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Neuroprotectin D1 Induces Dephosphorylation of Bcl-xL in a PP2A-dependent Manner during Oxidative Stress and Promotes Retinal Pigment Epithelial Cell Survival*

  • Rajee Antony
    Affiliations
    Neuroscience Center of Excellence, Louisiana State University Health Sciences Center, New Orleans, Louisiana 70112
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  • Walter J. Lukiw
    Affiliations
    Neuroscience Center of Excellence, Louisiana State University Health Sciences Center, New Orleans, Louisiana 70112
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  • Nicolas G. Bazan
    Correspondence
    To whom correspondence should be addressed: Neuroscience Center of Excellence, Louisiana State University Health Sciences Center, 2020 Gravier St., Ste. D, New Orleans, LA 70112. Tel.: 504-599-0831.
    Affiliations
    Neuroscience Center of Excellence, Louisiana State University Health Sciences Center, New Orleans, Louisiana 70112
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  • Author Footnotes
    * This work was supported, in whole or in part, by National Institutes of Health Grant R01 EY005121 through the NEI, United States Public Health Service.
Open AccessPublished:April 02, 2010DOI:https://doi.org/10.1074/jbc.M109.095232
      Retinal pigment epithelial (RPE) cell integrity is critical for the survival of photoreceptor cells. Bcl-xL is a major anti-apoptotic Bcl-2 protein required for RPE cell survival, and phosphorylation of Bcl-xL at residue Ser-62 renders this protein pro-apoptotic. In this study, we identify serine/threonine protein phosphatase 2A (PP2A) as a key regulator of Bcl-xL phosphorylation at residue Ser-62 in ARPE-19 cells, a spontaneously arising RPE cell line in which Bcl-xL is highly expressed. We found that either PP2A inhibitor okadaic acid or depletion of catalytic subunit α of PP2A (PP2A/Cα) by small interfering RNA enhanced Bcl-xL phosphorylation when activated with hydrogen peroxide and tumor necrosis factor α-induced oxidative stress. Disruption of PP2A/Cα exacerbated oxidative stress-induced apoptosis. PP2A/Cα colocalized and interacted with S62Bcl-xL in cells stressed with H2O2/tumor necrosis factor α. By contrast, the omega-3 fatty acid docosahexaenoic acid derivative, neuroprotectin D1 (NPD1), a potent activator of survival signaling, down-regulated oxidative stress-induced phosphorylation of Bcl-xL by increasing protein phosphatase activity. NPD1 also attenuated the oxidative stress-induced apoptosis by knockdown of PP2A/Cα and increased the association of PP2A/Cα with S62Bcl-xL as well as total Bcl-xL. NPD1 also enhanced the heterodimerization of Bcl-xL with its counterpart, pro-apoptotic protein Bax. Thus, NPD1 modulates the activation of this Bcl-2 family protein by dephosphorylating in a PP2A-dependent manner, suggesting a coordinated, NPD1-mediated regulation of cell survival in response to oxidative stress.

      Introduction

      Retinal pigment epithelial (RPE)
      The abbreviations used are: RPE
      retinal pigment epithelial
      Bcl-2
      B-cell lymphoma protein-2
      Bcl-xL
      Bcl-2-like 1
      BH
      Bcl-2 homology domain
      JNK
      c-Jun N-terminal protein kinase
      PP2A
      protein phosphatase 2A
      PP2A/Cα
      protein phosphatase 2A, catalytic subunit α
      NPD1
      neuroprotectin D1
      siRNA
      small interfering RNA
      TNFα
      tumor necrosis factor α
      PBS
      phosphate-buffered saline.
      cell integrity is necessary for the survival of rod and cone photoreceptors, and these cells accomplish a myriad of functions, including transport of retinol and of the essential omega-3 fatty acid, docosahexaenoic acid, and also transport of nutrients between photoreceptors and the choriocapillaries (
      • Bok D.
      ,
      • Strauss O.
