionine-dependent LCMT1 (leucine carboxyl methyltransferase 1) [19,20]. Demethylation Fig. 1. Structure and regulation of dimeric and trimeric cellular PP2A holoenzymes. A) fi Three-dimentional crystal structure of a trimeric PP2A holoenzyme bound to is catalyzed by a speci c phosphatase methylesterase, PME-1 [21,22]. microcystin-LR (ref [175] see also ref [11] for recent published crystal structure of the PP2A1 holoenzyme). The scaffold (Aα), catalytic (Cα), and regulatory B (Bα) subunits are shown in blue violet, pink and green respectively. (B) Regulations of 3.3. Regulation by PTPA dimeric and trimeric cellular PP2A holoenzymes: The catalytic subunit PP2Ac has never been found as a free molecule, but is usually associated to the A structural subunit The Phosphotyrosyl phosphatase activator (PTPA) was initially constituting the dimeric core A/C (illustration is based on 2ie3.pdb structure) or, considered as a B subunit that interacted with AC core to specifically alternatively, to mammalian a4 protein, an orthologue of the yeast Tap42 protein. The activate a phosphotyrosyl phosphatase (PTPase) activity [23]. How- binding of a structurally unrelated third variable (B/B′/B″/B‴’) subunit to PP2AD generates the diversity of trimeric holoenzymes. Reversible methylation of PP2A-C ever later findings indicate that PTPA, also renamed PP2A Phosphatase leu309 by leucine carboxyl methyltransferase (LCMT) and phosphatase methylesterase Activator, functions as a able to reactivate the Ser/Thr (PME-1) regulates the binding of the third variable type B subunits. In addition binding phosphatase activity of inactive PP2A [24,25]. Indeed, ATP–Mg2+ of the phosphatase regulator PTPA to the PP2A–PME-1 inactive complex, provokes a conformational change leading to dissociation of PME-1 allowing the formation of stimulated peptidyl prolyl cis/trans isomerase activity of PTPA is trimeric holoenzyme [161]. required to reactivate inactive PP2A and PTPA binding to the PP2A- PME–1 inactive complex, provokes a conformational change leading to dissociation of PME-1 that permits the formation of an active trimeric holoenzyme [26]. mammals the A and C subunits of the core enzyme are constitutively expressed and two distinct genes encode closely related versions of 3.4. Cellular interacting proteins the PP2A A (Aα and Aβ) [4,5] and C (Cα and Cβ) subunits [6,7].

Cells contain a pool of dimeric PP2AD enzyme that can exist alone In addition to type-B subunits a large and still growing number of [8] or also be associated with a third partner corresponding to a cellular proteins can interact with one or more PP2A subunits or can variable “type B” subunits. PP2A B-type subunits contain a split A associate with a specific holoenzyme. As illustrated in Table 1 [10,27– subunit-binding domain allowing competition with other B-type 85], several cellular partners including protein kinases, cytoskeleton, subunits for A subunit binding [9]. As illustrated in Fig. 1, four major structural, regulatory proteins such as receptors or transcription classes of “type B” subunits have been characterized and referred as B, factors controlling major intracellular pathways (Hox, Wnt, Ras,…), B′,B″, and B‴. In addition, each of the “type B” subunits of PP2A can can form stable active complexes with PP2A. The formation of such exist in several isoforms that potentially generate a multitude of complexes represents a major way to regulate PP2A activity and (NN75) holoenzyme compositions. PP2A-mediated intracellular pathways. 1500 J. Guergnon et al. / Biochimica et Biophysica Acta 1812 (2011) 1498–1507

Table 1 Cellular PP2A interacting proteins. Studies in the past decade allowed us to identify a plethora of cellular PP2A-interacting partners that define paradigms towards multiple PP2A-mediated biological processes.

Protein Effects Ref.

