Regulation of the DNA Repair Complex during Somatic Hypermutation and Class-Switch Recombination.

B lymphocytes optimize Ab responses by somatic hypermutation (SH), which introduces point mutations in the variable regions of the Ab genes and by class-switch recombination (CSR), which changes the expressed C region exon of the IgH. These Ab diversification processes are initiated by the deaminating enzyme activation-induced cytidine deaminase followed by many DNA repair enzymes, ultimately leading to deletions and a high mutation rate in the Ab genes, whereas DNA lesions made by activation-induced cytidine deaminase are repaired with low error rate on most other genes. This indicates an advanced regulation of DNA repair. In this study, we show that initiation of Ab diversification in B lymphocytes of mouse spleen leads to formation of a complex between many proteins in DNA repair. We show also that BCR activation, which signals the end of successful SH, reduces interactions between some proteins in the complex and increases other interactions in the complex with varying kinetics. Furthermore, we show increased localization of SH- and CSR-coupled proteins on switch regions of the Igh locus upon initiation of SH/CSR and differential changes in the localization upon BCR signaling, which terminates SH. These findings provide early evidence for a DNA repair complex or complexes that may be of functional significance for carrying out essential roles in SH and/or CSR in B cells.

ETS1 and PAX5 transcription factors recruit AID to Igh DNA.

B lymphocytes optimize antibody responses by class switch recombination (CSR), which changes the expressed constant region exon of the immunoglobulin heavy chain (IgH), and by somatic hypermutation (SH) that introduces point mutations in the variable regions of the antibody genes. Activation-induced cytidine deaminase (AID) is the key mutagenic enzyme that initiates both these antibody diversification processes by deaminating cytosine to uracil. Here we asked the question if transcription factors can mediate the specific targeting of the antibody diversification by recruiting AID. We have recently reported that AID is together with the transcription factors E2A, PAX5 and IRF4 in a complex on key sequences of the Igh locus. Here we report that also ETS1 is together with AID in this complex on key sequences of the Igh locus in splenic B cells of mice. Furthermore, we show that both ETS1 and PAX5 can directly recruit AID to DNA sequences from the Igh locus with the specific binding site for the transcription factor. Taken together, our findings support the notion of a targeting mechanism for the selective diversification of antibody genes with limited genome wide mutagenesis by recruitment of AID by PAX5 and ETS1 in a transcription factor complex.

Allelic exclusion of IgH through inhibition of E2A in a VDJ recombination complex.

A key feature of the immune system is the paradigm that one lymphocyte has only one Ag specificity that can be selected for or against. This requires that only one of the alleles of genes for AgR chains is made functional. However, the molecular mechanism of this allelic exclusion has been an enigma. In this study, we show that B lymphocytes with E2A that cannot be inhibited by calmodulin are dramatically defective in allelic exclusion of the IgH locus. Furthermore, we provide data supporting that E2A, PAX5, and the RAGs are in a VDJ recombination complex bound to key sequences on the Igh gene. We show that pre-BCR activation releases the VDJ recombination complex through calmodulin binding to E2A. We also show that pre-BCR signaling downregulates several components of the recombination machinery, including RAG1, RAG2, and PAX5, through calmodulin inhibition of E2A.

Negative feedback regulation of antigen receptors through calmodulin inhibition of E2A.

Signaling from the BCR is used to judge Ag-binding strengths of the Abs of B cells. BCR signaling enables the selection for successive improvements in the Ag affinity over an extremely broad range of affinities during somatic hypermutation. We show that the mouse BCR is subject to general negative feedback regulation of the receptor proteins, as well as many coreceptors and proteins in signal pathways from the receptor. Thus, the BCR can downregulate itself, which can enable sensitive detection of successive improvements in the Ag affinity over a very large span of affinities. Furthermore, the feedback inhibition of the BCR signalosome and most of its proteins, as well as most other regulations of genes by BCR stimulation, is to a large extent through inhibition of the transcription factor E2A by Ca(2+)/calmodulin.

The transcription factor E2A can bind to and cleave single-stranded immunoglobulin heavy chain locus DNA.

