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AIP1/DAB2IP, a Novel Member of the Ras-GAP Family, Transduces TRAF2-induced ASK1-JNK Activation

AIP1/DAB2IP, a Novel Member of the Ras-GAP Family, Transduces TRAF2-induced ASK1-JNK Activation THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 279, No. 43, Issue of October 22, pp. 44955–44965, 2004 © 2004 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. AIP1/DAB2IP, a Novel Member of the Ras-GAP Family, Transduces TRAF2-induced ASK1-JNK Activation* Received for publication, July 7, 2004, and in revised form, August 12, 2004 Published, JBC Papers in Press, August 13, 2004, DOI 10.1074/jbc.M407617200 Haifeng Zhang‡§, Rong Zhang‡§, Yan Luo‡ , Alessio D’Alessio‡ , Jordan S. Pober‡ , and Wang Min‡** From the ‡Interdepartmental Program in Vascular Biology and Transplantation, Boyer Center for Molecular Medicine, Department of Pathology and the Department of Immunology and Dermatology, Yale University School of Medicine, New Haven, Connecticut 06510 and the Zhongshan Ophthalmic Center, Sun Yat-Sen University, Guangzhou, Peoples Republic of China differentiation, and apoptosis. TNF signals are initiated by Previously we have shown that ASK-interacting pro- tein 1 (AIP1, also known as DAB2IP), a novel member of binding to either of two different cell surface receptors, the Ras-GAP protein family, mediates TNF-induced ac- known as TNFR1 (CD122a) and TNFR2 (CD122b). The best- tivation of ASK1-JNK signaling pathway. However, the characterized signaling pathways are initiated by TNFR1. A mechanism by which TNF signaling is coupled to AIP1 is current model postulates that in the absence of TNF, TNFR1 not known. Here we show that AIP1 is localized on the is kept in an inactive state through binding of an adaptor plasma membrane in resting endothelial cells (EC) in a protein called silencer of death-domains (SODD). The inter- complex with TNFR1. TNF binding induces release of action between TNFR1 and SODD is mediated by association AIP1 from TNFR1, resulting in cytoplasmic transloca- of death domains on both proteins. TNF induces dissociation tion and concomitant formation of an intracellular sig- of SODD from TNFR1 with concomitant recruitment of naling complex comprised of TRADD, RIP1, TRAF2, and TNFR-associated death-domain protein (TRADD) (3, 4). AIPl. A proline-rich region (amino acids 796–807) is crit- TRADD functions as a platform adaptor to initiate assembly ical for maintaining AIP1 in a closed form, which asso- of a multiprotein complex that activates downstream signal- ciates with a region of TNFR1 distinct from the death ing pathways. Specifically, TRADD recruits both receptor- domain, the site of TNFR1 association with TRADD. An interacting protein-1 (RIP1) and TNFR-associated factor AIP1 mutant with deletion of this proline-rich region (TRAF2), which in turn recruit the IB kinase complex (IKK) constitutively binds to TRAF2 and ASK1. A PERIOD-like and a mitogen-activated protein kinase kinase kinase domain (amino acids 591–719) of AIP1 binds to the intact (MAP3K), leading to activation of NF-B and JNK pathways, RING finger of TRAF2, and specifically enhances respectively (5–8). Recently it has been proposed that TRAF2-induced ASK1 activation. At the same time, the binding of AIP1 to TRAF2 inhibits TNF-induced IKK- TNFR1-TRADD-RIP-TRAF2 complex function as an initial NF-B signaling. Taken together, our data suggest that membrane bound complex (complex I) to specifically activate AIP1 is a novel transducer in TNF-induced TRAF2-de- the NF-B cascade. The role of the complex I in the JNK pendent activation of ASK1 that mediates a balance be- cascade has not been addressed (8). Complex I is rapidly tween JNK versus NF-B signaling. internalized and TRADD rapidly dissociates from TNFR1. After a lag of some several hours, internalized TRADD may recruit Fas-associated death domain protein (FADD) and pro- Cytokines of the TNF superfamily utilize receptors that caspase-8 to form a cytoplasmic complex (complex II). Com- are devoid of intrinsic catalytic activity (1). Vascular endo- plex II promotes autocatalytic activation of pro-caspase-8 to thelial cells (EC) are among the principal physiological tar- initiate apoptosis unless the NF-B-induced long isoform of gets of TNF (2). In EC, as in other cell types, TNF elicits a the component known as FLICE inhibitory protein (FLIP is L) broad spectrum of biological effects including proliferation, also present in the complex (9). The events between the dissociation of complex I and the formation of complex II are not well established. * This work was supported by National Institutes of Health Grants TRAF2 is a member of a family of signal transducing pro- HL-65978 (to W. M.) and HL-36003 and HV28286 (to J. S. P.). The costs teins. Six members of TRAF family have been extensively stud- of publication of this article were defrayed in part by the payment of ied (10) and TRAF7 has been recently described (11). JNK and page charges. This article must therefore be hereby marked “advertise- ment” in accordance with 18 U.S.C. Section 1734 solely to indicate this NF-B pathways can be activated by overexpression of TRAF 2, fact. 5, and 6, but not of TRAF 1, 3, and 4, suggesting that different § These authors contributed equally to this work. TRAFs, despite their structural homology, might perform very ** Established Investigator of the American Heart Association. To different functions. Studies from various cell types have iden- whom correspondence should be addressed: Interdepartmental Pro- gram in Vascular Biology and Transplantation and Department of tified TRAF2 as the bifurcation of two different kinase cascades Pathology, Yale University School of Medicine, BCMM 454, 295 Con- leading to activation of NF-B and JNK: TRADD-TRAF2/RIP- gress Ave., New Haven, CT 06510. Tel.: 203-785-6047; Fax: 203-737- IKK for NF-B activation, and TRADD-TRAF2-MAP3K- 2293; E-mail: [email protected]. MAP2K for JNK activation (5, 6). Recent studies of TRAF2 The abbreviations used are: TNF, tumor necrosis factor; EC, endo- thelial cell; GST, glutathione S-transferase; MES, 4-morpholineethane- knockout mice or transgenic mice expressing dominant nega- sulfonic acid; TRADD, TNFR-associated death domain; ASK1, apo- tive TRAF2 mutant protein have established that TRAF2 is ptosis signal-regulating kinase-1; AID, AIP1-binding domain; AIP, absolutely required for activation of JNK by TNF, although it ASK-interacting protein; TRAF, TNFR-associated factor; JNK, Jun may be redundant in some cell types for NF-B activation (12, N-terminal kinase; MAP, mitogen-activated protein; RIP, receptor- interacting protein-1. 13) due to presence of TRAF5 (14). Studies in transfected cells This paper is available on line at http://www.jbc.org 44955 This is an Open Access article under the CC BY license. 44956 AIP1 as a Transducer of TRAF2 MKK4, GST-IB fusion proteins as a substrate, respectively (24, 27). have shown that the intact RING finger of TRAF2 is critical for Transfection and Reporter Assay—Transfection of HUVEC was per- TRAF2 function since a TRAF2 mutant with deletion of the N formed by DEAE-Dextran method as described previously (27). BAEC terminus (87–501) behaves as a dominant negative mutant for and EAhy926 were transfected by LipofectAMINE 2000 (Invitrogen, both NF-B and JNK activation (15). The TRAF2 gene may Life Technologies). Luciferase activity followed by Renilla activity was code for an alternatively spliced form, designated as TRAF-2A, measured twice in duplicate using a Berthold luminometer. All data which differs from TRAF2 in that it contains a seven amino were normalized as relative luciferase light units/Renilla unit. Isolation of Caveolae-enriched Membranes—Purification of caveolae- acids insertion in its RING domain (16). TRAF-2A fails to enriched membrane fractions was performed as described (29) with activate NF-B pathway while still retains an ability to acti- minor modifications. In brief, EAhy.926 cells were treated with 10 vate the JNK pathway (17). These results suggest that NF-B ng/ml TNF for the indicated time point, washed twice with ice-cold and JNK pathways may be differentially regulated by minor Dulbecco’s phosphate-buffered saline and scraped into 1 ml of MBS (25 alterations in the effector domain of TRAF2 or by modulators mM MES, pH 6.5, 0.15 M NaCl, 5 mM EDTA, and 0,2% Triton X-100) that bind to different sites on the TRAF2 RING finger. with protease 15 inhibitors (10 g/ml aprotinin, 10 g/ml leupeptin, 1 mM sodium orthovanadate, 10 mM NaF, 1 mM Pefabloc) and left 20 min The mechanism by which TRAFs activates MAP3Ks remains on ice. The suspension was subjected to 10–15 strokes in a Dounce unclear, and there is some disagreement about which MAP3K homogenizer and centrifuged for 10 min at 2000 rpm at 4 °C to remove is critical for JNK activation. It has been shown that the TRAF high density debris. Clarified post-nuclear supernatants were combined domain of TRAF2 and TRAF6 interact with apoptosis signal- with 90% (w/v) sucrose prepared in MES, transferred to the bottom of a regulating kinase-1 (ASK1), a member of the MAP3K family Beckman 12.5 ml ultracentrifuge tubes and overlaid gently with 6 ml of that specifically activates a cascade ending with JNK (but not 35% and 3 ml of 5% sucrose respectively. The resulting 5–40% discon- tinuous sucrose gradients were centrifuged 18–20 h at 40,000 rpm in a NF-B) activation. The association of TRAFs with ASK1 is SW41 Beckman rotor at 4 °C to allow the separation of the low density required for ASK1 activation (18). ASK1 is a 170-kDa protein rafts/caveolae. After centrifugation, a floating light band, corresponding containing an inhibitory N-terminal domain, an internal ki- to the Triton X-100 insoluble material, was detectable at the interface nase domain, and the C-terminal domain interacting with between 35 and 5% of each gradient. Fractions were harvested from the TRAFs (18, 19). The current model for ASK1 activation by TNF top to the bottom of the gradients and analyzed either by SDS-PAGE involves several critical steps including release of inhibitors followed by immunoblotting for caveolin-1, TNFR1, TRAF2, or AIP1 or for binding of cholera toxin subunit B (CTxB) by dot blot analysis. (thioredoxin and 14-3-3) (20, 21), TRAF-dependent homodimer- Immunoprecipitation and Immunoblotting—EAhy926 cells or BAEC ization/polymerization (22), and ASK1 autophosphorylation at after various treatments were washed twice with cold PBS and lysed in Thr-845 (23). We have recently shown that TNF can induce a 1.5 ml of cold lysis buffer (50 mM Tris-HCl, pH 7.6, 150 mM NaCl, 0.1% TRAF2-dependent association of ASK1 with ASK-interacting Triton X-100, 0.75% Brij 96, 1 mM sodium orthovanadate, 1 mM sodium protein 1 (AIP1, also called DAB2IP for DAB2-interacting pro- fluoride, 1 mM sodium pyrophosphate, 10 g/ml aprotinin, 10 g/ml tein), a novel member of the Ras-GAP family (24, 25). AIP1 