CLAP, a novel caspase recruitment domain-containing protein in the tumor necrosis factor receptor pathway, regulates NF-kappaB activation and apoptosis.

Molecules that regulate NF-kappaB activation play critical roles in apoptosis and inflammation. We describe the cloning of the cellular homolog of the equine herpesvirus-2 protein E10 and show that both proteins regulate apoptosis and NF-kappaB activation. These proteins were found to contain N-terminal caspase-recruitment domains (CARDs) and novel C-terminal domains (CTDs) and were therefore named CLAPs (CARD-like apoptotic proteins). The cellular and viral CLAPs induce apoptosis downstream of caspase-8 by activating the Apaf-1-caspase-9 pathway and activate NF-kappaB by acting upstream of the NF-kappaB-inducing kinase, NIK, and the IkB kinase, IKKalpha. Deletion of either the CARD or the CTD domain inhibits both activities. The CARD domain was found to be important for homo- and heterodimerization of CLAPs. Substitution of the CARD domain with an inducible FKBP12 oligomerization domain produced a molecule that can induce NF-kappaB activation, suggesting that the CARD domain functions as an oligomerization domain, whereas the CTD domain functions as the effector domain in the NF-kappaB activation pathway. Expression of the CARD domain of human CLAP abrogates tumor necrosis factor-alpha-induced NF-kappaB activation, suggesting that cellular CLAP plays an essential role in this pathway of NF-kappaB activation.     

vCLAP, a caspase-recruitment domain-containing protein of equine Herpesvirus-2, persistently activates the Ikappa B kinases through oligomerization of IKKgamma

vCLAP, the E10 gene product of equine herpesvirus-2, is a caspase-recruitment domain (CARD)-containing protein that has been shown to induce both apoptosis and NF-kappaB activation in mammalian cells. vCLAP has a cellular counterpart, Bcl10/cCLAP, which is also an activator of apoptosis and NF-kappaB. Recent studies demonstrated that vCLAP activates NF-kappaB through an IkappaB kinase (IKK)-dependent pathway, but the underlying mechanism remains unknown. In this report, we demonstrate that vCLAP associates stably with the IKK complex through direct binding to the C-terminal region of IKKgamma. Consistent with this finding, IKKgamma was found to be essential for vCLAP-induced NF-kappaB activation, and the association between vCLAP and the IKK complex induced persistent activation of the IKKs. Moreover, enforced oligomerization of the isolated C-terminal region of vCLAP, which interacts with IKKgamma, can trigger NF-kappaB activation. Finally, substitution of the C-terminal region of IKKgamma, which interacts with vCLAP, with the CARD of vCLAP or Bcl10 produced a molecule that was able to activate NF-kappaB when ectopically expressed in IKKgamma-deficient cells. These data suggest that vCLAP-induced oligomerization of IKKgamma, which is mediated by the CARD of vCLAP, could be the mechanism by which vCLAP induces activation of NF-kappaB.     

CARD11 mediates factor-specific activation of NF-kappaB by the T cell receptor complex

NF-kappaB is a critical target of signaling downstream of the T cell receptor (TCR) complex, but how TCR signaling activates NF-kappaB is poorly understood. We have developed an expression cloning strategy that can identify catalytic and noncatalytic molecules that participate in different pathways of NF-kappaB activation. Screening of a mouse thymus cDNA library yielded CARD11, a membrane-associated guanylate kinase (MAGUK) family member containing CARD, PDZ, SH3 and GUK domains. Using a CARD-deleted variant of CARD11 and RNA interference (RNAi), we demonstrate that CARD11 mediates NF-kappaB activation by alphaCD3/alphaCD28 cross-linking and PMA/ionomycin treatment, but not by TNFalpha or dsRNA. CARD11 is not required for TCR-mediated induction of NFAT or AP-1. CARD11 functions upstream of the IkappaB-kinase (IKK) complex and cooperates with Bcl10 in a CARD domain-dependent manner. RNAi-rescue experiments suggest that the CARD, coiled-coil, SH3 and GUK domains of CARD11 are critical for its signaling function. These results implicate CARD11 in factor- specific activation of NF-kappaB by the TCR complex and establish a role for a MAGUK family member in antigen receptor signaling.     

