- Email: [email protected]
Interaction of calmodulin with Bcl10 modulates NF-κB activation
Molecular Immunology 47 (2010) 2057–2064
Contents lists available at ScienceDirect
Molecular Immunology journal homepage: www.elsevier.com/locate/molimm
Interaction of calmodulin with Bcl10 modulates NF-B activation Sofia Edin 1 , Sreenivasa Rao Oruganti, Christine Grundström, Thomas Grundström ∗ Department of Molecular Biology, Umeå University, SE-901 87 Umeå, Sweden
a r t i c l e
i n f o
Article history: Received 2 February 2010 Received in revised form 8 April 2010 Accepted 11 April 2010 Available online 1 May 2010 Keywords: Gene regulation Signal transduction NF-B Bcl10 Carma1 Calmodulin Calcium signaling
a b s t r a c t Calcium signals resulting from antigen receptor activation are important in determining the responses of a T or B lymphocyte to an antigen. Calmodulin (CaM), a multi-functional sensor of intracellular calcium (Ca2+ ) signals in cells, is required in the pathway from the T cell receptor (TCR) to activation of the key transcription factor NF-B. Here we searched for a partner in direct interaction with CaM in the pathway, and found that CaM interacts specifically with the signaling adaptor Bcl10. The binding is Ca2+ dependent and of high affinity, with a Kd of approximately 160 nM. Proximity of CaM and Bcl10 in vivo is induced by increases in the intracellular Ca2+ level. The interaction is localized to the CARD domain of Bcl10, which interacts with the CARD domain of the upstream signaling partner Carma1. Binding of CaM to Bcl10 is shown to inhibit the ability of Bcl10 to interact with Carma1, an interaction that is required for signaling from the TCR to NF-B. Furthermore, a mutant of Bcl10 with reduced binding to CaM shows increased activation of an NF-B reporter, which is further enhanced by activating stimuli. We propose a novel mechanism whereby the Ca2+ sensor CaM regulates T cell responses to antigens by binding to Bcl10, thereby modulating its interaction with Carma1 and subsequent activation of NF-B. © 2010 Elsevier Ltd. All rights reserved.
1. Introduction Antigen receptor activation results in the recruitment and activation of a number of signaling mediators and lipid metabolizing enzymes, building up an immunological synapse at the T cell receptor (TCR) or B cell receptor (BCR) (Schulze-Luehrmann and Ghosh, 2006; Weil and Israel, 2004). The immunological synapse triggers downstream signaling pathways, leading to activation of transcription factors such as the NF-B family, which is important for the regulation of activation, proliferation, and differentiation of T and B lymphocytes (Caamano and Hunter, 2002). In a resting cell, NF-B dimers mainly reside in the cytoplasm, sequestered by inhibitory IB proteins. Most signals to NF-B converge on the activation of the IB kinase (IKK), resulting in signal-induced phosphorylation and degradation of IB, which allows NF-B to enter the nucleus and activate gene transcription (Hayden and Ghosh, 2008).
Abbreviations: CaM, calmodulin; TCR, T cell receptor; BCR, B cell receptor; CBM, Carma1, Bcl10, Malt1 complex; IKK, IB kinase; PKC, protein kinase C; CARD, caspase recruitment domain; IP3 , inositol-1,4,5-triphosphate; CRAC, calcium release-activated Ca2+ channel; CaMK, calmodulin dependent kinase; PMA, phorbol12-myristate-13-acetate. ∗ Corresponding author. Tel.: +46 90 7852531; fax: +46 90 772630. E-mail address: [email protected] (T. Grundström). 1 Current address: Department of Medical Biosciences, Umeå University, SE-901 85 Umeå, Sweden. 0161-5890/$ – see front matter © 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.molimm.2010.04.005
T cell receptor-induced activation of NF-B is dependent on the activation of protein kinase C (PKC) and the subsequent recruitment and activation of a signaling complex containing the proteins Carma1, Bcl10, and Malt1, which is often referred to as the CBM complex. The formation of this complex is crucial for T cell activation, since deletion of any of the proteins impairs the NF-B responses (Thome, 2004). The molecular mechanisms by which these proteins promote IKK and NF-B activation are not fully understood, however. Carma 1 and Bcl10 belong to the Caspase recruitment domain (CARD) subfamily of the large Death domain family of proteins. The CARD domains and other domains found in this family are highly conserved and are characterized by their homotypic interactions with proteins within the same subfamily. They have been highly implicated in cell signaling to apoptosis and immunity (Bouchier-Hayes and Martin, 2002; Park et al., 2007). Bcl10 and Carma1 interact with each other through their CARD motifs, and this interaction is crucial for NF-B signaling after TCR stimulation, since deletion of the CARD domain of Carma1 or mutation of the CARD domain of Bcl10 prevents NF-B activation (Rawlings et al., 2006). In addition to activation of PKC-dependent pathways, triggering of the TCR also leads to production of inositol-1,4,5triphosphate (IP3 ), initiating the release of Ca2+ from intracellular stores. Depletion of intracellular reservoirs of Ca2+ activates the influx of Ca2+ through store-operated calcium release-activated Ca2+ (CRAC) channels in the plasma membrane, resulting in elevated levels of intracellular Ca2+ . Depending on the pattern and strength of these Ca2+ signals, signaling molecules and transcrip-
2058
S. Edin et al. / Molecular Immunology 47 (2010) 2057–2064
tion factors with different requirements for Ca2+ will be activated (Feske, 2007; Quintana et al., 2005). The activation of NF-B transcription factors after TCR engagement is sensitive to such changes in intracellular Ca2+ , but the mechanisms are not well characterized. The main transducer of Ca2+ signals in cells is the Ca2+ binding protein calmodulin (CaM). CaM belongs to the EF-hand family of Ca2+ binding proteins and changes conformation upon Ca2+ binding, allowing it to bind to a new set of targets. CaM is involved in the regulation of numerous important processes such as proliferation, signaling, and differentiation (Chin and Means, 2000; Yamniuk and Vogel, 2004). In the present study we investigated the role of CaM in TCR-induced activation of NF-B, and found that CaM interacts specifically with Bcl10. We present a novel mechanism by which CaM through interaction with Bcl10 can modulate the binding of Bcl10 to Carma1 and thereby NF-B activation. 2. Materials and methods 2.1. Plasmids and mutagenesis Expression vectors for Bcl10 and Carma1 were obtained by subcloning cDNA of mouse Bcl10, IMAGE ID 4976147, and mouse Carma1, IMAGE ID 5318165 (both from Mammalian Gene Collection), from pOTB7 to pCDNA1/Amp using EcoRI/NotI and EcoRV/NotI, respectively. Mutated derivatives of Bcl10 and Carma1 were constructed using standard cloning and PCR techniques. For expression in E. coli, Bcl10 and Carma1 cDNA were amplified by PCR and subcloned into the EcoRI/XhoI sites of the pET-20b + His expression vector (Onions et al., 1997) using the In FusionTM CF Liquid PCR cloning kit (Clontech). The NF-B luciferase reporter plasmid (Hughes et al., 1998) and the hCMV-gal normalization plasmid (Corneliussen et al., 1994) have been described previously. Co-transfection with a green fluorescent protein (GFP) expressing plasmid (Hughes et al., 2001) was used in transfections for proximity ligation assays. 2.2. Expression and purification of proteins C-terminally (His)6 -tagged Bcl10 or Carma1 constructs were expressed in E. coli strain Rosetta BL21. Cells were lysed by sonication in 100 mM NaH2 PO4 , 10 mM Tris (pH 8.0), 8 M urea, and 10 mM imidazol, and Bcl10 and Carma1 derivatives were purified from the insoluble fraction by Ni-NTA agarose chromatography (Qiagen) according to the manufacturer’s instructions. The purified preparations were dialyzed against 20 mM Tris (pH 8.0), 100 mM NaCl, 0.05% Triton X-100, 10% glycerol, and 1 mM dithiothreitol (DTT). 2.3. In vitro binding experiments For CaM Sepharose binding experiments, 0.5 g of purified proteins were incubated with 10 l CaM Sepharose (GE Healthcare) in binding buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 0.5% Triton X-100, 1 mM DTT, protease inhibitor cocktail tablet without EDTA (Roche)) supplemented with either 1 mM CaCl2 or 1 mM EGTA, with rotation overnight at 4 ◦ C. Where indicated, 5 M CaMKII peptide (amino acids 290–309) (Sigma) was added to the binding reaction. The CaM Sepharose was washed three times with binding buffer with 1% Triton X-100, and bound proteins were eluted with binding buffer supplemented with 2 mM EGTA. For binding of Bcl10 to Carma1, purified Carma1 (amino acids 1–160) was coupled to CNBr-activated Sepharose (GE Healthcare) according to the manufacturer’s instructions. Bcl10 was preincubated in binding buffer with or without CaM (Upstate), as indicated, using rotation overnight at 4 ◦ C before addition of 10 l of Carma1 (1–160)
Sepharose. Reactions were incubated for 4 additional hours at 4 ◦ C before washing as above and elution by boiling in sample buffer. Samples were separated by 10% SDS-PAGE and analyzed for Bcl10 or Carma1 by Western blot using ␣-Bcl10 N-term (H-197) or ␣Bcl10 C-term (331.3) (both from Santa Cruz), or ␣-Carma1 (AL-220) (Alexis) antibodies and the SuperSignal Chemiluminescence Substrate (Pierce). All binding experiments were performed at least three times. Where indicated, Western blots were quantified using the ChemidocTM XRS gel documentation system and Quantity One® software (BioRad). For spectrofluorimetric analysis, dansylated CaM (5(dimethylamino)naphthalene-1-sulfonyl-CaM) was prepared using standard procedures (Kincaid et al., 1982), and 20 nM dansylated CaM and increasing concentrations of Bcl10 protein were allowed to equilibrate for 2 h in a buffer containing 20 mM Tris (pH 8.0), 100 mM NaCl, and 100 M CaCl2 . The fluorescence emission was recorded at 490 nm following excitation at 340 nm with a SPEX FluoroMax-2 fluorescence spectrometer. Measurements were repeated three times for each solution. The concentration of bound Bcl10 was plotted against free Bcl10 and the data were fit by one-site-specific binding with Hill slope (GraphPad Prism version 5.00).
2.4. Cell culture and transient transfections The Jurkat-derived T cell line with Bcl10 down-regulated is stably expressing an shRNA against Bcl10 (Wu and Ashwell, 2008). Jurkat T cells and this Jurkat derivative with down-regulated Bcl10 were maintained in RPMI supplemented with 5% FCS and antibiotics. Cells were transiently transfected with 5 g of expression vector. The cells were stimulated 24 h after transfection. PMA (Sigma) was used at a concentration of 1 ng/ml and ionomycin (Calbiochem) at 1 g/ml. For proximity ligation assays (PLA), cells were co-transfected with the GFP expression vector to identify successfully electroporated cells. Cells were stimulated with the indicated drugs for 10 min and harvested. For reporter experiments, cells were co-transfected with the NF-B-dependent reporter and normalization plasmid by electroporation as described (Hughes et al., 1998). Cells were stimulated for 2 h and harvested. Luciferase activity was measured with the Luciferase assay system (Promega) and normalized to -galactosidase from the co-transfected hCMV-gal plasmid.
2.5. Proximity ligation assays The harvested lymphocytes were fixed with 4% paraformaldehyde, permeabilized with 0.2% Triton X-100 and blocked with 5% FCS in PBS. Proximity ligation assays (PLA) (Jarvius et al., 2007; Söderberg et al., 2006, 2008, 2007) were performed with Duolink in situ PLA kits purchased from Olink Bioscience (http://www.olink.com/). In brief, cells were stained for intracellular proteins using a goat polyclonal antibody against CaM (N-19) and a mouse monoclonal antibody against Bcl10 (331.3) (both from Santa Cruz Biotechnology). Anti-goat IgG and anti-mouse IgG secondary antibodies conjugated with oligonucleotides (PLA probes) were subsequently used according to the manufacturer’s protocol to generate fluorescence signals only when the two PLA probes were in close proximity (≤40 nm). The fluorescence signal from each detected pair of PLA probes was visualized as a distinct individual red dot (Jarvius et al., 2007; Söderberg et al., 2006, 2008, 2007). Nuclei were counterstained with Hoechst 33342 dye.