leupeptin, 2 mM phenylmethylsulfonyl fluoride, 1 mM EDTA) for 20 min on ice. Immunoprecipitation and immunoblotting were performed as causes 14-3-3 release from ASK1, initiating ASK1-dependent described previously (24, 27). A rabbit polyclonal antibody against AIP1 JNK activation (24). was generated by immunizing rabbits with GST-AIP1 protein through In the present study, we show that AIP1 is localized to the Cocalico Biologicals Inc. (Reamstown, PA) (24). Anti-TRAF2, anti- plasma membrane of EC where it associates with TNFR1 TRADD, anti-RIP1, anti-ASK1, and anti-GST were purchased from through a site distinct from the death domain involved in Santa Cruz Biotechnology. Anti-TNFR1 was purchased from R&D Sys- SODD and TRADD binding. In response to TNF, AIP1 dissoci- tem. Anti-FLAG (M2) was from Sigma. Indirect Immunofluorescence Confocal Microscopy—Fixation, perme- ates from TNFR1 with concomitant cytoplasmic translocation abilization, and staining of cultured EC were performed as described and formation of a complex comprised of TRADD, RIP1, previously (27). Alexa Fluor 488 (green) or 594 (red) conjugated-second- TRAF2, and AIP1, which is distinct from the complex I and ary antibodies (Molecular Probes, Eugene, OR) were used. Confocal complex II and which initiates specific activation of the ASK1- immunofluorescence microscopy was performed using an Olympus con- JNK pathway. Furthermore, we demonstrate that AIP1 specif- focal microscope and acquired images were transferred to Photoshop 6.0 ically interacts with the effector domain RING finger of TRAF2 to generate the final figures. to enhance TNF-induced ASK1-JNK but to inhibit IKK-NF-B RESULTS signaling. Our data suggest that AIP1 is a novel transducer of TNF Induces a Translocation of AIP1 from Plasma Mem- TRAF2 in TNF-induced ASK1-JNK signaling. brane to Cytoplasm in EC—We have recently shown that AIP1/ MATERIALS AND METHODS DAB2IP is highly expressed in EC but not in several common tumor cell lines including human prostate cancer and breast Plasmid Construction—Expression plasmids for truncated TNFR1 was generously provided by Dr. Martin Kronke (Institute for Medical cancer cell lines (24, 30). AIP1 contains a pleckstrin homology Microbiology, Immunology and Hygiene, Medical Center of the Univer- (PH) and a PKC-conserved region 2 (C2), two motifs implicated sity of Cologne, Cologne, Germany) (26). Expression plasmids for in plasma membrane targeting. To determine if AIP1 is local- TRAF2, ASK1, and AIP1 were described previously (24, 27). Deletion ized on EC plasma membrane, we first examined AIP1 local- constructs of TRAF2 and AIP1 were generated by PCR and cloning into ization in resting BAEC by indirect immunofluorescence mi- FLAG vectors. TRAF2-CA was generated by site-directed mutagenesis TM croscopy with an anti-AIP1 antibody. AIP1 showed a distinct using QuickChange site-directed mutagenesis kit (Stratagene) ac- cording to the protocol of the manufacturer. Constructs were confirma- plasma membrane staining (Fig. 1a, top panel). Control stain- tion by DNA sequencing and by Western blot for protein expression. ing with a normal rabbit serum did not show any staining. To Cells and Cytokines—Bovine ECs (BAECs) were purchased from further approve AIP1 membrane localization, an EC mem- Clonetics (San Diego, CA). Human umbilical vein EC (HUVEC) were brane protein CD31 was used as a positive control. AIP1 and isolated and cultured as described previously through a protocol ap- CD31 showed co-localization on EC (Fig. 1a, bottom panel). proved by the Yale Human Investigations Committee (27). The human AIP1 also showed plasma membrane localization in cultured EAhy926 cell line (28) (provided by Dr. W. Sessa, Yale University, New Haven, CT) was maintained in Dulbecco’s modified Eagle’s medium HUVEC (not shown). To determine the critical domain for AIP1 (Invitrogen, Life Technologies) containing 10% fetal calf serum, 2% for membrane localization, BAEC were transiently transfected (w/v) HAT (hypoxanthine/aminopterin/thymidine (Sigma), 200 ML- with expression constructs for FLAG-tagged AIP1 containing glutamine, 100 units/ml penicillin/streptomycin) (Invitrogen, Life Tech- various domains (AIP1-F for the full-length, AIP1-N for the nologies) at 37 C in a 5% CO humidified atmosphere. Human rTNF N-terminal domain containing the PH, C2, and GAP domains, was from R&D Systems (Minneapolis, MN) and used at 10 ng/ml. AIP1-PHC2, AIP1-PH, AIP1-PH for the mutant with a dele- The Kinase Assays for JNK, ASK1, and IKK—JNK, ASK1, and IKK assay was performed by kinase assays using GST-c-Jun-(1–80), GST- tion of the PH domain, and AIP1-C for the C-terminal domain). AIP1 as a Transducer of TRAF2 44957 FIG.1. TNF induces a translocation of AIP1 from plasma membrane to cy- toplasm in EC. a, AIP1 is localized on EC plasma membrane. Top panel, endog- enous AIP1 in BAEC was stained with anti-AIP1 followed by an Alexa Fluor 488- conjugated anti-rabbit secondary anti- body. Cells were then counterstained with 4,6-diamidino-2-phenylindole (DAPI) for nucleus. Bottom panel, AIP1 and EC membrane protein CD31 were costained with anti-AIP1 (rabbit) and anti-CD31 (mouse) followed by an Alexa Fluor 488 conjugated anti-rabbit and an Alexa Fluor 594 conjugated anti-mouse second- ary antibodies. Location of AIP1 and CD31 was visualized under a confocal flu- orescence microscope. b, critical domain of AIP1 for membrane localization. AIP1 constructs containing different domains (AIP1-F, N, PHC2, PH, and C) were transfected into BAEC, and localization of AIP1 proteins was determined by indirect immunofluorescence microscopy with an- ti-FLAG followed by an Alexa Fluor 488 (green)-conjugated anti-mouse secondary antibody. c, TNF induces AIP1 transloca- tion in a time-dependent manner. Human EC (HUVEC) were treated with TNF (10 ng/ml) for various time points (0, 5, 15, 30, 45, and 60 min). EC were stained with AIP1 followed by an Alexa Fluor 594-con- jugated anti-rabbit secondary antibody. Localization of AIP1 at 0, 15, and 60 min are shown. AIP1-F, AIP1-N, and PHC2 are predominantly localized on vesicles, and AIP1-PH also caused elongation of cells. In con- membrane vesicles (or microdomains). AIP1-PH showed trast, AIP1-C and AIP1-PH are detected in the cytoplasm, plasma membrane as well as cytoplasm with no detectable suggesting that the PH and C2 domain are critical for AIP1 44958 AIP1 as a Transducer of TRAF2 FIG.2. TNF induces release of AIP1 from TNFR1 with concomitant for- mation of AIP1 complex (TRADD- RIP1-TRAF2-AIP1-ASK1) in EC. a, hu- man EC EAhy926 were treated with TNF (10 ng/ml) for indicated times. Protein ex- pression was determined with respective antibodies. b, cell lysates were immuno- precipitated with indicated antibodies (TNFR1, TRADD, RIP1, TRAF2) followed by Western blot with anti-AIP1. As con- trols, TRAF2-ASK1 and AIP1-ASK1 com- plex were determined by immunoprecipi- tation with anti-TRAF2 or anti-AIP1 followed by Western blot with anti-ASK1. c, ASK1 activity was determined by an in vitro kinase assay using GST-MKK4 as a substrate. d, TNFR1-TRADD association precedes AIP1 complex. EAhy926 were treated with TNF for indicated time (0, 2, 5, 15, or 30 min) and association of TNFR1-TRADD was determined by im- munoprecipitation with anti-TNFR1 fol- lowed by Western blot with anti-TRADD. e, AIP1 is not localized in caveolae. EAhy926 cells were either untreated or treated with TNF (10 ng/ml for 15 min) and fractionated by sucrose gradient as described. No protein was detected in fractions 1–2 and 10 l of cell lysates from fractions 3–10 were subjected to Western blot with anti-TNFR1, anti-TRAF2 and anti-AIP1. TRADD, RIP1 and ASK1 were also determined. Anti-caveolin-1 was used as a control for caveolae. localization in membrane microdomains (Fig. 1b). EC were treated with TNF (10 ng/ml for 0, 15, and 60 min) and Previously we have shown that TNF induces association of association of AIP1 with TNFR1 signaling components by co- AIP1 with ASK1, which is predominantly localized in cyto- immunoprecipitation assays. TNF treatment did not alter pro- plasm, raising the possibility that TNF might induce a trans- tein expression of TNFR1, TRADD, RIP1, TRAF2, AIP1, or location of AIP1 from plasma membrane to cytoplasm. To test ASK1 (Fig. 2a). AIP1-TNFR1 complex is readily detected in this hypothesis, HUVEC were treated with TNF (10 ng/ml) for resting EC. In response to TNF, AIP1 is dissociated from various time points (0, 5, 15, 30, and 60 min) and localization of TNFR1 at 15 min but reassociates with TNFR1 at 60 min (Fig. AIP1 was determined as described. TNF treatment for 15 min 2b), consistent with the kinetics of AIP1 translocation. Forma- induces a translocation of AIP1 from plasma membrane to tion of AIP1-SODD complex shows a similar kinetics as that of cytoplasm. However, AIP1 returned to plasma membrane at 60 AIP1-TNFR1 complex (not shown). In contrast, TNF treatment min of post-treatment with TNF (Fig. 1c). The kinetics of in- induced a strong interaction of AIP1 with TRADD, RIP1, and ternalization is similar to that of TNF-induced AIP1-ASK1 TRAF2 at 15 min, which declined by 60 min of treatment (Fig. association and ASK1-JNK activation (Ref. 24, also see Fig. 2). 2b). Association of TRAF2 and AIP1 with ASK1 as well as These data suggest that TNF-induced AIP1 internalization ASK1 activation show a similar kinetics (Fig. 2c). These data correlates with formation of AIP1-ASK1 complex and activa- suggest that TNF induces formation of a complex comprising of tion of ASK1. TRADD-RIP1-TRAF2-AIP1-ASK1 (so named AIP1 complex) in TNF Induces Release of AIP1 from TNFR1 with Concomitant EC. Furthermore, we determined kinetics of TNFR1-TRADD Formation of AIP1 Complex (TRADD-RIP1-TRAF2-AIP1- complex, which peaks at 2 min, declines at 5 min and disap- ASK1) in EC—The membrane localization and rapid move- pears by 15 min (Fig. 2d), consistent with previous studies of ment of AIP1 in EC prompted us to examine association of HUVEC (31). Interestingly AIP1, which associates with the AIP1 with TNFR1 signaling components including TNFR1, unactivated TNFR1 molecule, is absent in the TNFR1-TRADD- TRADD, RIP1, and TRAF2. We found that AIP1 and TNFR1 RIP1-TRAF2 complex (complex I), which assembles on the are abundant in human EC cell line EAhy926 and we chose plasma membrane and has been shown to trigger activation of EAhy926 to determine endogenous AIP1-TNFR1 complexes. NF-B pathway (9). AIP1 as a Transducer of TRAF2 44959 EAhy926 displays an extensive caveolar system, which has AIP1 binding. The intracellular domain of TNFR1 comprises of been shown to be critical for signaling by TNFR1 and TRAF2 several conserved regions-a juxtamembrane (amino acids 205– (32, 33). To determine if AIP1 complex is localized in caveolae, 307), a NSD (amino acids 308–319), which binds to an adaptor EAhy926 were treated with TNF (10 ng/ml for 15) and cell protein FAN responsible for neutral sphingomyelinase activa- fractionations (fractions 1–10) by sucrose gradient centrifuga- tion domain (34), and the death-domain (amino acids 346– tion following Dounce homogenization in Triton X-100 buffer. 