INCA, a novel human caspase recruitment domain protein that inhibits interleukin-1beta generation

Using in silico methods for screening the human genome for new caspase recruitment domain (CARD) proteins, we have identified INCA (Inhibitory CARD) as a protein that shares 81% identity with the prodomain of caspase-1. The INCA gene is located on chromosome 11q22 between the genes of COP/Pseudo-ICE and ICEBERG, two other CARD proteins that arose from caspase-1 gene duplications. We show that INCA mRNA is expressed in many tissues. INCA is specifically upregulated by interferon-gamma in the monocytic cell lines THP-1 and U937. INCA physically interacts with procaspase-1 and blocks the release of mature IL-1beta from LPS-stimulated macrophages. Unlike COP/Pseudo-ICE and procaspase-1, INCA does not interact with RIP2 and does not induce NF-kappaB activation. Our data show that INCA is a novel intracellular regulator of procaspase-1 activation, involved in the regulation of pro-IL-1beta processing and its release during inflammation.     

ASC-mediated NF-kappaB activation leading to interleukin-8 production requires caspase-8 and is inhibited by CLARP

ASC is an adaptor molecule that mediates apoptotic and inflammatory signals from several Apaf-1-like molecules, including CARD12/Ipaf, cryopyrin/PYPAF1, PYPAF5, PYPAF7, and NALP1. To characterize the signaling pathway mediated by ASC, we established cell lines in which muramyl dipeptide, the bacterial component recognized by another Apaf-1-like molecule, Nod2, induced an interaction between a CARD12-Nod2 chimeric protein and ASC, and elicited cell autonomous NF-kappaB activation. This response required caspase-8, and was suppressed by CLARP/FLIP, an inhibitor of caspase-8. The catalytic activity of caspase-8 was required for the ASC-mediated NF-kappaB activation when caspase-8 was expressed at an endogenous level, although it was not essential when caspase-8 was overexpressed. In contrast, FADD, the adaptor protein linking Fas and caspase-8, was not required for this response. Consistently, ASC recruited caspase-8 and CLARP but not FADD and Nod2 to its speck-like aggregates in cells. Finally, muramyl dipeptide induced interleukin-8 production in MAIL8 cells. These results are the first to indicate that caspase-8 plays an important role in the ASC-mediated NF-kappaB activation, and that the ASC-mediated NF-kappaB activation actually induces physiologically relevant gene expression.     

Cloning of a novel human caspase-9 splice variant containing only the CARD domain

Caspase-9 plays a key role in the intrinsic apoptotic pathway and currently two splice variants (caspase-9-alpha and -beta) have been identified. The present study cloned and characterized a novel caspase-9 splice variant, hereby designated Casp9-gamma. Casp9-gamma is generated from an additional alternative 3' splice site in the fourth exon of caspase-9, resulting in a 58-nucleotide fragment insertion compared with the full-length caspase-9-alpha. The fragment introduces an in-frame stop codon, and the resulting open reading frame (ORF) is preterminated. The Casp9-gamma comprises the deduced 154 amino acid residues containing only the caspase recruitment domain (CARD) and does not contain the large and small subunits. The Casp9-gamma does not promote apoptosis when overexpressed in mammalian cells. Moreover, it inhibits the cleavage of procaspase-3 mediated by proapoptotic member Bax or apoptosis inductor staurosporine. Therefore, Casp9-gamma may function as an endogenous apoptotic inhibitor by interfering with the CARD-CARD interaction between Apaf-1 (apoptotic protease activating factor-1) and procaspase-9. In addition, Casp9-gamma does not enhance NF-kappaB activation in transfected 293T cells, conflicting with previous evidence that the isolated CARD of caspase-9 activates NF-kappaB in ND7 cells. This suggests that the procaspase-9-mediated NF-kappaB activation in response to cellular stresses is cell type-specific through an unidentified mechanism.     

