Sonia Sharma

Dual functions for the endoplasmic reticulum calcium sensors STIM1 and STIM2 in T cell activation and tolerance

Store-operated Ca2+ entry through calcium release–activated calcium channels is the chief mechanism for increasing intracellular Ca2+ in immune cells. Here we show that mouse T cells and fibroblasts lacking the calcium sensor STIM1 had severely impaired store-operated Ca2+ influx, whereas deficiency in the calcium sensor STIM2 had a smaller effect. However, T cells lacking either STIM1 or STIM2 had much less cytokine production and nuclear translocation of the transcription factor NFAT. T cell–specific ablation of both STIM1 and STIM2 resulted in a notable lymphoproliferative phenotype and a selective decrease in regulatory T cell numbers. We conclude that both STIM1 and STIM2 promote store-operated Ca2+ entry into T cells and fibroblasts and that STIM proteins are required for the development and function of regulatory T cells.

A genome-wide Drosophila RNAi screen identifies DYRK-family kinases as regulators of NFAT.

Precise regulation of the NFAT (nuclear factor of activated T cells) family of transcription factors (NFAT1–4) is essential for vertebrate development and function. In resting cells, NFAT proteins are heavily phosphorylated and reside in the cytoplasm; in cells exposed to stimuli that raise intracellular free Ca2+ levels, they are dephosphorylated by the calmodulin-dependent phosphatase calcineurin and translocate to the nucleus. NFAT dephosphorylation by calcineurin is countered by distinct NFAT kinases, among them casein kinase 1 (CK1) and glycogen synthase kinase 3 (GSK3). Here we have used a genome-wide RNA interference (RNAi) screen in Drosophila to identify additional regulators of the signalling pathway leading from Ca2+–calcineurin to NFAT. This screen was successful because the pathways regulating NFAT subcellular localization (Ca2+ influx, Ca2+–calmodulin–calcineurin signalling and NFAT kinases) are conserved across species, even though Ca2+-regulated NFAT proteins are not themselves represented in invertebrates. Using the screen, we have identified DYRKs (dual-specificity tyrosine-phosphorylation regulated kinases) as novel regulators of NFAT. DYRK1A and DYRK2 counter calcineurin-mediated dephosphorylation of NFAT1 by directly phosphorylating the conserved serine-proline repeat 3 (SP-3) motif of the NFAT regulatory domain, thus priming further phosphorylation of the SP-2 and serine-rich region 1 (SRR-1) motifs by GSK3 and CK1, respectively. Thus, genetic screening in Drosophila can be successfully applied to cross evolutionary boundaries and identify new regulators of a transcription factor that is expressed only in vertebrates.

Dephosphorylation of the nuclear factor of activated T cells (NFAT) transcription factor is regulated by an RNA-protein scaffold complex

Nuclear factor of activated T cells (NFAT) proteins are Ca2+-regulated transcription factors that control gene expression in many cell types. NFAT proteins are heavily phosphorylated and reside in the cytoplasm of resting cells; when cells are stimulated by a rise in intracellular Ca2+, NFAT proteins are dephosphorylated by the Ca2+/calmodulin-dependent phosphatase calcineurin and translocate to the nucleus to activate target gene expression. Here we show that phosphorylated NFAT1 is present in a large cytoplasmic RNA-protein scaffold complex that contains a long intergenic noncoding RNA (lincRNA), NRON [noncoding (RNA) repressor of NFAT]; a scaffold protein, IQ motif containing GTPase activating protein (IQGAP); and three NFAT kinases, casein kinase 1, glycogen synthase kinase 3, and dual specificity tyrosine phosphorylation regulated kinase. Combined knockdown of NRON and IQGAP1 increased NFAT dephosphorylation and nuclear import exclusively after stimulation, without affecting the rate of NFAT rephosphorylation and nuclear export; and both NRON-depleted T cells and T cells from IQGAP1-deficient mice showed increased production of NFAT-dependent cytokines. Our results provide evidence that a complex of lincRNA and protein forms a scaffold for a latent transcription factor and its regulatory kinases, and support an emerging consensus that lincRNAs that bind transcriptional regulators have a similar scaffold function.

