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
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 exon (alternative exon 15) containing an alternative stop codon. This transcript is predicted to encode for a polypeptide corresponding to amino acids 1–740 of CARMA2FL, and therefore we named this variant CARMA2short (CARMA2sh) (). The second variant mRNA skips exons 1–4, contains an alternative translational start codon in exon 5 and includes an additional exon containing an alternative stop codon (). The CARMA2 isoform encoded by this splice variant would lack the CARD domain, includes amino acids Met238-Val618 of CARMA2FL and contains a unique carboxy terminus (). We named this variant CARMA2cardless (CARMA2cl).
CARMA2 isoforms. A: Amino acid alignment of CARMA2FL, CARMA2sh, and CARMA2cl. The unique sequence of CARMA2cl is in bold. B: Schematic representation of CARMA2FL, CARMA2sh, and CARMA2cl isoforms.
To confirm the existence of these alternatively spliced transcripts of CARMA2, we performed RT-PCR on mRNAs isolated from different human normal and tumoral cell lines and normal tissues using internal primers that can distinguish each of the splice variants. Using a primer set specific for exon 15 and the alternative exon 15, we detected a PCR product that corresponds to the predicted size of a fragment derived from transcripts encoding CARMA2sh (). This transcript was expressed in most of the cell lines tested, in human fetal brain and human leucocytes ().
Fig. 2 CARMA2 isoform expression. A: RT-PCR was used to analyze CARMA2 transcripts in a part of cDNA from various human tissues and cell lines. These comprised human lung carcinoma (NCI-H226, DMS-79, CALU1, NCI-H526, SHP77, A549, NCI-H460, NCI-H1975, NCI-H292, (more ...)
We also confirmed the presence of the transcript coding for CARMA2cl using a primer within the 5′-untranslated region paired with a reverse primer located in exon 5. The CARMA2cl transcript was expressed only in HeLa cells ().
To study these CARMA2 splice variants, we generated a polyclonal rabbit antibody directed against a polypeptide corresponding to aa 108–407 of CARMA2 FL (). When probed on cell lysates prepared from HeLa and HEK-293 cells, the antisera detected bands corresponding to the predicted size of endogenous CARMA2FL, CARMA2sh, and CARMA2cl isoforms. The antisera also recognizes additional bands, indicating that other splice variants of CARMA2 may occur ().
Because CARMA proteins are implicated in NF-κB signaling pathways, we first determined whether CARMA2sh and CARMAcl can induce NF-κB activity using a luciferase reporter assay. When CARMA2sh was expressed in HEK-293 cells, NF-κB activity was induced at least 20- to 40-fold compared with empty vector (). In the same assay, CARMAcl was unable to promote NF-κB activity, thus confirming that the N-terminal CARD domain of CARMA proteins is essential for NF-κB signaling ().
Fig. 3 CARMA2sh interacts with BCL10. A: HEK-293 cells were transiently cotransfected with an expression vector encoding for the indicated polypeptides, together with pNF-κB-luc and pRSV-βgal reporter vectors. The total amount of transfected (more ...)
It has been shown that CARMA3, a member of the CARMA family of proteins, is unable to activate NF-κB in the absence of functional BCL10 (Wang et al., 2001
). To test whether also CARMA2sh
requires BCL10 for the NF-κB-inducing activity, CARMA2sh
was tested in HEK-293 cells expressing a short hairpin RNA (shRNA) designed to target BCL10 for degradation by the RNAi pathway. The results of these experiments indicate that, likewise CARMA3, CARMA2sh
as well depends on functional BCL10 to induce NF-κB (). Similarly to CARMA1 and CARMA3 (Stilo et al., 2008
), activation of NF-κB mediated by CARMA2sh
was inhibited by expression of A20 (data not shown).
Biochemical data confirmed our functional observation. In fact, to verify whether CARMA2 splice variants bind to BCL10, CARMA2sh
, and CARMA2cl
were co-expressed with BCL10, cell extracts were prepared, and were immunoprecipitated with anti-FLAG antibody. Immunoblotting experiments revealed that CARMA2sh
, but not CARMA2cl
, co-immunoprecipitated with BCL10 (). The binding of CARMA2sh
to BCL10 was dependent on an intact CARD of BCL10, because CARMA2sh
failed to co-precipitate variants of BCL10 containing a point mutation L41Q or G78R that disrupts CARD/CARD interactions (Srinivasula et al., 1999
; Guiet and Vito, 2000
) (). These results indicate that CARMA2sh
, but not CARMA2cl
, associates with BCL10 through a CARD/CARD homotypic interaction.
