CARP-2 is a novel regulator of TNF-induced NF-κB activation
To identify proteins that regulate TNF signaling in the endocytic pathway, we focused on proteins that associate with the endosomal and Golgi membrane vesicles with which fusion of internalized TNF receptor complexes occurs. Besides trans-membrane proteins, proteins with a particular motif known as FYVE (conserved in Fab1p/YOTB/Vac1p /EEA1) are known to localize to these vesicles [17
]. Therefore, we searched the NCBI gene bank for FYVE motif-containing proteins. Interestingly, two of the 45 FYVE domain-containing proteins found also contain RING domains, a sequence that confers ubiquitin protein ligase activity to many molecules [19
]. Reasoning that molecules containing such properties might be especially well-suited to interact with and modify components of early TNF-R1 associated proteins, we tested their ability to affect TNF-induced NF-κB activation. One of the molecules, CARP-2 (caspase-8 and –10-associated RING protein-2) (), is known to bind and negatively 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 containing proteins with no conserved phosphoinositide-binding motif [22
]. In spite of the missing PI3P binding property of the FYVE motif, this sequence has been shown in overexpression studies to target the protein to endocytic membrane vesicles [23
]. The RING domain is also functional and exhibits ubiquitin protein ligase (E3) activity in vitro
and ex vivo
CARP-2 negatively regulates TNF-induced NF-κB activation
To assess the effect of CARP-2 expression on TNF-induced NF-κB activation, we analyzed the endogenous IKK activity and IκBα degradation (). As expected, treatment of vector only expressing cells with TNF resulted in increased IKK activation (). Expression of CARP-2 wild type decreased IKK activity both at basal level and upon TNF stimulation. A substitution of alanine for a histidine in the RING domain (H333A) that abrogates E3 activity failed to reduce IKK activity suggesting that E3 activity is required for CARP-2 inhibitory function (). Consistent with these results expression of CARP-2 wild type () 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 ().
Additionally, NF-κB reporter assays were used to assess the effect of increased CARP-2 expression on NF-κB activation. CARP-2 reduced TNF-induced NF-κB reporter activity in a dose-responsive fashion (). At high concentrations the RING mutant also exhibited some 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 by CARP-2 was observed in all cell lines such as HT1080 (human fibrosarcoma), HeLa (human cervical carcinoma) and C2C12 (mouse myoblast) tested (data not shown). To investigate the effect of CARP-2 expression on NF-κB mediated cytokine production, we examined IL-6 secretion in response to TNF stimulation in mouse embryonic fibroblasts that express CARP-2 variants. Treatment of vector–only expressing cells with TNF resulted in increased production of IL-6, but cells that express wild type CARP-2 produced very little IL-6 (). In agreement with the requirement of E3 activity for CARP-2 inhibitory function, MEFs that express the RING mutant (H333A) exhibited increased IL-6 production both at the basal levels and upon TNF stimulation (). Therefore, in TNF stimulated cells CARP-2 inhibits activation of NF-κB in a largely RING dependent manner.
To investigate the physiologic function of endogenous CARP-2, we designed small hairpin RNA (ShRNA) specific for two different regions of CARP-2. Transfection of the siRNA hairpins in 293T cells resulted in a large reduction in the level of endogenous CARP-2 protein (). Knockdown of endogenous CARP-2 expression enhanced TNF-induced NF-κB reporter activity by approximately two fold (). Consistent with this, knockdown of CARP-2 prolonged the IKK activation to as late as 60 min () and delayed the recovery of IκBα (beginning at 30-45 min in control cells but occurring at 60-90 min in ShRNA-treated cells) (). The observed effects of ShRNAs are specific because co-expression of ShRNA-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 the physiological function of this molecule is to limit the intensity or duration of signaling.
