Regulation of gene expression by NFAT transcription factors in hibernating ground squirrels is dependent on the cellular environment

Calcineurin is a calmodulin-stimulated phosphatase that regulates the nuclear translocation of nuclear factor of activated T cell (NFAT) c1-4 through dephosphorylation. We believe that this mechanism plays various roles in the remodeling and maintenance of Ictidomys tridecemlineatus skeletal muscle. During hibernation, bouts of torpor and arousal take place, and squirrels do not lose muscle mass despite being inactive. Protein expression of Ca²⁺ signaling proteins were studied using immunoblotting. A DNA-protein interaction ELISA technique was created to test the binding of NFATs in the nucleus to DNA probes containing the NFAT response element under environmental conditions reflective of those during hibernation. Calcineurin protein levels increased by 3.08-fold during torpor (compared to euthermic control), whereas calpain1 levels also rose by 3.66-fold during torpor. Calmodulin levels were elevated upon entering torpor. NFATc4 binding to DNA showed a 1.4-fold increase during torpor, and we found that this binding was further enhanced when 600 nM of Ca²⁺ was supplemented. We also found that decreasing the temperature of ELISAs resulted in progressive decreases in the binding of NFATs c1, c3, and c4 to DNA. In summary, calmodulin and calpain1 appear to activate calcineurin and NFATc4 during torpor. NFAT binding to target promoters is affected by intranuclear [Ca²⁺] and environmental temperatures. Therefore, Ca²⁺ signaling and temperature changes play key roles in regulation of the NFAT-calcineurin pathway in skeletal muscle of hibernating 13-lined ground squirrels over the torpor-arousal cycle, and they may contribute to the avoidance of disuse-induced muscle atrophy that occurs naturally in these animals.     

The calcium/NFAT pathway: role in development and function of regulatory T cells

Calcium signals are essential for diverse cellular functions in the immune system. Sustained Ca²⁺ entry is necessary for complete and long-lasting activation of calcineurin/NFAT pathways. A growing number of studies have emphasized that Ca²⁺/calcineurin/NFAT pathway is crucial for both development and function of regulatory T cells.     

Critical role of divalent cations and Na+ efflux in Arabidopsis thaliana salt tolerance

Detrimental effects of salinity on plants are known to be partially alleviated by external Ca²⁺. Previous work demonstrated that the Arabidopsis SOS3 locus encodes a Ca²⁺‐binding protein with similarities to CnB, the regulatory subunit of protein phosphatase 2B (calcineurin). In this study, we further characterized the role of SOS3 in salt tolerance. We found that reduced root elongation of sos3 mutants in the presence of high concentrations of either NaCl or LiCl is specifically rescued by Ca²⁺ and not Mg²⁺, whereas root growth is rescued by both Ca²⁺ and Mg²⁺ in the presence of high concentrations of KCl. Phenocopies of sos3 mutants were obtained in wild‐type plants by the application of calmodulin and calcineurin inhibitors. These data provide further evidence that SOS3 is a calcineurin‐like protein and that calmodulin plays an important role in the signalling pathways involved in plant salt tolerance. The origin of the elevated Na : K ratio in sos3 mutants was investigated by comparing Na⁺ efflux and influx in both mutant and wild type. No difference in Na⁺ influx was recorded between wild type and sos3; however, sos3 plants showed a markedly lower Na⁺ efflux, a property that would contribute to the salt‐oversensitive phenotype of sos3 plants.     

Calcium Regulation in the Protozoan Model,Paramecium tetraurelia

Early in eukaryotic evolution, the cell has evolved a considerable inventory of proteins engaged in the regulation of intracellular Ca²⁺ concentrations, not only to avoid toxic effects but beyond that to exploit the signaling capacity of Ca²⁺ by small changes in local concentration. Among protozoa, the ciliate Paramecium may now be one of the best analyzed models. Ciliary activity and exo‐/endocytosis are governed by Ca²⁺, the latter by Ca²⁺ mobilization from alveolar sacs and a superimposed store‐operated Ca²⁺‐influx. Paramecium cells possess plasma membrane‐ and endoplasmic reticulum‐resident Ca²⁺‐ATPases/pumps (PMCA, SERCA), a variety of Ca²⁺ influx channels, including mechanosensitive and voltage‐dependent channels in the plasma membrane, furthermore a plethora of Ca²⁺‐release channels (CRC) of the inositol 1,4,5‐trisphosphate and ryanodine receptor type in different compartments, notably the contractile vacuole complex and the alveolar sacs, as well as in vesicles participating in vesicular trafficking. Additional types of CRC probably also occur but they have not been identified at a molecular level as yet, as is the equivalent of synaptotagmin as a Ca²⁺ sensor for exocytosis. Among established targets and sensors of Ca²⁺ in Paramecium are calmodulin, calcineurin, as well as Ca²⁺/calmodulin‐dependent protein kinases, all with multiple functions. Thus, basic elements of Ca²⁺ signaling are available for Paramecium.     

Analysis of CaM-kinase signaling in cells

A change in intracellular free calcium is a common signaling mechanism that modulates a wide array of physiological processes in most cells. Responses to increased intracellular Ca²⁺ are often mediated by the ubiquitous protein calmodulin (CaM) that upon binding Ca²⁺ can interact with and alter the functionality of numerous proteins including a family of protein kinases referred to as CaM-kinases (CaMKs). Of particular interest are multifunctional CaMKs, such as CaMKI, CaMKII, CaMKIV and CaMKK, that can phosphorylate multiple downstream targets. This review will outline several protocols we have used to identify which members and/or isoforms of this CaMK family mediate specific cellular responses with a focus on studies in neurons. Many previous studies have relied on a single approach such as pharmacological inhibitors or transfected dominant-negative kinase constructs. Since each of these protocols has its limitations, that will be discussed, we emphasize the necessity to use multiple, independent approaches in mapping out cellular signaling pathways.     