[PP2A]–[Protein–Kinases] Aurora-A Binding to PP2Ac is involved in Aurora degradation during mitosis. [27]

CaM Kinase IV Binds PP2A1 holoenzyme. Associates and regulates CaM Kinase IV. [28] Casein kinase 2 (CK2) Binds dimeric PP2A/C. Involved in Ras/raf/Mapk pathway. [29] JAK2 Interacts with PP2Ac (probably through dimeric AC) uppon IL-11 [30] induction in 3T3-L1 adipocyte cells. KSR (kinase suppressor of Raf) Binds to dimeric (non stimulated cells) or trimeric PP2A (stimulated cells). [31]

PAK1 (P21 activated kinase) PAK1 is a substrate for PP2A1 in a preexisting Kinase–phosphatase complex. [32]

P70-S6kinase P70-S6kinase is a substrate for PP2A1 in a preexisting Kinase–phosphatase complex. [33] PKCα PP2Ac and PKCα are physically associated in Mast cells. PP2A controls [34] PKCα phosphorylation. PKCδ PKCδ physically associates with the PP2Ac to increase its phosphatase activity. [35] PKR Interacts with B56 subunit of trimeric PP2A and regulates the activity of the [36] translation initiation factor eIF4. RAF-1 Interacts with dimeric core A/C to regulate RAF/Mek pathway. [37] Src Binds to dimeric core A/C. PP2A inhibits src activity. [38]

[PP2A]–[Mitosis/cell cycle/cytoskeleton] Cas (p130 Crk-associated Src substrate) Binds dimeric core A/C. Involved in cell cycle specific dephosphorylation of Cas. [39] Cdc6 Binds trimeric A/C/PR48 holoenzyme and is involved in cell cycle regulation. [40] Cdc25c Binds A/C/B56δ holoenzyme and is involved in mitosis regulation. [41] CG-NAP (AKAP 350) Binds PR130 subunit of trimeric A/C/PR130 holoenzyme, involved in cell cycle and [42] centrome dynamic regulation. Cyclin G1 P53-dependent binding to PP2A-B′ subunit. [43] Cyclin G2 Binds to B′ subunits and provokes G1/S cell cycle arrest. [44] DNA polymerase α primase (Pol-Prim) Cell cycle-dependent association of demethylated dimeric core A/C with Pol-Prim. [45] HOX 11 Binds to C-subunit to disrupt a G2/M cell cycle checkpoint. [46] P107 R3/PR59 subunit of trimeric PP2A binds P107 but not related pRB protein. Involved [47] in G1 cell cycle arrest.

NF-L, NF-M, and NF-H PP2A1 subunits associate with NF proteins. PP2A mediated dephosphorylation of NF [48,49] (Neurofilament proteins) proteins promotes assembly of NF proteins.

Vimentin Binds the Bα subunit and is dephosphorylated by PP2A1 in Hs68 fibroblasts. [50] Shugoshin Binds to trimeric AB′C holoenzyme to regulate centromeric protection of cohesion [51,52] during meiosis. The Shugoshins-PP2A complex dephosphorylates cohesin to prevent cleavage by separase during meiosis I. Greatwall Gwl is a promitotic kinase that phosphorylates α-endosulfine (Ensa). [53] Ensa suppresses antimitotic activity the trimeric PP2A-B55δ holoenzyme.

[PP2A]–[Receptor/transporter/membrane]

β-adrenergic receptor Binds PP2A1 holoenzyme. Regulates receptor resensitization and recycling to [54] plasma membrane. Estrogen receptor α Binds to C subunit. PP2Ac controls the phosphorylation of Erα. [55] (Erα) NMDA receptor (NMDAR) PP2Acβ associates with NR3A subunit of NMDAR. NMDAR stimulation regulates [56] both PP2A activity and PP2A association with the receptor. Shc adaptor protein PP2A associates with the PTB domain of Shc in the basal state and dissociates in [57] response to growth factors (insulin and EGF). Amine transporters Binds PP2Ac. Regulates heteromeric assemblies involving biogenic amine in presynaptic [58] transporter proteins. E-cadherin Binds PP2Ac. Required for stabilization of E-cadherin/β-catenin complexes at the [59] plasma membrane. Class C L-type calcium Binds dimeric core A/C. Antagonizes PKA-mediated chanel phosphorylation. [60] chanel STAT5 Binds to PP2Ac upon IL-3 stimulation of in myeloid progenitors. PP2A plays a [61] negative regulatory role in regulating the IL-3 signaling pathway via formation of complexes with JAK2 and Stat5.