Class switch recombination (CSR) changes the constant region of the immunoglobulin heavy chain (IgH), and somatic hypermutation (SH) introduces point mutations in the variable regions of the antibody genes. Both these processes that optimize antibody responses of B lymphocytes are initiated by the enzyme Activation Induced cytidine Deaminase (AID). Here we have searched for CSR or SH coupled activities of the transcription factor E2A, since E2A is in a complex with AID and the transcription factors PAX5, ETS1 and IRF4 on key sequences of the Igh locus in B lymphocytes activated to CSR and SH. We report that E2A in contrast to other described transcription factors binds sequence specifically also to single-stranded DNA. The binding of E2A to single-stranded DNA has a strong sequence preference for one strand of a site in the intronic enhancer of the Igh locus. Furthermore, E2A was also found to cleave single-stranded DNA. The sequence profile of substrates cleaved by E2A is coupled to the sequences of substrates and products of AID, suggesting that E2A has a role not only in targeting of AID to switch regions and SH parts of antibody genes but also in cleavage of DNA at these sites.

Induction of TLR4-target genes entails calcium/calmodulin-dependent regulation of chromatin remodeling.

Upon toll-like receptor 4 (TLR4) signaling in macrophages, the mammalian Swi/Snf-like BAF chromatin remodeling complex is recruited to many TLR4 target genes where it remodels their chromatin to promote transcription. Here, we show that, surprisingly, recruitment is not sufficient for chromatin remodeling; a second event, dependent on calcium/calmodulin (CaM), is additionally required. Calcium/CaM directly binds the HMG domain of the BAF57 subunit within the BAF complex. Calcium/CaM antagonists, including a CaM-binding peptide derived from BAF57, abolish BAF-dependent remodeling and gene expression without compromising BAF recruitment. BAF57 RNAi and BAF57 dominant negative mutants defective in CaM binding similarly impair the induction of BAF target genes. Our data implicate calcium/CaM in TLR4 signaling, and reveal a previously undescribed, recruitment-independent mode of regulation of the BAF complex that is probably achieved through a direct CaM-BAF interaction.

PIP5K1α is Required for Promoting Tumor Progression in Castration-Resistant Prostate Cancer.

PIP5K1α has emerged as a promising drug target for the treatment of castration-resistant prostate cancer (CRPC), as it acts upstream of the PI3K/AKT signaling pathway to promote prostate cancer (PCa) growth, survival and invasion. However, little is known of the molecular actions of PIP5K1α in this process. Here, we show that siRNA-mediated knockdown of PIP5K1α and blockade of PIP5K1α action using its small molecule inhibitor ISA-2011B suppress growth and invasion of CRPC cells. We demonstrate that targeted deletion of the N-terminal domain of PIP5K1α in CRPC cells results in reduced growth and migratory ability of cancer cells. Further, the xenograft tumors lacking the N-terminal domain of PIP5K1α exhibited reduced tumor growth and aggressiveness in xenograft mice as compared to that of controls. The N-terminal domain of PIP5K1α is required for regulation of mRNA expression and protein stability of PIP5K1α. This suggests that the expression and oncogenic activity of PIP5K1α are in part dependent on its N-terminal domain. We further show that PIP5K1α acts as an upstream regulator of the androgen receptor (AR) and AR target genes including CDK1 and MMP9 that are key factors promoting growth, survival and invasion of PCa cells. ISA-2011B exhibited a significant inhibitory effect on AR target genes including CDK1 and MMP9 in CRPC cells with wild-type PIP5K1α and in CRPC cells lacking the N-terminal domain of PIP5K1α. These results indicate that the growth of PIP5K1α-dependent tumors is in part dependent on the integrity of the N-terminal sequence of this kinase. Our study identifies a novel functional mechanism involving PIP5K1α, confirming that PIP5K1α is an intriguing target for cancer treatment, especially for treatment of CRPC.

FcγRIIIa receptor interacts with androgen receptor and PIP5K1α to promote growth and metastasis of prostate cancer.