S. Edin et al. / Molecular Immunology 47 (2010) 2057–2064
2059
of Bcl10. The fluorescence emitted was recorded and a binding curve was plotted (Fig. 1B). The stoichiometry of the interaction was predicted to be 1:1, since 20 nM of CaM gave a maximal Bcl10 binding of approximately 20 nM (Fig. 1B), and the sigmoidal binding curve indicated that the binding was cooperative in nature. The binding curve was fitted using the equation for one-site-specific binding with Hill slope, which gave a Hill coefficient of 2.2. CaM was found to bind Bcl10 with high affinity. The Kd was determined to be 158 ± 6 nM, which is a highly relevant binding strength, since many CaM targets have binding strengths of that level and CaM concentrations can be in the M range in vivo (Chin and Means, 2000). 3.2. Identification of the CaM binding domain of Bcl10
Fig. 1. CaM interacts with Bcl10. (A) Recombinant Bcl10 or Carma1 (amino acids 1–160) proteins were bound to CaM Sepharose in the presence of 1 mM Ca2+ (lanes 3 and 4) or 1 mM EGTA (lane 5), and eluted with 2 mM EGTA. Where indicated, the beads were pretreated with a CaM binding peptide (5 M) that corresponds to amino acids 290–309 of CaMKII (lane 4). Bound proteins were detected by Western blot; preload (lane 1) was 10% of total protein added to the binding reactions and Ctrl (lane 2) was protein eluted after binding to empty control Sepharose beads. (B) Increasing amounts of Bcl10 protein were added to dansylated CaM and fluorescence emission was recorded at 490 nm. A binding curve was plotted as bound Bcl10 against free Bcl10. The average binding ± s.d. is indicated (n = 3).
3. Results
Bcl10 is characterized by an N-terminal CARD domain and a Cterminal Ser/Thr-rich region of less known function (Rawlings et al., 2006) (Fig. 2A). To map the CaM binding sequence of Bcl10, we created a set of deletion mutants and expressed them in E. coli. The mutants were Bcl103–12, lacking the most N-terminal domain through which Bcl10 contacts the general transcription factor TFIIB (Liu et al., 2004), Bcl101–103 lacking the N-terminus including the entire CARD domain (Rawlings et al., 2006), Bcl10104–119 lacking sequences essential for Malt1 interaction (Lucas et al., 2001), and Bcl10120–233 lacking the sequences C-terminal to the CARD domain (Fig. 2A). The deletion mutants were analyzed for their ability to bind to CaM Sepharose. All mutants except one bound CaM Sepharose to similar extents. The exception was the Bcl101–103 mutant, lacking the CARD domain, where no binding was detected (Fig. 2B). The CaM binding of Bcl10 with a corresponding deletion of sequences C-terminal to the CARD domain as the utilized Carma1 derivative shows that there is a differential CaM binding between the CARD domains of the two proteins (cf. Figs. 1A and 2B). Thus, the differential CaM binding found between
3.1. CaM interacts with Bcl10 CaM is involved in the regulation of IKK and subsequently NF-B activation after TCR engagement or after mimicking this by activation of protein kinase C (PKC) with phorbol ester (Hughes et al., 1998). A part of the role of CaM in NF-B signaling from PKC was found to be mediated through the activation of CaM-dependent kinase II (CaMKII) (Hughes et al., 2001), but this could not explain the full effect of CaM in the pathway. Based on these previous findings, we decided to investigate whether CaM possibly also regulates TCR signaling to NF-B through direct interaction with a signaling protein in the pathway from PKC. Carma1 is a direct phosphorylation target of PKC, and this phosphorylation induces the interaction of Carma1 with Bcl10 (Thome, 2004). To start searching for a possible interaction partner for CaM, we therefore recombinantly expressed and purified full-length Bcl10 and a derivative (amino acids 1–160) of the much larger Carma1 in E. coli and determined their ability to bind to CaM Sepharose. The proteins were allowed to bind to the CaM Sepharose in the presence of Ca2+ or the Ca2+ chelator EGTA, and eluates were analyzed for bound proteins by Western blot. Interestingly, we could identify a Ca2+ -dependent interaction between Bcl10 and CaM Sepharose, whereas no interaction was seen for Carma1 (1–160) (Fig. 1A, cf. lanes 3 and 5). The specificity of the binding was verified both by the lack of binding to Sepharose without CaM and by inhibition of the interaction with a peptide representing the CaM binding domain of CaMKII (amino acids 290–309) (Fig. 1A, cf. lane 3 with lanes 2 and 4). We then addressed the question of whether this interaction had a binding strength that would make it likely to be of relevance in vivo. Changes in fluorescence of dansyl-labeled CaM can be used to determine the affinity of binding of CaM to a target (Kincaid et al., 1982). Dansyllabeled CaM was therefore titrated with increasing concentrations
Fig. 2. CaM binds to the CARD domain of Bcl10. (A) Schematic illustration of the domain structure of Bcl10 and of the deletion mutants analyzed. Shaded boxes indicate the CARD domain. (B) Recombinant wild-type Bcl10 or deletion mutants were bound to CaM Sepharose in the presence of Ca2+ and eluted with EGTA. Bound proteins were detected by Western blot using antibodies that recognize either the N-terminal part (left) or the C-terminal part (right) of Bcl10, as indicated. Preload was 10% of total protein added to the binding reactions and Ctrl was protein eluted after binding to empty control Sepharose beads.
2060
S. Edin et al. / Molecular Immunology 47 (2010) 2057–2064
ate a mutation of the potential CaM binding sequence in the sixth ␣-helix of Bcl10 without affecting the packing of the ␣-helices in the CARD domain, or the very important CARD–CARD interaction between Bcl10 and Carma1, we wanted to make as subtle a change as possible and not affect the hydrophobic core of the CARD domain. The mutation chosen, changing the basic amino acid arginine to alanine at position 88 (R88A) reduced the binding of Bcl10 to CaM Sepharose by approximately 65% (Fig. 3C). The mutation did not alter the CARD–CARD interaction, since the mutant protein was still able to bind Carma1 to an extent that was similar to that of the wild-type protein (Fig. 4B and data not shown). Although it cannot be excluded that the reduced CaM binding of the R88A mutant could be through an indirect effect on another part of the CARD domain, the results strongly suggest that CaM interacts with Bcl10 through the sequence identified, encompassing arginine 88 (see also below). A peptide corresponding to amino acids 81–100 was analyzed for its binding to CaM, but it did not show any strong binding (data not shown). This indicates that an interaction/interactions outside this sequence contributes to the CaM binding or that amino acids outside this sequence are needed for the right conformation of the binding site. Fig. 3. An R88A mutant of Bcl10 shows reduced CaM binding. (A) Sequence alignment of the CARD domains of mouse Bcl10 and mouse Carma1 (nucleotide sequence accession numbers BC024379 and AY135367, respectively). The sequences were aligned by comparison with other CARD proteins in the Conserved Domain database (NCBI). Identical amino acids are indicated by asterisks. (B) Detailed display of the sequence indicated. Hydrophobic and basic residues are indicated by open and shaded boxes, respectively, and the ␣-helix disrupting amino acid proline in Carma1 is circled. The position of mutagenesis is indicated by an arrow. (C) Recombinant wild-type Bcl10 or the Arg 88 to Ala (R88A) mutant protein was bound to CaM Sepharose in the presence of Ca2+ , and eluted with EGTA. Bound proteins were detected by Western blot. Preload was 10% of total protein added to the binding reactions and Ctrl was protein bound to empty control Sepharose beads. The CaM binding of the R88A mutant compared to the wild-type from three independent experiments is shown as percent bound protein ± s.d.
Bcl10 and the utilized derivative of Carma1 cannot be explained by any C-terminal sequences of full-length Bcl10. The CARD domain, which therefore contains the CaM binding sequence of Bcl10, consists of six anti-parallel ␣-helices with a hydrophobic core (Chou et al., 1998; Day et al., 1999; Park et al., 2007). Importantly, binding of CaM to its targets is dependent on ␣-helical segments containing basic and hydrophobic stretches in the target proteins (Ishida and Vogel, 2006; Yamniuk and Vogel, 2004), making an ␣-helical sequence of the CARD domain of Bcl10 a possible target for CaM.
3.4. Binding of CaM to Bcl10 inhibits the interaction between Bcl10 and Carma1 One very important step in T cell activation is the induced interaction between Carma1 and Bcl10 after PKC-induced phosphorylation and activation of Carma1 (Matsumoto et al., 2005; Rueda and Thome, 2005; Sommer et al., 2005). A possible way by which CaM binding to Bcl10 could affect NF-B signaling is through the interaction between Bcl10 and Carma1. Since the CaM binding CARD domain of Bcl10 also mediates the interaction of Bcl10 with Carma1, we wanted to investigate whether the presence of Ca2+ -loaded CaM affects the binding of Bcl10 to Carma1. Purified Bcl10 protein was therefore preincubated with CaM in the presence of Ca2+ and subsequently allowed to bind to Carma1 (1–160) Sepharose. The Western blot of eluted proteins from a representative experiment is shown in Fig. 4. The binding of CaM to Bcl10 was indeed found to inhibit the interaction between Bcl10 and Carma1, since increasing concentrations of CaM were shown to reduce this interaction in a dose-dependent manner (Fig. 4A). Interestingly, the
3.3. A Bcl10 mutant with reduced CaM binding We examined the homologous CARD domains of Bcl10 and Carma1, searching for differences between them that could allow binding of CaM to Bcl10 but not to Carma1. By aligning the CARD domain sequences of mouse Bcl10 and Carma1 (which both differ by only 2 amino acids between mice and humans), we could identify stretches of basic and hydrophobic amino acids in different parts of the CARD domain (Fig. 3A). Of special interest was the area indicated in Fig. 3B, where basic and hydrophobic stretches present in the Bcl10 sequence make it a putative CaM binding site, whereas the corresponding sequence in the CARD domain of Carma1 contains fewer basic and hydrophobic amino acids and also a proline, which—due to its geometry and poor flexibility—rarely takes part in an ␣-helix. These differences might explain why CaM binds to the CARD domain of Bcl10 but not to that of Carma1, if this indeed was the CaM binding site. Interestingly, from a comparison with other CARD domains (Koseki et al., 1999), this sequence is likely to include the sixth ␣-helix of the CARD domain of Bcl10. We made peptides corresponding to other sequences in the CARD domain with slight resemblances to CaM binding sites, but none of them showed any significant binding to CaM (data not shown). To cre-
Fig. 4. Binding of CaM to Bcl10 is inhibitory for the interaction between Bcl10 and Carma1. (A) Recombinant Bcl10 was preincubated with the indicated concentrations of CaM in the presence of Ca2+ , and subsequently bound to Carma1 (amino acids 1–160) Sepharose. Bound proteins were eluted and detected by Western blot. Ctrl is protein bound to empty control Sepharose beads. The amounts of Carma1 eluted from the beads are shown as a control below. (B) Binding of recombinant wild-type Bcl10 or the R88A mutant of Bcl10 to Carma1 (amino acids 1–160) Sepharose with and without preincubation with 3 M CaM was analyzed as in A.