[PP2A]–[Apoptosis/cell activation/diseases] Bcl-2 Bcl-2 interacts with PP2A. PP2A dephosphorylates Bcl-2. [62] Caspase 3 Binds and cleaves structural Aα subunit. Stimulates PP2A activity and apoptosis [63] induced by anti-Fas antibody. CD28 and CTLA-4 Two T lymphocyte cell surface receptors bind trimeric PP2A through their short [64] receptors intracytoplasmic domain. HRX Binds PP2A through I2PP2A involved in acute leukemias. [65] PP2A I2 (SET) Specific PP2A inhibitor involved in acute myeloid leukemiogenesis. [66] PP2A I1 (PHAP1, mapmodulin) Potent heat stable Inhibitor of PP2A. [67]

Tau Binds to Bα subunit of trimeric PP2A1 holoenzyme. Tau dephosphorylation promotes [10,68] microtubule binding and stabilization. Hyperphosphorylated tau provokes neurofibrillary tangles in brains of patients with Alzheimer's disease (AD) and related tauopathies. CIP2A Human oncoprotein that interacts with PP2A-A subunit and inhibits PP2A-mediated [69] dephosphorylation and subsequent proteolytic degradation of Myc protein. J. Guergnon et al. / Biochimica et Biophysica Acta 1812 (2011) 1498–1507 1501

Table 1 (continued) Protein Effects Ref.

[PP2A]–[Transcription-protein synthesis]

TRIP-Br1 Binds to Bα subunit of trimeric PP2A1 holoenzyme and regulates transcription and [70] cell cycle progression. eRF1 Binds to C subunit of dimeric core A/C. Regulates termination of protein synthesis. [71] IKK γ subunit (also named NEMO) Binding to PP2Ac is involved in the regulation of I-κB kinase. [72] RelA (member of the NF-κB family of Binds the A subunit of dimeric core A/C. PP2A dephosphorylates RelA. [73] transcription factors) SP1 Interacts with PP2Ac in T cells and remains dephosphorylated during cell cycle. [74,75] Heat Shock transcription HSF2 binds to structural A subunit and competes with binding of C-subunit. [76] factor 2 Carmal1 Binds structural Aα subunit (dimeric core A/C) involved in T-cell activation. [77]

[PP2A]–[Hox/Wnt/Alpha4/Ca2+ pathways] Adenomatous Polyposis Binds B56 subunit. Regulates Wnt signaling pathway. [78] Coli (APC) Axin Binds C subunit. Regulates Wnt signaling pathway. [79] Mid1 Interacts with α4 protein in a complex with the PP2Ac. Involved in ubiquitin-dependent regulation of [80] microtubule-associated phosphatase 2A. SCR (sex Combs Reduced) homologouus to human Binds B56 subunit of trimeric Holoenzyme. PP2A positively modulate homeotic function. [81] HOX5 and HOX6 Striatin and S/G(2) nuclear autoantigen (SG2NA), Two highly related WD40 repeat proteins that bind dimeric core A/C. May function as scaffolding proteins type B [82] proteins involved in Ca2+-dependent signal transduction pathways. Tap42/α4 Interacts with PP2Ac in Tor–Rapamycin pathway. Mammalian homolog α4 involved in B cell activation. [83] alpha4-b Interacts with PP2Ac.alpha4-related gene (66% identity in amino-acid sequence) expressed selectively in the [84] brain and testis. Type 2A-interacting protein (TIP) Binds PP2Ac and inhibits PP2A activity. Expressed in human cancer cells. [85]