426), which binds to SODD in resting state while recruits In resting EC, the majority of TNFR1 is detected in fractions 10 TRADD in response to TNF (Fig. 4a). To map the AIP1-binding containing heavy membranes and a small portion of TNFR1 domain (AID) in TNFR1, AIP1-F, and various TNFR1 trun- can be detected in fractions 4–5 where caveolin-1 is typically cates were transfected into BAEC. Expression of TNFR1 pro- teins was determined by an indirect fluorescence microscopy distributed. TNF decreases distribution of TNFR1 in fractions 4–5 at 15 min (Fig. 2e). TRAF2 is also detected in caveolae with anti-TNFR1 (Fig. 4b). Association of AIP1 with TNFR1 fractions in both resting and TNF-treated EC. However, AIP1 was determined by co-immunoprecipitation assay with anti- is only detected in fractions 10, but not in other fractions, TNFR1 followed by Western blot with anti-FLAG (for AIP1-F). suggesting that AIP1 is not localized in caveolae (Fig. 2e). AIP1 associates TNFR1-WT and D212–308/346, but not with Similar to AIP1, TRADD, RIP1, and ASK1 were not detected in TNFR1-D205, D244, or D308–346), suggesting that the se- caveolae fractions (Fig. 2e). Taken together, these data suggest quence between amino acids 309–346 of TNFR1 is critical for that that AIP1 associates with TNFR1 in resting EC whereas AIP1 interaction (Fig. 4c). AIP1 poorly binds to TNFR1-D320 associates with TRADD-RIP1-TRAF2-ASK1 in TNF-treated with the intact NSD, which has been shown to be a FAN- EC in different membrane microdomains other than caveolae. binding motif, suggesting that AIP1 and FAN bind to different AIP1 in a Closed Inactive Form Associates with TNFR1 sequences of TNFR1. As a control, TRADD interacts with the death domain-containing TNFR1 (TNFR1-WT and D308–346) Whereas in an Open Active Form Binds to TRAF2/ASK1—We have previously shown that in resting EC AIP1 is retained in a as determined by Western blot with anti-TRADD. The results closed conformation by intramolecular interactions and AIP1 were summarized in Fig. 4a. Taken together, these data sug- associates with ASK1 only after the disruption of these in- gest that AIP1 associates with TNFR1 at a site distinct from tramolecular interactions (24). To define the critical domains the TRADD-binding domain (DD) and the FAN-binding for AIP1 intramolecular loop formation, we first generated domain (NSD). deletion constructs of AIP1 at the N terminus or the C terminus AIP1 via a PERIOD-like Domain Binds to the Intact RING (AIP1-PH with a deletion of the N-terminal PH, amino acids Finger of TRAF2—TRAF2 associates with AIP1 in response to 81–1056), AIP1-LZ with deletion of the C-terminal LZ motif, TNF (Fig. 2) and constitutively binds to an open form of AIP1 amino acids 1–910), and AIP1-PR with a deletion of the PR (AIP1-PR) (Fig. 3). To further map the critical domain in AIP1 region, amino acids 1–796) (Fig. 3a). EC were transfected with for TRAF2 binding, we determined association of TRAF2 with various AIP1 mutants, and association of ASK1 with various various truncated AIP1 proteins. Since we have previously AIP1 was determined by co-immunoprecipitation assays. AIP1- shown that the C2 domain of AIP1 is critical for ASK1 associ- PR, but not AIP1-F, AIP1-PH or AIP1-LZ, binds to ASK1 in ation (24), and we first examined if TRAF2 binds to the N- the absence of TNF (Fig. 3b, TNF). However, TNF treatment terminal half of AIP1. Results show that TRAF2 did not inter- (10 ng/ml for 15 min) induced association of ASK1 with these act with the N-terminal domains (AIP1-N, PHC2, and PH) (Fig. AIP1 proteins (Fig. 3b, TNF). These data suggest that the PR 5a), indicating that TRAF2 and ASK1 bind to different sites on region, but not the LZ motif or the PH domain, is a critical AIP1. To determine TRAF2 binds to the C-terminal half of domain in retaining AIP1 in a closed inactive form. AIP1, we generated truncated AIP1-C (AIP1-C, C-LZ with a Association of AIP1 mutants with endogenous TRAF2 and deletion of the LZ motif, C-PR with a deletion of the PR TNFR1 in EC were then examined by co-immunoprecipitation region). Results show that TRAF2 binds to AIP1-C-PR, but assays. AIP1-PR, but not AIP1-F, AIP1-PH, or AIP1-LZ, not AIP1-C or C-LZ (Fig. 5a). These data suggest that TRAF2 binds to TRAF2 in the absence of TNF (Fig. 3b, TNF). Like binds to an upstream sequence of the PR region in the C- ASK1, TRAF2 associated with the AIP1 proteins in response to terminal half of AIP1, and that the PR region is also critical to TNF (Fig. 3b, TNF). In contrast, TNFR1 associates with maintain AIP1-C in a closed conformation. Domain searching AIP1-F, AIP1-PH, and AIP1-LZ, but not to AIP1-PR (Fig. indicated that AIP1-C-PR does not contain a TRAF2-binding 3b). These data suggest that TNFR1 binds to a closed inactive consensus site but has a PERIOD-like domain (PER, amino whereas TRAF2/ASK1 bind to an open active form of AIP1. To acids 591–719). We further generated AIP1-C-PER (amino further test this idea, we examined association of AIP1-F (the acids 522–719) and C-tPER (amino acids 522–620) with a closed form) and AIP1-PR (the open form) with TNFR1 and truncation of PER domain. Association of TRAF2 with C-PR, TRAF2 in response to TNF. BAEC were transfected with C-PER, and C-tPER was then determined by a co-immunopre- AIP1-F or AIP1-PR in the absence or presence of TNF (10 cipitation assay. TRAF2 binds to AIP1-C-PR and C-PER, but ng/ml for 15 min). Association of AIP1 proteins with endoge- not C-tPER (Fig. 5b), suggesting that the PER domain in the nous TNFR1 and TRAF2 were determined. AIP1-F (but not C-terminal-half is involved in TRAF2 binding. AIP1-PR) binds to TNFR1 in resting EC, and AIP1-F/ To define the critical domain of TRAF2 for AIP1 binding, we TNFR1complex was significantly reduced in the presence of generated a series of mutant forms of FLAG-tagged TRAF2. TNF (Fig. 3c). In contrast, association of AIP1-F withTRAF2 is DN-TRAF2 (amino acids 80–531) contains a deletion of the only weakly detected in resting EC but is strongly induced in whole RING finger whereas TRAF2-CA contains a single mu- response to TNF. AIP1-PR constitutively binds to TRAF2 and tation at C31 within the RING finger leading to disruption of TNF has no effects on their interaction (Fig. 3c). These data the RING structure (15). TRAF2-N (amino acids 1–249) con- strongly suggest that TNF induces an alteration of AIP1 con- tains the intact RING and the five zinc fingers of the N-termi- formation leading to release of AIP1 from TNFR1 with concom- nal half of TRAF2 whereas TRFA2-C (amino acids 265–531) itant binding of AIP1 to the TRAF2/ASK1 complex. consists of the C-terminal TRAF domains of TRAF2 (Fig. 5c). AIP1 Associates with a Non-death Domain Region of AIP1-PR was co-transfected with various TRAF2, and associ- TNFR1—We next determined the critical domain in TNFR1 for ation of AIP1-PR with TRAF2 molecules was determined. As 44960 AIP1 as a Transducer of TRAF2 FIG.3. AIP1 in a closed inactive form associates with TNFR1 whereas in an open active form binds to TRAF2/ASK1. a, schematic diagram of AIP1 domains and expression constructs (C2, protein kinase C-conserved domain 2; GAP, GTPase-activating protein; PER, period-like domain; PR, proline-rich; LZ, leucine-zipper; aa, amino acid). b, proline-rich region is critical for closed conformation of AIP1. BAEC were transfected with FLAG-tagged truncated AIP1 (AIP1-F, PH, LZ, PR), and cells were untreated or treated with TNF (10 ng/ml for 15 min). Association of endogenous ASK1, TRAF2, or TNFR1 with AIP1 domains was examined by immunoprecipitation with a respective antibody followed by Western blot with anti-FLAG. c, TNF responses of AIP1-TNFR1 and TRAF2-AIP1 complexes. BAEC were transfected with AIP1-F or PR and treated with TNF (10 ng/ml for 15 min). Association of AIP1 with TNFR1 and TRAF2 was determined as in b. previously described, TRAF2-N was detected in an insoluble or the presence of TNF as indicated. Reporter activity was fraction (27) and we cannot determine association of AIP1 with measured for a luciferase assay. As shown previously, expres- TRAF2-N. Results show that only TRAF2-WT binds to AIP1- sion of TRAF2 in EC-induced activation of both JNK and PR. However, DN-TRAF2, TRAF2-CA, or TRAF2-C did not NF-B reporter genes (Fig. 6a). Co-expression of AIP1 strongly interact with AIP1-PR (Fig. 5d). These data indicate that the induces JNK reporter gene activation. In contrast, AIP1 dra- intact RING finger (the effector domain) of TRAF2 is required matically inhibits TRAF2-induced activation of B-reporter for AIP1 association. gene. A similar effect of AIP1 on TNF-induced activation of AIP1 Is a Transducer of TRAF2 and Specifically Induces JNK and NF-B reporter genes was observed (data not shown). ASK1-JNK Activation While It Inhibits NF-B Activation— AIP1 on TNF-induced activation of ASK1 and IB kinase (IKK) TRAF2 has been shown to be a critical adaptor in TNF-induced were also determined by an in vitro kinase assay using GST- activation of both JNK and NF-B cascades. We first deter- MKK4 and GST-IB as a substrate, respectively. As shown mined effects of AIP1 in TRAF2-induced JNK and NF-B acti- previously, AIP1 enhanced TNF-induced ASK1 activation. In vation in reporter gene assays. BAEC were transfected with a contrast, AIP1 strongly inhibited TNF-induced IKK activation JNK- or NF-B-dependent reporter gene with AIP1 in absence (Fig. 6b). These data suggest that AIP1 interacts with the AIP1 as a Transducer of TRAF2 44961 FIG.4. AIP1 associates with a non-death domain region of TNFR1. a, schematic diagram of TNFR1 domains and deletion constructs (EM, extracellular domain; TM, transmembrane; JMD, jaxtamembrane; NSD, neutral sphingomyelinase domain; DD, death domain; aa, amino acid). b and c, AIP1-interacting domain (AID) is located between NSD and DD. BAEC were transfected with FLAG-tagged AIP1 and various TNFR1 deletion constructs. Expression of TNFR1 proteins was determined by indirect immunofluorescence microscopy with anti-TNFR1 (goat, recognizing the extracellular domain of TNFR1) followed by an Alexa Fluor 488-conjugated anti-goat secondary antibody (b). Association of AIP1 domains with TNFR1 was determined by immunoprecipitation with anti-TNFR1 followed by Western blot with anti-FLAG (c). TNFR1-associated TRADD was also determined by anti-TRADD. Associations of these TNFR1 proteins with TRADD and AIP1 are summarized on the right panel in a. effector domain of TRAF2 to reciprocally regulate TNF-induced binding abilities for ASK1 and TRAF2. JNK and NF-B cascades. We have previously shown that a short hairpin RNA We then determined the critical domains of AIP1 in regulat- (shRNA) down-regulated endogenous AIP1 level leading to en- ing TNF-induced JNK and NF-B activation. BAEC were hanced TNF-induced ASK1-JNK activation and apoptosis (24). transfected with a JNK- or NF-B-dependent reporter gene To determine a physiological role of AIP1 in TNF-induced with various AIP1 in absence or the presence of TNF as indi- NF-B signaling, we further examined TNF-induced ASK1 and cated. Reporter activity was measured for a luciferase assay. IKK activation in AIP1-knockdown cells by in vitro kinase As shown previously, expression of TRAF2 in EC induced ac- assays as described. Endogenous AIP1 expression was signifi- tivation of both JNK and NF-B reporter genes (Fig. 6c). Ex- cantly (80%) reduced in BAEC expressing a short hairpin RNA pression of AIP1-F, N, or C-PR (but not C-tPER) significantly of AIP1 (ShAIP1) while expression of TRAF2 or ASK1 was not augmented TNF-induced JNK activation. In contrast, AIP1-F, altered (Fig. 6e). As we have shown previously (24), knockdown NorC-PR (but not C-tPER) dramatically inhibited TRAF2- of AIP1 inhibited TNF-induced ASK1 activation, However, induced NF-B reporter gene activity (Fig. 6d). A similar effect AIP1 knockdown cells showed enhanced TNF-induced IKK ki- of AIP1 on TRAF2-induced JNK and NF-B was observed (data nase activity (Fig. 6f). These data further support that physi- not shown). These data suggest that effects of AIP1-F, N, ological AIP1 differentially regulates TNF-induced ASK1-JNK C-PR, C-tPER) on JNK and B reporter correlate with their and IKK-NF-B pathways. 