Author Manuscript NIH Public Access Qi Xiao RIP and regulates TNF … w... · 2009-06-23 · TNF...

CARP-2 is an endosome-associated ubiquitin protein ligase forRIP and regulates TNF-induced NF-κB activation

Wentao Liao1, Qi Xiao1, Vladimir Tchikov2, Ken-ichi Fujita1, Wensheng Yang3, StephenWincovitch1, Susan Garfield1, Dietrich Conze1, Wafik S. El-Deiry3, Stefan Schutze2, andSrinivasa M. Srinivasula1,*

1Laboratory of Immune Cell Biology, National Cancer Institute, National Institutes of Health, Bethesda,Maryland 20892, USA.

2Institute of Immunology, University hospital of Schleswig-Holstein, D-24105 Kiel, Germany.

3Laboratory of Molecular Oncology and Cell Cycle Regulation, Departments of Medicine (Hematology/Oncology), Genetics, Pharmacology, the Institute for Translational Medicine and Therapeutics and theAbramson Comprehensive Cancer Center, University of Pennsylvania School of Medicine, Philadelphia, PA19104, USA.

SummaryBackground—The proinflammatory cytokine Tumor Necrosis Factor–α (TNF) elicits cellularresponses by signaling through a receptor complex that includes the essential adaptor molecule RIP.One important consequence of signaling is activation of the transcription factor NF-κB, and failureto downregulate TNF-induced NF-κB transcriptional activity results in chronic inflammation anddeath. Internalization of the receptor complex plays an important regulatory role in TNF signaling.

Results—We report that CARP-2, a RING domain-containing ubiquitin protein ligase (E3), is anegative regulator of TNF-induced NF-κB activation. By virtue of its phospholipid-binding FYVEdomain, CARP-2 localized to endocytic vesicles where it interacted with internalized TNF-receptorcomplex, resulting in RIP ubiquitination and degradation. Knockdown of CARP-2 stabilized TNFR1-associated polyubiquitinated RIP levels after TNF simulation and enhanced activation of NF-κB.

Conclusions—CARP-2 acts at the level of endocytic vesicles to limit the intensity of TNF-inducedNF-κB activation by the regulated elimination of a necessary signaling component within the receptorcomplex.

KeywordsRIP; FYVE domain; ubiquitination; endocytic vesicles; signal transduction

IntroductionTNF is a multifunctional cytokine that elicits many biological responses that are critical forcellular homeostasis [1,2]. Deregulation of TNF signaling has been implicated in a variety ofpathological conditions such as cachexia, inflammation, osteoporosis, and rheumatoid arthritis.

*Correspondence should be addressed to SMS at Email: [email protected], Tel: 01-301-496-1648, Fax: 01-301-402-4844Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customerswe are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resultingproof before it is published in its final citable form. Please note that during the production process errors may be discovered which couldaffect the content, and all legal disclaimers that apply to the journal pertain.

NIH Public AccessAuthor ManuscriptCurr Biol. Author manuscript; available in PMC 2009 May 6.

Published in final edited form as:Curr Biol. 2008 May 6; 18(9): 641–649. doi:10.1016/j.cub.2008.04.017.

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TNF orchestrates most of its cellular functions, either cell survival and proliferation orapoptosis, via TNF-receptor-1 (TNF-R1) [3,4].

TNF-induced NF-κB activation is crucial for immune responses. Binding of TNF triggersrecruitment of many cytosolic proteins to TNF-R1 including TNF receptor associated deathdomain protein (TRADD), receptor interacting kinase (RIP), and TNF-receptor associatedfactor 2 (TRAF-2) [5]. The assembly of this complex (also known as complex I) takes placeimmediately after stimulation at the plasma membrane, where posttranslational modificationslike polyubiquitination of several complex-associated proteins occur, resulting in the activationof the IκB kinase (IKK) complex and subsequently NF-κB [6-9]. This receptor-associatedcomplex containing RIP internalizes within minutes of formation and fuse with the trans-golginetwork to form multivesicular endosomes and this event is believed to be critical to attenuateinflammatory responses [5,10]. However, the underlying mechanisms that regulate NF-κBsignaling during endocytosis of the activated TNF-receptor complexes remain unknown.

RIP, a death domain-containing serine/threonine kinase, is heavily ubiquitinated uponrecruitment to early cell surface TNF-R1 complexes in TNF-stimulated cells. RIP is essentialfor TNF pathway, because RIP-deficient Jurkat human T lymphoma cells and mousefibroblasts have no TNF-inducible NF-κB response [11-13]. NEMO, the regulatory subunit ofthe IKK complex, specifically recognizes K63-linked polyubiquitinated chains on RIP,triggering activation of IKK and NF-κB [6,8,14]. The domain structure of RIP is organizedinto three distinct regions, a C-terminal death domain (DD), an intermediate domain (ID) andan N-terminal kinase domain (KD). While the DD mediates RIP recruitment to the early TNF-R1 complex, Lysine 377 in the ID is the site of K63-linked polyubiquitination, probablymediated by TRAF-2 [6,11,14]. Both DD and ID are essential for TNF-induced IKK and NF-κB activation. Whereas no functional role for the RIP KD has been established, it is thoughtto be dispensable for downstream NF-κB signaling [12,13].