4. PP2A holoenzymes are specific targets of viral proteins variable subunits (B′/B56/PR61, B56γ), resulting in activation of PI3K/ Akt, Wnt, and c-Myc pathways involved in tumors [117]. Viral proteins encoded by different viruses can form stable Py middle T-antigen (PyMT) associates with cell membranes and complexes with PP2A holoenzymes or, with cellular PP2A interacting provokes tumors by mimicking an activated growth factor receptor proteins, in order to selectively subvert important cellular pathways. [118]. The interaction between PP2A dimeric core enzyme and PyMT The functional effects of PP2A association with a are is required for the subsequent assembly of the transformation- critical for the accomplishment of viral life cycle. As illustrated in competent PyMT complex that includes pp60c-src and phosphatidy- Table 2, and in Ref. [86–102], the diversity of already identified PP2A- linositol 3-kinase [118,119], in that later case PP2A associated to PyMT associated viral proteins suggests a widespread viral strategy that can carries a tyr-phosphatase activity sensitive to okadaic acid [96]. be schematized in the following points. Recent discovery of human polyomaviruses has yield a new interest in the role played by encoded proteins in human disease. (MCV) is the etiological 4.1. Viral oncoproteins encoded by DNA/RNA tumor viruses can interact agent of human Merkel cell carcinomas, a highly aggressive skin with PP2A to activate pathways involved in cell transformation and in cancer [120]. PP2A targeting by MCV small T-Antigen was found to be diseases required for enhanced viral replication [95]. JC virus (JCV) infects oligodendrocytes and is the etiological agent PP2A is a and pharmacological inhibitors of of a progressive multifocal leukoencephalopathy (PML), a rare PP2A such as microcystin and okadaic acid act as potent tumor demyelinating disease that affects elderly or immunocompromised promoters [103]. PP2A has been implicated in the regulation of patients. Two small JCV virally encoded proteins, and several major signaling pathways, including Wnt, PI3K/Akt, MAPK, small t antigen can interact with PP2A. Agnoprotein phosphorylation, and c-Myc, that result in cell transformation [104]. Consistently, which is antagonistically controlled by PKC and PP2A, is essential for inactivating mutations of PP2A A subunits have been detected in the viral life cycle. The PP2A inhibition by small t antigen inhibits the human cancers [105–108] and the inactivation of these subunits dephosphorylation of agnoprotein by PP2A. In addition, interplay contributes to cell transformation [109,110]. In this regard some DNA between PP2A with agnoprotein and small t antigen could be tumor viruses of the papovae family encode small proteins (around important for the PML disease progression [91]. 100 aa residues), such as small t antigens and polyomavirus middle T, BK virus (BKV) infects kidney epithelial cells and is the etiological that are found associated with dimeric core AC in infected or agent of a polyomavirus-associated nephropathy (PVN) that occa- transfected cells and behaves as potent [111]. Studies of sionally develops in renal transplant [121]. Based on the inactivation the transformation induced by the DNA tumor Simian virus 40 (SV40) of the functions of the tumor suppressor mediated by proteins p53 or Murine polyoma virus (Py) allowed a better understanding of the and pRB family, a role of BKV in oncogenesis processes has been pathways involved in cancers [112]. suggested [121]. Moreover the BKV small t antigen binds to the A/C Biochemical, mutational and more recently structural experiments PP2A core subunits suggesting a contribution of PP2A in the confirm that SV40 small t directly interacts with the PP2A scaffolding underlying mechanism of cell transformation [122]. A subunit and regulates PP2A activity by displacing regulatory B Human papillomaviruses (HPV), a major etiologic agent of cervical subunits from the A subunit [113,114]. Functional experiments have cancer [123] encode HPV-E7, a small oncogenic protein of approximately also clearly demonstrated that the binding of SV40 small t to PP2A is a 100 amino acids, that regulates viral replication and is involved in prerequisite for its cell transformation occurrence [115,116].In infected host cell transformation [124]. It has been recently reported that addition, as illustrated in Fig. 2 it has been demonstrated that SV40 binding of HPV-E7 to PP2A dimeric A/C core leads to the constitutive small t inhibits PP2A through a specific targeting and displacing of activation of the PI3K/PKB a survival/transforming pathway [94]. 1502 J. Guergnon et al. / Biochimica et Biophysica Acta 1812 (2011) 1498–1507