CARD9 is a novel caspase recruitment domain-containing protein that interacts with BCL10/CLAP and activates NF-kappa B

BCL10/CLAP is an activator of apoptosis and NF-kappaB signaling pathways and has been implicated in B cell lymphomas of mucosa-associated lymphoid tissue. Although its role in apoptosis remains to be determined, BCL10 likely activates NF-kappaB through the IKK complex in response to upstream stimuli. The N-terminal caspase recruitment domain (CARD) of BCL10 has been proposed to function as an activation domain that mediates homophilic interactions with an upstream CARD-containing NF-kappaB activator. To identify upstream signaling partners of BCL10, we performed a mammalian two-hybrid analysis and identified CARD9 as a novel CARD-containing protein that interacts selectively with the CARD activation domain of BCL10. When expressed in cells, CARD9 binds to BCL10 and activates NF-kappaB. Furthermore, endogenous CARD9 is found associated with BCL10 suggesting that both proteins form a pre-existing signaling complex within cells. CARD9 also self-associates and contains extensive coiled-coil motifs that may function as oligomerization domains. We propose here that CARD9 is an upstream activator of BCL10 and NF-kappaB signaling.     

Caspase-1 activates nuclear factor of the kappa-enhancer in B cells independently of its enzymatic activity

The proteolytic activity of caspases is involved in apoptosis and inflammation. In this regard, caspase-1 is required for pro-interleukin (IL)-1beta and pro-IL-18 maturation. We report here on a novel function of caspase-1 as an activator of nuclear factor of the kappa-enhancer in B-cells (NF-kappaB) and p38 mitogen-activated protein kinase (MAPK). This function is not shared by the murine caspase-1 homologues caspase-11 and -12. In contrast to pro-IL-1beta maturation, caspase-1-induced NF-kappaB activation is not inhibited by the virus-derived caspase-1 inhibitor cytokine response modifier A and is equally induced by the enzymatically inactive caspase-1 C285A mutant. Although the general NF-kappaB-inhibiting protein A20 inhibits caspase-1-derived activation of NF-kappaB, dominant-negative forms of TRAF2 and RIP1 have no effect. We demonstrate that caspase-1 interacts with RIP2 and that dominant-negative forms of RIP2 and IkappaB kinase complex-beta inhibit caspase-1-mediated NF-kappaB activation. Structure-function analysis shows that the caspase recruitment domain of caspase-1 mediates the activation of NF-kappaB and p38 MAPK. These data demonstrate that caspase-1 contributes to inflammation by two distinct pathways: proteolysis of pro-IL-1beta, and RIP2-dependent activation of NF-kappaB and p38 MAPK mediated by the caspase recruitment domain.     

Card10 is a novel caspase recruitment domain/membrane-associated guanylate kinase family member that interacts with BCL10 and activates NF-kappa B

BCL10 belongs to the caspase recruitment domain (CARD) family of proteins that regulate apoptosis and NF-kappaB signaling pathways. Analysis of BCL10-deficient mice has revealed that BCL10 mediates NF-kappaB activation by antigen receptors in B and T cells. We recently identified a subclass of CARD proteins (CARD9, CARD11, and CARD14) that may function to connect BCL10 to multiple upstream signaling pathways. We report here that CARD10 is a novel BCL10 interactor that belongs to the membrane-associated guanylate kinase family, a class of proteins that function to organize signaling complexes at plasma membranes. When expressed in cells, CARD10 binds to BCL10 and signals the activation of NF-kappaB through its N-terminal effector CARD domain. We propose that CARD10 functions as a molecular scaffold for the assembly of a BCL10 signaling complex that activates NF-kappaB.     

Nod1, an Apaf-1-like activator of caspase-9 and nuclear factor-kappaB.

Ced-4 and Apaf-1 belong to a major class of apoptosis regulators that contain caspase-recruitment (CARD) and nucleotide-binding oligomerization domains. Nod1, a protein with an NH2-terminal CARD-linked to a nucleotide-binding domain and a COOH-terminal segment with multiple leucine-rich repeats, was identified. Nod-1 was found to bind to multiple caspases with long prodomains, but specifically activated caspase-9 and promoted caspase-9-induced apoptosis. As reported for Apaf-1, Nod1 required both the CARD and P-loop for function. Unlike Apaf-1, Nod1 induced activation of nuclear factor-kappa-B (NF-kappaB) and bound RICK, a CARD-containing kinase that also induces NF-kappaB activation. Nod1 mutants inhibited NF-kappaB activity induced by RICK, but not that resulting from tumor necrosis factor-alpha stimulation. Thus, Nod1 is a leucine-rich repeat-containing Apaf-1-like molecule that can regulate both apoptosis and NF-kappaB activation pathways.     