Low-affinity immunoglobulin gamma Fc region receptor III-A (FcγRIIIa) is a cell surface protein that belongs to a family of Fc receptors that facilitate the protective function of the immune system against pathogens. However, the role of FcγRIIIa in prostate cancer (PCa) progression remained unknown. In this study, we found that FcγRIIIa expression was present in PCa cells and its level was significantly higher in metastatic lesions than in primary tumors from the PCa cohort (P = 0.006). PCa patients with an elevated level of FcγRIIIa expression had poorer biochemical recurrence (BCR)-free survival compared with those with lower FcγRIIIa expression, suggesting that FcγRIIIa is of clinical importance in PCa. We demonstrated that overexpression of FcγRIIIa increased the proliferative ability of PCa cell line C4-2 cells, which was accompanied by the upregulation of androgen receptor (AR) and phosphatidylinositol-4-phosphate 5-kinase alpha (PIP5Kα), which are the key players in controlling PCa progression. Conversely, targeted inhibition of FcγRIIIa via siRNA-mediated knockdown or using its inhibitory antibody suppressed growth of xenograft PC-3 and PC-3M prostate tumors and reduced distant metastasis in xenograft mouse models. We further showed that elevated expression of AR enhanced FcγRIIIa expression, whereas inhibition of AR activity using enzalutamide led to a significant downregulation of FcγRIIIa protein expression. Similarly, inhibition of PIP5K1α decreased FcγRIIIa expression in PCa cells. FcγRIIIa physically interacted with PIP5K1α and AR via formation of protein-protein complexes, suggesting that FcγRIIIa is functionally associated with AR and PIP5K1α in PCa cells. Our study identified FcγRIIIa as an important factor in promoting PCa growth and invasion. Further, the elevated activation of FcγRIII and AR and PIP5K1α pathways may cooperatively promote PCa growth and invasion. Thus, FcγRIIIa may serve as a potential new target for improved treatment of metastatic and castration-resistant PCa.

Initiation of antigen receptor-dependent differentiation into plasma cells by calmodulin inhibition of E2A.

Differentiation of B lymphocytes into Ab-secreting plasmablasts and plasma cells is Ag driven. The interaction of Ag with the membrane-bound Ab of the BCR is critical in determining which clones enter the plasma cell response. However, not much is known about the coupling between BCR activation and the shift in transcription factor network from that of a B cell to that of ASC differentiation. Our genome-wide analysis shows that Ab-secreting cell differentiation of mouse B cells is induced by BCR activation through very fast regulatory events from the BCR. We identify activation of IFN regulatory factor-4 and down-regulation of Pax5, Bcl-6, MITF, Ets-1, Fli-1, and Spi-B gene expression as immediate early events. Furthermore, the transcription factor E2A is required for the rapid key down-regulations after BCR activation, and the Ca(2+) sensor protein calmodulin has the corresponding regulatory effect as BCR activation. Moreover, mutants in the calmodulin binding site of E2A show that Ca(2+) signaling through calmodulin inhibition of E2A is essential for the rapid down-regulation of immediate early genes after BCR activation in initiation of plasma cell differentiation.

B-cell receptor activation inhibits AID expression through calmodulin inhibition of E-proteins.

Upon encountering antigens, B-lymphocytes can adapt to produce a highly specific and potent antibody response. Somatic hypermutation, which introduces point mutations in the variable regions of antibody genes, can increase the affinity for antigen, and antibody effector functions can be altered by class switch recombination (CSR), which changes the expressed constant region exons. Activation-induced cytidine deaminase (AID) is the mutagenic antibody diversification enzyme that is essential for both somatic hypermutation and CSR. The mutagenic AID enzyme has to be tightly controlled. Here, we show that engagement of the membrane-bound antibodies of the B-cell receptor (BCR), which signals that good antibody affinity has been reached, inhibits AID gene expression and that calcium (Ca(2+)) signaling is essential for this inhibition. Moreover, we show that overexpression of the Ca(2+) sensor protein calmodulin inhibits AID gene expression, and that the transcription factor E2A is required for regulation of the AID gene by the BCR. E2A mutated in the binding site for calmodulin, and thus showing calmodulin-resistant DNA binding, makes AID expression resistant to the inhibition through BCR activation. Thus, BCR activation inhibits AID gene expression through Ca(2+)/calmodulin inhibition of E2A.

Targeted inhibition of ERα signaling and PIP5K1α/Akt pathways in castration-resistant prostate cancer.