Recent reports indicate that a number of peripheral (as opposed to integral membrane) proteins,that localize to specific organelles through lipid-protein and protein-protein interactions, targetthe internalized receptor complexes and provide necessary spatial and temporal regulation forsignaling [15,16]. Because internalized TNF-R1 complexes are known to associate withendocytic membrane vesicles, we hypothesized that proteins in these intracellularcompartments might affect TNF-induced NF-κB activation by targeting signaling complexes.Here, we present evidence that CARP-2, a phospholipid-binding domain containing proteinwith ubiquitin protein ligase activity, localizes to endocytic vesicles, recruits to internalizedearly TNF-R1 complex, targets internalized RIP for proteasome-mediated degradation, andlimits TNF-induced NF-κB activation.

ResultsCARP-2 is a novel regulator of TNF-induced NF-κB activation

To identify proteins that regulate TNF signaling in the endocytic pathway, we focused onproteins that associate with the endosomal and Golgi membrane vesicles with which fusion ofinternalized TNF receptor complexes occurs. Besides trans-membrane proteins, proteins witha particular motif known as FYVE (conserved in Fab1p/YOTB/Vac1p /EEA1) are known tolocalize to these vesicles [17,18]. Therefore, we searched the NCBI gene bank for FYVE motif-containing proteins. Interestingly, two of the 45 FYVE domain-containing proteins found alsocontain RING domains, a sequence that confers ubiquitin protein ligase activity to manymolecules [19,20]. Reasoning that molecules containing such properties might be especiallywell-suited to interact with and modify components of early TNF-R1 associated proteins, wetested their ability to affect TNF-induced NF-κB activation. One of the molecules, CARP-2(caspase-8 and –10-associated RING protein-2) (Fig. 1A), is known to bind and negatively

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regulate DED caspases, but has not been reported to regulate TNF signaling [21]. CARP-2(also known as Rififylin/SAKURA) belongs to a minor subgroup of FYVE-domain containingproteins with no conserved phosphoinositide-binding motif [22-24]. In spite of the missingPI3P binding property of the FYVE motif, this sequence has been shown in overexpressionstudies to target the protein to endocytic membrane vesicles [23]. The RING domain is alsofunctional and exhibits ubiquitin protein ligase (E3) activity in vitro and ex vivo [21,22].

To assess the effect of CARP-2 expression on TNF-induced NF-κB activation, we analyzedthe endogenous IKK activity and IκBα degradation (Fig. 1B and C). As expected, treatmentof vector only expressing cells with TNF resulted in increased IKK activation (Fig. 1B).Expression of CARP-2 wild type decreased IKK activity both at basal level and upon TNFstimulation. A substitution of alanine for a histidine in the RING domain (H333A) thatabrogates E3 activity failed to reduce IKK activity suggesting that E3 activity is required forCARP-2 inhibitory function (Fig. 1B). Consistent with these results expression of CARP-2wild type (Fig. 1C) that did not affect the level of IκBα in unstimulated cells prevented its TNF-induced degradation. In contrast, the RING-mutant had shown no such effect (Fig. 1C).

Additionally, NF-κB reporter assays were used to assess the effect of increased CARP-2expression on NF-κB activation. CARP-2 reduced TNF-induced NF-κB reporter activity in adose-responsive fashion (Fig. 1D). At high concentrations the RING mutant also exhibitedsome inhibition, which may result from the ability of the RING mutant to bind to target protein(s) and affect its (their) function in a subtle way. This downregulation of NF-κB activity byCARP-2 was observed in all cell lines such as HT1080 (human fibrosarcoma), HeLa (humancervical carcinoma) and C2C12 (mouse myoblast) tested (data not shown). To investigate theeffect of CARP-2 expression on NF-κB mediated cytokine production, we examined IL-6secretion in response to TNF stimulation in mouse embryonic fibroblasts that express CARP-2variants. Treatment of vector–only expressing cells with TNF resulted in increased productionof IL-6, but cells that express wild type CARP-2 produced very little IL-6 (Fig. 1E). Inagreement with the requirement of E3 activity for CARP-2 inhibitory function, MEFs thatexpress the RING mutant (H333A) exhibited increased IL-6 production both at the basal levelsand upon TNF stimulation (Fig. 1E). Therefore, in TNF stimulated cells CARP-2 inhibitsactivation of NF-κB in a largely RING dependent manner.

