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.     

Protein kinase C-associated kinase (PKK) mediates Bcl10-independent NF-kappa B activation induced by phorbol ester

Protein kinase C-associated kinase (PKK) is a recently described kinase of unknown function that was identified on the basis of its specific interaction with PKC beta. PKK contains N-terminal kinase and C-terminal ankyrin repeats domains linked to an intermediate region. Here we report that the kinase domain of PKK is highly homologous to that of two mediators of nuclear factor-kappa B (NF-kappa B) activation, RICK and RIP, but these related kinases have different C-terminal domains for binding to upstream factors. We find that expression of PKK, like RICK and RIP, induces NF-kappa B activation. Mutational analysis revealed that the kinase domain of PKK is essential for NF-kappa B activation, whereas replacement of serine residues in the putative activation loop did not affect the ability of PKK to activate NF-kappa B. A catalytic inactive PKK mutant inhibited NF-kappa B activation induced by phorbol ester and Ca(2+)-ionophore, but it did not block that mediated by tumor necrosis factor alpha, interleukin-1 beta, or Nod1. Inhibition of NF-kappa B activation by dominant negative PKK was reverted by co-expression of PKC beta I, suggesting a functional association between PKK and PKC beta I. PKK-mediated NF-kappa B activation required IKK alpha and IKK beta but not IKK gamma, the regulatory subunit of the IKK complex. Moreover, NF-kappa B activation induced by PKK was not inhibited by dominant negative Bimp1 and proceeded in the absence of Bcl10, two components of a recently described PKC signaling pathway. These results suggest that PKK is a member of the RICK/RIP family of kinases, which is involved in a PKC-activated NF-kappa B signaling pathway that is independent of Bcl10 and IKK gamma.     

PDGF-induced Akt phosphorylation does not activate NF-kappa B in human vascular smooth muscle cells and fibroblasts

A recent report suggested that platelet-derived growth factor (PDGF) activates nuclear factor-kappa B (NF-kappa B) by phosphorylation of the protein kinase Akt [Romashkova and Makarov, Nature 401 (1999) 86-90]. The present study investigates the role of Akt in the activation of NF-kappa B by tumor necrosis factor-alpha (TNF alpha, 10 ng/ml) and PDGF-BB (20 ng/ml) in human vascular smooth muscle cells (SMC), skin and foreskin fibroblasts. TNF alpha stimulated serine phosphorylation and degradation of the inhibitory protein I kappa B alpha and strongly induced nuclear NF-kappa B translocation and binding activity. PDGF did not induce serine phosphorylation or degradation of I kappa B alpha and did not enhance binding activity of NF-kappa B. In contrast, stimulation with PDGF resulted in a marked phosphorylation of Akt, but no Akt phosphorylation occurred after stimulation with TNF alpha. These data suggest that Akt phosphorylation is not involved in NF-kappa B activation in human SMC and fibroblasts.     

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.     

CARD9 Mediates Dectin-2-induced IκBα Kinase Ubiquitination Leading to Activation of NF-κB in Response to Stimulation by the Hyphal Form of Candida albicans

The scaffold protein CARD9 plays an essential role in anti-fungus immunity and is implicated in mediating Dectin-1/Syk-induced NF-κB activation in response to Candida albicans infection. However, the molecular mechanism by which CARD9 mediates C. albicans-induced NF-κB activation is not fully characterized. Here we demonstrate that CARD9 is involved in mediating NF-κB activation induced by the hyphal form of C. albicans hyphae (Hyphae) but not by its heat-inactivated unicellular form. Our data show that inhibiting Dectin-2 expression selectively blocked Hyphae-induced NF-κB, whereas inhibiting Dectin-1 mainly suppressed zymosan-induced NF-κB, indicating that Hyphae-induced NF-κB activation is mainly through Dectin-2 and not Dectin-1. Consistently, we find that the hyphae stimulation induces CARD9 association with Bcl10, an adaptor protein that functions downstream of CARD9 and is also involved in C. albicans-induced NF-κB activation. This association is dependent on Dectin-2 but not Dectin-1 following the hyphae stimulation. Finally, we find that although both CARD9 and Syk are required for Hyphae-induced NF-κB activation, they regulate different signaling events in which CARD9 mediates IκBα kinase ubiquitination, whereas Syk regulates IκBα kinase phosphorylation. Together, our data demonstrated that CARD9 is selectively involved in Dectin-2-induced NF-κB activation in response to C. albicans hyphae challenging.     