Selective ERα modulator, tamoxifen, is well tolerated in a heavily pretreated castration-resistant prostate cancer (PCa) patient cohort. However, its targeted gene network and whether expression of intratumor ERα due to androgen deprivation therapy (ADT) may play a role in PCa progression is unknown. In this study, we examined the inhibitory effect of tamoxifen on castration-resistant PCa in vitro and in vivo. We found that tamoxifen is a potent compound that induced a high degree of apoptosis and significantly suppressed growth of xenograft tumors in mice, at a degree comparable to ISA-2011B, an inhibitor of PIP5K1α that acts upstream of PI3K/AKT survival signaling pathway. Moreover, depletion of tumor-associated macrophages using clodronate in combination with tamoxifen increased inhibitory effect of tamoxifen on aggressive prostate tumors. We showed that both tamoxifen and ISA-2011B exert their on-target effects on prostate cancer cells by targeting cyclin D1 and PIP5K1α/AKT network and the interlinked estrogen signaling. Combination treatment using tamoxifen together with ISA-2011B resulted in tumor regression and had superior inhibitory effect compared with that of tamoxifen or ISA-2011B alone. We have identified sets of genes that are specifically targeted by tamoxifen, ISA-2011B or combination of both agents by RNA-seq. We discovered that alterations in unique gene signatures, in particular estrogen-related marker genes are associated with poor patient disease-free survival. We further showed that ERα interacted with PIP5K1α through formation of protein complexes in the nucleus, suggesting a functional link. Our finding is the first to suggest a new therapeutic potential to inhibit or utilize the mechanisms related to ERα, PIP5K1α/AKT network, and MMP9/VEGF signaling axis, providing a strategy to treat castration-resistant ER-positive subtype of prostate cancer tumors with metastatic potential.

Establishment of Prostate Tumor Growth and Metastasis Is Supported by Bone Marrow Cells and Is Mediated by PIP5K1α Lipid Kinase.

Cancer cells facilitate growth and metastasis by using multiple signals from the cancer-associated microenvironment. However, it remains poorly understood whether prostate cancer (PCa) cells may recruit and utilize bone marrow cells for their growth and survival. Furthermore, the regulatory mechanisms underlying interactions between PCa cells and bone marrow cells are obscure. In this study, we isolated bone marrow cells that mainly constituted populations that were positive for CD11b and Gr1 antigens from xenograft PC-3 tumor tissues from athymic nu/nu mice. We found that the tumor-infiltrated cells alone were unable to form tumor spheroids, even with increased amounts and time. By contrast, the tumor-infiltrated cells together with PCa cells formed large numbers of tumor spheroids compared with PCa cells alone. We further utilized xenograft athymic nu/nu mice bearing bone metastatic lesions. We demonstrated that PCa cells were unable to survive and give rise to colony-forming units (CFUs) in media that were used for hematopoietic cell colony-formation unit (CFU) assays. By contrast, PC-3M cells survived when bone marrow cells were present and gave rise to CFUs. Our results showed that PCa cells required bone marrow cells to support their growth and survival and establish bone metastasis in the host environment. We showed that PCa cells that were treated with either siRNA for PIP5K1α or its specific inhibitor, ISA-2011B, were unable to survive and produce tumor spheroids, together with bone marrow cells. Given that the elevated expression of PIP5K1α was specific for PCa cells and was associated with the induced expression of VEGF receptor 2 in PCa cells, our findings suggest that cancer cells may utilize PIP5K1α-mediated receptor signaling to recruit growth factors and ligands from the bone marrow-derived cells. Taken together, our study suggests a new mechanism that enables PCa cells to gain proliferative and invasive advantages within their associated host microenvironment. Therapeutic interventions using PIP5K1α inhibitors may not only inhibit tumor invasion and metastasis but also enhance the host immune system.

Regulation of c-Rel nuclear localization by binding of Ca2+/calmodulin.

The NF-kappa B/Rel family of transcription factors participates in the control of a wide array of genes, including genes involved in embryonic development and regulation of immune, inflammation, and stress responses. In most cells, inhibitory I kappa B proteins sequester NF-kappa B/Rel in the cytoplasm. Cellular stimulation results in the degradation of I kappa B and modification of NF-kappa B/Rel proteins, allowing NF-kappa B/Rel to translocate to the nucleus and act on its target genes. Calmodulin (CaM) is a highly conserved, ubiquitously expressed Ca(2+) binding protein that serves as a key mediator of intracellular Ca(2+) signals. Here we report that two members of the NF-kappa B/Rel family, c-Rel and RelA, interact directly with Ca(2+)-loaded CaM. The interaction with CaM is greatly enhanced by cell stimulation, and this enhancement is blocked by addition of I kappa B. c-Rel and RelA interact with CaM through a similar sequence near the nuclear localization signal. Compared to the wild-type protein, CaM binding-deficient mutants of c-Rel exhibit increases in both nuclear accumulation and transcriptional activity on the interleukin 2 and granulocyte macrophage colony-stimulating factor promoters in the presence of a Ca(2+) signal. Conversely, for RelA neither nuclear accumulation nor transcriptional activity on these promoters is increased by mutation of the sequence interacting with CaM. Our results suggest that CaM binds c-Rel and RelA after their release from I kappa B and can inhibit nuclear import of c-Rel while letting RelA translocate to the nucleus and act on its target genes. CaM can therefore differentially regulate the activation of NF-kappa B/Rel proteins following stimulation.