Multiple protein domains mediate interaction between Bcl10 and MALT1

Bcl10 and MALT1 are essential mediators of NF-kappaB activation in response to the triggering of a diverse array of transmembrane receptors, including antigen receptors. Additionally, both proteins are translocation targets in MALT lymphoma. Thus, a detailed understanding of the interaction between these mediators is of considerable biological importance. Previous studies have indicated that a 13-amino acid region downstream of the Bcl10 caspase recruitment domain (CARD) is responsible for interacting with the immunoglobulin-like domains of MALT1. We now provide evidence that the death domain of MALT1 and the CARD of Bcl10 also contribute to Bcl10-MALT1 interactions. Although a direct interaction between the MALT1 death domain and Bcl10 cannot be detected via immunoprecipitation, FRET data strongly suggest that the death domain of MALT1 contributes significantly to the association between Bcl10 and MALT1 in T cells in vivo. Furthermore, analysis of point mutants of conserved residues of Bcl10 shows that the Bcl10 CARD is essential for interaction with the MALT1 N terminus. Mutations that disrupt proper folding of the Bcl10 CARD strongly impair Bcl10-MALT1 interactions. Molecular modeling and functional analyses of Bcl10 point mutants suggest that residues Asp(80) and Glu(84) of helix 5 of the Bcl10 CARD directly contact MALT1. Together, these data demonstrate that the association between Bcl10 and MALT1 involves a complex interaction between multiple protein domains. Moreover, the Bcl10-MALT1 interaction is the second reported example of interactions between a CARD and a non-CARD protein region, which suggests that many signaling cascades may utilize CARD interactions with non-CARD domains.     

CARD6 is a modulator of NF-kappa B activation by Nod1- and Cardiak-mediated pathways

We cloned a novel cDNA derived from the CARD6 gene locus on chromosome 5p12 of 311 amino acids in length. By immunoprecipitation we detected specific binding of this CARD6-encoding protein to Nod1 (CARD4), Cardiak (Rip2/Rick), NAC (NALP1/DEFCAP/CARD7), and TUCAN (CARD8/Cardinal/NDPP/Dakar), caspase recruitment domain (CARD)-containing proteins implicated in NF-kappa B and caspase-1 activation but not to other CARD family proteins. Cardiak and Nod1 (but not other CARD proteins) also exhibited opposing effects on CARD6 protein phosphorylation and expression, providing further evidence of functional interactions among these proteins in cells. In transfection experiments, the CARD6 protein suppressed NF-kappa B induction by Nod1 or Cardiak but did not interfere with NF-kappa B activation by the CARD-containing adapter protein Bcl10 or the cytokine tumor necrosis factor-alpha, demonstrating specificity of CARD6 for Nod-1 and Cardiak-dependent pathways. In contrast to its effects on Nod1- and Cardiak-dependent NF-kappa B activation, CARD6 did not interfere with caspase-1-dependent interleukin-1 beta secretion induced by Cardiak or Nod1. CARD6 also did not affect caspase activation and apoptosis induced by overexpression of Fas, Bax, or other pro-apoptotic stimuli. Thus, CARD6 represents a selective modulator of NF-kappa B activation by Cardiak and Nod1, adding to the repertoire of CARD-family proteins implicated in inflammatory responses and innate immunity.     

SPAK kinase is a substrate and target of PKCtheta in T-cell receptor-induced AP-1 activation pathway

Protein kinase C-theta (PKCtheta) plays an important role in T-cell activation via stimulation of AP-1 and NF-kappaB. Here we report the isolation of SPAK, a Ste20-related upstream mitogen-activated protein kinase (MAPK), as a PKCtheta-interacting kinase. SPAK interacted with PKCtheta (but not with PKCalpha) via its 99 COOH-terminal residues. TCR/CD28 costimulation enhanced this association and stimulated the catalytic activity of SPAK. Recombinant SPAK was phosphorylated on Ser-311 in its kinase domain by PKCtheta, but not by PKCalpha. The magnitude and duration of TCR/CD28-induced endogenous SPAK activation were markedly impaired in PKCtheta-deficient T cells. Transfected SPAK synergized with constitutively active PKCtheta to activate AP-1, but not NF-kappaB. This synergistic activity, as well as the receptor-induced SPAK activation, required the PKCtheta-interacting region of SPAK, and Ser-311 mutation greatly reduced these activities of SPAK. Conversely, a SPAK-specific RNAi or a dominant-negative SPAK mutant inhibited PKCtheta- and TCR/CD28-induced AP-1, but not NF-kappaB, activation. These results define SPAK as a substrate and target of PKCtheta in a TCR/CD28-induced signaling pathway leading selectively to AP-1 (but not NF-kappaB) activation.     

