Alternative Splicing of CARMA2/CARD14 Transcripts Generates Protein Variants With Differential Effect on NF-kB Activation and Endoplasmic Reticulum Stress-Induced Cell Death IVAN SCUDIERO, 1,2 TIZIANA ZOTTI, 1,2 ANGELA FERRAVANTE, 1,2 MARIANGELA VESSICHELLI, 1,2 PASQUALE VITO, 1,2 * AND ROMANIA STILO 1,2 1 Biogem, Ariano Irpino, Italy 2 Dipartimento di Scienze Biologiche ed Ambientali, Universita` degli Studi del Sannio, Benevento, Italy The caspase recruitment domain (CARD)-containing proteins CARMA1-3 share high degree of sequence, structure and functional homology. Whereas CARMA1 and CARMA3 have been identified as crucial components of signal transduction pathways that lead to activation of NF-kB transcription factor, little is known about the function of CARMA2. Here we report the identification of two splice variants of CARMA2. One transcript, named CARMA2short (CARMA2sh), is predicted to encode for a CARMA2 polypeptide containing the CARD, coiled coil, and a PDZ domains, but lacking the SH3 and the GuK domains. The second variant, CARMA2cardless (CARMA2cl), encodes for a polypeptide lacking the CARD domain and containing only a portion of the coiled coil domain and a linker region. Expression analysis confirmed the presence of the CARMA2 alternatively spliced transcripts in both human cell lines and tissues. Fluorescence microscopy data show that both splice variants localize in the cytosol. Biochemical experiments indicate that CARMA2sh interacts with TRAF2 and activates NF-kB in a TRAF2-dependent manner. Finally, CARMA2sh variant protects cells from apoptosis induced by different stimuli. Taken together, these results demonstrate that multiple transcripts encoding several CARMA2 isoforms exist in vivo and regulate NF-kB activation and apoptosis. J. Cell. Physiol. 226: 3121–3131, 2011. ß 2011 Wiley Periodicals, Inc. The caspase recruitment domain (CARD)-containing proteins CARMA1 (CARD11/Bimp2) (Bertin et al., 2001; Gaide et al., 2001; Wang et al., 2001), CARMA2 (CARD14/Bimp3) (Bertin et al., 2001), and CARMA3 (CARD10/Bimp1) (Wang et al., 2001; McAllister-Lucas et al., 2001) share a high degree of sequence, structure, and functional homology. CARMA proteins belong to the membrane-associated guanylate kinase (MAGUK) family of proteins, which can function as molecular scaffolds that assist recruitment and assembly of signal transduction molecules (Dimitratos et al., 1999). CARMA proteins contain a CARD domain, a Src-homology 3 (SH3) domain, one or several PDZ domains and a GuK domain (Bertin et al., 2001; Gaide et al., 2001; Wang et al., 2001; McAllister- Lucas et al., 2001). Genetic and biochemical studies have identified CARMA1 as crucial component of a complex of proteins that links antigen receptors on B and T lymphocytes to activation of NF-kB (Wang et al., 2002; Gaide et al., 2002; Pomerantz et al., 2002; Hara et al., 2003; Egawa et al., 2003; Newton and Dixit, 2003; Jun et al., 2003). This complex, which includes CARMA1, BCL10, MALT1, and TRAF6, has been shown to activate the IKK complex through an ubiquitylation- dependent pathway (Schulze-Luehrmann and Ghosh, 2006). A second member of the CARMA family, CARMA3, is required for activation of NF-kB induced by G-protein-coupled receptors (GPCRs) (Grabiner et al., 2007; McAllister-Lucas et al., 2007). In fact, it has been demonstrated that a complex of proteins, including CARMA3, BCL10, and MALT1, appears to be responsible for transmitting the signal from the GPCRs to the IKK complex (Grabiner et al., 2007; McAllister-Lucas et al., 2007). By contrast, although overexpression of CARMA2 also induces NF-kB activation in HEK-293 cells (Bertin et al., 2001), the signaling pathway(s) regulated by this protein is still unknown. Here we describe the identification of two splice variants of CARMA2 and show data indicating an involvement of these proteins in regulation of NF-kB activation and endoplasmic reticulum (ER)-stress induced cell death. Results Previous work identified CARMA2/CARD14 (CARMA2FL) as a 1,004 amino acidic residues protein belonging to the CARMA family of proteins (Bertin et al., 2001). To examine the occurrence of splice variants derived from the human CARMA2 gene, we performed an extensive reverse transcription- polymerase chain reaction (RT-PCR) analysis of mRNAs isolated from human cell lines using CARMA2/CARD14 exon-specific primers. Such analysis revealed the existence of two different splice variants of CARMA2/CARD14. One variant contains the same translational start of CARMA2FL but includes an additional *Correspondence to: Pasquale Vito, Dipartimento di Scienze Biologiche ed Ambientali, Universita ` degli Studi del Sannio, Via Port’Arsa 11, 82100 Benevento, Italy E-mail: vito@unisannio.it Received 20 August 2010; Accepted 19 January 2011 Published online in Wiley Online Library (wileyonlinelibrary.com), 2 February 2011. DOI: 10.1002/jcp.22667 ORIGINAL RESEARCH ARTICLE 3121 Journal of Journal of Cellular Physiology Cellular Physiology ß 2011 WILEY PERIODICALS, INC.