      ). Our laboratory has shown that RPE cells, when induced with oxidative stress, produce and release to the media a stereospecific oxygenation product of docosahexaenoic acid, named neuroprotectin D1 (NPD1) (
      • Mukherjee P.K.
      • Marcheselli V.L.
      • Serhan C.N.
      • Bazan N.G.
      ,
      • Mukherjee P.K.
      • Marcheselli V.L.
      • Barreiro S.
      • Hu J.
      • Bok D.
      • Bazan N.G.
      ,
      • Mukherjee P.K.
      • Marcheselli V.L.
      • de Rivero Vaccari J.C.
      • Gordon W.C.
      • Jackson F.E.
      • Bazan N.G.
      ,
      • Marcheselli V.L.
      • Mukherjee P.K.
      • Arita M.
      • Hong S.
      • Antony R.
      • Sheets K.
      • Winkler J.W.
      • Petasis N.A.
      • Serhan C.N.
      • Bazan N.G.
      ). Hence, NPD1 is a pleiotropic modulator of inflammation resolution (
      • Serhan C.N.
      • Yacoubian S.
      • Yang R.
      ). NPD1 up-regulates anti-apoptotic proteins (Bcl-2, Bcl-xL) and down-regulates pro-apoptotic proteins (Bax, Bad) in ARPE-19 cells upon exposure to hydrogen peroxide/tumor necrosis factor α (H2O2/TNFα)-induced oxidative stress (
      • Mukherjee P.K.
      • Marcheselli V.L.
      • Serhan C.N.
      • Bazan N.G.
      ,
      • Mukherjee P.K.
      • Marcheselli V.L.
      • Barreiro S.
      • Hu J.
      • Bok D.
      • Bazan N.G.
      ). Under these conditions, NPD1 inhibits cytokine-mediated pro-inflammatory gene induction (
      • Mukherjee P.K.
      • Marcheselli V.L.
      • Serhan C.N.
      • Bazan N.G.
      ,
      • Marcheselli V.L.
      • Hong S.
      • Lukiw W.J.
      • Tian X.H.
      • Gronert K.
      • Musto A.
      • Hardy M.
      • Gimenez J.M.
      • Chiang N.
      • Serhan C.N.
      • Bazan N.G.
      ) and oxidative stress-induced apoptosis and also promotes RPE cell survival (
      • Bazan N.G.
      ,
      • Lukiw W.J.
      • Cui J.G.
      • Marcheselli V.L.
      • Bodker M.
      • Botkjaer A.
      • Gotlinger K.
      • Serhan C.N.
      • Bazan N.G.
      ). Oxidative stress (leading to apoptosis), neovascularization, and lipid peroxidation are involved in neurodegenerative diseases, including age related-macular degeneration (
      • Hollyfield J.G.
      ,
      • Hollyfield J.G.
      • Bonilha V.L.
      • Rayborn M.E.
      • Yang X.
      • Shadrach K.G.
      • Lu L.
      • Ufret R.L.
      • Salomon R.G.
      • Perez V.L.
      ,
      • Bazan N.G.
      ,
      • Lotery A.
      • Trump D.
      ).
      Apoptotic pathway activation comprises well orchestrated interactions between the Bcl-2 anti-apoptotic (Bcl-2 and Bcl-xL) and pro-apoptotic (Bax and Bad) proteins. Bcl-2 and Bcl-xL protect cells from apoptosis, whereas Bax and Bad promote apoptosis (
      • Kim R.
      ,
      • Yang E.
      • Zha J.
      • Jockel J.
      • Boise L.H.
      • Thompson C.B.
      • Korsmeyer S.J.
      ). Bcl-xL regulates apoptosis by undergoing post-translational modifications, which include phosphorylation and heterodimerization with pro-apoptotic proteins (Bax and Bad), and by subcellularly localizing in the mitochondria (
      • Basu A.
      • Haldar S.
      ,
      • Sedlak T.W.
      • Oltvai Z.N.
      • Yang E.
      • Wang K.
      • Boise L.H.
      • Thompson C.B.
      • Korsmeyer S.J.
      ,
      • Minn A.J.