To investigate the physiologic function of endogenous CARP-2, we designed small hairpinRNA (ShRNA) specific for two different regions of CARP-2. Transfection of the siRNAhairpins in 293T cells resulted in a large reduction in the level of endogenous CARP-2 protein(Fig. 1F). Knockdown of endogenous CARP-2 expression enhanced TNF-induced NF-κBreporter activity by approximately two fold (Fig. 1F). Consistent with this, knockdown ofCARP-2 prolonged the IKK activation to as late as 60 min (Fig. 1G) and delayed the recoveryof IκBα (beginning at 30-45 min in control cells but occurring at 60-90 min in ShRNA-treatedcells) (Fig. 1H). The observed effects of ShRNAs are specific because co-expression ofShRNA-resistant CARP-2 but not wild type rescued TNF-induced NF-κB reporter activity(Fig. S1A-B). The increase in TNF signaling in cells with reduced CARP-2 suggests that thephysiological function of this molecule is to limit the intensity or duration of signaling.

CARP-2 localizes to membrane compartments and recruits to vesicles containingendocytosed TNF-receptor

Previous studies have shown that overexpressed mouse CARP-2 associates with membranecompartments in the perinuclear region that are positive for the endosomal markers, Rab5 andRab11[23]. Therefore, to determine if endogenous CARP-2 constitutively associates withendocytic membrane vesicles, we developed a monoclonal antibody that specificallyrecognizes CARP-2 (Fig. S2A and B). Confocal microscopic analysis of cells stained with thisantibody showed punctate staining for endogenous CARP-2, mostly in the cytosol (Fig. 2A).

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A subset of these cytosolic vesicles also stained positive with antibody specific for Rab5 (Fig.2B). No significant co-localization between CARP-2 and either Golgi marker GM130, or thelysosomal marker Lamp1 was observed (Fig. S2C). Collectively these results suggest thatendogenous CARP-2 associates constitutively with, albeit undefined, membrane vesicles thatfuse with internalized TNF-receptor complex (see below) and are referred as CARP-2 positivevesicles in this manuscript.

Given that internalized TNF-receptor complex fuse with endocytic vesicles to form multivesicular endosomes (also known as TNF receptosomes), we examined whether these isolatedTNF receptosomes include CARP-2 [10,25]. As expected all the receptosome preparationsfrom both HeLa and U937 cells contain TNF-R1 (Fig. 2C and Fig. S2D). After 5 minutes ofinternalization significant amounts of CARP-2 were found along with markers of otherendocytic vesicles such as Rab 5 and Rab 7. Of note, barely detectable in the cell lysate,considerable enrichment of these proteins in isolated receptosomes indicate that either CARP-2is directly recruited to endocytosed TNF complex, or CARP-2 vesicles along with that of Rab5 and 7 fuse with internalized receptor complex. While the recruitment of CARP-2 occurredin as early as 5 minutes, Lamp-1 (a lysosomal protein) is recruited around 30-60 minutessuggesting that the association of CARP-2 is an early event that occurs much before the fusionof the internalized TNF-receptor with lysosomal compartments.

Additionally, confocal microscopy was used to investigate the association between CARP-2and endocytosed TNF-receptor complex. The intracellular trafficking of TNF/TNF-R1 wasfollowed by first, incubating HeLa cells with biotin TNF/streptavidin FITC at a lowtemperature and then by increasing the temperature to 37°C. Confocal analysis of these cellsstained with anti-CARP-2 antibody showed (30 minutes after internalization) significant co-localization of the receptor with CARP-2 (Fig. 2D). The co-localization coefficient (Pearson'scoefficient) for TNF receptor after 30 minutes of internalization ranged from 0.314 to 0.956.Thus, these results demonstrate that internalized TNF-R1 complex indeed associates withCARP-2.

CARP-2 promotes endogenous RIP degradation in TNF-stimulated cellsThe association of the internalized TNF-R complexes with CARP-2 suggests that CARP-2inhibits NF-κB activation in TNF-stimulated cells possibly by targeting one or morecomponents of the early TNF-R1 complex. To test this possibility CARP-2 was co-expressedwith a number of gene products found in the early TNF-R1 complex whose overexpression incells is known to activate NF-κB. Co-expression of CARP-2 substantially inhibited the NF-κB activation induced by all the three proteins and the RING mutant was only slightly inhibitory(Fig. 3A). The NF-κB activity induced by IKKα was minimally inhibited by CARP-2,independent of its RING function. Therefore, CARP-2 likely inhibits NF-κB activation at thelevel of the early signaling complex.