44962 AIP1 as a Transducer of TRAF2 FIG.5. AIP1 via a PERIOD-like do- main binds to the intact RING finger of TRAF2. a and b, critical domains of AIP1 for TRAF2 binding. Truncated AIP-1N (N, PHC2, and PH) and AIP1-C (C, C-PR, C-LZ) were transfected into BAEC. Association of endogenous TRAF2 with AIP1 proteins was determined by immunoprecipitation with anti-TRAF2 followed by Western blot with anti-FLAG. b, period-like domain (PER, amino acids 591–719) in AIP1 is critical for TRAF2 binding. AIP1-PR, C-PER (amino acids 522–719), and C-tPER (amino acids 522– 620 with a truncated PER domain). TRAF2 was co-transfected with various AIP1-C constructs (C-PR, C-PER and C- tPER). Association of TRAF2 with AIP1-C was determined by co-immunoprecipita- tion assay as described. c, diagrammatic representation of the domain structure of TRAF2 and expression constructs. The numbers refer to amino acid number in- dicating the boundary of the RING finger, five zinc fingers and the TRAF domains. d, intact RING finger of TRAF2 is critical for AIP1 association. BAEC were trans- fected with AIP1-PR and various TRAF2 constructs. Association of AIP1-PR with TRAF2 was determined by immunopre- cipitation with anti-TRAF2 followed by Western blot with anti-FLAG. DISCUSSION plex structurally different from complex I. Second, AIP1 com- plex is formed at 15 min in response to TNF and is dissociated Based on the data presented in this study, we propose the by 60 min. However, complex II formation detected in certain following model for the role of AIP1 in TNF signaling (Fig. 7). cell types (HT1080) at 2–8 h post-treatment with TNF. More- AIP1 via its PH/C2 domain is localized on plasma membrane over, we could not detect association of AIP1 with FADD and microdomain where it exists in an inactive form complexed pro-caspase-8 in response to TNF in EC (not shown). These with TNFR1. In response to TNF, AIP is unfolded and dissoci- data suggest that AIP1 complex is also structurally different ated from TNFR1 with concomitant of translocation of AIP1 to from complex II. Most significantly, AIP1 overexpression en- cytoplasm where it associates with TRAF2 and ASK1. More hances, whereas knockdown of AIP1 inhibits, TNF/TRAF2- significantly, AIP1 associates with the effector domain (the induced ASK1-JNK activation. However, AIP1 has opposite RING finger) of TRAF2 and mediates TNF/TRAF2-induced effects on TNF/TRAF2-induced IKK-NF-B pathway. These ASK1-JNK activation while inhibits IKK-NF-B signaling. data suggest AIP1 complex is functionally different from com- These data suggest that AIP1 is a novel transducer of TRAF2 plex I. In cell types including EC, ASK1-JNK activation in- in TNF signaling. duces apoptotic signaling which is dependent on intrinsic but Intermediate Complex—Tschopp and co-workers (9) have re- not extrinsic pathways. We have recently shown that AIP1 cently dissected TNFR1 signaling complexes in more details enhances ASK1-mediated JNK activation and EC apoptosis. and has proposed a sequential signaling complex model: the AIP1-suppressed IKK-NF-B activation further support that initial plasma membrane bound complex (complex I comprising AIP1 is proapoptotic. Thus, AIP complex is functionally differ- of TNFR1, TRADD, RIP1 and TRAF2) for NF-B activation and ent from complex II. Whether or not complex II is present in EC a cytoplasmic complex (complex II) in which the internalized TRADD, RIP, TRAF2 recruits FADD and pro-caspase-8 for are under investigation. AIP1 Specifies TRAF2 toward to ASK1-JNK Pathway— apoptotic signaling. However, it has not been determined if either complex I or complex II is active in the initiation of JNK TRAF2 has been shown to be the bifurcation point in TNF- induced activation of NF-B and activation of JNK (8, 35, 36). signaling. Since TRAF2 is critical for JNK activation, it seemed reasonable to assume that complex I, which contains TRAF2, While the C-terminal TRAF domain is responsible for associa- tion with TNFR1-TRADD complex as well as the MAP3Ks, the would recruit one or more MAP3Ks to induce JNK activation (8, 9). Here we provide several lines of evidence to support the N-terminal RING finger of TRAF2 is an effector domain in activation of both JNK and NF-B. Deletion of the RING do- existence of an intermediate complex, which lacks TNFR1, as being responsible for TRAF2-AIP1-mediated ASK1-JNK acti- main (TRAF2-(87–501)) completely loses the ability to activate JNK and NF-B, and this mutant functions as a dominant vation. First, AIP1 forms a preexisting complex with TNFR1 in EC. However, in response to TNF AIP1 is dissociated from negative. Specific downstream signaling events from TRAF2 TNFR1 and in turn forms a complex with TRADD, RIP1, are mediated by RIP/IKK and MAP3Ks. For example, IKK is TRAF2, and ASK1 (so named AIP1 complex). Thus, AIP1 com- specific for NF-B signaling, while ASK1 appear to direct sig- AIP1 as a Transducer of TRAF2 44963 FIG.6. AIP1 is a transducer of TRAF2 and specifically induces ASK1-JNK activation while inhibits IKK-NF-B activation. a, AIP1 specifically enhances TRAF2-induced JNK (but inhibits NF-B) activation. AIP1 together with a JNK or NF-B-dependent reporter gene was transfected into BAEC in the presence or absence of TRAF2 as indicated. A constitutive expression vector for Renilla was used as an internal control for transfection efficiency. Reporter gene activity was determined by luciferase assay as described. Data are presented as fold increase by expression of TRAF2 or AIP1 compared with the vector (as 1). Similar results were obtained from additional three independent experiments. b, AIP1 specifically enhances ASK1 whereas inhibits IKK activation by TNF. BAEC were transfected with a control vector or AIP1 and treated with TNF as indicated (10 ng/ml for 15 min). ASK1 and IKK activities were determined by an in vitro kinase assay using GST-MKK4 and GST-IKB, respectively. Relative ASK1 and IKK activities are presented by taking TNF-treated VC as 1.0. Similar results were obtained from two additional independent experiments. c and d, critical domains of AIP1 in TNF-induced activation of JNK and NF-B. BAEC were transfected with a JNK- or NF-B-dependent reporter gene with various AIP1 (F, N, PR, or tPER) as indicated and a constitutive expression vector for Renilla was used as an internal control for transfection efficiency. 24 h post-transfection, cells were treated with TNF (10 ng/ml) for 12 h. Reporter gene activity was determined by luciferase assay as described. Data are presented as fold increase by AIP1 expression by taking untreated vector as 1 (). Similar results were obtained from additional three independent experiments. e, knockdown of AIP1 in BAEC. BAEC were transiently transfected with pShag or pSh-AIP1 encoding a short-hairpin RNA of AIP1 (Sh-AIP1) as described previously (24). 48 h post-transfection, total cell lysates were used to determine AIP1 expression by Western blot with anti-AIP1. As expected, expression of TRAF2 and ASK1 was not altered by Sh-AIP1. f, knockdown of AIP1 suppresses ASK1 activity whereas enhances IKK activation by TNF. BAEC expressing pShag or pSh-AIP1 were untreated or treated with TNF (10 ng/ml for 15 min). ASK1 and IKK activities were determined as described in b. Relative ASK1 and IKK activities are presented by taking untreated pShag as 1.0. Similar results were obtained from additional two experiments. naling exclusively to JNK. In contrast, TAK1 and MEKK1 can induced activation of JNK but not of NF-B (40). These data activate both NF-B and JNK (37, 38). Recent studies suggest suggest that TRAF2 modulation/translocation or interactions that TRAF2 engagement is not obligatory to trigger NF-B and with other proteins may provide a new layer of regulation to JNK cascades simultaneously. For example, sphingosine ki- determine specific downstream signaling (activation of JNK nase binds to TRAF domain of TRAF2 and specifically activates and NF-B). Our study shows that AIP1 associates with NF-B while inhibits JNK activation (39). In contrast, TNF- TRAF2 in response to TNF and the intact RING finger of induced TRAF2 ubiquitination coincides with its translocation TRAF2 is required for TRAF2-AIP1 association. Moreover, to an insoluble cellular fraction, which is critical for TNF- TRAF2, AIP1, and ASK1 form a complex in response to TNF 44964 AIP1 as a Transducer of TRAF2 FIG.7. A proposed model for AIP1 as a transducer of TRAF2 in TNF-induced ASK1-JNK activation. AIP1 via its PH and C2 domains anchors to EC plasma membrane where it associates with a non-death domain region of TNFR1. In response to TNF, AIP1 is dissociated from TNFR1 with concomitant cytoplasmic translocation of AIP1 and formation of AIP1 complex comprising of TRADD-RIP-TRAF2-AIP1, which specifically activates ASK1-JNK signaling. As demonstrated by Tschopp and co-workers (9), TNFR1 recruits TRADD, RIP1, and TRAF2 to form a membrane-bound complex I, which specifically activates NF-B signaling. Since TNFR1-TRADD association precedes formation of AIP1 complex, it is likely that AIP1 complex is derived from complex I upon ubiquitination () and internalization of TRADD, RIP1, TRAF2, and AIP1. As we described previously (9), unfolded AIP1 binds to and activates ASK1-JNK at least in part by facilitating dephosphorylation of ASK1 at Ser and dissociation of ASK1 from its inhibitor 14-3-3. Joberty, G., Neubauer, G., Rick, J., Kuster, B., and Superti-Furga, G. 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AIP1/DAB2IP, a Novel Member of the Ras-GAP Family, Transduces TRAF2-induced ASK1-JNK Activation