      • Kettlun C.S.
      • Liang H.
      • Kelekar A.
      • Vander Heiden M.G.
      • Chang B.S.
      • Fesik S.W.
      • Fill M.
      • Thompson C.B.
      ). Bcl-xL is phosphorylated at residue (Ser-62) through c-Jun N-terminal protein kinase (JNK) in cancer cell lines in response to anti-mitotic drugs (
      • Basu A.
      • Haldar S.
      ,
      • Schmitt E.
      • Beauchemin M.
      • Bertrand R.
      ,
      • Basu A.
      • Haldar S.
      ,
      • Upreti M.
      • Galitovskaya E.N.
      • Chu R.
      • Tackett A.J.
      • Terrano D.T.
      • Granell S.
      • Chambers T.C.
      ), and in turn, its anti-apoptotic function is abolished. Moreover, Bcl-xL phosphorylation is a universal response to microtubule damage. However, the involvement of a physiological protein phosphatase in the dephosphorylation of Bcl-xL, as well as mediators that modulate Ser-62 removal, remains unclear.
      Protein phosphatase 2A (PP2A), a serine/threonine protein phosphatase, has been implicated in the dephosphorylation of Bcl-2 family proteins (
      • Tamura Y.
      • Simizu S.
      • Osada H.
      ,
      • Ruvolo P.P.
      • Deng X.
      • Ito T.
      • Carr B.K.
      • May W.S.
      ,
      • Chiang C.W.
      • Harris G.
      • Ellig C.
      • Masters S.C.
      • Subramanian R.
      • Shenolikar S.
      • Wadzinski B.E.
      • Yang E.
      ). PP2A is a heterotrimer composed of a catalytic (C-subunit), a structural (A-subunit), and a regulatory (B-subunit) subunit. The A- and B-subunits are ubiquitously expressed and evolutionarily conserved. The B-subunit defines the specific PP2A substrate target and its cellular localization (
      • Parsons R.
      ). The B-subunit targets the catalytic complex (A- and C-subunits) to intracellular sites such as microtubules, the nucleus, and the cytoplasm (
      • McCright B.
      • Rivers A.M.
      • Audlin S.
      • Virshup D.M.
      ,
      • Sontag E.
      • Nunbhakdi-Craig V.
      • Bloom G.S.
      • Mumby M.C.
      ). PP2A has been shown to be the major phosphatase involved in the dephosphorylation of Bad (
      • Chiang C.W.
      • Harris G.
      • Ellig C.
      • Masters S.C.
      • Subramanian R.
      • Shenolikar S.
      • Wadzinski B.E.
      • Yang E.
      ), leading to apoptosis. Moreover, the catalytic subunit of PP2A alone is sufficient to dephosphorylate Bcl-2 (
      • Deng X.
      • Gao F.
      • May W.S.
      ) and Bax, triggering cell death (
      • Xin M.
      • Deng X.
      ).
      Although the involvement of PP2A in the regulation of Bcl-xL protein expression is known (
      • Boon-Unge K.
      • Yu Q.
      • Zou T.
      • Zhou A.
      • Govitrapong P.
      • Zhou J.
      ,
      • Benito A.
      • Lerga A.
      • Silva M.
      • Leon J.
      • Fernandez-Luna J.L.
      ), knowledge of protein phosphatases that dephosphorylates Bcl-xL is scarce, particularly in response to apoptotic stimuli (
      • Cook S.A.
      • Sugden P.H.
      • Clerk A.
      ,
      • Valks D.M.
      • Kemp T.J.
      • Clerk A.
      ). Thus, given the potent bioactivity of NPD1 on the Bcl-2 family proteins (
      • Mukherjee P.K.
      • Marcheselli V.L.
      • Serhan C.N.
      • Bazan N.G.
      ,
      • Mukherjee P.K.
      • Marcheselli V.L.
      • Barreiro S.
      • Hu J.
      • Bok D.
      • Bazan N.G.