Given that ubiquitin ligase (E3) activity of CARP-2 is essential for its function, we asked ifCARP-2 might affect the expression of the above signaling molecules. RIP protein level wasdownregulated by co-expression of CARP-2 (Fig. 3B). The downregulation was specific forRIP, as CARP-2 had no effect on the level of expression of either TNF-R1, or TRAF-2, orNEMO, another critical regulator of NF-κB activation, under the same experimental conditions(Fig. 3B). The function of CARP-2 appears to be specific for TNF, as co-expression of CARP-2had no effect on the expression of IRAK (Interleukin-1 receptor-associated kinase) a functionalhomologue of RIP in IL-1 signaling and an essential modulator of IL-1-induced NF-κbactivation (Fig. S3). The RING mutant did not affect RIP protein expression (Fig. 3B),demonstrating that the protein ubiquitin ligase activity of CARP-2 is critical for this effect.This was strongly supported by the finding that the proteasome inhibitor lactacystin reversesthe loss of RIP protein in the presence of CARP-2 (Fig. 3C). In order to determine whether

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CARP-2 binds directly to RIP, immunoprecipitates of FLAG-tagged CARP-2 from 293T cellswere probed for endogenous proteins with antibodies against TNF-R1, RIP, and TRAF-2 (Fig.3D). Consistent with the specific downregulation of RIP protein, immunoprecipitates from cellextracts expressing wild-type CARP-2 but not vector contained substantial amounts of RIPprotein and only a very little amount of TNF-R1 and TRAF-2. Interestingly the RING-domainmutant, which was unable to affect RIP expression or inhibit TNF signaling, also co-immunoprecipitated these proteins equally well as wild-type CARP-2 indicating that theRING-domain is dispensable for RIP-binding. These interactions were further confirmed asextracts of cells expressing FLAG-tagged RIP and V5-tagged CARP-2 contained RIP/CARP-2complexes (Fig. S4).

Because CARP-2 binds and downregulates RIP, we asked if stimulation with TNF promotesinteraction between these proteins at endogenous level. To answer this endogenous CARP-2was immunoprecipitated from cells stimulated with TNF in the presence or absence of MG-132,and the immunoprecipitated material was probed with anti-RIP (Fig. 3E). Some interactionbetween RIP and CARP-2 was detected in unstimulated cells. But in stimulated cells we foundno interaction in the absence of MG-132 between CARP-2 and RIP. However, whenproteasomal degradation is inhibited, anti-CARP-2 immunoprecipitates RIP from cellsstimulated by TNF for 30 min or more, suggesting that after TNF stimulation CARP-2 bindsand rapidly degrades RIP (Fig. 3E). In order to evaluate the direct interaction of endogenousCARP-2 with endogenous RIP in internalized TNF receptosomes, CARP-2 wasimmunoprecipitated from TNF receptosomes isolated from MG-132-treated HeLa cells after0 and 30 min of internalization and probed for CARP-2 and RIP (Fig. 3F). TNF receptosomesin 30 min contained both RIP and CARP-2 and RIP from lysed TNF receptosomes was foundto coimmunoprecipitate specifically with CARP-2 antibody. These data demonstrate thatCARP-2 directly interacts with RIP at internalized TNF receptosomes.

CARP-2 targets the internalized receptor associated RIP for degradationTo test the hypothesis that RIP degradation at endocytic vesicles is the mechanism to terminateNF-κB signaling, we investigated the effect of internalization on RIP degradation in TNF-stimulated cells. Plasma membrane TNF-R1 is reported to internalize through two distinctendocytic routes, namely, clathrin mediated endocytosis (CME) and raft/caveolar endocytosis(RCE) [26-28] . Results from a previous study that show reduced TNF-induced NF-κB targetgene expression upon inhibition of endocytosis indicate that active signaling continues duringTNF-receptor endocytosis [29]. While most cell types upon TNF stimulation exhibit rapiddegradation of receptor associated RIP, only NEMO deficient cells show significant decreasein total cellular RIP [8]. For this reason, to investigate the effect of internalization on RIPubiquitination and degradation we have also used NEMO deficient cells in our studies.

As reported earlier, TNF stimulation significantly reduced total cellular RIP in NEMO −/−MEFs (Fig. 4A). This decrease in RIP level is not the result of apoptosis, as pan caspaseinhibitor Z-VAD-FMK had failed to rescue the loss (Fig. 4B). Treatment with chlorpromazine(CPZ), a specific CME inhibitor, effectively reduced the loss in a dose dependent manner (Fig.4A). Inhibition of TNF-R1 endocytosis by monodansyl cadaverine (MDC) also prevented RIPloss (data not shown). However, the presence of another inhibitor Filipin at a concentrationthat specifically inhibits RCE, had very little effect suggesting that CME promotes thedegradation of RIP in these cells. Next, the effect of internalization on RIP ubiquitination wasassessed from cells treated with or without CPZ in the presence of proteasome inhibitorMG-132, and stimulated with TNF. In agreement with earlier published reports proteasomalinhibition efficiently blocked TNF-induced RIP loss (Fig. 4B). While TNF stimulation in thepresence of MG-132 promoted RIP ubiquitination, the presence of CPZ blocked RIP

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modification suggesting that the observed loss of RIP might be the consequence ofinternalization and subsequent ubiquitination (Fig. 4C).