ASC is an activating adaptor for NF-kappa B and caspase-8-dependent apoptosis

ASC is a pro-apoptotic protein containing a pyrin domain (PD) and a caspase-recruitment domain (CARD). A previous study suggests that ASC interacts with Ipaf, a member of the Apaf-1/Nod1 protein family. However, the functional relevance of the interaction has not been determined. Here, we report that co-expression of ASC with Ipaf or oligomerization of ASC induces both apoptosis and NF-kappa B activation. Apoptosis induced through ASC was inhibited by a mutant form of Caspase-8 but not by that of Caspase-1. The PD of ASC physically interacted with Caspase-8 as well as with pyrin, the familial Mediterranean fever gene product. Caspase-8 deficiency rescued mouse fibroblasts from apoptosis induced by ASC oligomerization. Pyrin disrupted the interaction between ASC and Caspase-8, and inhibited both apoptosis and NF-kappa B activation induced by ASC. These findings suggest that ASC is a mediator of NF-kappa B activation and Caspase-8-dependent apoptosis in an Ipaf signaling pathway.     

The protein kinase C-responsive inhibitory domain of CARD11 functions in NF-kappaB activation to regulate the association of multiple signaling cofactors that differentially depend on Bcl10 and MALT1 for association

The activation of NF-κB by T-cell receptor (TCR) signaling is critical for T-cell activation during the adaptive immune response. CARD11 is a multidomain adapter that is required for TCR signaling to the IκB kinase (IKK) complex. During TCR signaling, the region in CARD11 between the coiled-coil and PDZ domains is phosphorylated by protein kinase Cθ (PKCθ) in a required step in NF-κB activation. In this report, we demonstrate that this region functions as an inhibitory domain (ID) that controls the association of CARD11 with multiple signaling cofactors, including Bcl10, TRAF6, TAK1, IKKγ, and caspase-8, through an interaction that requires both the caspase recruitment domain (CARD) and the coiled-coil domain. Consistent with the ID-mediated control of their association, we demonstrate that TRAF6 and caspase-8 associate with CARD11 in T cells in a signal-inducible manner. Using an RNA interference rescue assay, we demonstrate that the CARD, linker 1, coiled-coil, linker 3, SH3, linker 4, and GUK domains are each required for TCR signaling to NF-κB downstream of ID neutralization. Requirements for the CARD, linker 1, and coiled-coil domains in signaling are consistent with their roles in the association of CARD11 with Bcl10, TRAF6, TAK1, caspase-8, and IKKγ. Using Bcl10- and MALT1-deficient cells, we show that CARD11 can recruit signaling cofactors independently of one another in a signal-inducible manner.     

Mice lacking the CARD of CARMA1 exhibit defective B lymphocyte development and impaired proliferation of their B and T lymphocytes

CARMA1 (originally called CARD11) is a membrane-associated guanylate kinase family member that is required for T cell receptor (TCR)-induced NF-kappa B activation in T cell leukemia lines. It uses its N-terminal caspase activation and recruitment domain (CARD) to interact with the CARD in the downstream adaptor Bcl-10. We show that primary B and T lymphocytes from knock-in mice expressing only a CARDless form of CARMA1 (Delta CARD) are defective at mitogen-induced NF-kappa B activation and fail to proliferate. CARMA1 mutant mice exhibited normal T but impaired B cell development; CD5(+) peritoneal B cells were absent, and serum immunoglobulin levels were markedly reduced. A lacZ reporter gene knocked into the CARMA1 locus confirmed lymphocyte-specific expression of CARMA1. Thus, CARMA1 has an essential role in mediating B and T lymphocyte proliferation and requires its CARD to engage downstream signaling components.     

Protein kinases negatively affect nuclear factor-kappa B activation by tumor necrosis factor-alpha at two different stages in promyelocytic HL60 cells.

HL60 and EL4 cells incubated with tumor necrosis factor-alpha (TNF-alpha) plus staurosporin, a potent inhibitor of protein kinases, showed at least 2-fold increased levels of nuclear factor-kappa B (NF-kappa B) activity compared with TNF-alpha alone both during rapid NF-kappa B activation from the cytosolic pool and protein synthesis-dependent NF-kappa B activation. NF-kappa B activation by phorbol 12-myristate 13-acetate (PMA) and interleukin-1 was inhibited by staurosporin. Staurosporin treatment hardly affected the TNF-alpha-induced increase in mRNA for the p51 subunit of NF-kappa B but interfered with any phorbol ester (PMA)-induced increase in p51 mRNA. Thus, induction of NF-kappa B and p51 mRNA by TNF-alpha was not mediated by a staurosporin-sensitive factor, but NF-kappa B activation by TNF-alpha was even reduced by action of a staurosporin-sensitive factor. Decreased levels of phosphorylation of TNF-R alpha (TNF receptor type alpha) after staurosporin-treatment correlated with increased induction of NF-kappa B by TNF-alpha. Staurosporin-treatment did not affect TNF-R levels. Although protein kinase C stimulation by PMA inhibited NF-kappa B activation by TNF-alpha, its action mechanism may be different from that of the staurosporin-sensitive factor. PMA induced disappearance of TNF-R alpha by shedding into the surrounding medium, with kinetics similar to those of its inhibition of NF-kappa B activation by TNF-alpha. Phosphorylation may not mediate receptor shedding, since PMA treatment did not detectably affect TNF-R alpha phosphorylation.     

Activation of NF-kappa B in vivo is regulated by multiple phosphorylations.