      ) during oxidative stress, it seems that NPD1 is key in ARPE-19 cell survival. The present study was undertaken to examine the effect of NPD1 in modulating the phosphorylation of Bcl-xL and to identify the specific species of protein phosphatase involved during induced oxidative stress in ARPE-19 cells.

      DISCUSSION

      Bcl-xL is a pro-survival member of the Bcl-2 family of proteins that inhibits cell death induced by a number of apoptotic stimuli (
      • Grillot D.A.
      • González-García M.
      • Ekhterae D.
      • Duan L.
      • Inohara N.
      • Ohta S.
      • Seldin M.F.
      • Nuñez G.
      ). The Bcl-2 family members each share homology within the BH1–BH4 regions. Bcl-2 and Bcl-xL contains all four homology regions, Bax lacks the BH4 domain, and Bad lacks all three domains except the BH3 death domain (
      • Zhou H.
      • Hou Q.
      • Chai Y.
      • Hsu Y.T.
      ). Bcl-xL is a close homolog of Bcl-2 and is proposed to exert its anti-apoptotic activity in a similar manner. In healthy cells, Bcl-xL is localized primarily to the mitochondria with a smaller fraction residing in the cytosol (
      • Hsu Y.T.
      • Wolter K.G.
      • Youle R.J.
      ,
      • Thuduppathy G.R.
      • Craig J.W.
      • Kholodenko V.
      • Schon A.
      • Hill R.B.
      ), and in some cells such as murine thymocytes, it is also localized in the nucleus (
      • Sedlak T.W.
      • Oltvai Z.N.
      • Yang E.
      • Wang K.
      • Boise L.H.
      • Thompson C.B.
      • Korsmeyer S.J.
      ). One of the pro-survival functions of Bcl-xL is the inhibition of pro-apoptotic Bax (
      • Yang E.
      • Zha J.
      • Jockel J.
      • Boise L.H.
      • Thompson C.B.
      • Korsmeyer S.J.
      ). Bax is found in both the cytosol and the mitochondria in many culture cells (
      • Hsu Y.T.
      • Wolter K.G.
      • Youle R.J.
      ,
      • Hsu Y.T.
      • Youle R.J.
      ). Upon induction of apoptosis, Bcl-xL and Bax are predominantly membrane-associated (
      • Hsu Y.T.
      • Wolter K.G.
      • Youle R.J.
      ). Moreover, phosphorylated forms of the Bcl-2 family proteins Bad and Bax are localized exclusively in the cytosol (
      • Deng X.
      • Gao F.
      • May W.S.
      ,
      • Xin M.
      • Deng X.
      ). In a spinal cord injury model (
      • Cittelly D.M.
      • Nesic-Taylor O.
      • Perez-Polo J.R.
      ), Bcl-xL was shown to be present in the endoplasmic reticulum, nuclei, and cytosol, whereas phosphorylated S62Bcl-xL was absent from the cytosolic fraction in uninjured spinal cord. As early as 15 min after contusion, an up-regulation of the phospho-Bcl-xL in the cytosol was observed. In PC-12 cells treated with vinblastine, there was a correlation between the cytoplasmic S62Bcl-xL levels and apoptosis, suggesting that Bcl-xL phosphorylation is pro-apoptotic (
      • Basu A.
      • Haldar S.
      ,
      • Cittelly D.M.
      • Nesic-Taylor O.
      • Perez-Polo J.R.
      ). In support of this, subcellular fractionation studies in ARPE-19 cells revealed that S62Bcl-xL was not localized in mitochondrial fractions or in the unstimulated cytosolic fractions. After induction of oxidative stress, S62Bcl-xL was detected as early as 15 min in the cytosol and gradually increased thereafter up to 60 min (Fig. 4). The overall protein level of Bcl-xL remained unchanged in the mitochondria, consistent with previous studies (
      • Hsu Y.T.
      • Wolter K.G.
      • Youle R.J.
      ). In unstimulated cells, Bax was localized in the cytosol, and a smaller fraction was localized in the mitochondria, and upon induction of oxidative stress, a smaller fraction was translocated to the mitochondria at 30 min (Fig. 4).