CARP-2 localizes to membrane compartments and recruits to vesicles containing endocytosed TNF-receptor
Previous studies have shown that overexpressed mouse CARP-2 associates with membrane compartments in the perinuclear region that are positive for the endosomal markers, Rab5 and Rab11[23
]. Therefore, to determine if endogenous CARP-2 constitutively associates with endocytic membrane vesicles, we developed a monoclonal antibody that specifically recognizes CARP-2 (Fig. S2A and B
). Confocal microscopic analysis of cells stained with this antibody showed punctate staining for endogenous CARP-2, mostly in the cytosol (). A subset of these cytosolic vesicles also stained positive with antibody specific for Rab5 (). No significant co-localization between CARP-2 and either Golgi marker GM130, or the lysosomal marker Lamp1 was observed (Fig. S2C
). Collectively these results suggest that endogenous CARP-2 associates constitutively with, albeit undefined, membrane vesicles that fuse with internalized TNF-receptor complex (see below) and are referred as CARP-2 positive vesicles in this manuscript.
CARP-2 localizes to endocytic vesicles
Given that internalized TNF-receptor complex fuse with endocytic vesicles to form multi vesicular endosomes (also known as TNF receptosomes), we examined whether these isolated TNF receptosomes include CARP-2 [10
]. As expected all the receptosome preparations from both HeLa and U937 cells contain TNF-R1 ( and Fig. S2D
). After 5 minutes of internalization significant amounts of CARP-2 were found along with markers of other endocytic 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-2 is directly recruited to endocytosed TNF complex, or CARP-2 vesicles along with that of Rab 5 and 7 fuse with internalized receptor complex. While the recruitment of CARP-2 occurred in as early as 5 minutes, Lamp-1 (a lysosomal protein) is recruited around 30-60 minutes suggesting that the association of CARP-2 is an early event that occurs much before the fusion of the internalized TNF-receptor with lysosomal compartments.
Additionally, confocal microscopy was used to investigate the association between CARP-2 and endocytosed TNF-receptor complex. The intracellular trafficking of TNF/TNF-R1 was followed by first, incubating HeLa cells with biotin TNF/streptavidin FITC at a low temperature and then by increasing the temperature to 37°C. Confocal analysis of these cells stained with anti-CARP-2 antibody showed (30 minutes after internalization) significant co-localization of the receptor with CARP-2 (). The co-localization coefficient (Pearson's coefficient) 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 with CARP-2.
CARP-2 promotes endogenous RIP degradation in TNF-stimulated cells
The association of the internalized TNF-R complexes with CARP-2 suggests that CARP-2 inhibits NF-κB activation in TNF-stimulated cells possibly by targeting one or more components of the early TNF-R1 complex. To test this possibility CARP-2 was co-expressed with a number of gene products found in the early TNF-R1 complex whose overexpression in cells 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 (). 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 the level of the early signaling complex.
CARP-2 functions at the level of the early TNF-R1 complex
Given that ubiquitin ligase (E3) activity of CARP-2 is essential for its function, we asked if CARP-2 might affect the expression of the above signaling molecules. RIP protein level was downregulated by co-expression of CARP-2 (). The downregulation was specific for RIP, as CARP-2 had no effect on the level of expression of either TNF-R1, or TRAF-2, or NEMO, another critical regulator of NF-κB activation, under the same experimental conditions (). The function of CARP-2 appears to be specific for TNF, as co-expression of CARP-2 had no effect on the expression of IRAK (Interleukin-1 receptor-associated kinase) a functional homologue of RIP in IL-1 signaling and an essential modulator of IL-1-induced NF-κb activation (Fig. S3
). The RING mutant did not affect RIP protein expression (), 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 reverses the loss of RIP protein in the presence of CARP-2 (). In order to determine whether CARP-2 binds directly to RIP, immunoprecipitates of FLAG-tagged CARP-2 from 293T cells were probed for endogenous proteins with antibodies against TNF-R1, RIP, and TRAF-2 (). Consistent with the specific downregulation of RIP protein, immunoprecipitates from cell extracts expressing wild-type CARP-2 but not vector contained substantial amounts of RIP protein and only a very little amount of TNF-R1 and TRAF-2. Interestingly the RING-domain mutant, 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 the RING-domain is dispensable for RIP-binding. These interactions were further confirmed as extracts of cells expressing FLAG-tagged RIP and V5-tagged CARP-2 contained RIP/CARP-2 complexes (Fig. S4
Because CARP-2 binds and downregulates RIP, we asked if stimulation with TNF promotes interaction between these proteins at endogenous level. To answer this endogenous CARP-2 was immunoprecipitated from cells stimulated with TNF in the presence or absence of MG-132, and the immunoprecipitated material was probed with anti-RIP (). Some interaction between RIP and CARP-2 was detected in unstimulated cells. But in stimulated cells we found no interaction in the absence of MG-132 between CARP-2 and RIP. However, when proteasomal degradation is inhibited, anti-CARP-2 immunoprecipitates RIP from cells stimulated by TNF for 30 min or more, suggesting that after TNF stimulation CARP-2 binds and rapidly degrades RIP (). In order to evaluate the direct interaction of endogenous CARP-2 with endogenous RIP in internalized TNF receptosomes, CARP-2 was immunoprecipitated from TNF receptosomes isolated from MG-132-treated HeLa cells after 0 and 30 min of internalization and probed for CARP-2 and RIP (). TNF receptosomes in 30 min contained both RIP and CARP-2 and RIP from lysed TNF receptosomes was found to coimmunoprecipitate specifically with CARP-2 antibody. These data demonstrate that CARP-2 directly interacts with RIP at internalized TNF receptosomes.