The activation of nuclear factor kappa B (NF-kappa B) in intact cells is mechanistically not well understood. Therefore we investigated the modifications imposed on NF-kappa B/I kappa B components following stimulation and show that the final step of NF-kappa B induction in vivo involves phosphorylation of several members of the NF-kappa B/I kappa B protein families. In HeLa cells as well as in B cells, TNF-alpha rapidly induced nuclear translocation primarily of p50-p65, but not of c-rel. Both NF-kappa B precursors and I kappa B alpha became strongly phosphorylated with the same kinetics. In addition to the inducible phosphorylation after stimulation, B lymphocytes containing constitutive nuclear NF-kappa B revealed constitutively phosphorylated p65 and I kappa B alpha. Phosphorylation was accompanied by induced processing of the precursors p100 and p105 and by degradation of I kappa B alpha. As an in vitro model we show that phosphorylation of p105 impedes its ability to interact with NF-kappa B, as has been shown before for I kappa B alpha. Surprisingly, even p65, but not c-rel, was phosphorylated after induction in vivo, suggesting that TNF-alpha selectively activates only specific NF-kappa B heteromers and that modifications regulate not only I kappa B molecules but also NF-kappa B molecules. In fact, cellular NF-kappa B activity was phosphorylation-dependent and the DNA binding activity of p65-containing NF-kappa B was enhanced by phosphorylation in vitro. Furthermore, we found that the induction by hydrogen peroxide of NF-kappa B translocation to the nucleus, which is assumed to be triggered by reactive oxygen intermediates, also coincided with incorporation of phosphate into the same subunits that were modified after stimulation by TNF-alpha. Thus, phosphorylation appears to be a general mechanism for activation of NF-kappa B in vivo.     

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.     

The dynamic distribution of CARD11 at the immunological synapse is regulated by the inhibitory kinesin GAKIN

T cell receptor (TCR) signaling to NF-κB is required for antigen-induced T cell activation. We conducted an expression-cloning screen for modifiers of CARD11, a critical adaptor in antigen receptor signaling, and identified the kinesin-3 family member GAKIN as a CARD11 inhibitor. GAKIN negatively regulates TCR signaling to NF-κB, associates with CARD11 in a signal-dependent manner and can compete with the required signaling protein, Bcl10, for association. In addition, GAKIN dynamically localizes to the immunological synapse and regulates the redistribution of CARD11 from the central region of the synapse to a distal region. We propose that CARD11 scaffold function and occupancy at the center of the synapse are negatively regulated by GAKIN to tune the output of antigen-receptor signaling.     

Negative Regulation of CARD11 Signaling and Lymphoma Cell Survival by the E3 Ubiquitin Ligase RNF181

NF-κB activation downstream of antigen receptor engagement is a highly regulated event required for lymphocyte activation during the adaptive immune response. The pathway is often dysregulated in lymphoma, leading to constitutive NF-κB activity that supports the aberrant proliferation of transformed lymphocytes. To identify novel regulators of antigen receptor signaling to NF-κB, we developed bioluminescence resonance energy transfer-based interaction cloning (BRIC), a screening strategy that can detect protein-protein interactions in live mammalian cells in a high-throughput manner. Using this strategy, we identified the RING finger protein RNF181 as an interactor of CARD11, a key signaling scaffold in the antigen receptor pathway. We present evidence that RNF181 functions as an E3 ubiquitin ligase to inhibit antigen receptor signaling to NF-κB downstream of CARD11. The levels of the obligate signaling protein Bcl10 are reduced by RNF181 even prior to signaling, and Bcl10 can serve as a substrate for RNF181 E3 ligase activity in vitro. Furthermore, RNF181 limits the proliferation of human diffuse large B cell lymphoma cells that depend upon aberrant CARD11 signaling to NF-κB for growth and survival in culture. Our results define a new regulatory checkpoint that can modulate the output of CARD11 signaling to NF-κB in both normal and transformed lymphocytes.     

Phosphorylation of Bcl10 negatively regulates T-cell receptor-mediated NF-kappaB activation

Bcl10 (B-cell lymphoma 10) is an adaptor protein comprised of an N-terminal caspase recruitment domain and a C-terminal serine/threonine-rich domain. Bcl10 plays a critical role in antigen receptor-mediated NF-kappaB activation and lymphocyte development and functions. Our current study has discovered that T-cell activation induced monophosphorylation and biphosphorylation of Bcl10 and has identified S138 within Bcl10 as one of the T-cell receptor-induced phosphorylation sites. Alteration of S138 to an alanine residue impaired T-cell activation-induced ubiquitination and subsequent degradation of Bcl10, ultimately resulting in prolongation of TCR-mediated NF-kappaB activation and enhancement of interleukin-2 production. Taken together, our findings demonstrate that phosphorylation of Bcl10 at S138 down-regulates Bcl10 protein levels and thus negatively regulates T-cell receptor-mediated NF-kappaB activation.