Antigen-specific stimulation of T helper (Th) cells initiates signaling cascades that ultimately result in the activation of the transcription factors NF-jB, NFAT, and AP-1 which regulate, together with other factors, many T-cell functions such as cytokine production, proliferation, and differentiation. Ordered assembly and different phosphorylation events, along with subcellular translocation of the CARMA1/Bcl-10/MALT1 complex, determine NF-jB activation after T-cell receptor (TCR) triggering. We now provide evidence that inhibition of the Ser/Thr phosphatase calcineurin (CaN) prevents dephosphorylation of Bcl-10. CaN, in constant interaction with the Bcl-10/MALT1 complex, is able to dephosphorylate Bcl-10. The CaN inhibitor cyclosporine A (CsA) converts a transient phosphorylation of Bcl-10 Ser138 during the immediate early phase of T-cell activation into a persistent state. Thus, subsequent processes such as IKKb phosphorylation, IjBa degradation, p65 nuclear translocation, and DNA binding are diminished. Consistently, CsA treatment does not affect the phosphorylation pattern of the upstream kinase PKCh. Together, our findings demonstrate that CaN functions as a critical signaling molecule during Th cell activation, regulating Bcl-10 phosphorylation and thereby NF-jB activation.Keywords: Calcineurin . Cell activation . T cell . TCR signaling . Transcription factors Supporting Information available online IntroductionAntigen-specific stimulation of T helper (Th) cells induces activation of the transcription factors nuclear factor of activated T cells (NFAT) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) via a complex signaling network. The cooperative action of these and other transcription factors results in gene expression profiles leading to the activation of Th cells, production of growth factors, in particular cytokines, and proliferation [1][2][3]. T-cell receptor (TCR) engagement triggers phosphorylation of membrane-associated tyrosine kinases that activate PLCg, which in turn generates the second messengers IP 3 and DAG. The IP 3 -induced Ca 21 influx triggers the Ser/Thr phosphatase calcineurin (CaN) which dephosphorylates NFAT. This dephosphorylation step is crucial for the nuclear translocation of NFAT and binding to promoters of target genes. The second messenger, DAG, activates the NF-kB signaling cascade by binding to the protein kinase C-y (PKCy) leading to a change in PKCy conformation into an open, active state. The precise nature of these changes is not entirely clear, but phosphorylation at different sites, subcellular translocation, and assembly of assorted molecules contributes to this process (reviewed in [4,5] [6,7]. The importance of Bcl-10 for NF-kB activation is underlined by studies in Bcl-10 knockout mice showing that Bcl-10 is essential for NF-kB-driven cytokine production, cell proliferation, and B-as well as T-cell development [8,9]. Furthermore, Bcl-10 overexpression in MALT B-cell lymphomas is characterized by constant NF-kB activation [10,11]. Bcl-1...

The cell biology of circadian clocks is still in its infancy. Here, we describe an efficient strategy for generating knock-in reporter cell lines using CRISPR technology that is particularly useful for genes expressed transiently or at low levels, such as those coding for circadian clock proteins. We generated single and double knock-in cells with endogenously expressed PER2 and CRY1 fused to fluorescent proteins allowing us to simultaneously monitor the dynamics of CRY1 and PER2 proteins in live single cells. Both proteins are highly rhythmic in the nucleus of human cells with PER2 showing a much higher amplitude than CRY1. Surprisingly, CRY1 protein is nuclear at all circadian times indicating the absence of circadian gating of nuclear import. Furthermore, in the nucleus of individual cells CRY1 abundance rhythms are phase-delayed (~5 hours), and CRY1 levels are much higher (>5 times) compared to PER2 questioning the current model of the circadian oscillator.