CARP-2 targets the internalized receptor associated RIP for degradation
To test the hypothesis that RIP degradation at endocytic vesicles is the mechanism to terminate NF-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 distinct endocytic routes, namely, clathrin mediated endocytosis (CME) and raft/caveolar endocytosis (RCE) [26
] . Results from a previous study that show reduced TNF-induced NF-κB target gene expression upon inhibition of endocytosis indicate that active signaling continues during TNF-receptor endocytosis [29
]. While most cell types upon TNF stimulation exhibit rapid degradation of receptor associated RIP, only NEMO deficient cells show significant decrease in total cellular RIP [8
]. For this reason, to investigate the effect of internalization on RIP ubiquitination 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 (). This decrease in RIP level is not the result of apoptosis, as pan caspase inhibitor Z-VAD-FMK had failed to rescue the loss (). Treatment with chlorpromazine (CPZ), a specific CME inhibitor, effectively reduced the loss in a dose dependent manner (). Inhibition of TNF-R1 endocytosis by monodansyl cadaverine (MDC) also prevented RIP loss (data not shown). However, the presence of another inhibitor Filipin at a concentration that specifically inhibits RCE, had very little effect suggesting that CME promotes the degradation of RIP in these cells. Next, the effect of internalization on RIP ubiquitination was assessed from cells treated with or without CPZ in the presence of proteasome inhibitor MG-132, and stimulated with TNF. In agreement with earlier published reports proteasomal inhibition efficiently blocked TNF-induced RIP loss (). While TNF stimulation in the presence of MG-132 promoted RIP ubiquitination, the presence of CPZ blocked RIP modification suggesting that the observed loss of RIP might be the consequence of internalization and subsequent ubiquitination ().
Endogenous CARP-2 targets internalized receptor-associated RIP for degradation
To investigate the role of CARP-2 in RIP degradation, we transfected NEMO−/−cells with ShRNA specific for mouse CARP-2. While the knockdown of CARP-2 protein expression had minimal effect on RIP level in unstimulated cells, it partially rescued the loss of RIP in stimulated cells () 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-132 results in accumulation of ubiquitinated RIP at endocytic vesicles. To address this we purified TNF-R1 associated endocytic vesicles from U937 cells treated with or without MG-132 and analyzed the vesicles for RIP by immunoblotting. MG-132 treatment did not alter the TNF-R1 complex fusion to endocytic vesicles (). Compared to control (- MG-132) cells, inhibition of proteasomal degradation resulted in increased amounts of both polyubiquitinated and unubiquitinated RIP (30 and 60 min samples). Not surprisingly, the presence of MG-132 also stabilized TNF-R1 protein (). Nevertheless, these results suggest that, in TNF stimulated cells, internalized RIP ubiquitination and degradation broadly occur at endocytic vesicles. While the initial ubiquitination of RIP that occurs immediately after stimulation at the plasma membrane (presumably mediated by TRAF-2) promotes NF-κB activation, the later ubiquitination at endocytic vesicles might be required for its degradation resulting in termination of NF-κB activation. If CARP-2, indeed, promotes RIP degradation we reasoned that the knockdown of CARP-2 expression should result in stabilization of this receptor associated RIP.