S. Edin et al. / Molecular Immunology 47 (2010) 2057–2064
2061
Fig. 5. In vivo proximity of CaM and Bcl10 that increases with increased intracellular Ca2+ level. Detection of proximal location of CaM and Bcl10 (shown as red dots) by in situ proximity ligation assays (PLA) was as described in Section 2. Jurkat cells were stimulated for 10 min with PMA, ionomycin or PMA plus ionomycin or left unstimulated. In controls either the ␣-Bcl10 or ␣-CaM primary antibody were omitted in PLA of unstimulated cells. All samples were counterstained with Hoechst 33342 dye (blue) to visualize nuclei.
inhibitory effect of CaM was lost in the R88A mutant (Fig. 4B), suggesting that Ca2+ signaling through CaM regulates the interaction between Bcl10 and Carma1, and thereby also activation of NF-B. 3.5. In vivo proximity of CaM and Bcl10 is induced by an increased Ca2+ level The strong Ca2+ -dependent binding between CaM and Bcl10 in vitro indicated that they would interact also in vivo. To analyze if Ca2+ signaling could induce proximity between CaM and Bcl10 also in vivo, we performed in situ proximity ligation assays (PLA). Jurkat T cells were stained using antibodies against CaM and Bcl10 and secondary antibodies conjugated with oligonucleotides (PLA
probes) to generate fluorescence signals only when the two PLA probe marked proteins are in close proximity (≤40 nm). The fluorescence signal results in a distinct individual red dot (Jarvius et al., 2007; Söderberg et al., 2006, 2008, 2007). The number of dots was significantly higher than in controls where either the ␣-Bcl10 or ␣CaM primary antibody was omitted (Fig. 5). To analyze if increased proximity was induced by stimulation, the Jurkat cells were treated for 10 min with PMA, with ionomycin—which increases the intracellular Ca2+ level—or with PMA plus ionomycin (Fig. 5). Treatment with PMA or PMA plus ionomycin had a small effect on the number of PLA dots, whereas the stimulation with ionomycin increased the number of dots 3.4 ± 1.0-fold (the P-value for the increase was 0.01). Thus, the number of proximities between CaM and Bcl10 is
2062
S. Edin et al. / Molecular Immunology 47 (2010) 2057–2064
Fig. 6. In vivo proximity of CaM and wild-type but not mutant Bcl10 is induced by increased intracellular Ca2+ level. PLA was performed in a derivative of the Jurkat T cell line with strongly down-regulated expression of endogenous Bcl10 transiently transfected with expression vector for wild-type Bcl10, the R88A mutant of Bcl10, or empty vector together with GFP expression vector to detect transfected cells. Cells were either left unstimulated or stimulated with ionomycin for 10 min as indicated and harvested. Controls were unstimulated cells transfected with wild-type Bcl10 stained using ␣-Bcl10 alone, ␣-CaM alone, or no primary antibodies. Values shown are average numbers of proximities per successfully transfected cell ± s.d. Unstimulated cells transfected with expression vector for wild-type Bcl10 were set as 100% (n ≥ 3).
increased by treatment that increases the intracellular Ca2+ level, whereas the increase is less pronounced when the Ca2+ signal is combined with a PKC activating stimuli. Proximity between CaM and Bcl10 that increased upon increased intracellular Ca2+ level was also found in PLA with another pair of ␣-CaM and ␣-Bcl10 antibodies raised in other species (data not shown). To determine whether the R88A mutant of Bcl10 with defect CaM interaction in vitro was defect also in vivo, we compared the proximities of the wild-type and mutant Bcl10 proteins to CaM. PLA was performed in a derivative of the Jurkat T cell line with strongly down-regulated expression of endogenous Bcl10. After transient transfection of expression vector for wild-type Bcl10, the R88A mutant of Bcl10 or empty vector control together with GFP expression vector to detect transfected cells, the cells were either left unstimulated or stimulated with ionomycin for 10 min. The stimulation with ionomycin increased the number of dots approximately fourfold in the cells transfected with wild-type Bcl10, whereas no increase was detected in the cells transfected with the R88A mutant or with empty vector (Fig. 6). The P-value for this difference between wild-type and R88A mutated Bcl10 was <0.05. Thus, in contrast to wild-type Bcl10, the number of CaM proximities of the R88A mutant did not increase by the treatment that increases the intracellular Ca2+ level. 3.6. Binding of CaM to Bcl10 is inhibitory for NF-B activation in Jurkat T cells To investigate the functional role of the binding between CaM and Bcl10, we expressed either wild-type Bcl10 or the R88A mutant of Bcl10 together with an NF-B-dependent reporter in a derivative of the Jurkat T cell line in which endogenous Bcl10 had been strongly down-regulated. We titrated the level of transfected Bcl10 plasmid so we restored the Bcl10 expression level of the parental Jurkat cells, as verified by Western, and the parental level of induction by the PKC activating phorbol ester PMA, as verified by the reporter assay (data not shown). This expression of wild-type Bcl10 activated NF-B and the increase of this activation by stimulation with PMA was further enhanced in combination with the calcium ionophore ionomycin, mimicking T cell receptor activation (Fig. 7). Importantly, expression of the R88A mutant of Bcl10 activated NF-
Fig. 7. The R88A mutant of Bcl10 gives increased activation of an NF-B-dependent reporter in T cells. Wild-type Bcl10, the R88A mutant of Bcl10, or empty vector were transiently expressed together with an NF-B-dependent reporter in a derivative of the Jurkat T cell line in which expression of endogenous Bcl10 had been strongly down-regulated. Twenty-four hours after transfection, the cells were either left unstimulated (U) or stimulated with PMA (P) or PMA plus ionomycin (P + I) for 2 h and harvested. Values shown are normalized fold activations ± s.d. (n = 3) compared to untreated cells transfected with empty vector.
B more potently than wild-type Bcl10, both in the presence or absence of cellular stimulation (Fig. 7). Equal expression level of wild-type and mutant Bcl10 was verified by Western blot (data not shown). The mutant was found to activate the NF-B reporter 1.5–2-fold more than wild-type protein, either without PMA, with PMA, or with PMA plus ionomycin (the P-values were 0.07, 0.03, and 0.04, respectively). The corresponding effect of the mutation on activation of NF-B was also obtained upon over-expression of Bcl10 in the parental Jurkat T cells, where endogenous levels of Bcl10 were intact (data not shown). Stimulation with ionomycin alone had no significant effect on activation of the NF-B reporter in cells transfected with either wild-type or R88A mutated Bcl10 or vector control (Suppl. Fig. 1). In summary, these results support the notion that binding of CaM to Bcl10 has a negative effect on NF-B activation in vivo, which probably occurs through inhibition of the interaction between Bcl10 and Carma1. 4. Discussion Here we have presented results that Ca2+ signals from the TCR can negatively regulate NF-B activation through binding of CaM to Bcl10. We have shown that CaM interacts specifically and in a Ca2+ -dependent manner with Bcl10, with a binding strength that is of high biological relevance (with a Kd of approximately 160 nM). The proximity of Bcl10 and CaM was induced in vivo by an increased intracellular Ca2+ level. However, the binding was found to be less evident when the Ca2+ signal was accompanied by PKC activating stimuli. Furthermore, the interaction of CaM with Bcl10 was found to have a negative effect on the interaction between the CARD domains of Bcl10 and Carma1, an interaction that is required for signaling from the TCR to NF-B. A Bcl10 mutant with reduced ability to bind CaM was still found to be capable of interacting with Carma1, but this interaction was unaffected by the presence of CaM. Importantly, the CaM binding mutant of Bcl10 activated NFB in T cells in a more potent way, supporting the notion that CaM binding to Bcl10 negatively regulates NF-B activation in T cells by preventing the Bcl10–Carma1 interaction. The interaction between CaM and Bcl10 showed a sigmoidal binding curve, indicating cooperative binding—something that is typically seen for the classical wrap-around mode of binding of CaM to its targets (Bhattacharya et al., 2004; Ishida and Vogel, 2006). It is common among classical CaM target motifs to have
S. Edin et al. / Molecular Immunology 47 (2010) 2057–2064
two hydrophobic residues acting as anchors at each end of the sequence. The length of the intervening sequence has been used to classify CaM targets into either 1–10, 1–14, or 1–16 recognition motifs (Ishida and Vogel, 2006; Yamniuk and Vogel, 2004). The sequence identified in Bcl10 contains several potential hydrophobic anchors with 1–14 spacing (amino acids L82–L95, V83–I96, I86–I99) or 1–16 spacing (L79–F94), and with several intervening basic and hydrophobic amino acids (Fig. 3B), which fits the features of known CaM target motifs. However, CaM has been shown to bind also to target sequences not resembling these motifs. Thus, the binding mode and detailed structure of the CaM–Bcl10 interaction will have to await future studies. However, a point mutation (R88A) within the basic patch of the sequence did indeed reduce the binding of CaM by about 65%, supporting the idea that this is the domain to which CaM binds. The binding of CaM is, as discussed above, likely to occur at the predicted sixth ␣-helix of the CARD domain of Bcl10. The structure of the CARD–CARD interaction between Bcl10 and Carma1 is not available at present, and how binding of CaM at the predicted sixth ␣-helix would be inhibitory to this interaction remains to be determined. In support of our finding that the sixth ␣-helix is important for regulation of CARD–CARD interactions, Marasco and co-workers have, by using inhibitory peptides, identified the amino acids 91–98 of Bcl10 as important for its self-association as well as its interaction with external partners in NF-B activation (Marasco et al., 2009). The CaM binding mutant of Bcl10 activated the NF-B reporter 1.5–2 times more potently than the wild-type protein in T cells. The effect on NF-B activity was seen in cells with or without intact endogenous level of Bcl10, and both in the presence and absence of PMA and ionomycin stimulation. This 1.5–2-fold effect of the mutant could represent the level of the negative effect of CaM binding. However, the mutant Bcl10 retained 35% of the CaM binding capacity (Fig. 3C), which makes it likely that a complete loss of CaM binding would lead to a greater increase in NF-B activation. In addition, the positive effect of losing the CaM binding could be underestimated if the R88A mutant leads to a hypothetical negative effect on another function of the protein in the activation process. The full extent of the negative effect of CaM binding on NF-B activation is therefore unknown. Stimulation with ionomycin alone had no effect on the ability of wild-type or CaM binding mutant of Bcl10 to activate the NF-B reporter. This supports that Ca2+ changes induced by ionomycin alone favor a negative role of CaM in NF-B signaling and that additional PKC activating stimuli are required for NF-B activation. When PKC is activated, the negative role of the CaM–Bcl10 interaction appears to be modulatory in character, regulating the degree of NF-B activation. An interaction of the CARD domain of Bcl10 with Malt1 has recently been reported (Langel et al., 2008). They searched for mutations of amino acids within the CARD domain that disrupted the binding of Bcl10 to Malt1. Interestingly, one of the mutations analyzed was on a neighbor of R88, R87. The R87A mutant was, similarly to our R88A mutant, found to increase NF-B activation by around 2-fold, whereas its ability to bind Carma1 and Malt1 was unaffected. Thus, the CARD domain of the R87A and R88A mutants is likely to be structurally intact, and the effect of the R87A mutant supports that this segment of the protein is subjected to negative regulation, with binding of CaM being the likely mechanism. Changes in intracellular Ca2+ levels have an important role in the regulation of T cell activation, and the pattern of Ca2+ responses influences T cell commitment towards activation or anergy (Feske, 2007; Randriamampita and Trautmann, 2004). We have previously reported a requirement for CaM and CaMKII in activation of IKK and NF-B after TCR stimulation (Hughes et al., 1998, 2001). The CaMKII dependence of IKK activation has been shown to be at least partly due to phosphorylation of Carma1, which improves its interaction with Bcl10 (Ishiguro et al., 2006). Thus, from the present results it