Table 2 A Viral PP2A interacting proteins. A mutations CIP2A Virus Protein Effects Ref. Adenovirus Adenovirus Binds to AC–R2 complex; formation of [86,87] T E4orf4 protein complex required for E4orf4- U mediated apoptosis. B Subunit BKV BKV small t Binds A and C subunits. Possible role [88] Deregulation M antigen in oncogenesis. O EBV EBNA-LP Truncated EBNA-LP interacts with [89] A PP2Ac to inhibit apoptosis. A possible R mechanism for EBV persistence. C S JCV JCV agnoprotein Binds PP2Ac. Co-precipitates with [90] small t Antigen and PP2A. Cell effectors JCV JCV small t May bind dimeric core A/C dimer. Co- [91] Methyl regulators antigen precipitates with PP2Ac and (SET-I2PP2A/ α4…) agnoprotein and inhibits PP2A- (PME1/PPM1) mediated dephosphorylation of agnoprotein. B HCV HCV NS5A Binds dimeric core A/C to up-regulate [92] B56/ PP2A activity to inhibits of IFN-α B56/ PR61γ Small t signaling. PR61γ HIV-1 HIV-1 Binds A subunit and PP2A1 [93] A A holoenzyme. Role in cell cycle arrest and apoptosis. HPV HPV-E7 Binds dimeric core A/C and inhibit [94] CC Akt/PKB dephosphorylation. Small t MCV MCV small t Binding to PP2A allows optimal [95] antigen replication. Polyoma Polyomavirus Binds to AC dimer; increases [96,97] Activation of survival &transforming pathways middle tumor phosphatase activity of PP2A. antigen RAS Polyoma Polyomavirus Binds to dimeric core A/C allowing [97,98] -c-Myc + Wnt -other transcription Factors PI3K/Akt/ small tumor activation of the c-src Mek pathway antigen and phosphatidylinositol 3-that (CREB-AP1-SP1-E2F-NF-κB) pathways facilitates host-cell transformation. SV40 SV40 small Binds to AC dimer; displaces R2 [99] tumor antigen subunits; inhibits PP2A activity. HTLV-1 Tax The human T-lymphotropic [100,101] TUMORS type-I transactivator Tax interacts via a tripartite protein interaction that also involves IKKγ. The association of Fig. 2. PP2A inhibition can induce oncogenic pathways. A) Several mechanisms can Tax with this complex inhibits PP2A regulate transforming pathways through PP2A: Mutations in structural A subunit that activity maintaining Tax-PP2A-IKK in decrease PP2A activity have been characterized in several human cancers [106]. ′γ an active state. Truncation of PR61/B provokes radio-resistance and increases metastasis [161]. PP2A Deregulation PP2A methylation [151] and upregulation of oncogenic PP2A inhibitors West Nile West Nile virus Binds I2 to increase PP2A activity. [102] α – protein such as SET, CIP2A or 4 [154 156]. B) PP2A and SV40 small t: an example of how viral (WNVC) deregulation of PP2A-mediated intracellular pathways can lead to cell transformation. SV40 small t displaces some regulatory subunits to activate transforming pathways. Interestingly the suppression of PR61/B′γ subunit mimics the transforming effect of SV40 small t [117]. Epstein–Barr virus (EBV), a human γ-herpes virus, is an etiologic agent of human B-cell cancers, including Burkitt's lymphoma (BL), nation of its progeny. E4orf4 protein induces this process by

Hodgkin's lymphoma, and in immunocompromised patients induces interacting with the regulatory Bα subunit of trimeric PP2A1 lymphoproliferation [125]. The EBV-encoded leader protein (EBNA- holoenzyme in infected or transformed cells but not in normal LP) is one of the six virally encoded nuclear antigens that have been human primary cells [87,128]. This p53 independent cell death involved in EBV-induced B-cell transformation. Interestingly, Garibal mediated by E4Orf4 resembles apoptosis in some human cancer cell et al., [89] have recently proposed a mechanism for EBV persistence, in lines [129,130]. However other reports suggest that E4Orf4 may infected cells, based on the inhibition of PP2A-mediated apoptotic induce multiple death processes, depending on the physiological state pathway resulting from the binding of EBNA-LP with PP2Ac. or type of cell. For example in human H1299 lung carcinoma cells, cell death is caspase independent and appears to occur by mitotic