      Apoptosis-promoting agents, including H2O2/TNFα, calcium ionophore, A-23187, and IL-1β, can induce the synthesis of NPD1 via the action of a 15-lipoxygenase-1 enzyme (15-LOX-1) (
      • Mukherjee P.K.
      • Marcheselli V.L.
      • Serhan C.N.
      • Bazan N.G.
      ,
      • Calandria J.M.
      • Marcheselli V.L.
      • Mukherjee P.K.
      • Uddin J.
      • Winkler J.W.
      • Petasis N.A.
      • Bazan N.G.
      ). However, our understanding of the signaling mechanism(s) by which NPD1 promotes cell survival remains fragmentary. Because NPD1 counteracts oxidative stress-induced apoptosis and also up-regulates the anti-apoptotic protein Bcl-xL in ARPE-19 cells (
      • Mukherjee P.K.
      • Marcheselli V.L.
      • Serhan C.N.
      • Bazan N.G.
      ), it was proposed that NPD1 may act at the transcriptional, translational, and post-translational level to regulate the anti-apoptotic activity of Bcl-xL. In a previous report, it had been shown that after 1 h of serum starvation and 6 h of 400 and 800 μm H2O2/TNFα induction, total Bcl-xL protein was down-regulated, and 50 nm NPD1 up-regulated this protein (
      • Mukherjee P.K.
      • Marcheselli V.L.
      • Barreiro S.
      • Hu J.
      • Bok D.
      • Bazan N.G.
      ,
      • Mukherjee P.K.
      • Marcheselli V.L.
      • de Rivero Vaccari J.C.
      • Gordon W.C.
      • Jackson F.E.
      • Bazan N.G.
      ). The activated (phosphorylated) form of this protein has until now not been studied. We report here that 200 μm H2O2/TNFα induced the phosphorylation of Bcl-xL in ARPE-19 cells after 18 h of serum starvation and that NPD1 down-regulated the same (Fig. 1, A and B). We further demonstrate that NPD1 was not able to down-regulate the phosphorylation of Bcl-xL at higher concentrations, such as 400 and 600 μm H2O2 (Fig. 1B). It has also been demonstrated that reactive oxygen species can influence the phosphorylation of Bcl-2 family proteins. An appropriate amount of reactive oxygen species, usually a small amount, activates phosphokinases and increases the intracellular calcium levels (
      • Rothstein E.C.
      • Byron K.L.
      • Reed R.E.
      • Fliegel L.
      • Lucchesi P.A.
      ,
      • Benhar M.
      • Dalyot I.
      • Engelberg D.
      • Levitzki A.
      ). However, a large amount induces peroxidation of DNA, proteins, and lipids, resulting in cell impairments (
      • Covacci V.
      • Torsello A.
      • Palozza P.
      • Sgambato A.
      • Romano G.
      • Boninsegna A.
      • Cittadini A.
      • Wolf F.I.
      ,
      • Dröge W.
      ). Hence, higher concentrations of H2O2/TNFα may increase reactive oxygen species levels in ARPE-19 cells, which might decrease the potency of NPD1. Therefore, the effect observed in Fig. 1B might be a concentration- and time-dependent phenomenon. Taken together, our data suggest that Bcl-xL phosphorylation is an early event (>5 min) with a significant up-regulation from 30 to 90 min under the present experimental conditions. After 60 min, the phosphorylation status was reversed to that observed at 30 min (Fig. 1A). These observations are consistent with phosphorylation and dephosphorylation events occurring much earlier than the appearance of apoptotic features. We cannot rule out the possibility that a much higher concentration of NPD1 might be effective with an increased concentration of H2O2, resulting in a more rapid activation of Bcl-xL.
      Other studies also demonstrate Bcl-xL phosphorylation at Ser-62 through activation of JNK, thereby inactivating its anti-apoptotic activity (
      • Basu A.
      • Haldar S.
      ,
      • Basu A.
      • Haldar S.
      ,
      • Upreti M.
      • Galitovskaya E.N.
      • Chu R.
      • Tackett A.J.