Zhang, Haifeng; Zhang, Rong; Luo, Yan; D'Alessio, Alessio; Pober, Jordan S.; Min, Wang
Journal of Biological Chemistry – Oct 1, 2004
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10.1074/jbc.m407617200
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Abstract

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 279, No. 43, Issue of October 22, pp. 44955–44965, 2004 © 2004 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. AIP1/DAB2IP, a Novel Member of the Ras-GAP Family, Transduces TRAF2-induced ASK1-JNK Activation* Received for publication, July 7, 2004, and in revised form, August 12, 2004 Published, JBC Papers in Press, August 13, 2004, DOI 10.1074/jbc.M407617200 Haifeng Zhang‡§, Rong Zhang‡§, Yan Luo‡ , Alessio D’Alessio‡ , Jordan S. Pober‡ , and Wang Min‡** From the ‡Interdepartmental Program in Vascular Biology and Transplantation, Boyer Center for Molecular Medicine, Department of Pathology and the Department of Immunology and Dermatology, Yale University School of Medicine, New Haven, Connecticut 06510 and the Zhongshan Ophthalmic Center, Sun Yat-Sen University, Guangzhou, Peoples Republic of China differentiation, and apoptosis. TNF signals are initiated by Previously we have shown that ASK-interacting pro- tein 1 (AIP1, also known as DAB2IP), a novel member of binding to either of two different cell surface receptors, the Ras-GAP protein family, mediates TNF-induced ac- known as TNFR1 (CD122a) and TNFR2 (CD122b). The best- tivation of ASK1-JNK signaling pathway. However, the characterized signaling pathways are initiated by TNFR1. A mechanism by which TNF signaling is coupled to AIP1 is current model postulates that in the absence of TNF, TNFR1 not known. Here we show that AIP1 is localized on the is kept in an inactive state through binding of an adaptor plasma membrane in resting endothelial cells (EC) in a protein called silencer of death-domains (SODD). The inter- complex with TNFR1. TNF binding induces release of action between TNFR1 and SODD is mediated by association AIP1 from TNFR1, resulting in cytoplasmic transloca- of death domains on both proteins. TNF induces dissociation tion and concomitant formation of an intracellular sig- of SODD from TNFR1 with concomitant recruitment of naling complex comprised of TRADD, RIP1, TRAF2, and TNFR-associated death-domain protein (TRADD) (3, 4). AIPl. A proline-rich region (amino acids 796–807) is crit- TRADD functions as a platform adaptor to initiate assembly ical for maintaining AIP1 in a closed form, which asso- of a multiprotein complex that activates downstream signal- ciates with a region of TNFR1 distinct from the death ing pathways. Specifically, TRADD recruits both receptor- domain, the site of TNFR1 association with TRADD. An interacting protein-1 (RIP1) and TNFR-associated factor AIP1 mutant with deletion of this proline-rich region (TRAF2), which in turn recruit the IB kinase complex (IKK) constitutively binds to TRAF2 and ASK1. A PERIOD-like and a mitogen-activated protein kinase kinase kinase domain (amino acids 591–719) of AIP1 binds to the intact (MAP3K), leading to activation of NF-B and JNK pathways, RING finger of TRAF2, and specifically enhances respectively (5–8). Recently it has been proposed that TRAF2-induced ASK1 activation. At the same time, the binding of AIP1 to TRAF2 inhibits TNF-induced IKK- TNFR1-TRADD-RIP-TRAF2 complex function as an initial NF-B signaling. Taken together, our data suggest that membrane bound complex (complex I) to specifically activate AIP1 is a novel transducer in TNF-induced TRAF2-de- the NF-B cascade. The role of the complex I in the JNK pendent activation of ASK1 that mediates a balance be- cascade has not been addressed (8). Complex I is rapidly tween JNK versus NF-B signaling. internalized and TRADD rapidly dissociates from TNFR1. After a lag of some several hours, internalized TRADD may recruit Fas-associated death domain protein (FADD) and pro- Cytokines of the TNF superfamily utilize receptors that caspase-8 to form a cytoplasmic complex (complex II). Com- are devoid of intrinsic catalytic activity (1). Vascular endo- plex II promotes autocatalytic activation of pro-caspase-8 to thelial cells (EC) are among the principal physiological tar- initiate apoptosis unless the NF-B-induced long isoform of gets of TNF (2). In EC, as in other cell types, TNF elicits a the component known as FLICE inhibitory protein (FLIP is L) broad spectrum of biological effects including proliferation, also present in the complex (9). The events between the dissociation of complex I and the formation of complex II are not well established. * This work was supported by National Institutes of Health Grants TRAF2 is a member of a family of signal transducing pro- HL-65978 (to W. M.) and HL-36003 and HV28286 (to J. S. P.). The costs teins. Six members of TRAF family have been extensively stud- of publication of this article were defrayed in part by the payment of ied (10) and TRAF7 has been recently described (11). JNK and page charges. This article must therefore be hereby marked “advertise- ment” in accordance with 18 U.S.C. Section 1734 solely to indicate this NF-B pathways can be activated by overexpression of TRAF 2, fact. 5, and 6, but not of TRAF 1, 3, and 4, suggesting that different § These authors contributed equally to this work. TRAFs, despite their structural homology, might perform very ** Established Investigator of the American Heart Association. To different functions. Studies from various cell types have iden- whom correspondence should be addressed: Interdepartmental Pro- gram in Vascular Biology and Transplantation and Department of tified TRAF2 as the bifurcation of two different kinase cascades Pathology, Yale University School of Medicine, BCMM 454, 295 Con- leading to activation of NF-B and JNK: TRADD-TRAF2/RIP- gress Ave., New Haven, CT 06510. Tel.: 203-785-6047; Fax: 203-737- IKK for NF-B activation, and TRADD-TRAF2-MAP3K- 2293; E-mail: [email protected]. MAP2K for JNK activation (5, 6). Recent studies of TRAF2 The abbreviations used are: TNF, tumor necrosis factor; EC, endo- thelial cell; GST, glutathione S-transferase; MES, 4-morpholineethane- knockout mice or transgenic mice expressing dominant nega- sulfonic acid; TRADD, TNFR-associated death domain; ASK1, apo- tive TRAF2 mutant protein have established that TRAF2 is ptosis signal-regulating kinase-1; AID, AIP1-binding domain; AIP, absolutely required for activation of JNK by TNF, although it ASK-interacting protein; TRAF, TNFR-associated factor; JNK, Jun may be redundant in some cell types for NF-B activation (12, N-terminal kinase; MAP, mitogen-activated protein; RIP, receptor- interacting protein-1. 13) due to presence of TRAF5 (14). Studies in transfected cells This paper is available on line at http://www.jbc.org 44955 This is an Open Access article under the CC BY license. 44956 AIP1 as a Transducer of TRAF2 MKK4, GST-IB fusion proteins as a substrate, respectively (24, 27). have shown that the intact RING finger of TRAF2 is critical for Transfection and Reporter Assay—Transfection of HUVEC was per- TRAF2 function since a TRAF2 mutant with deletion of the N formed by DEAE-Dextran method as described previously (27). BAEC terminus (87–501) behaves as a dominant negative mutant for and EAhy926 were transfected by LipofectAMINE 2000 (Invitrogen, both NF-B and JNK activation (15). The TRAF2 gene may Life Technologies). Luciferase activity followed by Renilla activity was code for an alternatively spliced form, designated as TRAF-2A, measured twice in duplicate using a Berthold luminometer. All data which differs from TRAF2 in that it contains a seven amino were normalized as relative luciferase light units/Renilla unit. Isolation of Caveolae-enriched Membranes—Purification of caveolae- acids insertion in its RING domain (16). TRAF-2A fails to enriched membrane fractions was performed as described (29) with activate NF-B pathway while still retains an ability to acti- minor modifications. In brief, EAhy.926 cells were treated with 10 vate the JNK pathway (17). These results suggest that NF-B ng/ml TNF for the indicated time point, washed twice with ice-cold and JNK pathways may be differentially regulated by minor Dulbecco’s phosphate-buffered saline and scraped into 1 ml of MBS (25 alterations in the effector domain of TRAF2 or by modulators mM MES, pH 6.5, 0.15 M NaCl, 5 mM EDTA, and 0,2% Triton X-100) that bind to different sites on the TRAF2 RING finger. with protease 15 inhibitors (10 g/ml aprotinin, 10 g/ml leupeptin, 1 mM sodium orthovanadate, 10 mM NaF, 1 mM Pefabloc) and left 20 min The mechanism by which TRAFs activates MAP3Ks remains on ice. The suspension was subjected to 10–15 strokes in a Dounce unclear, and there is some disagreement about which MAP3K homogenizer and centrifuged for 10 min at 2000 rpm at 4 °C to remove is critical for JNK activation. It has been shown that the TRAF high density debris. Clarified post-nuclear supernatants were combined domain of TRAF2 and TRAF6 interact with apoptosis signal- with 90% (w/v) sucrose prepared in MES, transferred to the bottom of a regulating kinase-1 (ASK1), a member of the MAP3K family Beckman 12.5 ml ultracentrifuge tubes and overlaid gently with 6 ml of that specifically activates a cascade ending with JNK (but not 35% and 3 ml of 5% sucrose respectively. The resulting 5–40% discon- tinuous sucrose gradients were centrifuged 18–20 h at 40,000 rpm in a NF-B) activation. The association of TRAFs with ASK1 is SW41 Beckman rotor at 4 °C to allow the separation of the low density required for ASK1 activation (18). ASK1 is a 170-kDa protein rafts/caveolae. After centrifugation, a floating light band, corresponding containing an inhibitory N-terminal domain, an internal ki- to the Triton X-100 insoluble material, was detectable at the interface nase domain, and the C-terminal domain interacting with between 35 and 5% of each gradient. Fractions were harvested from the TRAFs (18, 19). The current model for ASK1 activation by TNF top to the bottom of the gradients and analyzed either by SDS-PAGE involves several critical steps including release of inhibitors followed by immunoblotting for caveolin-1, TNFR1, TRAF2, or AIP1 or for binding of cholera toxin subunit B (CTxB) by dot blot analysis. (thioredoxin and 14-3-3) (20, 21), TRAF-dependent homodimer- Immunoprecipitation and Immunoblotting—EAhy926 cells or BAEC ization/polymerization (22), and ASK1 autophosphorylation at after various treatments were washed twice with cold PBS and lysed in Thr-845 (23). We have recently shown that TNF can induce a 1.5 ml of cold lysis buffer (50 mM Tris-HCl, pH 7.6, 150 mM NaCl, 0.1% TRAF2-dependent association of ASK1 with ASK-interacting Triton X-100, 0.75% Brij 96, 1 mM sodium orthovanadate, 1 mM sodium protein 1 (AIP1, also called DAB2IP for DAB2-interacting pro- fluoride, 1 mM sodium pyrophosphate, 10 g/ml aprotinin, 10 g/ml