Cell type-specific regulation of ITAM-mediated NF-kappaB activation by the adaptors, CARMA1 and CARD9

Activating NK cell receptors transduce signals through ITAM-containing adaptors, including FcRgamma and DAP12. Although the caspase recruitment domain (CARD)9-Bcl10 complex is essential for FcRgamma/DAP12-mediated NF-kappaB activation in myeloid cells, its involvement in NK cell receptor signaling is unknown. Herein we show that the deficiency of CARMA1 or Bcl10, but not CARD9, resulted in severe impairment of cytokine/chemokine production mediated by activating NK cell receptors due to a selective defect in NF-kappaB activation, whereas cytotoxicity mediated by the same receptors did not require CARMA1-Bcl10-mediated signaling. IkappaB kinase (IKK) activation by direct protein kinase C (PKC) stimulation with PMA plus ionomycin (P/I) was abrogated in CARMA1-deficient NK cells, similar to T and B lymphocytes, whereas CARD9-deficient dendritic cells (DCs) exhibited normal P/I-induced IKK activation. Surprisingly, CARMA1 deficiency also abrogated P/I-induced IKK activation in DCs, indicating that CARMA1 is essential for PKC-mediated NF-kappaB activation in all cell types, although the PKC-CARMA1 axis is not used downstream of myeloid ITAM receptors. Consistently, PKC inhibition abrogated ITAM receptor-mediated activation only in NK cells but not in DCs, suggesting PKC-CARMA1-independent, CARD9-dependent ITAM receptor signaling in myeloid cells. Conversely, the overexpression of CARD9 in CARMA1-deficient cells failed to restore the PKC-mediated NF-kappaB activation. Thus, NF-kappaB activation signaling through ITAM receptors is regulated by a cell type-specific mechanism depending on the usage of adaptors CARMA1 and CARD9, which determines the PKC dependence of the signaling.     

Inhibition of death receptor-mediated gene induction by a cycloheximide-sensitive factor occurs at the level of or upstream of Fas-associated death domain protein (FADD)

In HeLa cells, induction of apoptosis and nuclear factor kappaB (NF-kappaB) activation initiated by TRAIL/Apo2L or the agonistic Apo1/Fas-specific monoclonal antibody anti-APO-1 require the presence of cycloheximide (CHX). Inhibition of caspases prevented TRAIL/anti-APO-1-induced apoptosis, but not NF-kappaB activation, indicating that both pathways bifurcate upstream of the receptor-proximal caspase-8. Under these conditions, TRAIL and anti-APO-1 up-regulated the expression of the known NF-kappaB targets interleukin-6, cellular inhibitor of apoptosis 2 (cIAP2), and TRAF1 (TRAF, tumor necrosis factor receptor-associate factor). In the presence of CHX, the stable overexpression of a deletion mutant of the Fas-associated death domain molecule FADD comprising solely the death domain of the molecule but lacking its death effector domain (FADD-(80-208)) led to the same response pattern as TRAIL or anti-APO-1 treatment. Moreover, the ability of death receptors to induce NF-kappaB activation was drastically reduced in a FADD-deficient Jurkat cell line. TRAIL-, anti-APO-1-, and FADD-(80-208)-initiated gene induction was blocked by a dominant-negative mutant of TRAF2 or the p38 kinase inhibitor SB203580, similar to tumor necrosis factor receptor-1-induced NF-kappaB activation. CHX treatment rapidly down-regulated endogenous cFLIP protein levels, and overexpression of cellular FLICE inhibitory protein (cFLIP) inhibited death receptor-induced NF-kappaB activation. Thus, a novel functional role of cFLIP as a negative regulator of gene induction by death receptors became apparent.