Published online before print February 21, 2008, doi: 10.1161/CIRCRESAHA.107.168153
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© 2008 American Heart Association, Inc.
Molecular Medicine |
AIP1 Recruits Phosphatase PP2A to ASK1 in Tumor Necrosis Factor–Induced ASK1-JNK Activation
From the Interdepartmental Program in Vascular Biology and Therapeutics (W.M., Y.L., L.Y., H.Z., T.W., H.C.), Department of Pathology, Yale University School of Medicine, New Haven, Conn; State Key Laboratory of Ophthalmology (S.T., T.W.), Zhongshan Ophthalmic Center, Sun Yat-Sen University, Guangzhou, China; and Department of Pharmacology (T.L., H.F.), Emory University, Atlanta, Ga.
Correspondence to Dr. Wang Min, Vascular Biology and Therapeutics Program and Department of Pathology, Yale University School of Medicine, 10 Amistad St, New Haven, CT 06520. E-mail wang.min{at}yale.edu
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Abstract |
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Previously we have shown that AIP1 (apoptosis signal-regulating kinase [ASK]1-interacting protein 1), a novel member of the Ras-GAP protein family, facilitates dephosphorylation of ASK1 at pSer967 and subsequently 14-3-3 release from ASK1, leading to enhanced ASK1-JNK signaling. However, the phosphatase(s) responsible for ASK1 dephosphorylation at pSer967 has not been identified. In the present study, we identified protein phosphatase (PP)2A as a potential phosphatase in vascular endothelial cells (ECs). Tumor necrosis factor (TNF)-induced dephosphorylation of ASK1 pSer967 in ECs was blocked by PP2A inhibitor okadaic acid. Overexpression of PP2A catalytic subunit induced dephosphorylation of ASK1 pSer967 and activation of c-Jun N-terminal kinase (JNK). In contrast, a catalytic inactive form of PP2A or PP2A small interfering RNA blunted TNF-induced dephosphorylation of ASK1 pSer967 and activation of JNK without effects on NF-
B activation. Whereas AIP1, via its C2 domain, binds to ASK1, PP2A binds to the GAP domain of AIP1. Endogenous AIP1-PP2A complex can be detected in the resting state, and TNF induces a complex formation of AIP1-PP2A with ASK1. Furthermore, TNF-induced association of PP2A with ASK1 was diminished in AIP1-knockdown ECs, suggesting a critical role of AIP1 in recruiting PP2A to ASK1. TNF-signaling molecules TRAF2 and RIP1, known to be in complex with AIP1 and activate AIP1 by phosphorylating AIP1 at Ser604, are critical for TNF-induced ASK1 dephosphorylation. Finally, PP2A and AIP1 cooperatively induce activation of ASK1-JNK signaling and EC apoptosis, as demonstrated by both overexpression and small interfering RNA knockdown approaches. Taken together, our data support a critical role of PP2A-AIP1 complex in TNF-induced activation of ASK1-JNK apoptotic signaling.
Key Words: AIP1/DAB2IP • ASK1 • TNF • phosphatase • 14-3-3
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Introduction |
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Various extracellular stimuli, including growth factors, cytokines, and stress, trigger MAP3K-MAP2K-MAPK (mitogen-activated protein [MAP] kinase kinase kinase–MAP kinase kinase–MAP kinase) cascades, leading to cell growth, differentiation, and apoptosis.1 ASK1 is a MAP3K that is activated in response to a variety of stress signals, including proinflammatory cytokine tumor necrosis factor (TNF), Fas ligation, reactive oxygen species (ROS), antineoplastic agents, endoplasmic reticulum stress.2,3 In cultured cells, overexpression of ASK1 is sufficient to induce apoptosis in a number of cell lines. It has been shown that ASK1 induces apoptotic cell death through a mitochondria-dependent pathway.4,5 Data from ASK1-deficient mice demonstrate a specific role of ASK1 in stresses-induced c-Jun N-terminal kinase (JNK)/p38 activation and cell death.3,6–8
The mechanism by which stresses, including TNF, activate ASK1 is not fully understood. The identification of proteins associated with ASK1 has provided some insights. ASK1 is a 170-kDa protein that functionally is composed of an inhibitory N-terminal domain, an internal kinase domain, and a C-terminal regulatory domain.9 ASK1 activity appears to be enhanced by its binding to a number of important regulatory proteins such as TNF receptor–associated factors (TRAFs), AIP1, Daxx, and JSAP/JIP3.7,10–13 On the other hand, several cellular factors, including thioredoxin, glutaredoxin, Hsp72, Hsp90, Raf-1, and 14-3-3, have been reported to interact with different ASK1 domains and inhibit ASK1 activity.5,10,14–17 Consistently, a recent report suggests that ASK1 constitutively forms a high-molecular-mass complex (ASK1 signalosome), and H2O2 induces alterations of ASK1 signalosome components.18 However, the underlying mechanism for regulating ASK1 signalosome is not understood.