tein), a novel member of the Ras-GAP family (24, 25). AIP1 leupeptin, 2 mM phenylmethylsulfonyl fluoride, 1 mM EDTA) for 20 min on ice. Immunoprecipitation and immunoblotting were performed as causes 14-3-3 release from ASK1, initiating ASK1-dependent described previously (24, 27). A rabbit polyclonal antibody against AIP1 JNK activation (24). was generated by immunizing rabbits with GST-AIP1 protein through In the present study, we show that AIP1 is localized to the Cocalico Biologicals Inc. (Reamstown, PA) (24). Anti-TRAF2, anti- plasma membrane of EC where it associates with TNFR1 TRADD, anti-RIP1, anti-ASK1, and anti-GST were purchased from through a site distinct from the death domain involved in Santa Cruz Biotechnology. Anti-TNFR1 was purchased from R&D Sys- SODD and TRADD binding. In response to TNF, AIP1 dissoci- tem. Anti-FLAG (M2) was from Sigma. Indirect Immunofluorescence Confocal Microscopy—Fixation, perme- ates from TNFR1 with concomitant cytoplasmic translocation abilization, and staining of cultured EC were performed as described and formation of a complex comprised of TRADD, RIP1, previously (27). Alexa Fluor 488 (green) or 594 (red) conjugated-second- TRAF2, and AIP1, which is distinct from the complex I and ary antibodies (Molecular Probes, Eugene, OR) were used. Confocal complex II and which initiates specific activation of the ASK1- immunofluorescence microscopy was performed using an Olympus con- JNK pathway. Furthermore, we demonstrate that AIP1 specif- focal microscope and acquired images were transferred to Photoshop 6.0 ically interacts with the effector domain RING finger of TRAF2 to generate the final figures. to enhance TNF-induced ASK1-JNK but to inhibit IKK-NF-B RESULTS signaling. Our data suggest that AIP1 is a novel transducer of TNF Induces a Translocation of AIP1 from Plasma Mem- TRAF2 in TNF-induced ASK1-JNK signaling. brane to Cytoplasm in EC—We have recently shown that AIP1/ MATERIALS AND METHODS DAB2IP is highly expressed in EC but not in several common tumor cell lines including human prostate cancer and breast Plasmid Construction—Expression plasmids for truncated TNFR1 was generously provided by Dr. Martin Kronke (Institute for Medical cancer cell lines (24, 30). AIP1 contains a pleckstrin homology Microbiology, Immunology and Hygiene, Medical Center of the Univer- (PH) and a PKC-conserved region 2 (C2), two motifs implicated sity of Cologne, Cologne, Germany) (26). Expression plasmids for in plasma membrane targeting. To determine if AIP1 is local- TRAF2, ASK1, and AIP1 were described previously (24, 27). Deletion ized on EC plasma membrane, we first examined AIP1 local- constructs of TRAF2 and AIP1 were generated by PCR and cloning into ization in resting BAEC by indirect immunofluorescence mi- FLAG vectors. TRAF2-CA was generated by site-directed mutagenesis TM croscopy with an anti-AIP1 antibody. AIP1 showed a distinct using QuickChange site-directed mutagenesis kit (Stratagene) ac- cording to the protocol of the manufacturer. Constructs were confirma- plasma membrane staining (Fig. 1a, top panel). Control stain- tion by DNA sequencing and by Western blot for protein expression. ing with a normal rabbit serum did not show any staining. To Cells and Cytokines—Bovine ECs (BAECs) were purchased from further approve AIP1 membrane localization, an EC mem- Clonetics (San Diego, CA). Human umbilical vein EC (HUVEC) were brane protein CD31 was used as a positive control. AIP1 and isolated and cultured as described previously through a protocol ap- CD31 showed co-localization on EC (Fig. 1a, bottom panel). proved by the Yale Human Investigations Committee (27). The human AIP1 also showed plasma membrane localization in cultured EAhy926 cell line (28) (provided by Dr. W. Sessa, Yale University, New Haven, CT) was maintained in Dulbecco’s modified Eagle’s medium HUVEC (not shown). To determine the critical domain for AIP1 (Invitrogen, Life Technologies) containing 10% fetal calf serum, 2% for membrane localization, BAEC were transiently transfected (w/v) HAT (hypoxanthine/aminopterin/thymidine (Sigma), 200 ML- with expression constructs for FLAG-tagged AIP1 containing glutamine, 100 units/ml penicillin/streptomycin) (Invitrogen, Life Tech- various domains (AIP1-F for the full-length, AIP1-N for the nologies) at 37 C in a 5% CO humidified atmosphere. Human rTNF N-terminal domain containing the PH, C2, and GAP domains, was from R&D Systems (Minneapolis, MN) and used at 10 ng/ml. AIP1-PHC2, AIP1-PH, AIP1-PH for the mutant with a dele- The Kinase Assays for JNK, ASK1, and IKK—JNK, ASK1, and IKK assay was performed by kinase assays using GST-c-Jun-(1–80), GST- tion of the PH domain, and AIP1-C for the C-terminal domain). AIP1 as a Transducer of TRAF2 44957 FIG.1. TNF induces a translocation of AIP1 from plasma membrane to cy- toplasm in EC. a, AIP1 is localized on EC plasma membrane. Top panel, endog- enous AIP1 in BAEC was stained with anti-AIP1 followed by an Alexa Fluor 488- conjugated anti-rabbit secondary anti- body. Cells were then counterstained with 4,6-diamidino-2-phenylindole (DAPI) for nucleus. Bottom panel, AIP1 and EC membrane protein CD31 were costained with anti-AIP1 (rabbit) and anti-CD31 (mouse) followed by an Alexa Fluor 488 conjugated anti-rabbit and an Alexa Fluor 594 conjugated anti-mouse second- ary antibodies. Location of AIP1 and CD31 was visualized under a confocal flu- orescence microscope. b, critical domain of AIP1 for membrane localization. AIP1 constructs containing different domains (AIP1-F, N, PHC2, PH, and C) were transfected into BAEC, and localization of AIP1 proteins was determined by indirect immunofluorescence microscopy with an- ti-FLAG followed by an Alexa Fluor 488 (green)-conjugated anti-mouse secondary antibody. c, TNF induces AIP1 transloca- tion in a time-dependent manner. Human EC (HUVEC) were treated with TNF (10 ng/ml) for various time points (0, 5, 15, 30, 45, and 60 min). EC were stained with AIP1 followed by an Alexa Fluor 594-con- jugated anti-rabbit secondary antibody. Localization of AIP1 at 0, 15, and 60 min are shown. AIP1-F, AIP1-N, and PHC2 are predominantly localized on vesicles, and AIP1-PH also caused elongation of cells. In con- membrane vesicles (or microdomains). AIP1-PH showed trast, AIP1-C and AIP1-PH are detected in the cytoplasm, plasma membrane as well as cytoplasm with no detectable suggesting that the PH and C2 domain are critical for AIP1 44958 AIP1 as a Transducer of TRAF2 FIG.2. TNF induces release of AIP1 from TNFR1 with concomitant for- mation of AIP1 complex (TRADD- RIP1-TRAF2-AIP1-ASK1) in EC. a, hu- man EC EAhy926 were treated with TNF (10 ng/ml) for indicated times. Protein ex- pression was determined with respective antibodies. b, cell lysates were immuno- precipitated with indicated antibodies (TNFR1, TRADD, RIP1, TRAF2) followed by Western blot with anti-AIP1. As con- trols, TRAF2-ASK1 and AIP1-ASK1 com- plex were determined by immunoprecipi- tation with anti-TRAF2 or anti-AIP1 followed by Western blot with anti-ASK1. c, ASK1 activity was determined by an in vitro kinase assay using GST-MKK4 as a substrate. d, TNFR1-TRADD association precedes AIP1 complex. EAhy926 were treated with TNF for indicated time (0, 2, 5, 15, or 30 min) and association of TNFR1-TRADD was determined by im- munoprecipitation with anti-TNFR1 fol- lowed by Western blot with anti-TRADD. e, AIP1 is not localized in caveolae. EAhy926 cells were either untreated or treated with TNF (10 ng/ml for 15 min) and fractionated by sucrose gradient as described. No protein was detected in fractions 1–2 and 10 l of cell lysates from fractions 3–10 were subjected to Western blot with anti-TNFR1, anti-TRAF2 and anti-AIP1. TRADD, RIP1 and ASK1 were also determined. Anti-caveolin-1 was used as a control for caveolae. localization in membrane microdomains (Fig. 1b). EC were treated with TNF (10 ng/ml for 0, 15, and 60 min) and Previously we have shown that TNF induces association of association of AIP1 with TNFR1 signaling components by co- AIP1 with ASK1, which is predominantly localized in cyto- immunoprecipitation assays. TNF treatment did not alter pro- plasm, raising the possibility that TNF might induce a trans- tein expression of TNFR1, TRADD, RIP1, TRAF2, AIP1, or location of AIP1 from plasma membrane to cytoplasm. To test ASK1 (Fig. 2a). AIP1-TNFR1 complex is readily detected in this hypothesis, HUVEC were treated with TNF (10 ng/ml) for resting EC. In response to TNF, AIP1 is dissociated from various time points (0, 5, 15, 30, and 60 min) and localization of TNFR1 at 15 min but reassociates with TNFR1 at 60 min (Fig. AIP1 was determined as described. TNF treatment for 15 min 2b), consistent with the kinetics of AIP1 translocation. Forma- induces a translocation of AIP1 from plasma membrane to tion of AIP1-SODD complex shows a similar kinetics as that of cytoplasm. However, AIP1 returned to plasma membrane at 60 AIP1-TNFR1 complex (not shown). In contrast, TNF treatment min of post-treatment with TNF (Fig. 1c). The kinetics of in- induced a strong interaction of AIP1 with TRADD, RIP1, and ternalization is similar to that of TNF-induced AIP1-ASK1 TRAF2 at 15 min, which declined by 60 min of treatment (Fig. association and ASK1-JNK activation (Ref. 24, also see Fig. 2). 2b). Association of TRAF2 and AIP1 with ASK1 as well as These data suggest that TNF-induced AIP1 internalization ASK1 activation show a similar kinetics (Fig. 2c). These data correlates with formation of AIP1-ASK1 complex and activa- suggest that TNF induces formation of a complex comprising of tion of ASK1. TRADD-RIP1-TRAF2-AIP1-ASK1 (so named AIP1 complex) in TNF Induces Release of AIP1 from TNFR1 with Concomitant EC. Furthermore, we determined kinetics of TNFR1-TRADD Formation of AIP1 Complex (TRADD-RIP1-TRAF2-AIP1- complex, which peaks at 2 min, declines at 5 min and disap- ASK1) in EC—The membrane localization and rapid move- pears by 15 min (Fig. 2d), consistent with previous studies of ment of AIP1 in EC prompted us to examine association of HUVEC (31). Interestingly AIP1, which associates with the AIP1 with TNFR1 signaling components including TNFR1, unactivated TNFR1 molecule, is absent in the TNFR1-TRADD- TRADD, RIP1, and TRAF2. We found that AIP1 and TNFR1 RIP1-TRAF2 complex (complex I), which assembles on the are abundant in human EC cell line EAhy926 and we chose plasma membrane and has been shown to trigger activation of EAhy926 to determine endogenous AIP1-TNFR1 complexes. NF-B pathway (9). AIP1 as a Transducer of TRAF2 44959 EAhy926 displays an extensive caveolar system, which has AIP1 binding. The intracellular domain of TNFR1 comprises of been shown to be critical for signaling by TNFR1 and TRAF2 several conserved regions-a juxtamembrane (amino acids 205– (32, 33). To determine if AIP1 complex is localized in caveolae, 307), a NSD (amino acids 308–319), which binds to an adaptor EAhy926 were treated with TNF (10 ng/ml for 15) and cell protein FAN responsible for neutral sphingomyelinase activa- fractionations (fractions 1–10) by sucrose gradient centrifuga- tion domain (34), and the death-domain (amino acids 346– tion following Dounce homogenization in Triton X-100 buffer. 