An siRNA screen for NFAT activation identifies septins as coordinators of store-operated Ca2+ entry

The STIM1–ORAI1 pathway of store-operated Ca2+ entry is an essential component of cellular Ca2+ signalling. STIM1 senses depletion of intracellular Ca2+ stores in response to physiological stimuli, and relocalizes within the endoplasmic reticulum to plasma-membrane-apposed junctions, where it recruits and gates open plasma membrane ORAI1 Ca2+ channels. Here we use a genome-wide RNA interference screen in HeLa cells to identify filamentous septin proteins as crucial regulators of store-operated Ca2+ entry. Septin filaments and phosphatidylinositol-4,5-bisphosphate (also known as PtdIns(4,5)P2) rearrange locally at endoplasmic reticulum–plasma membrane junctions before and during formation of STIM1–ORAI1 clusters, facilitating STIM1 targeting to these junctions and promoting the stable recruitment of ORAI1. Septin rearrangement at junctions is required for PtdIns(4,5)P2 reorganization and efficient STIM1–ORAI1 communication. Septins are known to demarcate specialized membrane regions such as dendritic spines, the yeast bud and the primary cilium, and to serve as membrane diffusion barriers and/or signalling hubs in cellular processes such as vesicle trafficking, cell polarity and cytokinesis. Our data show that septins also organize the highly localized plasma membrane domains that are important in STIM1–ORAI1 signalling, and indicate that septins may organize membrane microdomains relevant to other signalling processes.

Requirement for balanced Ca/NFAT signaling in hematopoietic and embryonic development

NFAT transcription factors are highly phosphorylated proteins residing in the cytoplasm of resting cells. Upon dephosphorylation by the phosphatase calcineurin, NFAT proteins translocate to the nucleus, where they orchestrate developmental and activation programs in diverse cell types. NFAT is rephosphorylated and inactivated through the concerted action of at least 3 different kinases: CK1, GSK-3, and DYRK. The major docking sites for calcineurin and CK1 are strongly conserved throughout vertebrate evolution, and conversion of either the calcineurin docking site to a high-affinity version or the CK1 docking site to a low-affinity version results in generation of hyperactivable NFAT proteins that are still fully responsive to stimulation. In this study, we generated transgenic mice expressing hyperactivable versions of NFAT1 from the ROSA26 locus. We show that hyperactivable NFAT increases the expression of NFAT-dependent cytokines by differentiated T cells as expected, but exerts unexpected signal-dependent effects during T cell differentiation in the thymus, and is progressively deleterious for the development of B cells from hematopoietic stem cells. Moreover, progressively hyperactivable versions of NFAT1 are increasingly deleterious for embryonic development, particularly when normal embryos are also present in utero. Forced expression of hyperactivable NFAT1 in the developing embryo leads to mosaic expression in many tissues, and the hyperactivable proteins are barely tolerated in organs such as brain, and cardiac and skeletal muscle. Our results highlight the need for balanced Ca/NFAT signaling in hematopoietic stem cells and progenitor cells of the developing embryo, and emphasize the evolutionary importance of kinase and phosphatase docking sites in preventing inappropriate activation of NFAT.

The NFAT Family: Structure, Regulation, and Biological Functions

Publisher Summary This chapter sheds light on the structure, regulation, and biological functions of the NFAT family, which is a family of transcription factors. The primordial NFAT family member NFAT5/TonEBP is expressed ubiquitously in mammalian cells and regulates the response to hypertonic stress. NFAT5 is also likely to be involved in regulating diverse other biological programs. The DNA binding domains of all NFAT and NFкB/Rel family members have two domains, an N-terminal specificity domain involved in making base specific DNA contacts, and a C-terminal domain involved in dimer formation. Together these domains constitute the Rel homology region (RHR) common to all members of the extended NFAT/NFкB/Rel family. NFAT proteins can also function as dimeric transcription factors at quasi-palindromic sites that resemble NFкB binding sites. The calcium regulated NFAT proteins are activated by ligand binding to a variety of cell surface receptors. The common feature of the receptors is their ability to activate phosphatidylinositol specific phospholipase C (PLC), thereby inducing calcium influx across the plasma membrane. NFAT dependent gene transcription is exquisitely sensitive to changes in intracellular calcium concentration. Even in the continuous presence of stimulus, levels may oscillate depending on specific parameters of receptor occupancy and desensitization. Interaction with calcineurin is central to the calcium responsiveness of NFAT1-4. The major calcineurin docking site on NFAT is located at the N-terminus of the regulatory domain and has the consensus sequence PxIxIT. Substitution of the PxIxIT sequence of NFAT1 with a higher affinity version obtained by peptide selection increases the basal sensitivity of NFAT.

