CARMA1 coiled-coil domain is involved in the oligomerization and subcellular localization of CARMA1 and is required for T cell receptor-induced NF-kappaB activation

T lymphocyte (T cell) activation and proliferation is induced by the activation of multiple signal transduction pathways. Earlier studies indicate that CARMA1, a Caspase Recruitment Domain (CARD) and Membrane-associated GUanylate Kinase domain (MAGUK)-containing scaffold protein, plays an essential role in NF-kappaB activation induced by the costimulation of T cell receptor (TCR) and CD28 molecules. However, the molecular mechanism by which CARMA1 mediates TCR-CD28 costimulation-induced NF-kappaB activation is not fully understood. Here we show that CARMA1 is constitutively oligomerized. This oligomerization of CARMA1 is through its Coiled-coil domain. Disruption of the predicted structure of the Coiled-coil domain of CARMA1 impaired its oligomerization and, importantly, abrogated CARMA1-mediated NF-kappaB activation. Interestingly, disruption of the CC1 domain abrogates CARMA1 localization, whereas disruption of the CC2 domain seems to inhibit CARMA1 self-association. Together, our results demonstrate that the oligomerization of CARMA1 is required for TCR-induced NF-kappaB activation.     

Novel Disulfide Bond-Mediated Dimerization of the CARD Domain Was Revealed by the Crystal Structure of CARMA1 CARD

CARMA1, BCL10 and MALT1 form a large molecular complex known as the CARMA1 signalosome during lymphocyte activation. Lymphocyte activation via the CARMA1 signalosome is critical to immune response and linked to many immune diseases. Despite the important role of the CARMA1 signalosome during lymphocyte activation and proliferation, limited structural information is available. Here, we report the dimeric structure of CARMA1 CARD at a resolution of 3.2 Å. Interestingly, although CARMA1 CARD has a canonical six helical-bundles structural fold similar to other CARDs, CARMA1 CARD shows the first homo-dimeric structure of CARD formed by a disulfide bond and reveals a possible biologically important homo-dimerization mechanism.     

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.     

Ca2+/calmodulin-dependent protein kinase II is a modulator of CARMA1-mediated NF-kappaB activation

CARMA1 is a central regulator of NF-kappaB activation in lymphocytes. CARMA1 and Bcl10 functionally interact and control NF-kappaB signaling downstream of the T-cell receptor (TCR). Computational analysis of expression neighborhoods of CARMA1-Bcl10MALT 1 for enrichment in kinases identified calmodulin-dependent protein kinase II (CaMKII) as an important component of this pathway. Here we report that Ca(2+)/CaMKII is redistributed to the immune synapse following T-cell activation and that CaMKII is critical for NF-kappaB activation induced by TCR stimulation. Furthermore, CaMKII enhances CARMA1-induced NF-kappaB activation. Moreover, we have shown that CaMKII phosphorylates CARMA1 on Ser109 and that the phosphorylation facilitates the interaction between CARMA1 and Bcl10. These results provide a novel function for CaMKII in TCR signaling and CARMA1-induced NF-kappaB activation.     

Cell type-specific regulation of RhoA activity during cytokinesis

Rho family GTPases play pivotal roles in cytokinesis. By using probes based on the principle of fluorescence resonance energy transfer (FRET), we have shown that in HeLa cells RhoA activity increases with the progression of cytokinesis. Here we show that in Rat1A cells RhoA activity remained suppressed during most of the cytokinesis. Consistent with this observation, the expression of C3 toxin inhibited cytokinesis in HeLa cells but not in Rat1A cells. Furthermore, the expression of a dominant negative mutant of Ect2, a Rho GEF, or Y-27632, an inhibitor of the Rho-dependent kinase ROCK, inhibited cytokinesis in HeLa cells but not in Rat1A cells. In contrast to the activity of RhoA, the activity of Rac1 was suppressed during cytokinesis and started increasing at the plasma membrane of polar sides before the abscission of the daughter cells in both HeLa and Rat1A cells. This type of Rac1 suppression was shown to be essential for cytokinesis because a constitutively active mutant of Rac1 induced a multinucleated phenotype in both HeLa and Rat1A cells. Moreover, the involvement of MgcRacGAP/CYK-4 in this suppression of Rac1 during cytokinesis was shown by the use of a dominant negative mutant. Because ML-7, an inhibitor of myosin light chain kinase, delayed the cytokinesis of Rat1A cells and because Pak, a Rac1 effector, is known to suppress myosin light chain kinase, the suppression of the Rac1-Pak pathway by MgcRacGAP may play a pivotal role in the cytokinesis of Rat1A cells.     