      • Terrano D.T.
      • Granell S.
      • Chambers T.C.
      ). Our results demonstrated JNK-mediated activation of S62Bcl-xL in our model system (data not shown) as well. It is possible that dephosphorylation of Bcl-xL mediated by a protein phosphatase may also play a role in regulating its anti-apoptotic activity. Our results further suggest that PP2A is able to function as a Bcl-xL phosphatase because specific disruption of PP2A activity by treatment of ARPE-19 cells with okadaic acid enhanced Bcl-xL phosphorylation (Fig. 2A). Furthermore PP2A expression was observed in the cytosol, consistent with the absence of S62Bcl-xL. In oxidative stress-induced cells, a down-regulation in the expression pattern of PP2A/C was observed as a function of time, correlating with increased phosphorylation of Bcl-xL (Fig. 4). PP2A/C also was found to be localized in the mitochondria of control cells, and oxidative stress enhanced the translocation. Importantly, two homologous proteins that noncompetitively inhibit the phosphatase activity of PP2A, I1PP2A and I2PP2A (
      • Li M.
      • Makkinje A.
      • Damuni Z.
      ,
      • Kovacech B.
      • Kontsekova E.
      • Zilka N.
      • Novak P.
      • Skrabana R.
      • Filipcik P.
      • Iqbal K.
      • Novak M.
      ), were not found to be altered in abundance between control or stressed ARPE-19 cells when compared with actin levels in the same sample. These observations are consistent with previous studies demonstrating that C2-ceramide, an activator of PP2A, mediates dephosphorylation of the Bcl-xL homolog Bcl-2 by translocating PP2A to the mitochondria (
      • Tamura Y.
      • Simizu S.
      • Osada H.
      ,
      • Ruvolo P.P.
      • Deng X.
      • Ito T.
      • Carr B.K.
      • May W.S.
      ). Confirmation of PP2A as a Bcl-xL phosphatase was obtained from the results of transfection studies that demonstrated specific knockdown of PP2A/Cα expression by siRNA-increased Bcl-xL phosphorylation (Fig. 2C), which promoted apoptotic cell death (Fig. 3C). In contrast, NPD1 attenuated PP2A/C-mediated cell death (Fig. 3C). Moreover, NPD1 enhanced the interaction between S62Bcl-xL and PP2A/Cα in H2O2/TNFα-induced ARPE-19 cells (Fig. 5B), supporting the notion that NPD1-stimulated Bcl-xL/Bax interaction (Fig. 5A) is associated with the increased availability of total Bcl-xL, presumably on the mitochondrial membrane. It is possible that these interactions disable the pro-apoptotic protein Bax from forming oligomers, thus inhibiting the formation of ion channels on the mitochondria and thus releasing cytochrome c into the cytosol.
      It has been also demonstrated that PP2A, specifically PP2A/C, can dephosphorylate the Bcl-xL homolog Bcl-2 by directly binding to its BH4 domain (
      • Deng X.
      • Gao F.
      • May W.S.
      ). Pro-survival Bcl-2 family members possess a conserved BH4 domain, and deletion of this domain presumably converts Bcl-2 to a multidomain Bax-like death effector (
      • Cheng E.H.
      • Kirsch D.G.
      • Clem R.J.
      • Ravi R.
      • Kastan M.B.
      • Bedi A.
      • Ueno K.
      • Hardwick J.M.
      ,
      • Reed J.C.
      • Zha H.
      • Aime-Sempe C.
      • Takayama S.
      • Wang H.G.
      ), suggesting that the BH4 domain is critical for anti-apoptotic activity. Taken together, we propose that the BH4 domain of Bcl-xL might function as the PP2A “docking site” and a potential bridge for PP2A to access the Ser-62 phosphorylation site located in the loop region adjacent to the N-terminal BH4 domain. This mechanism may contribute to removal of the phosphate of Bcl-xL, leading to inhibition of its pro-apoptotic function.
      In related studies, the sphingolipid ceramide, which exerts its effect at the subcellular site of production, has been shown to stimulate PP2A activity by binding to the catalytic domain of PP2A (
      • Ruvolo P.P.
