Jean-Luc Poyet

CARD11 and CARD14 are novel caspase recruitment domain (CARD)/membrane-associated guanylate kinase (MAGUK) family members that interact with BCL10 and activate NF-kappa B.

The caspase recruitment domain (CARD) is a protein-binding module that mediates the assembly of CARD-containing proteins into apoptosis and NF-κB signaling complexes. We report here that CARD protein 11 (CARD11) and CARD protein 14 (CARD14) are novel CARD-containing proteins that belong to the membrane-associated guanylate kinase (MAGUK) family, a class of proteins that functions as molecular scaffolds for the assembly of multiprotein complexes at specialized regions of the plasma membrane. CARD11 and CARD14 have homologous structures consisting of an N-terminal CARD domain, a central coiled-coil domain, and a C-terminal tripartite domain comprised of a PDZ domain, an Src homology 3 domain, and a GUK domain with homology to guanylate kinase. The CARD domains of both CARD11 and CARD14 associate specifically with the CARD domain of BCL10, a signaling protein that activates NF-κB through the IκB kinase complex in response to upstream stimuli. When expressed in cells, CARD11 and CARD14 activate NF-κB and induce the phosphorylation of BCL10. These findings suggest that CARD11 and CARD14 are novel MAGUK family members that function as upstream activators of BCL10 and NF-κB signaling.

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

BCL10/CLAP is an activator of apoptosis and NF−κB 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-κB 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-κB 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-κB. 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-κB signaling.

CLAP, a Novel Caspase Recruitment Domain-containing Protein in the Tumor Necrosis Factor Receptor Pathway, Regulates NF-κB Activation and Apoptosis

Molecules that regulate NF-κB 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-κB 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 apoptoticproteins). The cellular and viral CLAPs induce apoptosis downstream of caspase-8 by activating the Apaf-1-caspase-9 pathway and activate NF-κB by acting upstream of the NF-κB-inducing kinase, NIK, and the IkB kinase, IKKα. 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-κB activation, suggesting that the CARD domain functions as an oligomerization domain, whereas the CTD domain functions as the effector domain in the NF-κB activation pathway. Expression of the CARD domain of human CLAP abrogates tumor necrosis factor-α-induced NF-κB activation, suggesting that cellular CLAP plays an essential role in this pathway of NF-κB activation.

Expression and transcriptional regulation of caspase-14 in simple and complex epithelia

Caspase-14 is a recent addition to the caspase family of aspartate proteases involved in apoptotic processes. Human caspase-14 appears to be only weakly processed during apoptosis, and it does not cleave classical caspase substrates. Post partum, caspase-14 is prominently expressed by human keratinocytes and reportedly participates in terminal differentiation of complex epithelia. Here we provide evidence challenging the view that caspase-14 expression or processing is linked exclusively to terminal keratinocyte differentiation. We demonstrate that caspase-14 expression extended to multiple cell lines derived from simple epithelia of the breast, prostate, and stomach. In keratinocytes and breast epithelial cells, caspase-14 expression was upregulated in high-density cultures and during forced suspension culture. These effects were primarily due to transcriptional activation as indicated by reporter gene assays using a 2 kb caspase-14 promoter fragment. Importantly, caspase-14 was not cleaved during forced suspension culture of either cell type although this treatment induced caspase-dependent apoptosis (anoikis). Forced expression of caspase-14 in immortalized human keratinocytes had no effect on cell death in forced suspension nor was the transfected caspase-14 processed in this setting. In contrast to postconfluent and forced suspension culture, terminal differentiation of keratinocytes induced in vitro by Ca2+ treatment was not associated with increased caspase-14 expression or promoter activity. Our results indicate that (1) caspase-14 is expressed not only in complex but also simple epithelia; (2) cells derived from complex and simple epithelia upregulate caspase-14 expression in conditions of high cell density or lack of matrix interaction and; (3) in both cell types this phenomenon is due to transcriptional regulation.