To investigate the role of CARP-2 in RIP degradation, we transfected NEMO−/−cells withShRNA specific for mouse CARP-2. While the knockdown of CARP-2 protein expression hadminimal effect on RIP level in unstimulated cells, it partially rescued the loss of RIP instimulated cells (Fig. 4D) suggesting a possible role for CARP-2 in the degradation of RIP.

This finding prompted us to examine whether inhibition of RIP degradation with MG-132results in accumulation of ubiquitinated RIP at endocytic vesicles. To address this we purifiedTNF-R1 associated endocytic vesicles from U937 cells treated with or without MG-132 andanalyzed the vesicles for RIP by immunoblotting. MG-132 treatment did not alter the TNF-R1complex fusion to endocytic vesicles (Fig. 4E). Compared to control (- MG-132) cells,inhibition of proteasomal degradation resulted in increased amounts of both polyubiquitinatedand unubiquitinated RIP (30 and 60 min samples). Not surprisingly, the presence of MG-132also stabilized TNF-R1 protein (Fig. 4E). Nevertheless, these results suggest that, in TNFstimulated cells, internalized RIP ubiquitination and degradation broadly occur at endocyticvesicles. While the initial ubiquitination of RIP that occurs immediately after stimulation atthe plasma membrane (presumably mediated by TRAF-2) promotes NF-κB activation, the laterubiquitination at endocytic vesicles might be required for its degradation resulting intermination of NF-κB activation. If CARP-2, indeed, promotes RIP degradation we reasonedthat the knockdown of CARP-2 expression should result in stabilization of this receptorassociated RIP.

To test this, we immunoprecipitated TNF-R1 from HeLa cells expressing CARP-2 ShRNAand treated with TNF, and immunoblotted for associated endogenous RIP. Consistent withearlier reports, ubiquitinated and unubiquitinated RIP was found to coimmunoprecipitate withTNF-R1 from TNF-stimulated cells (Fig. 4F). Knockdown of CARP-2 resulted in a substantialincrease in receptor-associated RIP, both polyubiquitinated and unubiquitinated, indicatingthat CARP-2 targets RIP within the early TNF-R1 complex for degradation. Hence, an increasein TNF-induced IKK/NF-κB activation in CARP-2 ShRNA expressing cells (Fig. 1F - H) couldbe the result of stabilization of receptor-associated RIP.

CARP-2 is an ubiquitin protein ligase for RIPGiven that CARP-2 promotes RIP degradation, we investigated whether CARP-2 functions asan ubiquitin protein ligase (E3) for RIP. We first assessed the effect of CARP-2 on endogenousRIP ubiquitination in TNF-stimulated cells transfected with CARP-2 and His-ubiquitin. Whileendogenous RIP was ubiquitinated on CARP-2 overexpression, this was significantly increasedupon TNF-stimulation (Fig. 5A left panel). Furthermore, in co-transfection experiments V5-CARP-2 wild type but not RING mutant markedly enhanced the ubiquitination of FLAG RIP(Fig. 5A right panel). In these experiments overexpression of FLAG-RIP alone resulted inmoderate ubiquitination. Since, CARP-2 ubiquitination of RIP effectively results in itsdegradation, we hypothesized that CARP-2 promoted ubiquitination of RIP was K48-linked.To test this, we co-transfected cells with CARP-2 variants along with HA-ubiquitin mutantsin which only K-48 or K-63 are available for polymerization and analyzed ubiquitination statusof endogenous RIP from TNF stimulated cells. As expected, CARP-2 wild type markedlypromoted the K48-linked ubiquitination of RIP and very little K63-linked RIP ubiquitinationwas observed under these conditions (Fig. 5B). In agreement with this expression of mutantRIP that is unable to undergo K63-linked polyubiquitination (K377F) was reduced in RING-dependent manner by CARP-2 (Fig. S5). Additionally, the effect of CARP-2 variants on theturnover of FLAG-RIP was assessed by pulse chase analysis. Consistent with its function asan E3 ligase, the degradation of transfected FLAG-RIP was rapid in the presence CARP-2 wildtype (Fig. 5C). To determine if CARP-2 can directly ubiquitinate RIP, in vitro ubiquitination

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assays were performed using methylated (blocked) ubiquitin. In the presence of ATP, wild-type but not the RING-domain mutant of CARP-2 was found to ubiquitinate RIP protein (Fig.5D). Taken together, the results demonstrate that CARP-2 binds and ubiquitinates RIP, leadingto its degradation.