The NFAT Family

Publisher Summary This chapter sheds light on the structure, regulation, and biological functions of the NFAT family, which is a family of transcription factors. The primordial NFAT family member NFAT5/TonEBP is expressed ubiquitously in mammalian cells and regulates the response to hypertonic stress. NFAT5 is also likely to be involved in regulating diverse other biological programs. The DNA binding domains of all NFAT and NFкB/Rel family members have two domains, an N-terminal specificity domain involved in making base specific DNA contacts, and a C-terminal domain involved in dimer formation. Together these domains constitute the Rel homology region (RHR) common to all members of the extended NFAT/NFкB/Rel family. NFAT proteins can also function as dimeric transcription factors at quasi-palindromic sites that resemble NFкB binding sites. The calcium regulated NFAT proteins are activated by ligand binding to a variety of cell surface receptors. The common feature of the receptors is their ability to activate phosphatidylinositol specific phospholipase C (PLC), thereby inducing calcium influx across the plasma membrane. NFAT dependent gene transcription is exquisitely sensitive to changes in intracellular calcium concentration. Even in the continuous presence of stimulus, levels may oscillate depending on specific parameters of receptor occupancy and desensitization. Interaction with calcineurin is central to the calcium responsiveness of NFAT1-4. The major calcineurin docking site on NFAT is located at the N-terminus of the regulatory domain and has the consensus sequence PxIxIT. Substitution of the PxIxIT sequence of NFAT1 with a higher affinity version obtained by peptide selection increases the basal sensitivity of NFAT.

Immunological applications of genomics

A report of the Cold Spring Harbor Laboratory meeting Gene Expression and Signaling in the Immune System, Cold Spring Harbor, New York, USA, 26-30 April 2006.

Chapter 254 – The NFAT Family: Structure, Regulation, and Biological Functions

Publisher Summary This chapter sheds light on the structure, regulation, and biological functions of the NFAT family, which is a family of transcription factors. The primordial NFAT family member NFAT5/TonEBP is expressed ubiquitously in mammalian cells and regulates the response to hypertonic stress. NFAT5 is also likely to be involved in regulating diverse other biological programs. The DNA binding domains of all NFAT and NFкB/Rel family members have two domains, an N-terminal specificity domain involved in making base specific DNA contacts, and a C-terminal domain involved in dimer formation. Together these domains constitute the Rel homology region (RHR) common to all members of the extended NFAT/NFкB/Rel family. NFAT proteins can also function as dimeric transcription factors at quasi-palindromic sites that resemble NFкB binding sites. The calcium regulated NFAT proteins are activated by ligand binding to a variety of cell surface receptors. The common feature of the receptors is their ability to activate phosphatidylinositol specific phospholipase C (PLC), thereby inducing calcium influx across the plasma membrane. NFAT dependent gene transcription is exquisitely sensitive to changes in intracellular calcium concentration. Even in the continuous presence of stimulus, levels may oscillate depending on specific parameters of receptor occupancy and desensitization. Interaction with calcineurin is central to the calcium responsiveness of NFAT1-4. The major calcineurin docking site on NFAT is located at the N-terminus of the regulatory domain and has the consensus sequence PxIxIT. Substitution of the PxIxIT sequence of NFAT1 with a higher affinity version obtained by peptide selection increases the basal sensitivity of NFAT.