426), which binds to SODD in resting state while recruits In resting EC, the majority of TNFR1 is detected in fractions 10 TRADD in response to TNF (Fig. 4a). To map the AIP1-binding containing heavy membranes and a small portion of TNFR1 domain (AID) in TNFR1, AIP1-F, and various TNFR1 trun- can be detected in fractions 4–5 where caveolin-1 is typically cates were transfected into BAEC. Expression of TNFR1 pro- teins was determined by an indirect fluorescence microscopy distributed. TNF decreases distribution of TNFR1 in fractions 4–5 at 15 min (Fig. 2e). TRAF2 is also detected in caveolae with anti-TNFR1 (Fig. 4b). Association of AIP1 with TNFR1 fractions in both resting and TNF-treated EC. However, AIP1 was determined by co-immunoprecipitation assay with anti- is only detected in fractions 10, but not in other fractions, TNFR1 followed by Western blot with anti-FLAG (for AIP1-F). suggesting that AIP1 is not localized in caveolae (Fig. 2e). AIP1 associates TNFR1-WT and D212–308/346, but not with Similar to AIP1, TRADD, RIP1, and ASK1 were not detected in TNFR1-D205, D244, or D308–346), suggesting that the se- caveolae fractions (Fig. 2e). Taken together, these data suggest quence between amino acids 309–346 of TNFR1 is critical for that that AIP1 associates with TNFR1 in resting EC whereas AIP1 interaction (Fig. 4c). AIP1 poorly binds to TNFR1-D320 associates with TRADD-RIP1-TRAF2-ASK1 in TNF-treated with the intact NSD, which has been shown to be a FAN- EC in different membrane microdomains other than caveolae. binding motif, suggesting that AIP1 and FAN bind to different AIP1 in a Closed Inactive Form Associates with TNFR1 sequences of TNFR1. As a control, TRADD interacts with the death domain-containing TNFR1 (TNFR1-WT and D308–346) Whereas in an Open Active Form Binds to TRAF2/ASK1—We have previously shown that in resting EC AIP1 is retained in a as determined by Western blot with anti-TRADD. The results closed conformation by intramolecular interactions and AIP1 were summarized in Fig. 4a. Taken together, these data sug- associates with ASK1 only after the disruption of these in- gest that AIP1 associates with TNFR1 at a site distinct from tramolecular interactions (24). To define the critical domains the TRADD-binding domain (DD) and the FAN-binding for AIP1 intramolecular loop formation, we first generated domain (NSD). deletion constructs of AIP1 at the N terminus or the C terminus AIP1 via a PERIOD-like Domain Binds to the Intact RING (AIP1-PH with a deletion of the N-terminal PH, amino acids Finger of TRAF2—TRAF2 associates with AIP1 in response to 81–1056), AIP1-LZ with deletion of the C-terminal LZ motif, TNF (Fig. 2) and constitutively binds to an open form of AIP1 amino acids 1–910), and AIP1-PR with a deletion of the PR (AIP1-PR) (Fig. 3). To further map the critical domain in AIP1 region, amino acids 1–796) (Fig. 3a). EC were transfected with for TRAF2 binding, we determined association of TRAF2 with various AIP1 mutants, and association of ASK1 with various various truncated AIP1 proteins. Since we have previously AIP1 was determined by co-immunoprecipitation assays. AIP1- shown that the C2 domain of AIP1 is critical for ASK1 associ- PR, but not AIP1-F, AIP1-PH or AIP1-LZ, binds to ASK1 in ation (24), and we first examined if TRAF2 binds to the N- the absence of TNF (Fig. 3b, TNF). However, TNF treatment terminal half of AIP1. Results show that TRAF2 did not inter- (10 ng/ml for 15 min) induced association of ASK1 with these act with the N-terminal domains (AIP1-N, PHC2, and PH) (Fig. AIP1 proteins (Fig. 3b, TNF). These data suggest that the PR 5a), indicating that TRAF2 and ASK1 bind to different sites on region, but not the LZ motif or the PH domain, is a critical AIP1. To determine TRAF2 binds to the C-terminal half of domain in retaining AIP1 in a closed inactive form. AIP1, we generated truncated AIP1-C (AIP1-C, C-LZ with a Association of AIP1 mutants with endogenous TRAF2 and deletion of the LZ motif, C-PR with a deletion of the PR TNFR1 in EC were then examined by co-immunoprecipitation region). Results show that TRAF2 binds to AIP1-C-PR, but assays. AIP1-PR, but not AIP1-F, AIP1-PH, or AIP1-LZ, not AIP1-C or C-LZ (Fig. 5a). These data suggest that TRAF2 binds to TRAF2 in the absence of TNF (Fig. 3b, TNF). Like binds to an upstream sequence of the PR region in the C- ASK1, TRAF2 associated with the AIP1 proteins in response to terminal half of AIP1, and that the PR region is also critical to TNF (Fig. 3b, TNF). In contrast, TNFR1 associates with maintain AIP1-C in a closed conformation. Domain searching AIP1-F, AIP1-PH, and AIP1-LZ, but not to AIP1-PR (Fig. indicated that AIP1-C-PR does not contain a TRAF2-binding 3b). These data suggest that TNFR1 binds to a closed inactive consensus site but has a PERIOD-like domain (PER, amino whereas TRAF2/ASK1 bind to an open active form of AIP1. To acids 591–719). We further generated AIP1-C-PER (amino further test this idea, we examined association of AIP1-F (the acids 522–719) and C-tPER (amino acids 522–620) with a closed form) and AIP1-PR (the open form) with TNFR1 and truncation of PER domain. Association of TRAF2 with C-PR, TRAF2 in response to TNF. BAEC were transfected with C-PER, and C-tPER was then determined by a co-immunopre- AIP1-F or AIP1-PR in the absence or presence of TNF (10 cipitation assay. TRAF2 binds to AIP1-C-PR and C-PER, but ng/ml for 15 min). Association of AIP1 proteins with endoge- not C-tPER (Fig. 5b), suggesting that the PER domain in the nous TNFR1 and TRAF2 were determined. AIP1-F (but not C-terminal-half is involved in TRAF2 binding. AIP1-PR) binds to TNFR1 in resting EC, and AIP1-F/ To define the critical domain of TRAF2 for AIP1 binding, we TNFR1complex was significantly reduced in the presence of generated a series of mutant forms of FLAG-tagged TRAF2. TNF (Fig. 3c). In contrast, association of AIP1-F withTRAF2 is DN-TRAF2 (amino acids 80–531) contains a deletion of the only weakly detected in resting EC but is strongly induced in whole RING finger whereas TRAF2-CA contains a single mu- response to TNF. AIP1-PR constitutively binds to TRAF2 and tation at C31 within the RING finger leading to disruption of TNF has no effects on their interaction (Fig. 3c). These data the RING structure (15). TRAF2-N (amino acids 1–249) con- strongly suggest that TNF induces an alteration of AIP1 con- tains the intact RING and the five zinc fingers of the N-termi- formation leading to release of AIP1 from TNFR1 with concom- nal half of TRAF2 whereas TRFA2-C (amino acids 265–531) itant binding of AIP1 to the TRAF2/ASK1 complex. consists of the C-terminal TRAF domains of TRAF2 (Fig. 5c). AIP1 Associates with a Non-death Domain Region of AIP1-PR was co-transfected with various TRAF2, and associ- TNFR1—We next determined the critical domain in TNFR1 for ation of AIP1-PR with TRAF2 molecules was determined. As 44960 AIP1 as a Transducer of TRAF2 FIG.3. AIP1 in a closed inactive form associates with TNFR1 whereas in an open active form binds to TRAF2/ASK1. a, schematic diagram of AIP1 domains and expression constructs (C2, protein kinase C-conserved domain 2; GAP, GTPase-activating protein; PER, period-like domain; PR, proline-rich; LZ, leucine-zipper; aa, amino acid). b, proline-rich region is critical for closed conformation of AIP1. BAEC were transfected with FLAG-tagged truncated AIP1 (AIP1-F, PH, LZ, PR), and cells were untreated or treated with TNF (10 ng/ml for 15 min). Association of endogenous ASK1, TRAF2, or TNFR1 with AIP1 domains was examined by immunoprecipitation with a respective antibody followed by Western blot with anti-FLAG. c, TNF responses of AIP1-TNFR1 and TRAF2-AIP1 complexes. BAEC were transfected with AIP1-F or PR and treated with TNF (10 ng/ml for 15 min). Association of AIP1 with TNFR1 and TRAF2 was determined as in b. previously described, TRAF2-N was detected in an insoluble or the presence of TNF as indicated. Reporter activity was fraction (27) and we cannot determine association of AIP1 with measured for a luciferase assay. As shown previously, expres- TRAF2-N. Results show that only TRAF2-WT binds to AIP1- sion of TRAF2 in EC-induced activation of both JNK and PR. However, DN-TRAF2, TRAF2-CA, or TRAF2-C did not NF-B reporter genes (Fig. 6a). Co-expression of AIP1 strongly interact with AIP1-PR (Fig. 5d). These data indicate that the induces JNK reporter gene activation. In contrast, AIP1 dra- intact RING finger (the effector domain) of TRAF2 is required matically inhibits TRAF2-induced activation of B-reporter for AIP1 association. gene. A similar effect of AIP1 on TNF-induced activation of AIP1 Is a Transducer of TRAF2 and Specifically Induces JNK and NF-B reporter genes was observed (data not shown). ASK1-JNK Activation While It Inhibits NF-B Activation— AIP1 on TNF-induced activation of ASK1 and IB kinase (IKK) TRAF2 has been shown to be a critical adaptor in TNF-induced were also determined by an in vitro kinase assay using GST- activation of both JNK and NF-B cascades. We first deter- MKK4 and GST-IB as a substrate, respectively. As shown mined effects of AIP1 in TRAF2-induced JNK and NF-B acti- previously, AIP1 enhanced TNF-induced ASK1 activation. In vation in reporter gene assays. BAEC were transfected with a contrast, AIP1 strongly inhibited TNF-induced IKK activation JNK- or NF-B-dependent reporter gene with AIP1 in absence (Fig. 6b). These data suggest that AIP1 interacts with the AIP1 as a Transducer of TRAF2 44961 FIG.4. AIP1 associates with a non-death domain region of TNFR1. a, schematic diagram of TNFR1 domains and deletion constructs (EM, extracellular domain; TM, transmembrane; JMD, jaxtamembrane; NSD, neutral sphingomyelinase domain; DD, death domain; aa, amino acid). b and c, AIP1-interacting domain (AID) is located between NSD and DD. BAEC were transfected with FLAG-tagged AIP1 and various TNFR1 deletion constructs. Expression of TNFR1 proteins was determined by indirect immunofluorescence microscopy with anti-TNFR1 (goat, recognizing the extracellular domain of TNFR1) followed by an Alexa Fluor 488-conjugated anti-goat secondary antibody (b). Association of AIP1 domains with TNFR1 was determined by immunoprecipitation with anti-TNFR1 followed by Western blot with anti-FLAG (c). TNFR1-associated TRADD was also determined by anti-TRADD. Associations of these TNFR1 proteins with TRADD and AIP1 are summarized on the right panel in a. effector domain of TRAF2 to reciprocally regulate TNF-induced binding abilities for ASK1 and TRAF2. JNK and NF-B cascades. We have previously shown that a short hairpin RNA We then determined the critical domains of AIP1 in regulat- (shRNA) down-regulated endogenous AIP1 level leading to en- ing TNF-induced JNK and NF-B activation. BAEC were hanced TNF-induced ASK1-JNK activation and apoptosis (24). transfected with a JNK- or NF-B-dependent reporter gene To determine a physiological role of AIP1 in TNF-induced with various AIP1 in absence or the presence of TNF as indi- NF-B signaling, we further examined TNF-induced ASK1 and cated. Reporter activity was measured for a luciferase assay. IKK activation in AIP1-knockdown cells by in vitro kinase As shown previously, expression of TRAF2 in EC induced ac- assays as described. Endogenous AIP1 expression was signifi- tivation of both JNK and NF-B reporter genes (Fig. 6c). Ex- cantly (80%) reduced in BAEC expressing a short hairpin RNA pression of AIP1-F, N, or C-PR (but not C-tPER) significantly of AIP1 (ShAIP1) while expression of TRAF2 or ASK1 was not augmented TNF-induced JNK activation. In contrast, AIP1-F, altered (Fig. 6e). As we have shown previously (24), knockdown NorC-PR (but not C-tPER) dramatically inhibited TRAF2- of AIP1 inhibited TNF-induced ASK1 activation, However, induced NF-B reporter gene activity (Fig. 6d). A similar effect AIP1 knockdown cells showed enhanced TNF-induced IKK ki- of AIP1 on TRAF2-induced JNK and NF-B was observed (data nase activity (Fig. 6f). These data further support that physi- not shown). These data suggest that effects of AIP1-F, N, ological AIP1 differentially regulates TNF-induced ASK1-JNK C-PR, C-tPER) on JNK and B reporter correlate with their and IKK-NF-B pathways. 