RIP kinase domain is essential for CARP-2 mediated RIP degradationEarlier reports have shown that the RIP intermediate domain and death domain are essentialbut the kinase domain is dispensable for NF-κB activation [12,13]. To determine which portionof RIP interacts with CARP-2, we tested its binding to individual regions of RIP. Because thedifferent constructs were not expressed in transiently transfected cells to the same level, weexpressed FLAG-tagged RIP constructs and V5-tagged CARP-2 constructs individually in293T cells and used different amounts of the extracts from these cells for immunoprecipitationexperiments. Interestingly the RIP kinase domain, but not RIP lacking the KD, was found tospecifically co-immunoprecipitate with CARP-2 (Fig. 6A), suggesting a requirement of RIPKD for CARP-2 mediated RIP degradation. In order to test this possibility we examined theeffect of CARP-2 on the degradation of RIP without KD in co-transfection and pulse-chaseexperiments. In agreement with its inability to bind to RIP without its kinase domain, CARP-2did not affect RIPΔKD expression but reduced the full length RIP protein level (Fig. 6B).Consistent with this, in pulse-chase experiments the degradation of FLAG-RIPΔKD remainedunchanged in the presence of wild type or RING mutant CARP-2 (Fig. 6C). Thus, these resultsprove that the kinase domain of RIP is essential for its binding and subsequent degradation byCARP-2.

Given this apparent paradox on the role of the kinase domain in NF-κB signaling wehypothesized that this domain might regulate the termination of NF-κB activation by targetinginternalized RIP to CARP-2 in intracellular compartments. In such a scenario, cells expressingonly RIP without the kinase domain should exhibit increased or prolonged NF-κB activation.In fact, earlier it has been shown that expression of a construct containing the ID-DD regionof RIP in RIP-deficient Jurkat cells has increased NF-κB activity as compared to full-lengthRIP-expressing cells [13]. Indeed, TNF stimulation of RIP −/− MEFs expressing RIP lackingthe kinase domain are consistently found to have an increase in basal as well as TNF-inducibleNF-κB activity (Fig. 6D). Thus CARP-2 negatively regulates NF-κB signaling by binding toRIP of the internalized early complex via the kinase domain leading to RIP ubiquitination anddegradation.

DiscussionEndocytic organelles are organized as a transport network consisting of physically andbiochemically distinct membranous domains, which serve as intracellular stations that integratediverse signals to provide a high order of specificity and regulation to signaling [16,30-32].The end result of a signaling process is determined by factors, presumably associated withmembrane vesicles that regulate the duration and intensity of the signal. While duration ofsignaling is an important parameter that ultimately determines the biological outcome, to ourknowledge, no factor(s) at the vesicles that terminate TNF signaling have been reported.

In this report, we propose that degradation of internalized RIP by CARP-2 is the mechanismto dampening TNF-induced IKK/NF-κB activation (Fig. 7). Endocytosed TNF-receptorcomplex that includes RIP is known to fuse with intracellular compartments to form TNF-receptosomes [10]. We now present evidence that CARP-2 localizes to compartments thatassociate with receptosomes and targets endocytosed RIP for K-48 linked ubiquitination andproteosomal degradation. Whereas, results from pulse-chase experiments showed marginaldifferences between wild-type and mutant CARP-2, collective evidence from severalexperiments including in vivo and in vitro ubiquitination assays proves that CARP-2 is an

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ubiquitin protein ligase (E3) for RIP. In knockdown experiments, we observed only small effectin RIP ubiquitination/ IKK/NF-κB activation possibly owing to the presence of some residualprotein in our shRNA experiments, and/or other molecules that could functionally offset theeffect of loss of CARP-2. A20, an ubiquitin-editing enzyme that negatively regulates TNF-induced NF-κB activation targets, like CARP-2, TNFR1-associated ubiquitinated RIP fordegradation [7]. Knockdown of either CARP-2 or A20 stabilized RIP in a similar manner (Fig.S6A), and suppression of A20 expression together with CARP-2 further increased RIPubiquitination and slightly decreased the level of IκBα compared to reduction of A20 alone(Fig. S6B). These results indicate that CARP-2 might function co-operatively with A20 inregulating TNF-induced NF-κB activation with CARP-2 acting at the endocytic compartments.Notwithstanding of these similarities the level of CARP-2, unlike A20 whose expression issubstantially upregulated by NF-κB, remained unchanged upon TNF treatment (Fig. S6C).More experiments are needed to understand the relative contribution of A20 and CARP-2 inthe regulation of TNF signaling.