4.2. Adenoviral protein E4orf4 can interact with PP2A1 to induce catastrophe following mitotic arrest [131,132]. Recent studies have apoptosis reported that E4orf4 binding reduced the activity of B55 containing PP2A holoenzymes suggesting that E4orf4 toxicity may result from Adenovirus early region 4 open reading frame 4 (E4orf4) is an the failure to dephosphorylate substrates involved in cell cycle early viral gene transcribed from the E4 promoter of human progression [131]. adenoviruses. The small 114-residue E4orf4 protein is a highly multifunctional viral regulator that persists during infection [126]. 4.3. Flaviviral proteins interact with PP2A partners to upregulate PP2A E4orf4 counteracts the effect of oncogenic viral protein E1A, down activity and to deregulate specific PP2A-mediated pathways regulates cellular transcription factors (AP1, E2F) and also negatively regulates expression of the E4 promoter [127]. Interestingly during virus (HCV), a small hepatotropic virus classified within early steps of infection the products of the viral gene (E1) (E1B55K the Flaviviridae family, is the major etiological agent of chronic hepatitis and E1B19K) inactivate p53 and prevent activation of the apoptotic which often causes two major chronic liver diseases: cirrhosis and machinery. Later during the viral replication phase, the virus evolved hepatocellular carcinoma [133]. The HCV-encoded non-structural a p53-independent apoptotic process that could allow the dissemi- protein NS5A is a multifunctional serine phosphoprotein of 56– J. Guergnon et al. / Biochimica et Biophysica Acta 1812 (2011) 1498–1507 1503