Cell type-specific activation of the cytomegalovirus promoter by dimethylsulfoxide and 5-aza-2'-deoxycytidine

The cytomegalovirus promoter is a very potent promoter commonly used for driving the expression of transgenes, though it gradually becomes silenced in stably transfected cells. We examined the methylation status of the cytomegalovirus promoter in two different cell lines and characterized its mechanisms of activation by dimethylsulfoxide and 5-Aza-2'-deoxycytidine. The cytomegalovirus promoter stably transfected into Chinese hamster ovary cells is suppressed by DNA methylation-independent mechanisms, which is different from the rat embryonic cardiomyoblast H9c2-Fluc.3 cells in which the cytomegalovirus promoter is silenced by methylation. Dimethylsulfoxide and 5-Aza-2'-deoxycytidine can activate the cytomegalovirus promoter in both cell types by overlapping mechanisms. Dimethylsulfoxide activates the cytomegalovirus promoter in Chinese hamster ovary cells by promoting histone acetylation and the activation of p38 mitogen-activated protein kinase and nuclear factor kappaB (NFkappaB) signaling pathways, while 5-Aza-2'-deoxycytidine increases histone acetylation and activates the nuclear factor kappaB but not the p38 mitogen-activated protein kinase pathway. In H9c2-Fluc.3 cells, both agents promote demethylation of the cytomegalovirus promoter, and enhance its activity exclusively through activation of the nuclear factor kappaB pathway and to a lesser extent of the p38 mitogen-activated protein kinase pathway. Our findings suggest that suppression and activation of the cytomegalovirus promoter are cell type-specific. These results may be used for developing strategies to enhance the expression of transgenes and the production of recombinant proteins encoded by transgenes controlled by a cytomegalovirus promoter.     

Caspase-8 and c-FLIPL associate in lipid rafts with NF-kappaB adaptors during T cell activation

Humans and mice lacking functional caspase-8 in T cells manifest a profound immunodeficiency syndrome due to defective T cell antigen receptor (TCR)-induced NF-kappaB signaling and proliferation. It is unknown how caspase-8 is activated following T cell stimulation, and what is the caspase-8 substrate(s) that is necessary to initiate T cell cycling. We observe that following TCR ligation, a small portion of total cellular caspase-8 and c-FLIP(L) rapidly migrate to lipid rafts where they associate in an active caspase complex. Activation of caspase-8 in lipid rafts is followed by rapid cleavage of c-FLIP(L) at a known caspase-8 cleavage site. The active caspase.c-FLIP complex forms in the absence of Fas (CD95/APO1) and associates with the NF-kappaB signaling molecules RIP1, TRAF2, and TRAF6, as well as upstream NF-kappaB regulators PKC theta, CARMA1, Bcl-10, and MALT1, which connect to the TCR. The lack of caspase-8 results in the absence of MALT1 and Bcl-10 in the active caspase complex. Consistent with this observation, inhibition of caspase activity attenuates NF-kappaB activation. The current findings define a link among TCR, caspases, and the NF-kappaB pathway that occurs in a sequestered lipid raft environment in T cells.     

CD3/CD28 costimulation-induced NF-kappaB activation is mediated by recruitment of protein kinase C-theta, Bcl10, and IkappaB kinase beta to the immunological synapse through CARMA1