2063
appears that CaM can have both positive and negative roles in signaling from the TCR to NF-B activation. What could be the function of CaM binding to Bcl10, considering that this negatively affects its interaction with Carma1 and thereby its ability to activate NF-B? Different patterns of Ca2+ signaling can lead to quite distinct outcomes, in particular the activation of different transcription factors. Complete TCR stimulations result in high Ca2+ transients that activate NF-B, whereas TCR activation without co-stimulatory signals results in sustained but weaker Ca2+ signals. These more modest Ca2+ signals are sufficient to activate other factors but not NF-B, thereby inducing a state of T cell anergy (Jun and Goodnow, 2003; Macian et al., 2004; Quintana et al., 2005; Randriamampita and Trautmann, 2004). The negative effect of CaM on NF-B activation reported here might therefore be of importance during incomplete TCR stimulation, where an anergic state of the cell is induced and activation of NF-B therefore should not be allowed. In contrast, when a proper immunological synapse is formed, the concert of signaling events induced—which includes high fluctuations in Ca2+ —allows CaM to positively regulate NF-B, at least in part through activation of CaMKII, which is highly regulated by such Ca2+ changes. In line with the multiple modes of Ca2+ /CaM modulation of NF-B activation, CaM has also been shown to regulate the transcriptional activity of NF-B both by direct interaction with the NF-B proteins c-Rel and RelA (and thereby differential regulation of their nuclear translocation and activation) (Antonsson et al., 2003) and by activation of CaMKIV, which, through phosphorylation of RelA (p65), increases the activity of this transcription factor (Bae et al., 2003; Jang et al., 2001). Ca2+ and CaM are thus involved in the regulation of NF-B at several different levels. Taken together, the results in this report support the notion that by sensing certain changes in intracellular Ca2+ , CaM can interact with Bcl10 and thereby negatively regulate TCR-induced NF-B activation. Acknowledgments We thank Göran Larsson and Peter Gimeson at the Department of Medical Chemistry, Umeå University, for excellent guidance during the spectrofluorimetric analysis of the CaM–Bcl10 interaction. The Bcl10 knockdown cell line was a kind gift from Drs. Chuan-Jin Wu and Jonathan D. Ashwell. This work was supported by grants from the Swedish Research Council and the Swedish Cancer Society to T.G. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.molimm.2010.04.005. References Antonsson, Å., Hughes, K., Edin, S., Grundström, T., 2003. Regulation of c-Rel nuclear localization by binding of Ca2+ /calmodulin. Mol. Cell. Biol. 23, 1418–1427. Bae, J.S., Jang, M.K., Hong, S., An, W.G., Choi, Y.H., Kim, H.D., Cheong, J., 2003. Phosphorylation of NF-B by calmodulin-dependent kinase IV activates anti-apoptotic gene expression. Biochem. Biophys. Res. Commun. 305, 1094–1098. Bhattacharya, S., Bunick, C.G., Chazin, W.J., 2004. Target selectivity in EF-hand calcium binding proteins. Biochim. Biophys. Acta 1742, 69–79. Bouchier-Hayes, L., Martin, S.J., 2002. CARD games in apoptosis and immunity. EMBO Rep. 3, 616–621. Caamano, J., Hunter, C.A., 2002. NF-B family of transcription factors: central regulators of innate and adaptive immune functions. Clin. Microbiol. Rev. 15, 414–429. Chin, D., Means, A.R., 2000. Calmodulin: a prototypical calcium sensor. Trends Cell Biol. 10, 322–328. Chou, J.J., Matsuo, H., Duan, H., Wagner, G., 1998. Solution structure of the RAIDD CARD and model for CARD/CARD interaction in caspase-2 and caspase-9 recruitment. Cell 94, 171–180. Corneliussen, B., Holm, M., Waltersson, Y., Onions, J., Hallberg, B., Thornell, A., Grundström, T., 1994. Calcium/calmodulin inhibition of basic-helix–loop–helix transcription factor domains. Nature 368, 760–764.
2064
S. Edin et al. / Molecular Immunology 47 (2010) 2057–2064
Day, C.L., Dupont, C., Lackmann, M., Vaux, D.L., Hinds, M.G., 1999. Solution structure and mutagenesis of the caspase recruitment domain (CARD) from Apaf-1. Cell Death Differ. 6, 1125–1132. Feske, S., 2007. Calcium signalling in lymphocyte activation and disease. Nat. Rev. Immunol. 7, 690–702. Hayden, M.S., Ghosh, S., 2008. Shared principles in NF-B signaling. Cell 132, 344–362. Hughes, K., Antonsson, Å., Grundström, T., 1998. Calmodulin dependence of NF-B activation. FEBS Lett. 441, 132–136. Hughes, K., Edin, S., Antonsson, Å., Grundström, T., 2001. Calmodulin-dependent kinase II mediates T cell receptor/CD3- and phorbol ester-induced activation of IB kinase. J. Biol. Chem. 276, 36008–36013. Ishida, H., Vogel, H.J., 2006. Protein–peptide interaction studies demonstrate the versatility of calmodulin target protein binding. Protein Pept. Lett. 13, 455–465. Ishiguro, K., Green, T., Rapley, J., Wachtel, H., Giallourakis, C., Landry, A., Cao, Z., Lu, N., Takafumi, A., Goto, H., Daly, M.J., Xavier, R.J., 2006. Ca2+ /calmodulin-dependent protein kinase II is a modulator of CARMA1-mediated NF-B activation. Mol. Cell. Biol. 26, 5497–5508. Jang, M.K., Goo, Y.H., Sohn, Y.C., Kim, Y.S., Lee, S.K., Kang, H., Cheong, J., Lee, J.W., 2001. Ca2+ /calmodulin-dependent protein kinase IV stimulates nuclear factorB transactivation via phosphorylation of the p65 subunit. J. Biol. Chem. 276, 20005–20010. Jarvius, M., Paulsson, J., Weibrecht, I., Leuchowius, K.J., Andersson, A.C., Wählby, C., Gullberg, M., Botling, J., Sjöblom, T., Markova, B., Östman, A., Landegren, U., Söderberg, O., 2007. In situ detection of phosphorylated platelet-derived growth factor receptor beta using a generalized proximity ligation method. Mol. Cell. Proteomics 6, 1500–1509. Jun, J.E., Goodnow, C.C., 2003. Scaffolding of antigen receptors for immunogenic versus tolerogenic signaling. Nat. Immunol. 4, 1057–1064. Kincaid, R.L., Vaughan, M., Osborne Jr., J.C., Tkachuk, V.A., 1982. Ca2+ -dependent interaction of 5-dimethylaminonaphthalene-1-sulfonyl-calmodulin with cyclic nucleotide phosphodiesterase, calcineurin, and troponin I. J. Biol. Chem. 257, 10638–10643. Koseki, T., Inohara, N., Chen, S., Carrio, R., Merino, J., Hottiger, M.O., Nabel, G.J., Nunez, G., 1999. CIPER, a novel NF-B-activating protein containing a caspase recruitment domain with homology to Herpesvirus-2 protein E10. J. Biol. Chem. 274, 9955–9961. Langel, F.D., Jain, N.A., Rossman, J.S., Kingeter, L.M., Kashyap, A.K., Schaefer, B.C., 2008. Multiple protein domains mediate interaction between Bcl10 and MALT1. J. Biol. Chem. 283, 32419–32431. Liu, Y., Dong, W., Chen, L., Zhang, P., Qi, Y., 2004. Characterization of Bcl10 as a potential transcriptional activator that interacts with general transcription factor TFIIB. Biochem. Biophys. Res. Commun. 320, 1–6. Lucas, P.C., Yonezumi, M., Inohara, N., McAllister-Lucas, L.M., Abazeed, M.E., Chen, F.F., Yamaoka, S., Seto, M., Nunez, G., 2001. Bcl10 and MALT1, independent targets of chromosomal translocation in malt lymphoma, cooperate in a novel NF-B signaling pathway. J. Biol. Chem. 276, 19012–19019. Macian, F., Im, S.H., Garcia-Cozar, F.J., Rao, A., 2004. T-cell anergy. Curr. Opin. Immunol. 16, 209–216.
Marasco, D., Stilo, R., Sandomenico, A., Monti, S.M., Tizzano, B., de Capua, A., Varricchio, E., Liguoro, D., Zotti, T., Formisano, S., Ruvo, M., Vito, P., 2009. Generation and functional characterization of a BCL10-inhibitory peptide that represses NF-kappaB activation. Biochem. J. 422, 553–561. Matsumoto, R., Wang, D., Blonska, M., Li, H., Kobayashi, M., Pappu, B., Chen, Y., Lin, X., 2005. Phosphorylation of CARMA1 plays a critical role in T cell receptormediated NF-B activation. Immunity 23, 575–585. Onions, J., Hermann, S., Grundström, T., 1997. Basic helix–loop–helix protein sequences determining differential inhibition by calmodulin and S-100 proteins. J. Biol. Chem. 272, 23930–23937. Park, H.H., Lo, Y.C., Lin, S.C., Wang, L., Yang, J.K., Wu, H., 2007. The death domain superfamily in intracellular signaling of apoptosis and inflammation. Annu. Rev. Immunol. 25, 561–586. Quintana, A., Griesemer, D., Schwarz, E.C., Hoth, M., 2005. Calcium-dependent activation of T-lymphocytes. Pflugers Arch. 450, 1–12. Randriamampita, C., Trautmann, A., 2004. Ca2+ signals and T lymphocytes; “new mechanisms and functions in Ca2+ signalling”. Biol. Cell 96, 69–78. Rawlings, D.J., Sommer, K., Moreno-Garcia, M.E., 2006. The CARMA1 signalosome links the signalling machinery of adaptive and innate immunity in lymphocytes. Nat. Rev. Immunol. 6, 799–812. Rueda, D., Thome, M., 2005. Phosphorylation of CARMA1: the link(er) to NF-B activation. Immunity 23, 551–553. Schulze-Luehrmann, J., Ghosh, S., 2006. Antigen–receptor signaling to nuclear factor B. Immunity 25, 701–715. Sommer, K., Guo, B., Pomerantz, J.L., Bandaranayake, A.D., Moreno-Garcia, M.E., Ovechkina, Y.L., Rawlings, D.J., 2005. Phosphorylation of the CARMA1 linker controls NF-B activation. Immunity 23, 561–574. Söderberg, O., Gullberg, M., Jarvius, M., Ridderstråle, K., Leuchowius, K.J., Jarvius, J., Wester, K., Hydbring, P., Bahram, F., Larsson, L.G., Landegren, U., 2006. Direct observation of individual endogenous protein complexes in situ by proximity ligation. Nat. Methods 3, 995–1000. Söderberg, O., Leuchowius, K.J., Gullberg, M., Jarvius, M., Weibrecht, I., Larsson, L.G., Landegren, U., 2008. Characterizing proteins and their interactions in cells and tissues using the in situ proximity ligation assay. Methods 45, 227– 232. Söderberg, O., Leuchowius, K.J., Kamali-Moghaddam, M., Jarvius, M., Gustafsdottir, S., Schallmeiner, E., Gullberg, M., Jarvius, J., Landegren, U., 2007. Proximity ligation: a specific and versatile tool for the proteomic era. Genet. Eng. (N. Y.) 28, 85–93. Thome, M., 2004. CARMA1, BCL-10 and MALT1 in lymphocyte development and activation. Nat. Rev. Immunol. 4, 348–359. Weil, R., Israel, A., 2004. T-cell-receptor- and B-cell-receptor-mediated activation of NF-B in lymphocytes. Curr. Opin. Immunol. 16, 374–381. Wu, C.J., Ashwell, J.D., 2008. NEMO recognition of ubiquitinated Bcl10 is required for T cell receptor-mediated NF-B activation. Proc. Natl. Acad. Sci. U.S.A. 105, 3023–3028. Yamniuk, A.P., Vogel, H.J., 2004. Calmodulin’s flexibility allows for promiscuity in its interactions with target proteins and peptides. Mol. Biotechnol. 27, 33–57.