58 kDa, involved in the control of viral replication and in viral-induced capacity and apoptotic properties [93]. In conclusion PP2A targeting by pathogenesis [134]. Recent work indicates that NS5A is a new type B viral proteins can deregulate proliferative and survival pathways. PP2A subunit that specifically interacts in vivo and in vitro with PP2A A/ HPV-E7, HTLV Tax and SV40 T antigens interact with PP2A to activate C core to up-regulate PP2A activity [92]. Up-regulation of PP2A increases survival pathways and to promote cell proliferation and cell NS3 helicase activity and inhibits of IFN-α-induced Jak-STAT signaling, transformation. In contrast adenoviral E4Orf4 and HIV-1 Vpr interact two events associated to efficient HCV replication [135]. with PP2A to induce apoptosis and cell cycle arrest [132,142,145]. West Nile virus (WNV), another virus of the Flaviviridae family, can cause fatal neurological diseases [136]. WNV capsid protein (WNVC) 4.6. Viral complementary strategies to deregulate PP2A is a pathogenic determinant and the non-structural functions of this protein involve specific interactions with host cell-encoded proteins. Deregulation of PP2A activity in the absence of characterized In this regard, WNVC interaction with the host cell-encoded virally-encoded PP2A interacting proteins also exists in infected cells. phosphatase inhibitor I2PP2A, up regulates PP2A activity. WNVC- For instance, human cytomegalovirus (CMV), an Herpesviridae that induced up regulation of PP2A, is also involved in down regulation of infects more than 50% population in western countries and can cause AP1 activity, and could eventually play a role in virus-induced congenital malformation, carries a cellular PP2A holoenzyme proba- neurodegeneration [102]. bly involved in early cellular phosphorylation shut off [146]. (HBV), a human hepadnavirus that causes acute and chronic 4.4. Viral proteins encoded by interact with PP2A and can hepatitis associated with hepatocarcinogenesis, also induces a deregulate cell cycle and cell survival significant up-regulation of PP2Ac that correlates to inhibition of IFN-α signaling [147]. Human T-lymphotropic virus type I (HTLV-1) is the etiological agent of adult T-cell leukemia/lymphoma (ATL) [137]. The viral 5. PP2A: viruses and human diseases transactivator/oncoprotein Tax, which regulates viral replication, is a potent constitutive activator of NF-κB and is also critically required to 5.1. PP2A dysfunction and human diseases deregulate cell cycle and to initiate cell transformation. The holo-IκB kinase (IKK) comprises two catalytic subunits, IKKα and IKKβ, Consistently with a key role of PP2A in the control of multiple associated with a regulatory subunit, IKKγ/NEMO (NF-κB essential cellular processes, a deregulation of some PP2A-regulated signaling modulator) regulates NF-κB activity [138]. IKK binds PP2A and via a pathways, such as the PI3K/Akt survival pathways, can be associated tripartite protein–protein interaction, Tax, IKKγ/NEMO, and PP2A with several major human diseases including diabetes/obesity form a stable ternary complex [100]. PP2A inhibition by NEMO-bound [148,149], cancer [118] and neurodegeneration [150]. Interestingly, Tax maintains IKK in the phosphorylated/active state that is required as illustrated in Fig. 2A, several mechanisms involved in PP2A for phosphorylation/degradation of I-κB and subsequent activation of dysfunction such as genetic alterations of A PP2A subunit [104], NF-κB [100,139]. deregulation of PP2Ac methylation [151] or over-expression of endogenous cellular proteins [69] can lead to human diseases. 4.5. Tat and Vpr, two viral proteins encoded by human Concerning cancer, several endogenous PP2A inhibitor proteins immunodeficiency virus type 1 (HIV-1) interfere with PP2A pathways have been recently involved in human malignancies. In this regard, the human oncoprotein named Cancerous Inhibitor of PP2A (CIP2A) is HIV-1, a retrovirus of the lentivirus family, is the etiologic agent of a specific PP2A inhibitor which is over expressed in several types of AIDS. HIV-1 transactivator Tat (HIV-1 Tat) is a small regulatory human cancer tissues and prevents PP2A-mediated degradation of c- protein, containing between 86 and 101 residues, that plays a pivotal Myc protein [69,152–154]. CIP2A expression is also upregulated by role in both viral replication and pathogenesis [140]. Interestingly bacterial oncoprotein CagA, which is produced by Helicobacter pylori, HIV-1 Tat modulates the expression of several cellular genes and an etiologic factor of duodenal and stomach ulcers, and gastric cancer displays pro-apoptotic properties. Moreover, Tat association with the [155]. Furthermore, upregulation of SET/I2PP2A, by the BCR/ABL promoters of PPP2R1B (structural subunit Aβ) and PPP2R5E (regu- contributes to leukemogenesis [17]. In addition to SET/ latory subunit B′ε) activates their transcription resulting in over I2PP2A and CIP2A, other endogenous proteins were found to inhibit expression of PP2A subunits and up regulation of PP2A activity in PP2A activity in cancer cells. For example, a recent study by Chen et al. lymphocytes. These results suggest that the increased PP2A-activity [156] indicates that upregulation of α4 in several types of primary induced by Tat could participate to Tat-mediated apoptotic mecha- human cancers contributes to cell transformation. nism [141]. In the case of neurodegenerative diseases, the aberrant hyperpho- HIV-1 Vpr is a small virally encoded 14 kDa accessory virion- sphorylation of Tau protein, leading to neurofibrillary destabilization associated protein containing 96 residues that performs multiple is one of the hallmarks of tauopathies including Alzheimer disease functions in vitro and plays an important role in viral pathogenesis [157]. It has been clearly established that such abnormal hyperpho-