Associations of ASK1 with thioredoxin and 14-3-3 have been best characterized. Both cytosolic and mitochondrial forms of thioredoxin directly associate with ASK1 in the N-terminal domain of ASK1. Interestingly, only the reduced form of thioredoxin binds to the N-terminal part of ASK1 and blocks activation of ASK1 by TNF.5,10,19,20 In contrast, an oxidized form or a redox-inactive form (a double mutation at catalytic sites Cys32 and Cys35) of thioredoxin fails to associate with ASK1. Similarly, reduced Cys residues on ASK1 appear to be critical for ASK1–thioredoxin association.5,20 Thus, it has been proposed that TNF and ROS activate ASK1 by oxidizing thioredoxin to release it from ASK1.5,10,14,20
In contrast, the underlying mechanism for the release of 14-3-3 from ASK1 is less understood. 14-3-3 is a phosphoserine-binding protein and binds to pSer967 located at the C-terminal domain of ASK1. ASK1 is basally phosphorylated at Ser967, and TNF/ROS induce dephosphorylation of pSer967, leading to a release of 14-3-3. We have previously identified AIP1, a novel Ras-GAP protein that forms a complex with ASK1 and facilitates dissociation of 14-3-3 from ASK1.11,21–23 Because AIP1 itself is a nonphosphatase protein, the phosphatase specifically responsible for dephosphorylation of ASK1 pSer967 is not known. Serine/threonine phosphatase family includes PP1, PP2A, PP2B, PP2C, PP4, and PP5. In the present study, we have identified PP2A as a phosphatase in TNF-induced dephosphorylation of ASK1 pSer967. More importantly, we show that AIP1 is critical in recruiting PP2A to ASK1, leading to dephosphorylation of ASK1 at pSer967 and activation of ASK1-JNK signaling.
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Materials and Methods |
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Constructs
Mammalian expression plasmids for ASK1 and AIP1 were described previously.11,21–23 The PP2A template for the PCR reaction was from Dave Pallas (Emory University), and wild-type (PP2A-WT) and a catalytic inactive form of the PP2A catalytic subunit (PP2A-L199P [PP2A-LP]) were in the Flag expression vector (pSCM167).
Antibodies
A rabbit polyclonal antibody against phospho-specific antibodies against phopsho-ASK1 (pSer967) and phospho-JNK were from Cell Signaling. We obtained anti-ASK1 (H300) from Santa Cruz Biotechnology; mouse monoclonal anti-PP2A, anti-PP5, and anti–β-tubulin from BD Pharmingen; and anti-Flag from Sigma.