An unexpected outcome of our studies is the requirement of the kinase domain of RIP forCARP-2 function. The dispensability of the KD for TNF-induced NF-κB activation has longbeen known [12,13,33]. Our data suggests, however, that the kinase domain may have aninhibitory effect on RIP function. Increased NF-κB activity observed in our experiments inRIP −/− MEFs expressing RIP KD deletion mutant is in agreement with earlier reports usingRIP deficient Jurkat cells [13]. In principle it is possible that kinase activity might negativelyregulate TNF-induced NF-κB activity by affecting RIP/CARP-2 interaction. Such a notion issupported by earlier data that showed increased NF-κB transcriptional activity in TNF-stimulated RIP deficient cells expressing kinase inactive version of RIP (K45R or D138N)[13,33].

Unlike other FYVE proteins that localize to Rab 5 or EEA-1 positive endosomes endogenousCARP-2 mostly associates with non-conventional endocytic vesicles. This could partly bebecause of the differences in FYVE domains' affinity to phospholipids [24]. We speculate thatthe FYVE domain restricts the function of CARP-2 to a specific set of endocytic vesicles alongthe endocytic pathway of TNF signaling. Knowledge of pathways such as this may haveimportant implications in understanding the constitutive activation of NF-κB in tumor cells.

Materials and methodsDetails of materials and methods can be found in supplementary information.

Supplementary MaterialRefer to Web version on PubMed Central for supplementary material.

AcknowledgementsThe research was supported by the Intramural Research Program of the NIH, Center for Cancer research, NationalCancer Institute. This work was supported in part by NIH grant CA97100 to W.S.E-D and by DFG grands SCHU733/7-1 and SFB 415, project A11 to S.S. We thank Jonathan Ashwell, Remy Bosselut and Stanley Lipkowitz forhelpful discussions, Michelle A. Kelliher for RIP −/− MEFs and MARC Schmidt-Supprian for NEMO −/− MEFs.

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Fig. 1. CARP-2 negatively regulates TNF-induced NF-κB activation(A). Schematic diagram shows the domain structure and position of conserved Histidine 333in the RING domain of CARP-2.(B) and (C). CARP-2 inhibits TNF-induced IKK activation and IκB α degradation. Extracts of293T cells transfected with indicated plasmids and treated with TNF for 5 min (B), or asindicated (C) were subjected to in vitro IKK kinase assay (B), or probed for expression ofindicated proteins (B and C).(D) and (F). CARP-2 inhibits TNF-induced NF-κB reporter activity. Extracts of TNF-stimulated 293T cells expressing reporter plasmids along with different amounts of FLAG-CARP-2 variants (D), or ShRNA constructs (F) were assayed for reporter activities and probedfor CARP-2.(E). CARP-2 inhibits TNF-induced IL-6 production. IL-6 secreted from mouse embryonicfibroblasts (MEFs) expressing CARP-2 variants was measured by ELISA and expression ofCARP-2 and β-actin in the extracts is shown.(G) and (H). Knockdown of CARP-2 prolongs TNF-induced IKK activation and IκBαdegradation. Extracts of TNF-treated 293T cells expressing ShRNA constructs weresubjected to in vitro IKK kinase assay (G), or probed for expression of indicated proteins (Gand H).

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Fig. 2. CARP-2 localizes to endocytic vesicles(A) and (B). CARP-2 localizes to endocytic membrane vesicles. Confocal images of HeLacells immunostained with control or CARP-2 (Red) (A), or CARP-2 (Red) and Rab 5 (Green)antibodies (B). Scale bar is 10 μm. Areas of colocalization was shown by line intensity profile.(C). CARP-2 is recruited to TNF-R1 receptosomes. Magnetic TNF-R1 fractions and post-nuclear extracts (lysate) from HeLa cells were probed for indicated proteins.(D). Internalized TNF-R1 vesicles associate with CARP-2 positive vesicles. Confocal imagesof HeLa cells labeled with Biotin-TNF/FITC (Green) conjugated streptavidine and anti-CARP-2 (Red). Colocalization analyses were performed with Zeiss AIM software v3.2 to givethe overlay image of colocalized pixels, scatter plots and Pearson's coefficients. Ch 3 is FITC(green) and Ch4 is CY3 (Red). Scale bar is10 μm.

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Fig. 3. CARP-2 functions at the level of the early TNF-R1 complex(A). CARP-2 inhibits NF-κB activation induced by early TNF-R1 complex components.Extracts from 293T cells transfected with indicated constructs along with reporter plasmidswere assayed for reporter activities. The fold increase in the activity relative to vectorbackground is shown in parenthesis. Expression of signaling molecules and one representativeset of CARP-2 are shown.(B) and (C). CARP-2 downregulates, and lactacystin rescues RIP expression. Expression ofexogenous proteins in the extracts of 293T cells transfected with indicated constructs andtreated with proteasomal inhibitor lactacystin are shown.(D). CARP-2 binds to endogenous RIP. Extracts from 293T cells expressing FLAG-CARP-2variants were immunoprecipitated with anti-FLAG antibody and probed for indicated proteins.(E). Endogenous CARP-2 binds and promotes endogenous RIP degradation in TNF-stimulatedcells. CARP-2 from extracts of TNF-stimulated HeLa cells was immunoprecipitated andprobed for RIP and CARP-2.(F). Endogenous CARP-2 binds endogenous RIP in TNF receptosomes. TNF receptosomesisolated from MG-132 treated HeLa cells were subjected to immunoprecipitation with anti-CARP-2 antibody coupled to magnetic MACS microbeads or with anti-rabbit IgG magneticmicrobeads. RIP and CARP-2 in total receptosomes are shown.