Contents lists available at ScienceDirect
Molecular Immunology journal homepage: www.elsevier.com/locate/molimm
Interaction of calmodulin with Bcl10 modulates NF-B activation Sofia Edin 1 , Sreenivasa Rao Oruganti, Christine Grundström, Thomas Grundström ∗ Department of Molecular Biology, Umeå University, SE-901 87 Umeå, Sweden
a r t i c l e
i n f o
Article history: Received 2 February 2010 Received in revised form 8 April 2010 Accepted 11 April 2010 Available online 1 May 2010 Keywords: Gene regulation Signal transduction NF-B Bcl10 Carma1 Calmodulin Calcium signaling
a b s t r a c t Calcium signals resulting from antigen receptor activation are important in determining the responses of a T or B lymphocyte to an antigen. Calmodulin (CaM), a multi-functional sensor of intracellular calcium (Ca2+ ) signals in cells, is required in the pathway from the T cell receptor (TCR) to activation of the key transcription factor NF-B. Here we searched for a partner in direct interaction with CaM in the pathway, and found that CaM interacts specifically with the signaling adaptor Bcl10. The binding is Ca2+ dependent and of high affinity, with a Kd of approximately 160 nM. Proximity of CaM and Bcl10 in vivo is induced by increases in the intracellular Ca2+ level. The interaction is localized to the CARD domain of Bcl10, which interacts with the CARD domain of the upstream signaling partner Carma1. Binding of CaM to Bcl10 is shown to inhibit the ability of Bcl10 to interact with Carma1, an interaction that is required for signaling from the TCR to NF-B. Furthermore, a mutant of Bcl10 with reduced binding to CaM shows increased activation of an NF-B reporter, which is further enhanced by activating stimuli. We propose a novel mechanism whereby the Ca2+ sensor CaM regulates T cell responses to antigens by binding to Bcl10, thereby modulating its interaction with Carma1 and subsequent activation of NF-B. © 2010 Elsevier Ltd. All rights reserved.
1. Introduction Antigen receptor activation results in the recruitment and activation of a number of signaling mediators and lipid metabolizing enzymes, building up an immunological synapse at the T cell receptor (TCR) or B cell receptor (BCR) (Schulze-Luehrmann and Ghosh, 2006; Weil and Israel, 2004). The immunological synapse triggers downstream signaling pathways, leading to activation of transcription factors such as the NF-B family, which is important for the regulation of activation, proliferation, and differentiation of T and B lymphocytes (Caamano and Hunter, 2002). In a resting cell, NF-B dimers mainly reside in the cytoplasm, sequestered by inhibitory IB proteins. Most signals to NF-B converge on the activation of the IB kinase (IKK), resulting in signal-induced phosphorylation and degradation of IB, which allows NF-B to enter the nucleus and activate gene transcription (Hayden and Ghosh, 2008).
Abbreviations: CaM, calmodulin; TCR, T cell receptor; BCR, B cell receptor; CBM, Carma1, Bcl10, Malt1 complex; IKK, IB kinase; PKC, protein kinase C; CARD, caspase recruitment domain; IP3 , inositol-1,4,5-triphosphate; CRAC, calcium release-activated Ca2+ channel; CaMK, calmodulin dependent kinase; PMA, phorbol12-myristate-13-acetate. ∗ Corresponding author. Tel.: +46 90 7852531; fax: +46 90 772630. E-mail address: [email protected] (T. Grundström). 1 Current address: Department of Medical Biosciences, Umeå University, SE-901 85 Umeå, Sweden. 0161-5890/$ – see front matter © 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.molimm.2010.04.005
T cell receptor-induced activation of NF-B is dependent on the activation of protein kinase C (PKC) and the subsequent recruitment and activation of a signaling complex containing the proteins Carma1, Bcl10, and Malt1, which is often referred to as the CBM complex. The formation of this complex is crucial for T cell activation, since deletion of any of the proteins impairs the NF-B responses (Thome, 2004). The molecular mechanisms by which these proteins promote IKK and NF-B activation are not fully understood, however. Carma 1 and Bcl10 belong to the Caspase recruitment domain (CARD) subfamily of the large Death domain family of proteins. The CARD domains and other domains found in this family are highly conserved and are characterized by their homotypic interactions with proteins within the same subfamily. They have been highly implicated in cell signaling to apoptosis and immunity (Bouchier-Hayes and Martin, 2002; Park et al., 2007). Bcl10 and Carma1 interact with each other through their CARD motifs, and this interaction is crucial for NF-B signaling after TCR stimulation, since deletion of the CARD domain of Carma1 or mutation of the CARD domain of Bcl10 prevents NF-B activation (Rawlings et al., 2006). In addition to activation of PKC-dependent pathways, triggering of the TCR also leads to production of inositol-1,4,5triphosphate (IP3 ), initiating the release of Ca2+ from intracellular stores. Depletion of intracellular reservoirs of Ca2+ activates the influx of Ca2+ through store-operated calcium release-activated Ca2+ (CRAC) channels in the plasma membrane, resulting in elevated levels of intracellular Ca2+ . Depending on the pattern and strength of these Ca2+ signals, signaling molecules and transcrip-
2058
S. Edin et al. / Molecular Immunology 47 (2010) 2057–2064
tion factors with different requirements for Ca2+ will be activated (Feske, 2007; Quintana et al., 2005). The activation of NF-B transcription factors after TCR engagement is sensitive to such changes in intracellular Ca2+ , but the mechanisms are not well characterized. The main transducer of Ca2+ signals in cells is the Ca2+ binding protein calmodulin (CaM). CaM belongs to the EF-hand family of Ca2+ binding proteins and changes conformation upon Ca2+ binding, allowing it to bind to a new set of targets. CaM is involved in the regulation of numerous important processes such as proliferation, signaling, and differentiation (Chin and Means, 2000; Yamniuk and Vogel, 2004). In the present study we investigated the role of CaM in TCR-induced activation of NF-B, and found that CaM interacts specifically with Bcl10. We present a novel mechanism by which CaM through interaction with Bcl10 can modulate the binding of Bcl10 to Carma1 and thereby NF-B activation. 2. Materials and methods 2.1. Plasmids and mutagenesis Expression vectors for Bcl10 and Carma1 were obtained by subcloning cDNA of mouse Bcl10, IMAGE ID 4976147, and mouse Carma1, IMAGE ID 5318165 (both from Mammalian Gene Collection), from pOTB7 to pCDNA1/Amp using EcoRI/NotI and EcoRV/NotI, respectively. Mutated derivatives of Bcl10 and Carma1 were constructed using standard cloning and PCR techniques. For expression in E. coli, Bcl10 and Carma1 cDNA were amplified by PCR and subcloned into the EcoRI/XhoI sites of the pET-20b + His expression vector (Onions et al., 1997) using the In FusionTM CF Liquid PCR cloning kit (Clontech). The NF-B luciferase reporter plasmid (Hughes et al., 1998) and the hCMV-gal normalization plasmid (Corneliussen et al., 1994) have been described previously. Co-transfection with a green fluorescent protein (GFP) expressing plasmid (Hughes et al., 2001) was used in transfections for proximity ligation assays. 2.2. Expression and purification of proteins C-terminally (His)6 -tagged Bcl10 or Carma1 constructs were expressed in E. coli strain Rosetta BL21. Cells were lysed by sonication in 100 mM NaH2 PO4 , 10 mM Tris (pH 8.0), 8 M urea, and 10 mM imidazol, and Bcl10 and Carma1 derivatives were purified from the insoluble fraction by Ni-NTA agarose chromatography (Qiagen) according to the manufacturer’s instructions. The purified preparations were dialyzed against 20 mM Tris (pH 8.0), 100 mM NaCl, 0.05% Triton X-100, 10% glycerol, and 1 mM dithiothreitol (DTT). 2.3. In vitro binding experiments For CaM Sepharose binding experiments, 0.5 g of purified proteins were incubated with 10 l CaM Sepharose (GE Healthcare) in binding buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 0.5% Triton X-100, 1 mM DTT, protease inhibitor cocktail tablet without EDTA (Roche)) supplemented with either 1 mM CaCl2 or 1 mM EGTA, with rotation overnight at 4 ◦ C. Where indicated, 5 M CaMKII peptide (amino acids 290–309) (Sigma) was added to the binding reaction. The CaM Sepharose was washed three times with binding buffer with 1% Triton X-100, and bound proteins were eluted with binding buffer supplemented with 2 mM EGTA. For binding of Bcl10 to Carma1, purified Carma1 (amino acids 1–160) was coupled to CNBr-activated Sepharose (GE Healthcare) according to the manufacturer’s instructions. Bcl10 was preincubated in binding buffer with or without CaM (Upstate), as indicated, using rotation overnight at 4 ◦ C before addition of 10 l of Carma1 (1–160)
Sepharose. Reactions were incubated for 4 additional hours at 4 ◦ C before washing as above and elution by boiling in sample buffer. Samples were separated by 10% SDS-PAGE and analyzed for Bcl10 or Carma1 by Western blot using ␣-Bcl10 N-term (H-197) or ␣Bcl10 C-term (331.3) (both from Santa Cruz), or ␣-Carma1 (AL-220) (Alexis) antibodies and the SuperSignal Chemiluminescence Substrate (Pierce). All binding experiments were performed at least three times. Where indicated, Western blots were quantified using the ChemidocTM XRS gel documentation system and Quantity One® software (BioRad). For spectrofluorimetric analysis, dansylated CaM (5(dimethylamino)naphthalene-1-sulfonyl-CaM) was prepared using standard procedures (Kincaid et al., 1982), and 20 nM dansylated CaM and increasing concentrations of Bcl10 protein were allowed to equilibrate for 2 h in a buffer containing 20 mM Tris (pH 8.0), 100 mM NaCl, and 100 M CaCl2 . The fluorescence emission was recorded at 490 nm following excitation at 340 nm with a SPEX FluoroMax-2 fluorescence spectrometer. Measurements were repeated three times for each solution. The concentration of bound Bcl10 was plotted against free Bcl10 and the data were fit by one-site-specific binding with Hill slope (GraphPad Prism version 5.00).
2.4. Cell culture and transient transfections The Jurkat-derived T cell line with Bcl10 down-regulated is stably expressing an shRNA against Bcl10 (Wu and Ashwell, 2008). Jurkat T cells and this Jurkat derivative with down-regulated Bcl10 were maintained in RPMI supplemented with 5% FCS and antibiotics. Cells were transiently transfected with 5 g of expression vector. The cells were stimulated 24 h after transfection. PMA (Sigma) was used at a concentration of 1 ng/ml and ionomycin (Calbiochem) at 1 g/ml. For proximity ligation assays (PLA), cells were co-transfected with the GFP expression vector to identify successfully electroporated cells. Cells were stimulated with the indicated drugs for 10 min and harvested. For reporter experiments, cells were co-transfected with the NF-B-dependent reporter and normalization plasmid by electroporation as described (Hughes et al., 1998). Cells were stimulated for 2 h and harvested. Luciferase activity was measured with the Luciferase assay system (Promega) and normalized to -galactosidase from the co-transfected hCMV-gal plasmid.
2.5. Proximity ligation assays The harvested lymphocytes were fixed with 4% paraformaldehyde, permeabilized with 0.2% Triton X-100 and blocked with 5% FCS in PBS. Proximity ligation assays (PLA) (Jarvius et al., 2007; Söderberg et al., 2006, 2008, 2007) were performed with Duolink in situ PLA kits purchased from Olink Bioscience (http://www.olink.com/). In brief, cells were stained for intracellular proteins using a goat polyclonal antibody against CaM (N-19) and a mouse monoclonal antibody against Bcl10 (331.3) (both from Santa Cruz Biotechnology). Anti-goat IgG and anti-mouse IgG secondary antibodies conjugated with oligonucleotides (PLA probes) were subsequently used according to the manufacturer’s protocol to generate fluorescence signals only when the two PLA probes were in close proximity (≤40 nm). The fluorescence signal from each detected pair of PLA probes was visualized as a distinct individual red dot (Jarvius et al., 2007; Söderberg et al., 2006, 2008, 2007). Nuclei were counterstained with Hoechst 33342 dye.