Cells, Cytokines, and Inhibitors and Transfection and Reporter Assay
Human umbilical vein endothelial cells (HUVECs) and bovine aortic ECs (BAECs) were purchased from Clonetics Corp (San Diego, Calif) and cultured as previously described.11,21–23 Human recombinant TNF was from R&D Systems Inc (Minneapolis, Minn). Chemical inhibitors including okadaic acid were purchased from Calbiochem. Transfection of ECs was performed by Lipofectamine 2000 according to the protocol of the manufacturer (Invitrogen Corp, San Diego, Calif). Reporter gene assay was performed as described previously.11,21–23
Immunoprecipitation and Immunoblotting
HUVECs or BAECs after various treatments were washed twice with cold PBS and lysed in cold lysis buffer (50 mmol/L Tris-HCl, pH 7.6, 150 mmol/L NaCl, 0.1% Triton X-100, 0.75% Brij 96, 1 mmol/L sodium orthovanadate, 1 mmol/L sodium fluoride, 1 mmol/L sodium pyrophosphate, 10 µg/mL aprotinin, 10 µg/mL leupeptin, 2 mmol/L phenylmethylsulfonyl fluoride, 1 mmol/L EDTA). Immunoprecipitation to analyze protein interaction in vivo was performed as described previously.11
RNA Interference for PP2A and AIP1
A specific small interfering (si)RNA for human PP2A (NM_002715) and a control siRNA were purchased from Upstate (PP2A Ca subunit siRNA/siAb assay kit). We designed a pair of siRNA oligonucleotides for human AIP1 (5'-GGAGCGCAACAGUUACCUGdTdT-3'; the corresponding scramble siRNA oligonucleotide, 5'GGAGCGCAUCUGUUACCUGdTdT3'). 5'GGACCAGCAUACGCUUUCUdTdT3' with 2 nucleotide mutations (underlined) was synthesized from Ambion (Austin, Tex). siRNA (20 µmol/L) was transfected into cells by Oligofectamine following protocols provided by the manufacturer (Invitrogen).
Quantitation of Apoptosis
Cell killing assay was performed as described previously with a modification.21
Statistical Analysis
Data are presented as means(±SD). Experiments were performed at least twice with duplicates. Analysis of densitometry was performed using NIH Image 1.60. Results were then normalized for comparison among different experimental groups by arbitrarily setting the value of control cells to 1.0. Statistical analyses were performed with StatView 4.0 package (ABACUS Concepts). Differences were analyzed by unpaired 2-tailed Student’s t test. Values of P<0.05 were taken as significant.
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Results |
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Overexpression of Wild-Type but Not a Catalytic Inactive Form of PP2A Induces Dephosphorylation of ASK1 pSer967 and Activation of the ASK1-JNK Signaling Cascade
Previously, we have shown that oxidative stress triggers dephosphorylation of ASK1 at Ser-967 through an okadaic acid-sensitive phosphatase, resulting in dissociation of ASK1 from 14-3-3 complex with concomitant increase of ASK1 catalytic activity and ASK1-mediated activation of JNK/p38 pathways.24 To determine whether okadaic acid–sensitive phosphatase PP2A is a potential pSer967 phosphatase, we first determined whether overexpression of the PP2A catalytic subunit induces ASK1 dephosphorylation at pSer967. Flag-tagged wild-type (PP2A-WT) or a catalytic inactive form of the PP2A catalytic subunit (PP2A-LP) was transfected into bovine aortic endothelial cells (BAECs), a highly transfectable primary EC, in the presence of ASK1. Expression of PP2A-WT, but not PP2A-LP, significantly reduced the phosphorylation of ASK1 at pSer967 (Figure 1a, top, with quantification in 1b) concomitant with an increase of ASK1 pThr845 (an active form of ASK1) (Figure 1a, top, with quantification in 1c). Dephosphorylation of ASK1 at pSer967 was further determined by a glutathione S-transferase (GST)–14-3-3 pull-down assay. Consistent with the effects of PP2A on pSer967, PP2A-WT (not PP2A-LP) significantly reduced the binding of ASK1 to GST–14-3-3 (Figure 1a, bottom, with quantification in 1b).
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We then determined the effects of PP2A on ASK1 downstream signaling by a JNK reporter gene assay. BAECs were transfected with the reporter gene in the presence of PP2A-WT or PP2A-LP, followed by TNF treatment. Expression of PP2A-WT strongly enhanced, whereas PP2A-LP reduced, TNF-induced JNK reporter gene activity (Figure 1d). To assess a direct effect of PP2A on ASK1 activity, the reporter gene and PP2A were cotransfected with ASK1-WT or ASK1-S967A in BAECs. We have previously shown that a mutation of serine 967 to alanine (ASK1-S967A) renders ASK1 defective in phosphorylation at the 14-3-3-binding site, leading to increased ASK1 activity. Overexpression of ASK1-WT induced activation of a JNK-dependent reporter gene, and ASK1 activity was enhanced by coexpression of PP2A-WT (but not PP2A-LP). In contrast, PP2A had no effects on ASK1-S967A–induced JNK reporter gene activity (Figure 1e), suggesting that the effect of PP2A on ASK1 is dependent on the pSer967 site.