To test this, we immunoprecipitated TNF-R1 from HeLa cells expressing CARP-2 ShRNA and treated with TNF, and immunoblotted for associated endogenous RIP. Consistent with earlier reports, ubiquitinated and unubiquitinated RIP was found to coimmunoprecipitate with TNF-R1 from TNF-stimulated cells (). Knockdown of CARP-2 resulted in a substantial increase in receptor-associated RIP, both polyubiquitinated and unubiquitinated, indicating that CARP-2 targets RIP within the early TNF-R1 complex for degradation. Hence, an increase in TNF-induced IKK/NF-κB activation in CARP-2 ShRNA expressing cells () could be the result of stabilization of receptor-associated RIP.
CARP-2 is an ubiquitin protein ligase for RIP
Given that CARP-2 promotes RIP degradation, we investigated whether CARP-2 functions as an ubiquitin protein ligase (E3) for RIP. We first assessed the effect of CARP-2 on endogenous RIP ubiquitination in TNF-stimulated cells transfected with CARP-2 and His-ubiquitin. While endogenous RIP was ubiquitinated on CARP-2 overexpression, this was significantly increased upon TNF-stimulation (). Furthermore, in co-transfection experiments V5-CARP-2 wild type but not RING mutant markedly enhanced the ubiquitination of FLAG RIP (). In these experiments overexpression of FLAG-RIP alone resulted in moderate ubiquitination. Since, CARP-2 ubiquitination of RIP effectively results in its degradation, 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 mutants in which only K-48 or K-63 are available for polymerization and analyzed ubiquitination status of endogenous RIP from TNF stimulated cells. As expected, CARP-2 wild type markedly promoted the K48-linked ubiquitination of RIP and very little K63-linked RIP ubiquitination was observed under these conditions (). In agreement with this expression of mutant RIP 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 the turnover of FLAG-RIP was assessed by pulse chase analysis. Consistent with its function as an E3 ligase, the degradation of transfected FLAG-RIP was rapid in the presence CARP-2 wild type (). To determine if CARP-2 can directly ubiquitinate RIP, in vitro ubiquitination 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 (). Taken together, the results demonstrate that CARP-2 binds and ubiquitinates RIP, leading to its degradation.
CARP2 is an ubiquitin ligase (E3) for RIP
RIP kinase domain is essential for CARP-2 mediated RIP degradation
Earlier reports have shown that the RIP intermediate domain and death domain are essential but the kinase domain is dispensable for NF-κB activation [12
]. To determine which portion of RIP interacts with CARP-2, we tested its binding to individual regions of RIP. Because the different constructs were not expressed in transiently transfected cells to the same level, we expressed FLAG-tagged RIP constructs and V5-tagged CARP-2 constructs individually in 293T cells and used different amounts of the extracts from these cells for immunoprecipitation experiments. Interestingly the RIP kinase domain, but not RIP lacking the KD, was found to specifically co-immunoprecipitate with CARP-2 (), suggesting a requirement of RIP KD for CARP-2 mediated RIP degradation. In order to test this possibility we examined the effect of CARP-2 on the degradation of RIP without KD in co-transfection and pulse-chase experiments. In agreement with its inability to bind to RIP without its kinase domain, CARP-2 did not affect RIPΔKD expression but reduced the full length RIP protein level (). Consistent with this, in pulse-chase experiments the degradation of FLAG-RIPΔKD remained unchanged in the presence of wild type or RING mutant CARP-2 (). Thus, these results prove that the kinase domain of RIP is essential for its binding and subsequent degradation by CARP-2.
CARP-2 interacts with RIP kinase domain
Given this apparent paradox on the role of the kinase domain in NF-κB signaling we hypothesized that this domain might regulate the termination of NF-κB activation by targeting internalized RIP to CARP-2 in intracellular compartments. In such a scenario, cells expressing only 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 region of RIP in RIP-deficient Jurkat cells has increased NF-κB activity as compared to full-length RIP-expressing cells [13
]. Indeed, TNF stimulation of RIP −/− MEFs expressing RIP lacking the kinase domain are consistently found to have an increase in basal as well as TNF-inducible NF-κB activity (). Thus CARP-2 negatively regulates NF-κB signaling by binding to RIP of the internalized early complex via the kinase domain leading to RIP ubiquitination and degradation.