S. Edin et al. / Molecular Immunology 47 (2010) 2057–2064
2059
of Bcl10. The fluorescence emitted was recorded and a binding curve was plotted (Fig. 1B). The stoichiometry of the interaction was predicted to be 1:1, since 20 nM of CaM gave a maximal Bcl10 binding of approximately 20 nM (Fig. 1B), and the sigmoidal binding curve indicated that the binding was cooperative in nature. The binding curve was fitted using the equation for one-site-specific binding with Hill slope, which gave a Hill coefficient of 2.2. CaM was found to bind Bcl10 with high affinity. The Kd was determined to be 158 ± 6 nM, which is a highly relevant binding strength, since many CaM targets have binding strengths of that level and CaM concentrations can be in the M range in vivo (Chin and Means, 2000). 3.2. Identification of the CaM binding domain of Bcl10
Fig. 1. CaM interacts with Bcl10. (A) Recombinant Bcl10 or Carma1 (amino acids 1–160) proteins were bound to CaM Sepharose in the presence of 1 mM Ca2+ (lanes 3 and 4) or 1 mM EGTA (lane 5), and eluted with 2 mM EGTA. Where indicated, the beads were pretreated with a CaM binding peptide (5 M) that corresponds to amino acids 290–309 of CaMKII (lane 4). Bound proteins were detected by Western blot; preload (lane 1) was 10% of total protein added to the binding reactions and Ctrl (lane 2) was protein eluted after binding to empty control Sepharose beads. (B) Increasing amounts of Bcl10 protein were added to dansylated CaM and fluorescence emission was recorded at 490 nm. A binding curve was plotted as bound Bcl10 against free Bcl10. The average binding ± s.d. is indicated (n = 3).
3. Results
Bcl10 is characterized by an N-terminal CARD domain and a Cterminal Ser/Thr-rich region of less known function (Rawlings et al., 2006) (Fig. 2A). To map the CaM binding sequence of Bcl10, we created a set of deletion mutants and expressed them in E. coli. The mutants were Bcl103–12, lacking the most N-terminal domain through which Bcl10 contacts the general transcription factor TFIIB (Liu et al., 2004), Bcl101–103 lacking the N-terminus including the entire CARD domain (Rawlings et al., 2006), Bcl10104–119 lacking sequences essential for Malt1 interaction (Lucas et al., 2001), and Bcl10120–233 lacking the sequences C-terminal to the CARD domain (Fig. 2A). The deletion mutants were analyzed for their ability to bind to CaM Sepharose. All mutants except one bound CaM Sepharose to similar extents. The exception was the Bcl101–103 mutant, lacking the CARD domain, where no binding was detected (Fig. 2B). The CaM binding of Bcl10 with a corresponding deletion of sequences C-terminal to the CARD domain as the utilized Carma1 derivative shows that there is a differential CaM binding between the CARD domains of the two proteins (cf. Figs. 1A and 2B). Thus, the differential CaM binding found between
3.1. CaM interacts with Bcl10 CaM is involved in the regulation of IKK and subsequently NF-B activation after TCR engagement or after mimicking this by activation of protein kinase C (PKC) with phorbol ester (Hughes et al., 1998). A part of the role of CaM in NF-B signaling from PKC was found to be mediated through the activation of CaM-dependent kinase II (CaMKII) (Hughes et al., 2001), but this could not explain the full effect of CaM in the pathway. Based on these previous findings, we decided to investigate whether CaM possibly also regulates TCR signaling to NF-B through direct interaction with a signaling protein in the pathway from PKC. Carma1 is a direct phosphorylation target of PKC, and this phosphorylation induces the interaction of Carma1 with Bcl10 (Thome, 2004). To start searching for a possible interaction partner for CaM, we therefore recombinantly expressed and purified full-length Bcl10 and a derivative (amino acids 1–160) of the much larger Carma1 in E. coli and determined their ability to bind to CaM Sepharose. The proteins were allowed to bind to the CaM Sepharose in the presence of Ca2+ or the Ca2+ chelator EGTA, and eluates were analyzed for bound proteins by Western blot. Interestingly, we could identify a Ca2+ -dependent interaction between Bcl10 and CaM Sepharose, whereas no interaction was seen for Carma1 (1–160) (Fig. 1A, cf. lanes 3 and 5). The specificity of the binding was verified both by the lack of binding to Sepharose without CaM and by inhibition of the interaction with a peptide representing the CaM binding domain of CaMKII (amino acids 290–309) (Fig. 1A, cf. lane 3 with lanes 2 and 4). We then addressed the question of whether this interaction had a binding strength that would make it likely to be of relevance in vivo. Changes in fluorescence of dansyl-labeled CaM can be used to determine the affinity of binding of CaM to a target (Kincaid et al., 1982). Dansyllabeled CaM was therefore titrated with increasing concentrations
Fig. 2. CaM binds to the CARD domain of Bcl10. (A) Schematic illustration of the domain structure of Bcl10 and of the deletion mutants analyzed. Shaded boxes indicate the CARD domain. (B) Recombinant wild-type Bcl10 or deletion mutants were bound to CaM Sepharose in the presence of Ca2+ and eluted with EGTA. Bound proteins were detected by Western blot using antibodies that recognize either the N-terminal part (left) or the C-terminal part (right) of Bcl10, as indicated. Preload was 10% of total protein added to the binding reactions and Ctrl was protein eluted after binding to empty control Sepharose beads.
2060
S. Edin et al. / Molecular Immunology 47 (2010) 2057–2064
ate a mutation of the potential CaM binding sequence in the sixth ␣-helix of Bcl10 without affecting the packing of the ␣-helices in the CARD domain, or the very important CARD–CARD interaction between Bcl10 and Carma1, we wanted to make as subtle a change as possible and not affect the hydrophobic core of the CARD domain. The mutation chosen, changing the basic amino acid arginine to alanine at position 88 (R88A) reduced the binding of Bcl10 to CaM Sepharose by approximately 65% (Fig. 3C). The mutation did not alter the CARD–CARD interaction, since the mutant protein was still able to bind Carma1 to an extent that was similar to that of the wild-type protein (Fig. 4B and data not shown). Although it cannot be excluded that the reduced CaM binding of the R88A mutant could be through an indirect effect on another part of the CARD domain, the results strongly suggest that CaM interacts with Bcl10 through the sequence identified, encompassing arginine 88 (see also below). A peptide corresponding to amino acids 81–100 was analyzed for its binding to CaM, but it did not show any strong binding (data not shown). This indicates that an interaction/interactions outside this sequence contributes to the CaM binding or that amino acids outside this sequence are needed for the right conformation of the binding site. Fig. 3. An R88A mutant of Bcl10 shows reduced CaM binding. (A) Sequence alignment of the CARD domains of mouse Bcl10 and mouse Carma1 (nucleotide sequence accession numbers BC024379 and AY135367, respectively). The sequences were aligned by comparison with other CARD proteins in the Conserved Domain database (NCBI). Identical amino acids are indicated by asterisks. (B) Detailed display of the sequence indicated. Hydrophobic and basic residues are indicated by open and shaded boxes, respectively, and the ␣-helix disrupting amino acid proline in Carma1 is circled. The position of mutagenesis is indicated by an arrow. (C) Recombinant wild-type Bcl10 or the Arg 88 to Ala (R88A) mutant protein was bound to CaM Sepharose in the presence of Ca2+ , and eluted with EGTA. Bound proteins were detected by Western blot. Preload was 10% of total protein added to the binding reactions and Ctrl was protein bound to empty control Sepharose beads. The CaM binding of the R88A mutant compared to the wild-type from three independent experiments is shown as percent bound protein ± s.d.
Bcl10 and the utilized derivative of Carma1 cannot be explained by any C-terminal sequences of full-length Bcl10. The CARD domain, which therefore contains the CaM binding sequence of Bcl10, consists of six anti-parallel ␣-helices with a hydrophobic core (Chou et al., 1998; Day et al., 1999; Park et al., 2007). Importantly, binding of CaM to its targets is dependent on ␣-helical segments containing basic and hydrophobic stretches in the target proteins (Ishida and Vogel, 2006; Yamniuk and Vogel, 2004), making an ␣-helical sequence of the CARD domain of Bcl10 a possible target for CaM.
3.4. Binding of CaM to Bcl10 inhibits the interaction between Bcl10 and Carma1 One very important step in T cell activation is the induced interaction between Carma1 and Bcl10 after PKC-induced phosphorylation and activation of Carma1 (Matsumoto et al., 2005; Rueda and Thome, 2005; Sommer et al., 2005). A possible way by which CaM binding to Bcl10 could affect NF-B signaling is through the interaction between Bcl10 and Carma1. Since the CaM binding CARD domain of Bcl10 also mediates the interaction of Bcl10 with Carma1, we wanted to investigate whether the presence of Ca2+ -loaded CaM affects the binding of Bcl10 to Carma1. Purified Bcl10 protein was therefore preincubated with CaM in the presence of Ca2+ and subsequently allowed to bind to Carma1 (1–160) Sepharose. The Western blot of eluted proteins from a representative experiment is shown in Fig. 4. The binding of CaM to Bcl10 was indeed found to inhibit the interaction between Bcl10 and Carma1, since increasing concentrations of CaM were shown to reduce this interaction in a dose-dependent manner (Fig. 4A). Interestingly, the
3.3. A Bcl10 mutant with reduced CaM binding We examined the homologous CARD domains of Bcl10 and Carma1, searching for differences between them that could allow binding of CaM to Bcl10 but not to Carma1. By aligning the CARD domain sequences of mouse Bcl10 and Carma1 (which both differ by only 2 amino acids between mice and humans), we could identify stretches of basic and hydrophobic amino acids in different parts of the CARD domain (Fig. 3A). Of special interest was the area indicated in Fig. 3B, where basic and hydrophobic stretches present in the Bcl10 sequence make it a putative CaM binding site, whereas the corresponding sequence in the CARD domain of Carma1 contains fewer basic and hydrophobic amino acids and also a proline, which—due to its geometry and poor flexibility—rarely takes part in an ␣-helix. These differences might explain why CaM binds to the CARD domain of Bcl10 but not to that of Carma1, if this indeed was the CaM binding site. Interestingly, from a comparison with other CARD domains (Koseki et al., 1999), this sequence is likely to include the sixth ␣-helix of the CARD domain of Bcl10. We made peptides corresponding to other sequences in the CARD domain with slight resemblances to CaM binding sites, but none of them showed any significant binding to CaM (data not shown). To cre-
Fig. 4. Binding of CaM to Bcl10 is inhibitory for the interaction between Bcl10 and Carma1. (A) Recombinant Bcl10 was preincubated with the indicated concentrations of CaM in the presence of Ca2+ , and subsequently bound to Carma1 (amino acids 1–160) Sepharose. Bound proteins were eluted and detected by Western blot. Ctrl is protein bound to empty control Sepharose beads. The amounts of Carma1 eluted from the beads are shown as a control below. (B) Binding of recombinant wild-type Bcl10 or the R88A mutant of Bcl10 to Carma1 (amino acids 1–160) Sepharose with and without preincubation with 3 M CaM was analyzed as in A.