CARMA1 (also known as CARD11) is a scaffold molecule and contains a caspase-recruitment domain (CARD) and a membrane-associated guanylate kinase-like (MAGUK) domain. It plays an essential role in mediating CD3/CD28 costimulation-induced NF-kappaB activation. However, the molecular mechanism by which CARMA1 mediates costimulatory signals remains to be determined. Here, we show that CARMA1 is constitutively associated with the cytoplasmic membrane. This membrane association is essential for the function of CARMA1, since a mutant of CARMA1, CARMA1(L808P), that is defective in the membrane association cannot rescue CD3/CD28 costimulation-induced NF-kappaB activation in JPM50.6 CARMA1-deficient T cells. Although CD3/CD28 costimulation effectively induces the formation of the immunological synapse in CARMA1-deficient T cells, the recruitment of protein kinase C-theta (PKC-theta), Bcl10, and IkappaB kinase beta (IKKbeta) into lipid rafts of the immunological synapse is defective. Moreover, expression of wild-type CARMA1, but not CARMA1(L808P), restores the recruitment of PKC-theta, Bcl10, and IKKbeta into lipid rafts in CARMA1-deficient T cells. Consistently, expression of a mutant CARMA1, CARMA1(DeltaCD), that cannot associate with Bcl10 failed to restore CD3/CD28 costimulation-induced NF-kappaB activation in JPM50.6 cells, whereas expression of Bcl10-CARMA(DeltaCD) fusion protein effectively restored this NF-kappaB activation. Together, these results indicate that CARMA1 mediates CD3/CD28 costimulation-induced NF-kappaB activation by recruiting downstream signaling components into the immunological synapse.     

Cell type-specific regulation of ion channels within the maize stomatal complex

The stomatal complex of Zea mays is composed of two pore-forming guard cells and two adjacent subsidiary cells. For stomatal movement, potassium ions and anions are thought to shuttle between these two cell types. As potential cation transport pathways, K(+)-selective channels have already been identified and characterized in subsidiary cells and guard cells. However, so far the nature and regulation of anion channels in these cell types have remained unclear. In order to bridge this gap, we performed patch-clamp experiments with subsidiary cell and guard cell protoplasts. Voltage-independent anion channels were identified in both cell types which, surprisingly, exhibited different, cell-type specific dependencies on cytosolic Ca(2+) and pH. After impaling subsidiary cells of intact maize plants with microelectrodes and loading with BCECF [(2',7'-bis-(2-carboxyethyl)-5(and6)carboxyflurescein] as a fluorescent pH indicator, the regulation of ion channels by the cytosolic pH and the membrane voltage was further examined. Stomatal closure was found to be accompanied by an initial hyperpolarization and cytosolic acidification of subsidiary cells, while opposite responses were observed during stomatal opening. Our findings suggest that specific changes in membrane potential and cytosolic pH are likely to play a role in determining the direction and capacity of ion transport in subsidiary cells.     

Immune regulation by SLAM family receptors and SAP-related adaptors

The signalling lymphocytic activation molecule (SLAM) family of receptors is expressed by a wide range of immune cells. Through their cytoplasmic domain, SLAM family receptors associate with SLAM-associated protein (SAP)-related molecules, a group of cytoplasmic adaptors composed almost exclusively of an SRC homology 2 domain. SAP, the prototype of the SAP family, is mutated in a human immunodeficiency named X-linked lymphoproliferative (XLP) disease. Recent observations indicate that SLAM family receptors, in association with SAP family adaptors, have crucial roles during normal immune reactions in innate and adaptive immune cells. The latest progress in this field is reviewed here.     

Cell type-specific regulation of B-Raf kinase by cAMP and 14-3-3 proteins

Cyclic AMP can either activate or inhibit the mitogen-activated protein kinase (MAPK) pathway in different cell types; MAPK activation has been observed in B-Raf-expressing cells and has been attributed to Rap1 activation with subsequent B-Raf activation, whereas MAPK inhibition has been observed in cells lacking B-Raf and has been attributed to cAMP-dependent protein kinase (protein kinase A)-mediated phosphorylation and inhibition of Raf-1 kinase. We found that cAMP stimulated MAPK activity in CHO-K1 and PC12 cells but inhibited MAPK activity in C6 and NB2A cells. In all four cell types, cAMP activated Rap1, and the 95- and 68-kDa isoforms of B-Raf were expressed. cAMP activation or inhibition of MAPK correlated with activation or inhibition of endogenous and transfected B-Raf kinase. Although all cell types expressed similar amounts of 14-3-3 proteins, approximately 5-fold less 14-3-3 was associated with B-Raf in cells in which cAMP was inhibitory than in cells in which cAMP was stimulatory. We found that the cell type-specific inhibition of B-Raf could be completely prevented by overexpression of 14-3-3 isoforms, whereas expression of a dominant negative 14-3-3 mutant resulted in partial loss of B-Raf activity. Our data suggest that 14-3-3 bound to B-Raf protects the enzyme from protein kinase A-mediated inhibition; the amount of 14-3-3 associated with B-Raf may explain the tissue-specific effects of cAMP on B-Raf kinase activity.