Calcium regulation of GM-CSF by calmodulin-dependent kinase II phosphorylation of Ets1.

The multipotent cytokine granulocyte macrophage-colony stimulating factor (GM-CSF) is involved in particular in the physiological response to infection and in inflammatory responses. GM-CSF is produced by many cell types, including T lymphocytes responding to T-cell receptor activation and mantle zone B lymphocytes. B-cell receptor and T-cell receptor activation generates two major signals: an increase in intracellular Ca(2+) concentration and a protein kinase cascade. Previous studies have shown that the Ca(2+)/calmodulin-dependent phosphatase calcineurin mediates stimulation of GM-CSF transcription in response to Ca(2+). In this study, we show that Ca(2+) signaling also regulates GM-CSF transcription negatively through Ca(2+)/calmodulin-dependent kinase II (CaMK II) phosphorylation of serines in the autoinhibitory domain for DNA binding of the transcription factor Ets1. Wild-type Ets1 negatively affects GM-CSF transcription on Ca(2+) stimulation in the presence of cyclosporin A, which inhibits calcineurin. Conversely, Ets1 with mutated CaMK II target serines showed an increase in transactivation of the GM-CSF promoter/enhancer. Moreover, constitutively active CaMK II inhibited transactivation of GM-CSF by wild-type Ets1 but not by Ets1 with mutated CaMK II sites. Mutation of CaMK II target serines in Ets1 also relieves inhibition of cooperative transactivation of GM-CSF with the Runx1/AML1 transcription factor. In addition, the Ca(2+)-dependent phosphorylation of Ets1 reduces the binding of Ets1 to the GM-CSF promoter in vivo.

Regulated localization of an AID complex with E2A, PAX5 and IRF4 at the Igh locus.

Activation-induced cytidine deaminase (AID) is the key mutagenic enzyme that initiates somatic hypermutation (SH) and class switch recombination (CSR) by deaminating cytosine to uracil. The targeting of AID and therefore SH and CSR to Ig genes is a central process of the immune system, but the trans-acting factors mediating the specific targeting have remained elusive. Here we show that defective calmodulin inhibition of the transcription factor E2A after activation of the B cell receptor (BCR) leads to reduced BCR, IL4 plus CD40 ligand stimulated CSR to IgE and instead CSR to other Ig classes. AID that initiates CSR is shown to be in a complex with the transcription factors E2A, PAX5 and IRF4 on key sequences of the Igh locus. Calmodulin shows proximity with each of them after BCR stimulation. BCR signaling reduces binding of the proteins to some of the target sites on the Igh locus, and calmodulin resistance of E2A blocks these reductions. AID binds directly to the bHLH domain of E2A and to the PD domain of PAX5. E2A, AID, PAX5 and IRF4 are components of a CSR complex that is redistributed on the Igh locus by BCR signaling through calmodulin binding.

The RUNX1 Runt domain at 1.25A resolution: a structural switch and specifically bound chloride ions modulate DNA binding.

The evolutionarily conserved Runt homology domain is characteristic of the RUNX family of heterodimeric eukaryotic transcription factors, including RUNX1, RUNX2 and RUNX3. The genes for RUNX1, also termed acute myeloid leukemia protein 1, AML1, and its dimerization partner core-binding factor beta, CBFbeta, are essential for hematopoietic development and are together the most common targets for gene rearrangements in acute human leukemias. Here, we describe the crystal structure of the uncomplexed RUNX1 Runt domain at 1.25A resolution and compare its conformation to previously published structures in complex with DNA, CBFbeta or both. We find that complex formation induces significant structural rearrangements in this immunoglobulin (Ig)-like DNA-binding domain. Most pronounced is the movement of loop L11, which changes from a closed conformation in the free Runt structure to an open conformation in the CBFbeta-bound and DNA-bound forms. This transition, which we refer to as the S-switch, and accompanying structural movements that affect other parts of the Runt domain are crucial for sustained DNA binding. The closed to open transition can be induced by CBFbeta alone; suggesting that one role of CBFbeta is to trigger the S-switch and to stabilize the Runt domain in a conformation enhanced for DNA binding.A feature of the Runt domain hitherto unobserved in any Ig-like DNA-binding domain is the presence of two specifically bound chloride ions. One chloride ion is coordinated by amino acid residues that make direct DNA contact. In a series of electrophoretic mobility-shift analyses, we demonstrate a chloride ion concentration-dependent stimulation of the DNA-binding activity of Runt in the physiological range. A comparable DNA-binding stimulation was observed for negatively charged amino acid residues. This suggests a regulatory mechanism of RUNX proteins through acidic amino acid residues provided by activation domains during cooperative interaction with other transcription factors.