S. Edin et al. / Molecular Immunology 47 (2010) 2057–2064
2061
Fig. 5. In vivo proximity of CaM and Bcl10 that increases with increased intracellular Ca2+ level. Detection of proximal location of CaM and Bcl10 (shown as red dots) by in situ proximity ligation assays (PLA) was as described in Section 2. Jurkat cells were stimulated for 10 min with PMA, ionomycin or PMA plus ionomycin or left unstimulated. In controls either the ␣-Bcl10 or ␣-CaM primary antibody were omitted in PLA of unstimulated cells. All samples were counterstained with Hoechst 33342 dye (blue) to visualize nuclei.
inhibitory effect of CaM was lost in the R88A mutant (Fig. 4B), suggesting that Ca2+ signaling through CaM regulates the interaction between Bcl10 and Carma1, and thereby also activation of NF-B. 3.5. In vivo proximity of CaM and Bcl10 is induced by an increased Ca2+ level The strong Ca2+ -dependent binding between CaM and Bcl10 in vitro indicated that they would interact also in vivo. To analyze if Ca2+ signaling could induce proximity between CaM and Bcl10 also in vivo, we performed in situ proximity ligation assays (PLA). Jurkat T cells were stained using antibodies against CaM and Bcl10 and secondary antibodies conjugated with oligonucleotides (PLA
probes) to generate fluorescence signals only when the two PLA probe marked proteins are in close proximity (≤40 nm). The fluorescence signal results in a distinct individual red dot (Jarvius et al., 2007; Söderberg et al., 2006, 2008, 2007). The number of dots was significantly higher than in controls where either the ␣-Bcl10 or ␣CaM primary antibody was omitted (Fig. 5). To analyze if increased proximity was induced by stimulation, the Jurkat cells were treated for 10 min with PMA, with ionomycin—which increases the intracellular Ca2+ level—or with PMA plus ionomycin (Fig. 5). Treatment with PMA or PMA plus ionomycin had a small effect on the number of PLA dots, whereas the stimulation with ionomycin increased the number of dots 3.4 ± 1.0-fold (the P-value for the increase was 0.01). Thus, the number of proximities between CaM and Bcl10 is
2062
S. Edin et al. / Molecular Immunology 47 (2010) 2057–2064
Fig. 6. In vivo proximity of CaM and wild-type but not mutant Bcl10 is induced by increased intracellular Ca2+ level. PLA was performed in a derivative of the Jurkat T cell line with strongly down-regulated expression of endogenous Bcl10 transiently transfected with expression vector for wild-type Bcl10, the R88A mutant of Bcl10, or empty vector together with GFP expression vector to detect transfected cells. Cells were either left unstimulated or stimulated with ionomycin for 10 min as indicated and harvested. Controls were unstimulated cells transfected with wild-type Bcl10 stained using ␣-Bcl10 alone, ␣-CaM alone, or no primary antibodies. Values shown are average numbers of proximities per successfully transfected cell ± s.d. Unstimulated cells transfected with expression vector for wild-type Bcl10 were set as 100% (n ≥ 3).
increased by treatment that increases the intracellular Ca2+ level, whereas the increase is less pronounced when the Ca2+ signal is combined with a PKC activating stimuli. Proximity between CaM and Bcl10 that increased upon increased intracellular Ca2+ level was also found in PLA with another pair of ␣-CaM and ␣-Bcl10 antibodies raised in other species (data not shown). To determine whether the R88A mutant of Bcl10 with defect CaM interaction in vitro was defect also in vivo, we compared the proximities of the wild-type and mutant Bcl10 proteins to CaM. PLA was performed in a derivative of the Jurkat T cell line with strongly down-regulated expression of endogenous Bcl10. After transient transfection of expression vector for wild-type Bcl10, the R88A mutant of Bcl10 or empty vector control together with GFP expression vector to detect transfected cells, the cells were either left unstimulated or stimulated with ionomycin for 10 min. The stimulation with ionomycin increased the number of dots approximately fourfold in the cells transfected with wild-type Bcl10, whereas no increase was detected in the cells transfected with the R88A mutant or with empty vector (Fig. 6). The P-value for this difference between wild-type and R88A mutated Bcl10 was <0.05. Thus, in contrast to wild-type Bcl10, the number of CaM proximities of the R88A mutant did not increase by the treatment that increases the intracellular Ca2+ level. 3.6. Binding of CaM to Bcl10 is inhibitory for NF-B activation in Jurkat T cells To investigate the functional role of the binding between CaM and Bcl10, we expressed either wild-type Bcl10 or the R88A mutant of Bcl10 together with an NF-B-dependent reporter in a derivative of the Jurkat T cell line in which endogenous Bcl10 had been strongly down-regulated. We titrated the level of transfected Bcl10 plasmid so we restored the Bcl10 expression level of the parental Jurkat cells, as verified by Western, and the parental level of induction by the PKC activating phorbol ester PMA, as verified by the reporter assay (data not shown). This expression of wild-type Bcl10 activated NF-B and the increase of this activation by stimulation with PMA was further enhanced in combination with the calcium ionophore ionomycin, mimicking T cell receptor activation (Fig. 7). Importantly, expression of the R88A mutant of Bcl10 activated NF-
Fig. 7. The R88A mutant of Bcl10 gives increased activation of an NF-B-dependent reporter in T cells. Wild-type Bcl10, the R88A mutant of Bcl10, or empty vector were transiently expressed together with an NF-B-dependent reporter in a derivative of the Jurkat T cell line in which expression of endogenous Bcl10 had been strongly down-regulated. Twenty-four hours after transfection, the cells were either left unstimulated (U) or stimulated with PMA (P) or PMA plus ionomycin (P + I) for 2 h and harvested. Values shown are normalized fold activations ± s.d. (n = 3) compared to untreated cells transfected with empty vector.
B more potently than wild-type Bcl10, both in the presence or absence of cellular stimulation (Fig. 7). Equal expression level of wild-type and mutant Bcl10 was verified by Western blot (data not shown). The mutant was found to activate the NF-B reporter 1.5–2-fold more than wild-type protein, either without PMA, with PMA, or with PMA plus ionomycin (the P-values were 0.07, 0.03, and 0.04, respectively). The corresponding effect of the mutation on activation of NF-B was also obtained upon over-expression of Bcl10 in the parental Jurkat T cells, where endogenous levels of Bcl10 were intact (data not shown). Stimulation with ionomycin alone had no significant effect on activation of the NF-B reporter in cells transfected with either wild-type or R88A mutated Bcl10 or vector control (Suppl. Fig. 1). In summary, these results support the notion that binding of CaM to Bcl10 has a negative effect on NF-B activation in vivo, which probably occurs through inhibition of the interaction between Bcl10 and Carma1. 4. Discussion Here we have presented results that Ca2+ signals from the TCR can negatively regulate NF-B activation through binding of CaM to Bcl10. We have shown that CaM interacts specifically and in a Ca2+ -dependent manner with Bcl10, with a binding strength that is of high biological relevance (with a Kd of approximately 160 nM). The proximity of Bcl10 and CaM was induced in vivo by an increased intracellular Ca2+ level. However, the binding was found to be less evident when the Ca2+ signal was accompanied by PKC activating stimuli. Furthermore, the interaction of CaM with Bcl10 was found to have a negative effect on the interaction between the CARD domains of Bcl10 and Carma1, an interaction that is required for signaling from the TCR to NF-B. A Bcl10 mutant with reduced ability to bind CaM was still found to be capable of interacting with Carma1, but this interaction was unaffected by the presence of CaM. Importantly, the CaM binding mutant of Bcl10 activated NFB in T cells in a more potent way, supporting the notion that CaM binding to Bcl10 negatively regulates NF-B activation in T cells by preventing the Bcl10–Carma1 interaction. The interaction between CaM and Bcl10 showed a sigmoidal binding curve, indicating cooperative binding—something that is typically seen for the classical wrap-around mode of binding of CaM to its targets (Bhattacharya et al., 2004; Ishida and Vogel, 2006). It is common among classical CaM target motifs to have
S. Edin et al. / Molecular Immunology 47 (2010) 2057–2064
two hydrophobic residues acting as anchors at each end of the sequence. The length of the intervening sequence has been used to classify CaM targets into either 1–10, 1–14, or 1–16 recognition motifs (Ishida and Vogel, 2006; Yamniuk and Vogel, 2004). The sequence identified in Bcl10 contains several potential hydrophobic anchors with 1–14 spacing (amino acids L82–L95, V83–I96, I86–I99) or 1–16 spacing (L79–F94), and with several intervening basic and hydrophobic amino acids (Fig. 3B), which fits the features of known CaM target motifs. However, CaM has been shown to bind also to target sequences not resembling these motifs. Thus, the binding mode and detailed structure of the CaM–Bcl10 interaction will have to await future studies. However, a point mutation (R88A) within the basic patch of the sequence did indeed reduce the binding of CaM by about 65%, supporting the idea that this is the domain to which CaM binds. The binding of CaM is, as discussed above, likely to occur at the predicted sixth ␣-helix of the CARD domain of Bcl10. The structure of the CARD–CARD interaction between Bcl10 and Carma1 is not available at present, and how binding of CaM at the predicted sixth ␣-helix would be inhibitory to this interaction remains to be determined. In support of our finding that the sixth ␣-helix is important for regulation of CARD–CARD interactions, Marasco and co-workers have, by using inhibitory peptides, identified the amino acids 91–98 of Bcl10 as important for its self-association as well as its interaction with external partners in NF-B activation (Marasco et al., 2009). The CaM binding mutant of Bcl10 activated the NF-B reporter 1.5–2 times more potently than the wild-type protein in T cells. The effect on NF-B activity was seen in cells with or without intact endogenous level of Bcl10, and both in the presence and absence of PMA and ionomycin stimulation. This 1.5–2-fold effect of the mutant could represent the level of the negative effect of CaM binding. However, the mutant Bcl10 retained 35% of the CaM binding capacity (Fig. 3C), which makes it likely that a complete loss of CaM binding would lead to a greater increase in NF-B activation. In addition, the positive effect of losing the CaM binding could be underestimated if the R88A mutant leads to a hypothetical negative effect on another function of the protein in the activation process. The full extent of the negative effect of CaM binding on NF-B activation is therefore unknown. Stimulation with ionomycin alone had no effect on the ability of wild-type or CaM binding mutant of Bcl10 to activate the NF-B reporter. This supports that Ca2+ changes induced by ionomycin alone favor a negative role of CaM in NF-B signaling and that additional PKC activating stimuli are required for NF-B activation. When PKC is activated, the negative role of the CaM–Bcl10 interaction appears to be modulatory in character, regulating the degree of NF-B activation. An interaction of the CARD domain of Bcl10 with Malt1 has recently been reported (Langel et al., 2008). They searched for mutations of amino acids within the CARD domain that disrupted the binding of Bcl10 to Malt1. Interestingly, one of the mutations analyzed was on a neighbor of R88, R87. The R87A mutant was, similarly to our R88A mutant, found to increase NF-B activation by around 2-fold, whereas its ability to bind Carma1 and Malt1 was unaffected. Thus, the CARD domain of the R87A and R88A mutants is likely to be structurally intact, and the effect of the R87A mutant supports that this segment of the protein is subjected to negative regulation, with binding of CaM being the likely mechanism. Changes in intracellular Ca2+ levels have an important role in the regulation of T cell activation, and the pattern of Ca2+ responses influences T cell commitment towards activation or anergy (Feske, 2007; Randriamampita and Trautmann, 2004). We have previously reported a requirement for CaM and CaMKII in activation of IKK and NF-B after TCR stimulation (Hughes et al., 1998, 2001). The CaMKII dependence of IKK activation has been shown to be at least partly due to phosphorylation of Carma1, which improves its interaction with Bcl10 (Ishiguro et al., 2006). Thus, from the present results it