We next determined the cellular localization of CARMA2sh and CARMA2cl. For this, we transfected HEK-293 cells with a FLAG-tagged vector encoding for these splice variants of CARMA2, and the expressed protein was detected using a monoclonal anti-FLAG antibody. The results of these experiments, shown in , indicate that both CARMA2sh and CARMA2cl exhibit a clear pattern of diffuse cytoplasmic distribution.
Fig. 4 Subcellular localization of CARMA2 isoforms. HEK-293 (upper parts) and HeLa cells (lower parts) were transfected with mammalian FLAG-tagged vector, empty (vector) or expressing CARMA2sh or CARMA2cl. Sixteen hours after transfection, cells were stained (more ...)
Interestingly, in a set of association experiments, we also found that immunocomplexes containing endogenous TRAF2, a scaffold protein that transduces signals from membrane receptors and the ER membrane (Rothe et al., 1994
; Yoneda et al., 2001
; Mauro et al., 2006
), also contain CARMA2 isoforms (). To confirm association of CARMA2sh
and TRAF2 in a different experimental system, a polypeptide corresponding to Met1
of CARMA2FL was expressed as His-tagged protein in bacteria and tested for binding to TRAF2 endogenously expressed. The results of these pull-down assays, shown in , indicate that recombinant CARMA2 Met1
binds to TRAF2 in lysates prepared from HEK-293 and Jurkat cells. Similar results were observed when lysates were prepared from HeLa cell (data not shown). In addition, coprecipitation experiments demonstrated that CARMA2sh
also binds to TRAF3, TRAF6 but not TRAF7 (). To further define the biological significance of CARMA2sh
interaction with TRAF2, we tested the NF-κB inducing activity of CARMA2sh
in embryonic fibroblasts (MEFs) derived from TRAF2−/−
mice and wt MEFs. As shown in , when CARMA2sh
was expressed in wt MEFs, NF-κB was induced 10- to 15-fold compared with empty vector. However, CARMA2sh
was barely able to induce NF-κB signaling in TRAF2−/−
MEFs. Thus, a functional TRAF2 protein is required for activation of NF-κB induced by CARMA2sh
Fig. 5 CARMA2sh binds to and activates NF-κB through TRAF2. A: Lysates prepared from HeLa, HEK-293, or Jurkat cells were immunoprecipitated with anti-TRAF2 antibody or an isotype-matched antibody. Immunoprecipitating material was separated by SDS–PAGE (more ...)
Since TRAF2 regulates apoptotic signal transduction pathways starting from membrane receptors and the ER membrane (Rothe et al., 1994
; Yoneda et al., 2001
; Mauro et al., 2006
), we investigated the involvement of CARMA2sh
in cell death induced by different stimuli. For this, we established HEK-293 cell lines that stably expressed FLAG-tagged CARMA2sh
through a lentiviral expression system and then examined their responses to inducers of programmed cell death. The expression of ectopic CARMA2sh
was assumed by immunoblot experiments; GFP is an irrelevant protein and here was used as a control. As shown in , expression of CARMA2sh
, and partially that of CARMA2cl
, confers resistance to apoptosis induced by thapsigargin, tunicamycin, and anisomycin treatment both in HEK-293 cells () and HeLa cells (). Induction of ER stress was monitored by assessing the expression level of ER stress marker protein BiP (Dudek et al., 2009
). At a lesser extent, CARMA2sh
, but not CARMA2cl
, protects cells also from death induced by etoposide, staurosporine, and TNFα treatment (data not shown).
Fig. 6 CARMA2sh expression protects cells from apoptosis. A: HEK-293 cells infected with a lentiviral vector expressing the indicated cDNAs were exposed to the indicated drugs for 24 h and then analyzed for apoptosis using ATPLite™ kit analysis. Each (more ...)
To further assess the biological significance of CARMA2 in programmed cell death, CARMA2 expression was silenced by siRNA. We identified a siRNA which partially silenced CARMA2 expression in HeLa and HEK-293 cells (data not shown). When CARMA2 expression was silenced by this siRNA, apoptotic response was significantly increased compared to control siRNA (). These results indicated that increased expression of CARMA2 has a protective role in apoptotic pathways.