A Critical Role of PP2A in ASK1-JNK Activation
To determine the role of endogenous PP2A in TNF-induced ASK1-JNK signaling, BAECs were pretreated with okadaic acid, followed by TNF treatment. ASK1 dephosphorylation at Ser967 was determined by Western blot with a phospho-specific antibody (pSer967). Activation of ASK1 and JNK was also determined by Western blot with respective phospho-specific antibodies. As shown in Figure 2a, TNF induced dephosphorylation of ASK1 pSer967, concomitant with phosphorylation of ASK1 at Thr845 and JNK activation. However, pretreatment with okadaic acid blunted TNF-induced dephosphorylation of ASK1 pSer967, as well as activation of ASK1-JNK. Similar data were observed in human ECs (HUVECs).
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We then used the siRNA approach to knock down the catalytic subunit of PP2A protein in ECs. Human ECs were transfected with siRNA oligonucleotides (against human PP2A), followed by treatment with TNF for indicated times. Dephosphorylation of ASK1 at Ser967, phosphorylation of ASK1 at Thr845, and JNK activation were determined. Transfection of PP2A siRNA, but not the control siRNA, dramatically reduced the protein level of endogenous PP2A in HUVECs without effects on the protein expression of PP5 (Figure 2b), another phosphatase negatively regulating ASK1 by dephosphorylating Thr845.25 Knockdown of PP2A significantly blocked TNF-induced dephosphorylation of ASK1-pSer967, phosphorylation of ASK1-Thr845, and activation of downstream JNK (Figure 2b, with quantification in 2c and 2d). In contrast, TNF-induced IB
degradation (an indicator of NF-B activation pathway) was not altered (Figure 2b). These data support a critical role of PP2A in TNF-induced ASK1-JNK activation.
TNF Induces a Formation of AIP1-PP2A-ASK1 Complex
Previously, we have shown that TNF induces a formation of AIP1-ASK1 complex to facilitate dephosphorylation of ASK1 pSer967 and subsequent release of 14-3-3 from ASK1. To determine whether TNF induces a recruitment of PP2A to AIP1-ASK1 complex, we detected associations of PP2A with AIP1 and ASK1 by coimmunoprecipitation assay. BAECs were untreated or treated with TNF (10 ng/mL for 5 and 15 minutes), and association of PP2A with AIP1 and ASK1 by was determined by immunoprecipitation with PP2A, followed by Western blot with anti-AIP1 or anti-ASK1. PP2A-AIP1 complex was detected in resting cells and was further enhanced in TNF-treated ECs at 5 minutes. In contrast, PP2A-ASK1 association was only detected in the TNF-treated ECs (Figure 3a) with a similar kinetics of AIP1-ASK1 complex formation as well as that of ASK1-JNK activation, which peaked at 15 minutes (Figure 3b and 3c) and declined at 60 minutes.11,21,26 These results indicate that PP2A-AIP1 association precedes the formation of PP2A-ASK1 complex and that AIP1 may recruit PP2A to ASK1 complex.
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To further determine the kinetics and the specificity of AIP1-PP2A complex, BAECs were treated with TNF for longer times (0 to 120 minutes). AIP1-PP2A association declined at 30 minutes. Interestingly, association of AIP1 with PP5, a phosphatase dephosphorylating ASK1 at pT845 to negatively feedback regulate ASK1 activity,25 was detected at 30 to 60 minutes (Figure 3d) when phosphorylation of Thr845 declines (see Figure 2b). These data demonstrate that TNF induces AIP1-PP2A-ASK1 complex formation at an early phase but AIP1-PP5-ASK1 complex formation at a late phase.
Critical Role of AIP1 in Recruiting PP2A to ASK1 Complex
To further define the role of AIP1 in recruiting PP2A to ASK1, we first determined whether AIP1 overexpression enhances PP2A-ASK1 interaction. Overexpression of AIP1 in BAECs significantly enhanced TNF-induced formation of endogenous PP2A-ASK1 complex, as determined by a coimmunoprecipitation assay (Figure 4a). We then determined whether AIP1 knockdown blocks associations of PP2A with ASK1. HUVECs were transfected with a control or AIP1-specific siRNA followed by TNF treatment. Knockdown of AIP1 had no significant effects on ASK1 expression (Figure 4a, top). TNF induced an association of PP2A with ASK1 in HUVECs, as observed in BAECs. However, AIP1 knockdown abolished the formation of PP2A-ASK1 complex (Figure 4b, middle, with quantification in 4c). As controls, effects of AIP1 on ASK1-JNK signaling were determined. Consistent with our previously data,11,21,26 TNF-induced activation of ASK1 (dephosphorylation at Ser967 and phosphorylation at Thr845) and JNK were significantly reduced in AIP1-knockdown cells (Figure 4b, bottom). These data strongly support a critical role of AIP1 in bridging the association between PP2A and ASK1 and subsequent activation of ASK1-JNK signaling.