Expression and recruitment of uracil-DNA glycosylase are regulated by E2A during antibody diversification.

B-lymphocytes can modify their immunoglobulin (Ig) genes to generate specific antibodies with a new isotype and enhanced affinity against an antigen. Activation-induced cytidine deaminase (AID), which is positively regulated by the transcription factor E2A, is the key enzyme that initiates these processes by deaminating cytosine to uracil in Ig genes. Nuclear uracil-DNA glycosylase (UNG2) is subsequently required for uracil processing in the generation of high affinity antibodies of different isotypes. Here we show that the transcription factor E2A binds to the UNG2 promoter and represses UNG2 expression. Inhibition of E2A by binding of Ca(2+)-activated calmodulin alleviates this repression. Furthermore, we demonstrate that UNG2 preferentially accumulates in regions of the Ig heavy chain (IgH) gene containing AID hotspots. Calmodulin inhibition of E2A strongly enhances this UNG2 accumulation, indicating that it is negatively regulated by E2A as well. We show also that over-expression of E2A can suppress class switch recombination. The results suggest that E2A is a key factor in regulating the balance between AID and UNG2, both at expression and Ig targeting levels, to stimulate Ig diversification and suppress normal DNA repair processes.

Broad feedback inhibition of pre-B-cell receptor signaling components.

During B lymphocyte development, first immunoglobulin heavy chain gene segments and then immunoglobulin light chain gene segments are rearranged to create antibody diversity. Early in the development, expression of a pre-B-cell receptor (pre-BCR) that has membrane-bound Ig heavy chain protein associated with surrogate light chain (SLC) proteins serves as a critical checkpoint that monitors for functional heavy chain rearrangement. Signaling from the pre-BCR induces survival and clonal expansion to select cells with good heavy chains, but it also down-regulates transcription of the genes for the SLC proteins and CD19 and limits its own proliferative signaling. Here we have analyzed whether the down-regulation is limited to the SLC proteins and CD19, and we show that the pre-BCR of primary mouse pre-B-cells instead is subject to a broad feedback inhibition of pre-BCR signaling components. Activation of signaling leads to down-regulation of the receptor proteins, many co-receptors and proteins participating in signal pathways from the receptor. Thus the down-regulation of the pre-BCR is much broader than previously assumed. We also show that Ca(2+)/calmodulin inhibition of the transcription factor E2A is required for the feedback inhibition of the pre-BCR signaling proteins.

CaMKII targets Bcl10 in T-cell receptor induced activation of NF-κB.

Recognition of antigen by T- or B-cell receptors leads to formation of an immunological synapse and initiation of signalling events that collaborate to determine the nature of the adaptive immune response. Activation of NF-κB transcription factors has a key role in regulation of numerous genes with important functions in immune responses and inflammation and is of great importance for lymphocyte activation and differentiation. The activation of NF-κB depends on changes in intracellular Ca(2+) levels, and both calmodulin (CaM) and a CaM-dependent kinase, CaMKII, help regulate NF-κB activation after T-cell receptor (TCR) stimulation, but the mechanisms are not well characterized. Here we have analyzed the functional role of CaMKII in the signalling pathway from the TCR to activation of IKK, the kinase that phosphorylates the NF-κB inhibitor IκB. We show that CaMKII is recruited to the immunological synapse where it interacts with and phosphorylates the signalling adaptor protein Bcl10. Furthermore, phosphorylation of the CARD domain of Bcl10 by CaMKII regulates the interactions within the important Carma1, Bcl10, Malt1 signalling complex and the essential signal induced ubiquitinations of Bcl10 and IKKγ. We propose a novel mechanism whereby Ca(2+) signals can be integrated at the immunological synapse through CaMKII-dependent phosphorylation of Bcl10.