[142]. Similarly to HIV-1 Tat, HIV-1 Vpr is also a cell penetrating sphorylated tau can result from specificPP2A1 deregulation in protein that can display pro-apoptotic properties [142]. Interestingly response to upregulation of endogenous inhibitors [158] or to soluble entire protein has been identified in the serum and inhibition of PP2Ac carboxy-methylation [159]. cerebrospinal fluid of HIV-1 infected individuals suggesting that Vpr could penetrate into cells acting by a bystander effect in non infected 5.2. Exploration of PP2A dysfunction and viral proteins: lessons from cells [143]. It has been firstly reported that Vpr induces cell cycle G2 PP2A-pathways involved in human diseases arrest by interfering with the PP2A activities [144]. In addition we have recently demonstrated that the non helical but flexible C- Viruses represent simple genetic tools that encode a limited terminal domain Vpr77–92 originated from HIV-1 89.6 isolate, but not number of proteins able to target critical cellular players such as PP2A. from NL4.3 isolate, interacts with the A structural subunit of PP2A1 The two last decades multiple studies of the functional interaction of holoenzyme to trigger cell transducing and apoptotic effects. Mutation diverse viral proteins with PP2A allowed us to identify key pathways experiments and comparative studies of HIV-1 89.6 Vpr77–92 for viral biology that also play a role in human diseases such as domain in homologous and the routinely used NL4.3 strain, suggest spontaneously arising cancers or neurodegenerative disorders. In- that sequence variability within Vpr77–92 sequences might regulate deed, PP2A appears as a privileged target for proteins encoded by the functional activities of the domain, including PP2A1 binding some DNA/RNA viruses or retroviruses such as HIV-1. For example, 1504 J. Guergnon et al. / Biochimica et Biophysica Acta 1812 (2011) 1498–1507 studies of PP2A-interacting and virally encoded oncoproteins, such as specifically deregulate intra-cellular pathways controlled by PP1/ SV40 small t, have allowed the better understanding of several PP2A proteins, we previously validated and published an approach, pathways involving PP2A inhibition and cell transformation (for named Drug Phosphatase Technology (DPT). This approach is based illustration see Fig. 2B and reviews [160,161]). Alternatively, studies on the design of cell penetrating peptides mimicking PP1/PP2A- of pro-apoptotic PP2A-interacting and virally-encoded proteins, such interacting motifs that disrupt or modulate specifically phosphatase- as adenoviral E4orf4 or HIV-1 Vpr proteins, provide new insights in mediated pathways [174]. Interestingly, a penetrating peptide named PP2A-mediated apoptotic pathways. DPT-5 with a short sequence deduced from human CD28 that,

Concerning E4orf4, a toxic protein that could facilitate release of similarly to E4orf4 interacts with the PP2A1 Bα subunit, provokes viral progeny, its ectopic expression in cultured human cells apoptosis in HeLa cells [174]. Furthermore we have also recently specifically induces a PP2A1-dependent and p53-independent cell found that a PP2A1 binding and cell penetrating sequence, deduced death in transformed or in tumor cells but not in healthy cells from canine adenovirus E4orf4, induces apoptosis in tumor cells [162,163]. These results clearly suggest that reproducing, or mimick- (Galioot et al. in preparation). ing, the anti-cancer effects resulting from PP2A1-E4orf4 interaction In conclusion, the emerging wealth of functional data from viral could be a promising strategy for the development of new cancer proteins improved our knowledge of PP2A-pathways involved in therapies. In this regard, direct electroporation of E4orf4 DNA human diseases. While DNA tumor viruses teach us how to activate provoked growth inhibition of murine B16 tumors. However this transforming pathways through inhibiting PP2A, others such as effect only occurs during the treatment, suggesting that electroporat- adenoviruses or HIV-1 tell us how to inhibit survival pathways ing gene transfer is inefficient to obtain a permanent therapeutic through activating PP2A. The dissection of these opposite pathways effect [164]. and the design of appropriate PP2A competitors that could deregulate In the case of HIV-1 Vpr, this protein can induce apoptosis in viral and pathogenic effects controlled by PP2A is clearly a challenging infected and non infected cells, and can also cause in vivo question for future treatments. neurodegeneration [165]. Interestingly, results by Godet et al. [93] suggest that polymorphism in PP2A1 binding region located in the C- Acknowledgements terminal sequence of Vpr proteins could play a role in Vpr-mediated and AIDS progression, including neurodegeneration. In this regard, it This research was supported by Institut Pasteur and INRA. We wish is noteworthy that WNVC and Vpr, two distinct viral proteins that to thank Alain Israël for the continuous support and for critically bind PP2A [93,102], may represent molecular tools for neurodegen- reviewing the manuscript. eration studies. Furthermore, in addition to studies concerning neurological disorders, Vpr has also already been proposed as a promising agent for cancer therapy [166]. References

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