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Previously, we have shown that AIP1 functions a scaffolding protein to recruit the TNF signaling complex (TRADD-RIP1-TRAF2) to ASK1, and AIP1 is a transducer in TRAF2-dependent activation of ASK1 induced by TNF.11 To determine the role of TRAF2 in TNF-induced dephosphorylation of ASK1, HUVECs were transfected with a control or TRAF2-specific siRNA, followed by TNF treatment. Knockdown of TRAF2 completely abrogated TNF-induced dephosphorylation of ASK1 at Ser967 (Figure 4d). Recently, we have shown that RIP1-mediated AIP1 phosphorylation at Ser604 is critical for TNF-induced ASK1-JNK activation.26 To determine whether AIP1 pSer604 is involved in TNF/PP2A-induced ASK1 dephosphorylation, BAECs were transfected with AIP1-WT or AIP1-S604A (a mutant form with a mutation at Ser604) in the presence of ASK1. Cells were treated with TNF (10 ng/mL for 15 minutes), and ASK1 pSer967 was determined. TNF induced dephosphorylation of ASK1 at Ser967 in the absence of overexpressed AIP1. However, coexpression of AIP1-WT with ASK1 induced ASK1 dephosphorylation before TNF treatment. In contrast, coexpression of AIP1-S604A with ASK1 blunted TNF-induced ASK1 dephosphorylation (Figure 4e). These results suggest that TRAF2 and RIP1-mediated AIP1 Ser604 phosphorylation plays a critical role in TNF-induced ASK1 dephosphorylation at Ser967.
The GAP Domain of AIP1 Is Critical for PP2A Binding
To map the critical domain of AIP1 for PP2A binding, we generated Flag-tagged AIP1 mutants containing various domains: AIP1-N for the N-terminal half containing a PH, a C2, and a GAP domain; AIP1-PHC2 with the PH and C2 domain; and AIP1-PH with the PH domain. We also generated AIP1-C for the C-terminal half containing the period-like domain (PER), the proline-rich domain (PR), and leucine-zipper motif (LZ), as well as truncated AIP1-C (C-PR with the PR domain and C-
PR with a deletion of the PR region) (Figure 5a). Various AIP1 truncates were transfected into BAECs, followed by treatment with TNF (10 ng/mL for 5 minutes). PP2A-AIP1 complex was determined by a coimmunoprecipitation assay. PP2A associated with AIP1-N, which contains the PH, C2, and GAP domain, but not with AIP1-PHC2, with a deletion of the GAP domain. PP2A did not interact with AIP1-C domain truncates (C, PR, and PR) (Figure 5b). These data suggest that the GAP domain of AIP1 is critical for PP2A binding.
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Critical Function of PP2A-AIP1 Complex in Activation of ASK1-JNK Signaling and EC Apoptosis
Activation of ASK1-JNK signaling has been implicated in EC apoptosis.11,20,21,27 To determine the role of PP2A-AIP1 complex in ASK1-mediated EC apoptosis, we first determined whether coexpression of PP2A and AIP1 induces EC apoptosis. BAECs were transfected with AIP1-N in the presence of PP2A-WT or PP2A-LP, and dephosphorylation of ASK1 at pSer967 was determined by Western blot. We chose AIP1-N because it can bind to ASK1 in the absence of TNF stimulation and it also contains the PP2A-binding domain. Expression of AIP1-N alone significantly induced dephosphorylation of ASK1, as shown previously.21 Coexpression PP2A-WT enhanced, whereas PP2A-LP blocked, AIP1-N–induced dephosphorylation of ASK1 pSer967 (Figure 6a). EC apoptosis characterized by nuclear fragmentation was determined by 4',6-diamidino-2-phenylindole (DAPI) staining, followed by fluorescence microscopy. Similar to ASK1 activation, coexpression of AIP1-N with PP2A-WT, but not LP, synergistically induced EC apoptosis (Figure 6b). To determine the role of endogenous PP2A-AIP1 in EC apoptosis, AIP1 was knocked down by siRNA, followed by treatment with TNF (10 ng/mL) in the presence of cycloheximide (CHX) (10 µg/mL) for 6 hours when EC apoptosis peaks.11 Consistent with previous observations,11 TNF(+CHX) strongly induced EC apoptosis (
24%). Knockdown of either PP2A (15%) or AIP1 (7.5%) significantly reduced, and knockdown of both synergistically reduced (4%), TNF(+CHX)-induced EC apoptosis (Figure 6c). These results support a critical role of PP2A-AIP1 complex in activation of ASK1-JNK signaling and EC apoptosis.