2063
appears that CaM can have both positive and negative roles in signaling from the TCR to NF-B activation. What could be the function of CaM binding to Bcl10, considering that this negatively affects its interaction with Carma1 and thereby its ability to activate NF-B? Different patterns of Ca2+ signaling can lead to quite distinct outcomes, in particular the activation of different transcription factors. Complete TCR stimulations result in high Ca2+ transients that activate NF-B, whereas TCR activation without co-stimulatory signals results in sustained but weaker Ca2+ signals. These more modest Ca2+ signals are sufficient to activate other factors but not NF-B, thereby inducing a state of T cell anergy (Jun and Goodnow, 2003; Macian et al., 2004; Quintana et al., 2005; Randriamampita and Trautmann, 2004). The negative effect of CaM on NF-B activation reported here might therefore be of importance during incomplete TCR stimulation, where an anergic state of the cell is induced and activation of NF-B therefore should not be allowed. In contrast, when a proper immunological synapse is formed, the concert of signaling events induced—which includes high fluctuations in Ca2+ —allows CaM to positively regulate NF-B, at least in part through activation of CaMKII, which is highly regulated by such Ca2+ changes. In line with the multiple modes of Ca2+ /CaM modulation of NF-B activation, CaM has also been shown to regulate the transcriptional activity of NF-B both by direct interaction with the NF-B proteins c-Rel and RelA (and thereby differential regulation of their nuclear translocation and activation) (Antonsson et al., 2003) and by activation of CaMKIV, which, through phosphorylation of RelA (p65), increases the activity of this transcription factor (Bae et al., 2003; Jang et al., 2001). Ca2+ and CaM are thus involved in the regulation of NF-B at several different levels. Taken together, the results in this report support the notion that by sensing certain changes in intracellular Ca2+ , CaM can interact with Bcl10 and thereby negatively regulate TCR-induced NF-B activation. Acknowledgments We thank Göran Larsson and Peter Gimeson at the Department of Medical Chemistry, Umeå University, for excellent guidance during the spectrofluorimetric analysis of the CaM–Bcl10 interaction. The Bcl10 knockdown cell line was a kind gift from Drs. Chuan-Jin Wu and Jonathan D. Ashwell. This work was supported by grants from the Swedish Research Council and the Swedish Cancer Society to T.G. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.molimm.2010.04.005. References Antonsson, Å., Hughes, K., Edin, S., Grundström, T., 2003. Regulation of c-Rel nuclear localization by binding of Ca2+ /calmodulin. Mol. Cell. Biol. 23, 1418–1427. Bae, J.S., Jang, M.K., Hong, S., An, W.G., Choi, Y.H., Kim, H.D., Cheong, J., 2003. Phosphorylation of NF-B by calmodulin-dependent kinase IV activates anti-apoptotic gene expression. Biochem. Biophys. Res. Commun. 305, 1094–1098. Bhattacharya, S., Bunick, C.G., Chazin, W.J., 2004. Target selectivity in EF-hand calcium binding proteins. Biochim. Biophys. Acta 1742, 69–79. Bouchier-Hayes, L., Martin, S.J., 2002. CARD games in apoptosis and immunity. EMBO Rep. 3, 616–621. Caamano, J., Hunter, C.A., 2002. NF-B family of transcription factors: central regulators of innate and adaptive immune functions. Clin. Microbiol. Rev. 15, 414–429. Chin, D., Means, A.R., 2000. Calmodulin: a prototypical calcium sensor. Trends Cell Biol. 10, 322–328. Chou, J.J., Matsuo, H., Duan, H., Wagner, G., 1998. Solution structure of the RAIDD CARD and model for CARD/CARD interaction in caspase-2 and caspase-9 recruitment. Cell 94, 171–180. Corneliussen, B., Holm, M., Waltersson, Y., Onions, J., Hallberg, B., Thornell, A., Grundström, T., 1994. Calcium/calmodulin inhibition of basic-helix–loop–helix transcription factor domains. Nature 368, 760–764.
2064
S. Edin et al. / Molecular Immunology 47 (2010) 2057–2064
Day, C.L., Dupont, C., Lackmann, M., Vaux, D.L., Hinds, M.G., 1999. Solution structure and mutagenesis of the caspase recruitment domain (CARD) from Apaf-1. Cell Death Differ. 6, 1125–1132. Feske, S., 2007. Calcium signalling in lymphocyte activation and disease. Nat. Rev. Immunol. 7, 690–702. Hayden, M.S., Ghosh, S., 2008. Shared principles in NF-B signaling. Cell 132, 344–362. Hughes, K., Antonsson, Å., Grundström, T., 1998. Calmodulin dependence of NF-B activation. FEBS Lett. 441, 132–136. Hughes, K., Edin, S., Antonsson, Å., Grundström, T., 2001. Calmodulin-dependent kinase II mediates T cell receptor/CD3- and phorbol ester-induced activation of IB kinase. J. Biol. Chem. 276, 36008–36013. Ishida, H., Vogel, H.J., 2006. Protein–peptide interaction studies demonstrate the versatility of calmodulin target protein binding. Protein Pept. Lett. 13, 455–465. Ishiguro, K., Green, T., Rapley, J., Wachtel, H., Giallourakis, C., Landry, A., Cao, Z., Lu, N., Takafumi, A., Goto, H., Daly, M.J., Xavier, R.J., 2006. Ca2+ /calmodulin-dependent protein kinase II is a modulator of CARMA1-mediated NF-B activation. Mol. Cell. Biol. 26, 5497–5508. Jang, M.K., Goo, Y.H., Sohn, Y.C., Kim, Y.S., Lee, S.K., Kang, H., Cheong, J., Lee, J.W., 2001. Ca2+ /calmodulin-dependent protein kinase IV stimulates nuclear factorB transactivation via phosphorylation of the p65 subunit. J. Biol. Chem. 276, 20005–20010. Jarvius, M., Paulsson, J., Weibrecht, I., Leuchowius, K.J., Andersson, A.C., Wählby, C., Gullberg, M., Botling, J., Sjöblom, T., Markova, B., Östman, A., Landegren, U., Söderberg, O., 2007. In situ detection of phosphorylated platelet-derived growth factor receptor beta using a generalized proximity ligation method. Mol. Cell. Proteomics 6, 1500–1509. Jun, J.E., Goodnow, C.C., 2003. Scaffolding of antigen receptors for immunogenic versus tolerogenic signaling. Nat. Immunol. 4, 1057–1064. Kincaid, R.L., Vaughan, M., Osborne Jr., J.C., Tkachuk, V.A., 1982. Ca2+ -dependent interaction of 5-dimethylaminonaphthalene-1-sulfonyl-calmodulin with cyclic nucleotide phosphodiesterase, calcineurin, and troponin I. J. Biol. Chem. 257, 10638–10643. Koseki, T., Inohara, N., Chen, S., Carrio, R., Merino, J., Hottiger, M.O., Nabel, G.J., Nunez, G., 1999. CIPER, a novel NF-B-activating protein containing a caspase recruitment domain with homology to Herpesvirus-2 protein E10. J. Biol. Chem. 274, 9955–9961. Langel, F.D., Jain, N.A., Rossman, J.S., Kingeter, L.M., Kashyap, A.K., Schaefer, B.C., 2008. Multiple protein domains mediate interaction between Bcl10 and MALT1. J. Biol. Chem. 283, 32419–32431. Liu, Y., Dong, W., Chen, L., Zhang, P., Qi, Y., 2004. Characterization of Bcl10 as a potential transcriptional activator that interacts with general transcription factor TFIIB. Biochem. Biophys. Res. Commun. 320, 1–6. Lucas, P.C., Yonezumi, M., Inohara, N., McAllister-Lucas, L.M., Abazeed, M.E., Chen, F.F., Yamaoka, S., Seto, M., Nunez, G., 2001. Bcl10 and MALT1, independent targets of chromosomal translocation in malt lymphoma, cooperate in a novel NF-B signaling pathway. J. Biol. Chem. 276, 19012–19019. Macian, F., Im, S.H., Garcia-Cozar, F.J., Rao, A., 2004. T-cell anergy. Curr. Opin. Immunol. 16, 209–216.
Marasco, D., Stilo, R., Sandomenico, A., Monti, S.M., Tizzano, B., de Capua, A., Varricchio, E., Liguoro, D., Zotti, T., Formisano, S., Ruvo, M., Vito, P., 2009. Generation and functional characterization of a BCL10-inhibitory peptide that represses NF-kappaB activation. Biochem. J. 422, 553–561. Matsumoto, R., Wang, D., Blonska, M., Li, H., Kobayashi, M., Pappu, B., Chen, Y., Lin, X., 2005. Phosphorylation of CARMA1 plays a critical role in T cell receptormediated NF-B activation. Immunity 23, 575–585. Onions, J., Hermann, S., Grundström, T., 1997. Basic helix–loop–helix protein sequences determining differential inhibition by calmodulin and S-100 proteins. J. Biol. Chem. 272, 23930–23937. Park, H.H., Lo, Y.C., Lin, S.C., Wang, L., Yang, J.K., Wu, H., 2007. The death domain superfamily in intracellular signaling of apoptosis and inflammation. Annu. Rev. Immunol. 25, 561–586. Quintana, A., Griesemer, D., Schwarz, E.C., Hoth, M., 2005. Calcium-dependent activation of T-lymphocytes. Pflugers Arch. 450, 1–12. Randriamampita, C., Trautmann, A., 2004. Ca2+ signals and T lymphocytes; “new mechanisms and functions in Ca2+ signalling”. Biol. Cell 96, 69–78. Rawlings, D.J., Sommer, K., Moreno-Garcia, M.E., 2006. The CARMA1 signalosome links the signalling machinery of adaptive and innate immunity in lymphocytes. Nat. Rev. Immunol. 6, 799–812. Rueda, D., Thome, M., 2005. Phosphorylation of CARMA1: the link(er) to NF-B activation. Immunity 23, 551–553. Schulze-Luehrmann, J., Ghosh, S., 2006. Antigen–receptor signaling to nuclear factor B. Immunity 25, 701–715. Sommer, K., Guo, B., Pomerantz, J.L., Bandaranayake, A.D., Moreno-Garcia, M.E., Ovechkina, Y.L., Rawlings, D.J., 2005. Phosphorylation of the CARMA1 linker controls NF-B activation. Immunity 23, 561–574. Söderberg, O., Gullberg, M., Jarvius, M., Ridderstråle, K., Leuchowius, K.J., Jarvius, J., Wester, K., Hydbring, P., Bahram, F., Larsson, L.G., Landegren, U., 2006. Direct observation of individual endogenous protein complexes in situ by proximity ligation. Nat. Methods 3, 995–1000. Söderberg, O., Leuchowius, K.J., Gullberg, M., Jarvius, M., Weibrecht, I., Larsson, L.G., Landegren, U., 2008. Characterizing proteins and their interactions in cells and tissues using the in situ proximity ligation assay. Methods 45, 227– 232. Söderberg, O., Leuchowius, K.J., Kamali-Moghaddam, M., Jarvius, M., Gustafsdottir, S., Schallmeiner, E., Gullberg, M., Jarvius, J., Landegren, U., 2007. Proximity ligation: a specific and versatile tool for the proteomic era. Genet. Eng. (N. Y.) 28, 85–93. Thome, M., 2004. CARMA1, BCL-10 and MALT1 in lymphocyte development and activation. Nat. Rev. Immunol. 4, 348–359. Weil, R., Israel, A., 2004. T-cell-receptor- and B-cell-receptor-mediated activation of NF-B in lymphocytes. Curr. Opin. Immunol. 16, 374–381. Wu, C.J., Ashwell, J.D., 2008. NEMO recognition of ubiquitinated Bcl10 is required for T cell receptor-mediated NF-B activation. Proc. Natl. Acad. Sci. U.S.A. 105, 3023–3028. Yamniuk, A.P., Vogel, H.J., 2004. Calmodulin’s flexibility allows for promiscuity in its interactions with target proteins and peptides. Mol. Biotechnol. 27, 33–57.