44962 AIP1 as a Transducer of TRAF2 FIG.5. AIP1 via a PERIOD-like do- main binds to the intact RING finger of TRAF2. a and b, critical domains of AIP1 for TRAF2 binding. Truncated AIP-1N (N, PHC2, and PH) and AIP1-C (C, C-PR, C-LZ) were transfected into BAEC. Association of endogenous TRAF2 with AIP1 proteins was determined by immunoprecipitation with anti-TRAF2 followed by Western blot with anti-FLAG. b, period-like domain (PER, amino acids 591–719) in AIP1 is critical for TRAF2 binding. AIP1-PR, C-PER (amino acids 522–719), and C-tPER (amino acids 522– 620 with a truncated PER domain). TRAF2 was co-transfected with various AIP1-C constructs (C-PR, C-PER and C- tPER). Association of TRAF2 with AIP1-C was determined by co-immunoprecipita- tion assay as described. c, diagrammatic representation of the domain structure of TRAF2 and expression constructs. The numbers refer to amino acid number in- dicating the boundary of the RING finger, five zinc fingers and the TRAF domains. d, intact RING finger of TRAF2 is critical for AIP1 association. BAEC were trans- fected with AIP1-PR and various TRAF2 constructs. Association of AIP1-PR with TRAF2 was determined by immunopre- cipitation with anti-TRAF2 followed by Western blot with anti-FLAG. DISCUSSION plex structurally different from complex I. Second, AIP1 com- plex is formed at 15 min in response to TNF and is dissociated Based on the data presented in this study, we propose the by 60 min. However, complex II formation detected in certain following model for the role of AIP1 in TNF signaling (Fig. 7). cell types (HT1080) at 2–8 h post-treatment with TNF. More- AIP1 via its PH/C2 domain is localized on plasma membrane over, we could not detect association of AIP1 with FADD and microdomain where it exists in an inactive form complexed pro-caspase-8 in response to TNF in EC (not shown). These with TNFR1. In response to TNF, AIP is unfolded and dissoci- data suggest that AIP1 complex is also structurally different ated from TNFR1 with concomitant of translocation of AIP1 to from complex II. Most significantly, AIP1 overexpression en- cytoplasm where it associates with TRAF2 and ASK1. More hances, whereas knockdown of AIP1 inhibits, TNF/TRAF2- significantly, AIP1 associates with the effector domain (the induced ASK1-JNK activation. However, AIP1 has opposite RING finger) of TRAF2 and mediates TNF/TRAF2-induced effects on TNF/TRAF2-induced IKK-NF-B pathway. These ASK1-JNK activation while inhibits IKK-NF-B signaling. data suggest AIP1 complex is functionally different from com- These data suggest that AIP1 is a novel transducer of TRAF2 plex I. In cell types including EC, ASK1-JNK activation in- in TNF signaling. duces apoptotic signaling which is dependent on intrinsic but Intermediate Complex—Tschopp and co-workers (9) have re- not extrinsic pathways. We have recently shown that AIP1 cently dissected TNFR1 signaling complexes in more details enhances ASK1-mediated JNK activation and EC apoptosis. and has proposed a sequential signaling complex model: the AIP1-suppressed IKK-NF-B activation further support that initial plasma membrane bound complex (complex I comprising AIP1 is proapoptotic. Thus, AIP complex is functionally differ- of TNFR1, TRADD, RIP1 and TRAF2) for NF-B activation and ent from complex II. Whether or not complex II is present in EC a cytoplasmic complex (complex II) in which the internalized TRADD, RIP, TRAF2 recruits FADD and pro-caspase-8 for are under investigation. AIP1 Specifies TRAF2 toward to ASK1-JNK Pathway— apoptotic signaling. However, it has not been determined if either complex I or complex II is active in the initiation of JNK TRAF2 has been shown to be the bifurcation point in TNF- induced activation of NF-B and activation of JNK (8, 35, 36). signaling. Since TRAF2 is critical for JNK activation, it seemed reasonable to assume that complex I, which contains TRAF2, While the C-terminal TRAF domain is responsible for associa- tion with TNFR1-TRADD complex as well as the MAP3Ks, the would recruit one or more MAP3Ks to induce JNK activation (8, 9). Here we provide several lines of evidence to support the N-terminal RING finger of TRAF2 is an effector domain in activation of both JNK and NF-B. Deletion of the RING do- existence of an intermediate complex, which lacks TNFR1, as being responsible for TRAF2-AIP1-mediated ASK1-JNK acti- main (TRAF2-(87–501)) completely loses the ability to activate JNK and NF-B, and this mutant functions as a dominant vation. First, AIP1 forms a preexisting complex with TNFR1 in EC. However, in response to TNF AIP1 is dissociated from negative. Specific downstream signaling events from TRAF2 TNFR1 and in turn forms a complex with TRADD, RIP1, are mediated by RIP/IKK and MAP3Ks. For example, IKK is TRAF2, and ASK1 (so named AIP1 complex). Thus, AIP1 com- specific for NF-B signaling, while ASK1 appear to direct sig- AIP1 as a Transducer of TRAF2 44963 FIG.6. AIP1 is a transducer of TRAF2 and specifically induces ASK1-JNK activation while inhibits IKK-NF-B activation. a, AIP1 specifically enhances TRAF2-induced JNK (but inhibits NF-B) activation. AIP1 together with a JNK or NF-B-dependent reporter gene was transfected into BAEC in the presence or absence of TRAF2 as indicated. A constitutive expression vector for Renilla was used as an internal control for transfection efficiency. Reporter gene activity was determined by luciferase assay as described. Data are presented as fold increase by expression of TRAF2 or AIP1 compared with the vector (as 1). Similar results were obtained from additional three independent experiments. b, AIP1 specifically enhances ASK1 whereas inhibits IKK activation by TNF. BAEC were transfected with a control vector or AIP1 and treated with TNF as indicated (10 ng/ml for 15 min). ASK1 and IKK activities were determined by an in vitro kinase assay using GST-MKK4 and GST-IKB, respectively. Relative ASK1 and IKK activities are presented by taking TNF-treated VC as 1.0. Similar results were obtained from two additional independent experiments. c and d, critical domains of AIP1 in TNF-induced activation of JNK and NF-B. BAEC were transfected with a JNK- or NF-B-dependent reporter gene with various AIP1 (F, N, PR, or tPER) as indicated and a constitutive expression vector for Renilla was used as an internal control for transfection efficiency. 24 h post-transfection, cells were treated with TNF (10 ng/ml) for 12 h. Reporter gene activity was determined by luciferase assay as described. Data are presented as fold increase by AIP1 expression by taking untreated vector as 1 (). Similar results were obtained from additional three independent experiments. e, knockdown of AIP1 in BAEC. BAEC were transiently transfected with pShag or pSh-AIP1 encoding a short-hairpin RNA of AIP1 (Sh-AIP1) as described previously (24). 48 h post-transfection, total cell lysates were used to determine AIP1 expression by Western blot with anti-AIP1. As expected, expression of TRAF2 and ASK1 was not altered by Sh-AIP1. f, knockdown of AIP1 suppresses ASK1 activity whereas enhances IKK activation by TNF. BAEC expressing pShag or pSh-AIP1 were untreated or treated with TNF (10 ng/ml for 15 min). ASK1 and IKK activities were determined as described in b. Relative ASK1 and IKK activities are presented by taking untreated pShag as 1.0. Similar results were obtained from additional two experiments. naling exclusively to JNK. In contrast, TAK1 and MEKK1 can induced activation of JNK but not of NF-B (40). These data activate both NF-B and JNK (37, 38). Recent studies suggest suggest that TRAF2 modulation/translocation or interactions that TRAF2 engagement is not obligatory to trigger NF-B and with other proteins may provide a new layer of regulation to JNK cascades simultaneously. For example, sphingosine ki- determine specific downstream signaling (activation of JNK nase binds to TRAF domain of TRAF2 and specifically activates and NF-B). Our study shows that AIP1 associates with NF-B while inhibits JNK activation (39). In contrast, TNF- TRAF2 in response to TNF and the intact RING finger of induced TRAF2 ubiquitination coincides with its translocation TRAF2 is required for TRAF2-AIP1 association. Moreover, to an insoluble cellular fraction, which is critical for TNF- TRAF2, AIP1, and ASK1 form a complex in response to TNF 44964 AIP1 as a Transducer of TRAF2 FIG.7. A proposed model for AIP1 as a transducer of TRAF2 in TNF-induced ASK1-JNK activation. AIP1 via its PH and C2 domains anchors to EC plasma membrane where it associates with a non-death domain region of TNFR1. In response to TNF, AIP1 is dissociated from TNFR1 with concomitant cytoplasmic translocation of AIP1 and formation of AIP1 complex comprising of TRADD-RIP-TRAF2-AIP1, which specifically activates ASK1-JNK signaling. As demonstrated by Tschopp and co-workers (9), TNFR1 recruits TRADD, RIP1, and TRAF2 to form a membrane-bound complex I, which specifically activates NF-B signaling. Since TNFR1-TRADD association precedes formation of AIP1 complex, it is likely that AIP1 complex is derived from complex I upon ubiquitination () and internalization of TRADD, RIP1, TRAF2, and AIP1. As we described previously (9), unfolded AIP1 binds to and activates ASK1-JNK at least in part by facilitating dephosphorylation of ASK1 at Ser and dissociation of ASK1 from its inhibitor 14-3-3. Joberty, G., Neubauer, G., Rick, J., Kuster, B., and Superti-Furga, G. 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