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Discussion |
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In the present study, we have investigated the mechanism by which TNF induces dephosphorylation of ASK1 pSer967 and subsequent release of 14-3-3 from ASK1, a critical step in TNF-induced ASK1-JNK signaling. Our data suggest that PP2A is a phosphatase critical for TNF-induced dephosphorylation of ASK1 pSer967 and activation of ASK1-JNK signaling pathway. Several lines of evidence support this conclusion. First, the PP2A-sensitive inhibitor okadaic acid inhibits TNF-induced dephosphorylation of ASK1 pSer967. Second, overexpression of PP2A wild type, but not a catalytic inactive form, induces dephosphorylation of ASK1 pSer967. More importantly, PP2A siRNA blunts TNF-induced dephosphorylation of ASK1 pSer967 and JNK activation. A similar role of PP2A in facilitating 14-3-3 release from other proapoptotic proteins has also been reported.28 For example, the proapoptotic protein BAD is phosphorylated at the 14-3-3 binding sites Ser112 and Ser136 under survival conditions. Following apoptotic stimuli, PP2A dephosphorylates the 14-3-3 binding sites, dissociating 14-3-3 from BAD and leading to the binding of BAD to Bcl-XL and induction of apoptosis.29However, our study suggests that PP2A appears not to directly bind ASK1, and AIP1 is required to bridge PP2A-ASK1 association.
We have previously shown that AIP1 via a lysine-rich cluster within the N-terminal C2 domain binds to ASK1. Interestingly, AIP1 binds to a sequence surrounding the 14-3-3 binding site on ASK1. In contrast to 14-3-3, AIP1 binds preferentially to dephosphorylated form of ASK1, facilitating the release of 14-3-3 from ASK1. These data had prompted us to propose that AIP1 functions together with a phosphatase(s) in regulating dephosphorylation of ASK1 pSer967.21 Our present study has provided evidence supporting this model. In resting ECs, PP2A-AIP1 complex can be detected in resting ECs, and is further enhanced by TNF stimulation. Kinetics studies suggest that PP2A-AIP1 complex formation precedes TNF-induced dephosphorylation of ASK1 pSer967 and activation of ASK1-JNK signaling. More importantly, we show that association of PP2A with ASK1 and ASK1-JNK activation are diminished in AIP1-knockdown ECs. Domain-mapping shows that PP2A binds to the GAP domain of AIP1. We have previously shown that ASK1 binds to the C2 domain, an immediate upstream domain of the GAP domain, suggesting that AIP1 brings PP2A and ASK1 in a close proximity, so that PP2A can dephosphorylate ASK1 (Figure 7). Furthermore, the functional significance of PP2A-AIP1 complex is demonstrated by a synergistic effect of PP2A and AIP1 on activation of ASK1-JNK signaling and EC apoptosis. Based on these data, we propose that AIP1 functions as a scaffolding protein in TNF-induced recruitment of PP2A to ASK1 complex, leading to dephosphorylation of ASK1 at pSer967 and activation of ASK1-JNK signaling. Previously, we have shown that AIP1 functions a scaffolding protein to recruit the TNF signaling complex (TRADD-RIP1-TRAF2) to ASK1,11 and TRAF2/RIP1-mediated AIP1 phosphorylation at Ser604 is critical for TNF-induced ASK1-JNK activation.26 Our present data demonstrate that an AIP1 mutant defective in phosphorylation at Ser604 fails to mediate TNF-induced ASK1, supporting that TRAF2 and RIP1 are critical for AIP1/PP2A-mediated ASK1 dephosphorylation (Figure 7).