      • Deng X.
      • Ito T.
      • Carr B.K.
      • May W.S.
      ,
      • Li M.
      • Makkinje A.
      • Damuni Z.
      ,
      • Law B.
      • Rossie S.
      ,
      • Kolesnick R.N.
      • Krönke M.
      ). We demonstrate that the omega-3 fatty acid derivative, NPD1, increases PP2A activity (Fig. 3A) and also that NPD1 synthesis is a near universal response to oxidative stress because of its ability to inactivate apoptotic signaling at the mitochondrial level (
      • Mukherjee P.K.
      • Marcheselli V.L.
      • Serhan C.N.
      • Bazan N.G.
      ,
      • Mukherjee P.K.
      • Marcheselli V.L.
      • Barreiro S.
      • Hu J.
      • Bok D.
      • Bazan N.G.
      ). The present study shows that NPD1 can induce dephosphorylation of Bcl-xL in a PP2A-dependent manner and promote ARPE-19 cell survival. Our results also indicate that NPD1 facilitation of PP2A interaction with S62Bcl-xL (Fig. 5B) might increase the dephosphorylation of Bcl-xL and promote Bax/Bcl-xL heterodimerization (Fig. 5A). That NPD1 may facilitate a direct Bax/Bcl-xL interaction through additional regulatory mechanisms can, at this time, not be excluded.
      Our findings and the demonstration of the involvement of the BH4 domain (a feature inherent only with the anti-apoptotic proteins) in PP2A docking (
      • Deng X.
      • Gao F.
      • May W.S.
      ) indicate a novel mechanism for NPD1-induced cell survival. NPD1-stimulated Bcl-xL dephosphorylation and/or PP2A-induced dephosphorylation might mediate a conformational change in Bcl-xL resulting in exposure of its BH3 domain, which is normally hidden in the hydrophobic domain of Bcl-xL (
      • Muchmore S.W.
      • Sattler M.
      • Liang H.
      • Meadows R.P.
      • Harlan J.E.
      • Yoon H.S.
      • Nettesheim D.
      • Chang B.S.
      • Thompson C.B.
      • Wong S.L.
      • Ng S.L.
      • Fesik S.W.
      ). Thus, NPD1-induced Bcl-xL dephosphorylation may activate Bcl-xL via a conformational change in the BH3 domain that promotes Bax and Bcl-xL association, and NPD1-activated PP2A may more efficiently facilitate heterodimerization of Bax and Bcl-xL.
      Taken together, these findings are significant for several reasons. First, the observation of NPD1-induced dephosphorylation of S62Bcl-xL defines a mechanism of controlling the expression of anti- and pro-apoptotic factors in response to oxidative stress. Second, a specific and direct mechanism mediated by an NPD1-activated protein phosphatase has been established in RPE cells. Moreover, NPD1 attenuates laser-induced choroidal neovascularization in vivo in an experimental model of age-related macular degeneration (
      • Sheets K.G.
      • Zhou Y.
      • Ertel M.K.
      • Knott E.J.
      • Regan Jr., C.E.
      • Elison J.R.
      • Gordon W.C.
      • Gjorstrup P.
      • Bazan N.G.
      ).
      In summary, we demonstrate that Bcl-xL phosphorylation in ARPE-19 cells is induced by oxidative stress and that NPD1 enhances PP2A activity, in turn attenuating oxidative stress-induced phosphorylation of the anti-apoptotic protein Bcl-xL. These studies, for the first time, identify NPD1 as a novel regulator of the PP2A/C/Bcl-xL interaction. Hence, our results establish a stimulus-specific response in NPD1-dependent phosphorylation of Bcl-xL. NPD1 modulates the activation of this Bcl-2 family member by changing the phosphorylation status in a PP2A-dependent manner and also by enhancing the interaction of its counterpart, pro-apoptotic protein Bax, suggesting a highly coordinated, NPD1-mediated regulation of cell survival in response to oxidative stress.

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