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Fig. 4. Endogenous CARP-2 targets internalized receptor-associated RIP for degradation(A) and (B). Inhibitors of internalization and MG-132 but not Z-VAD FMK rescue TNF–induced RIP degradation. Expression of endogenous RIP in TNF-stimulated NEMO −/− MEFs,pretreated with medium or FILIPIN (2 μg/ml) or chlorpromazine (CPZ) (1.6 μg/ml, 3.2 μg/ml,6.4 μg/ml) (A), or with indicated compounds (B) is shown.(C). CPZ inhibits TNF–induced RIP ubiquitination. RIP from extracts of TNF-stimulatedNEMO −/− MEFs pretreated with MG-132 and medium (UT) or CPZ (10 μg/ml) wasimmunoprecipitated under denaturing conditions and probed for ubiquitin and RIP.(D). Knockdown of CARP-2 rescues TNF-induced RIP loss. Extracts of TNF-stimulatedNEMO−/− MEFs expressing ShRNA constructs were probed for indicated proteins.(E). Ubiquitinated RIP accumulates on TNF-R1 endocytic vesicles in the presence of MG-132.Magnetic TNF-R1 fractions from U937 cells were probed for indicated proteins.(F). CARP-2 targets TNF receptor-associated RIP for degradation. Extracts from TNF-stimulated HeLa cells expressing ShRNA constructs were precipitated with hamster anti TNF-R1 antibody and probed for indicated proteins.

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Fig 5. CARP2 is an ubiquitin ligase (E3) for RIP(A). CARP-2 promotes TNF-induced endogenous RIP ubiquitination. PurifiedHisubiquitinated proteins from TNF-stimulated 293T cells transfected with indicated plasmidsand treated with MG-132 were analyzed with anti-RIP antibody (left panel). 293T cellsexpressing pcDNA3-HA-ubiquitin and pCMV-tag2B-RIP-His along with indicated plasmidswere pretreated with MG-132. His-RIP was purified and analyzed for ubiquitination (rightpanel).(B). CARP-2 mediates K48-linked polyubiquitination of endogenous RIP. Endogenous RIPfrom TNF-stimulated 293T cells expressing indicated plasmids, preincubated with MG-132,was immunoprecipitated under denaturing conditions and probed for ubiquitin and RIP.(C). CARP-2 promotes RIP degradation. 293T cells transfected with pFLAG-CMV-RIP andpcDNA3-nV5-CARP2 variants were incubated with DMEM containing [35S] ProMix for 30minutes and chased by growing in complete serum medium. RIP from the extracts wasimmunoprecipitated and analyzed by autoradiography and PhosphorImager.(D). In vitro ubiquitination of RIP by CARP-2. Modified RIP band is indicated.

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Fig. 6. CARP-2 interacts with RIP kinase domain(A). CARP-2 binds RIP kinase domain. Extracts from 293T cells expressing FLAG taggedRIP kinase domain (KD) or without kinase domain (ΔKD) were incubated with extracts fromcells expressing vector or wild type V5-CARP-2. RIP was immuno-precipitated and probedfor CARP-2.(B). Kinase domain is essential for CARP-2 mediated RIP loss. Extracts from HeLa cellstransfected with pcDNA3-nV5-RIP Full length (FL) or ΔKD together with CARP-2 wereprobed for indicated proteins.(C). Kinase domain is essential for CARP-2 mediated RIP degradation. 293T cells transfectedwith pcDNA3-nV5 -RIPΔKD and pFLAG-CMV-CARP2 variants were analyzed by proceduredescribed in figure 5C.(D). RIP kinase domain negatively regulates TNF-induced NF-κB activation. Extracts ofmedium (Med) or TNF treated RIP−/− MEFs transfected with indicated constructs in pMSCVvector along with reporter plasmids were assayed for the reporter activities.

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Fig. 7.Model for regulation of TNF mediated NF-κB activation by CARP-2

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Author Manuscript NIH Public Access Qi Xiao RIP and regulates TNF … w... · 2009-06-23 · TNF...

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Transcript of Author Manuscript NIH Public Access Qi Xiao RIP and regulates TNF … w... · 2009-06-23 · TNF...