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PP2A is a heterotrimer composed of catalytic C subunit, a structural A subunit, and 1 of several regulatory B-type subunits. In the present study, we have only examined the association of PP2A catalytic subunit with AIP1 and ASK1. We cannot exclude a possibility that ASK1 interacts with a regulatory subunit of PP2A to facilitate a complex formation of ASK1 with PP2A catalytic subunit. Nevertheless, ASK1-PP2A complex was diminished in AIP1-knockdown cells, highlighting a critical role of AIP1 in the formation of ASK1-PP2A complex.
Serine/threonine phosphatase family includes PP1, PP2A, PP2B, PP2C, PP4, and PP5, among these PP1 is also okadaic acid-sensitive. PP1 exists in 3 isoforms (, β, and 
), and all of these isoforms have been shown to be expressed in endothelial cells, in which they may play differential functions.30 The role of PP1 in TNF-induced ASK1 dephosphorylation needs to be further investigated. Similar to PP2A, PP2B (also called calcineurin) consists of 3 subunits: catalytic subunit, calcium-binding subunit, and calmodulin. A recent report shows that in cardiomyocytes calcineurin through its calcium-binding subunit binds to and directly dephosphorylates ASK1 pSer967.31 Moreover, the authors demonstrate that calcium–calcineurin–mediated (by overexpression of the catalytic subunit or treatment with ionomycin) dephosphorylation of ASK1 pSer967 is blocked by cyclophilin A but not by PP2A/PP1 inhibitor okadaic acid. Conversely, H2O2-mediated dephosphorylation of ASK1 pSer967 is blocked with okadaic acid but not by cyclophilin A, consistent with our previous observation24 and our present findings. It appears that different stress stimuli use different phosphatases (calcium via calcineurin and TNF/ROS via PP2A) in dephosphorylation of ASK1 pSer967.
ASK1 is phosphorylated at several Ser/Thr sites (pSer83, pThr845, pSer967, and pSer1034)25,32,33 as well as Tyr718.34 Regulation of phosphorylation (by kinase) and dephosphorylation (by phosphatase) at Thr845 has been investigated.25 In response to upstream signals, ASK1 is activated by autophosphorylation at Thr845.35 We have recently shown that ASK1 is also autophosphorylation at several other sites within the activation loop, leading to an enhanced ASK1 activity.36 PP5 and a member of PP2C (PP2C
) have been implicated in dephosphorylation of ASK1 Thr845 to negatively regulate ASK1 activity. PP5 binds to ASK1 In response to H2O2, functioning as a negative-feedback inhibitor of ASK1 signaling.25 Interestingly, we show that PP5 associates with AIP1 at a late phase in response to TNF, consistent with the kinetics of PP5-ASK1 complex formation.25 It is plausible that AIP1 may reciprocally regulate the recruitment of PP2A and PP5 to ASK1. PP2 bound to ASK1 under nonstress conditions and dissociated from ASK1 in response to H2O2 treatment, suggesting PP2C contributes to keeping the ASK1 signaling pathway in an inactive state.37 However, the regulation of ASK1 phosphorylation at other sites is less understood. It is known that the protein kinase Akt in complexed with Hsp90 phosphorylate ASK1 at Ser83 to inhibit ASK1-induced apoptosis.27,32 The kinases responsible for Ser967, Ser1034, and Tyr718 have not been identified. Similarly, the phosphatases specific to these sites have not been characterized. Our present study indicates that a phosphatase may need a specific scaffolding protein(s) to be recruited to ASK1, and may provide a new approach to identify the kinases and phosphatases involved in stress-activated ASK1 activation.
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Acknowledgments |
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Sources of Funding
This work was supported by NIH grants R01 HL-65978-5, R01 HL077357-1, and P01HL070295-6, American Heart Association Established Investigator Award 0440172N (to W.M.) and National Natural Science Foundation of China 30772342 (to S.T.).
Disclosures
None.
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Footnotes |
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Original received November 18, 2007; revision received February 8, 2008; accepted February 12, 2008.
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References |
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