A cellular atlas of calcineurin signaling

Intracellular Ca²⁺ signals are temporally controlled and spatially restricted. Signaling occurs adjacent to sites of Ca²⁺ entry and/or release, where Ca²⁺-dependent effectors and their substrates co-localize to form signaling microdomains. Here we review signaling by calcineurin, the Ca²⁺/calmodulin regulated protein phosphatase and target of immunosuppressant drugs, Cyclosporin A and FK506. Although well known for its activation of the adaptive immune response via NFAT dephosphorylation, systematic mapping of human calcineurin substrates and regulators reveals unexpected roles for this versatile phosphatase throughout the cell. We discuss calcineurin function, with an emphasis on where signaling occurs and mechanisms that target calcineurin and its substrates to signaling microdomains, especially binding of cognate short linear peptide motifs (SLiMs). Calcineurin is ubiquitously expressed and regulates events at the plasma membrane, other intracellular membranes, mitochondria, the nuclear pore complex and centrosomes/cilia. Based on our expanding knowledge of localized CN actions, we describe a cellular atlas of Ca²⁺/calcineurin signaling.     

Degranulation in RBL-2H3 cells: regulation by calmodulin pathway

Involvement of the calmodulin pathway in Ca2+-induced degranulation was evaluated in RBL-2H3 mast cells. Pretreatment of RBL-2H3 cells with a calmodulin antagonist, W-13, blocked ionomycin-dependent release of beta-hexosaminidase into the supernatant, although W-13 treatment alone slightly but significantly increased the release. Ca2+/calmodulin activates various protein kinases and phosphatases including myosin-light chain kinase (MLCK), calmodulin-dependent protein kinases (CaMKs), and calcineurin. When RBL-2H3 cells were pretreated with a MLCK inhibitor, ML-7, or a CaMKs inhibitor, KN-93, the ionomycin-dependent release of beta-hexosaminidase into the supernatant was inhibited. In addition, pretreatment with calcineurin inhibitors, cyclosporin A and FR901725, resulted in blockage of the ionomycin-dependent release of beta-hexosaminidase into the supernatant. Our results indicate that Ca2+/calmodulin, activated calmodulin, is indispensable for Ca2+-induced degranulation, and that within the calmodulin pathways, at least MLCK, CaMKs and calcineurin positively regulate the release of granules initiated by increasing cytosolic Ca2+ concentrations in RBL-2H3 cells.     

Pull-down of Calmodulin-binding Proteins

Calcium (Ca²⁺) is an ion vital in regulating cellular function through a variety of mechanisms. Much of Ca²⁺ signaling is mediated through the calcium-binding protein known as calmodulin (CaM)^1,2. CaM is involved at multiple levels in almost all cellular processes, including apoptosis, metabolism, smooth muscle contraction, synaptic plasticity, nerve growth, inflammation and the immune response. A number of proteins help regulate these pathways through their interaction with CaM. Many of these interactions depend on the conformation of CaM, which is distinctly different when bound to Ca²⁺ (Ca²⁺-CaM) as opposed to its Ca²⁺-free state (ApoCaM)³.While most target proteins bind Ca²⁺-CaM, certain proteins only bind to ApoCaM. Some bind CaM through their IQ-domain, including neuromodulin⁴, neurogranin (Ng)⁵, and certain myosins⁶. These proteins have been shown to play important roles in presynaptic function⁷, postsynaptic function⁸, and muscle contraction⁹, respectively. Their ability to bind and release CaM in the absence or presence of Ca²⁺ is pivotal in their function. In contrast, many proteins only bind Ca²⁺-CaM and require this binding for their activation. Examples include myosin light chain kinase¹⁰, Ca²⁺/CaM-dependent kinases (CaMKs)¹¹ and phosphatases (e.g. calcineurin)¹², and spectrin kinase¹³, which have a variety of direct and downstream effects¹⁴.The effects of these proteins on cellular function are often dependent on their ability to bind to CaM in a Ca²⁺-dependent manner. For example, we tested the relevance of Ng-CaM binding in synaptic function and how different mutations affect this binding. We generated a GFP-tagged Ng construct with specific mutations in the IQ-domain that would change the ability of Ng to bind CaM in a Ca²⁺-dependent manner. The study of these different mutations gave us great insight into important processes involved in synaptic function^8,15. However, in such studies, it is essential to demonstrate that the mutated proteins have the expected altered binding to CaM.Here, we present a method for testing the ability of proteins to bind to CaM in the presence or absence of Ca²⁺, using CaMKII and Ng as examples. This method is a form of affinity chromatography referred to as a CaM pull-down assay. It uses CaM-Sepharose beads to test proteins that bind to CaM and the influence of Ca²⁺ on this binding. It is considerably more time efficient and requires less protein relative to column chromatography and other assays. Altogether, this provides a valuable tool to explore Ca²⁺/CaM signaling and proteins that interact with CaM.     

植物体内钙信号及其在调节干旱胁迫中的作用

钙作为植物体内第二信使广泛参与了植物响应的各种非生物和生物胁迫的信号传导。胁迫信号通过激活位于细胞质膜上的钙离子通道,产生胞质内特异性的钙信号,传递至钙信号感受蛋白,如钙调素(calmodulin,CaM)、钙依赖蛋白激酶(Ca2+-dependent protein kinases,CDPK)和类钙调磷酸酶B蛋白(calcineurin B-like protein,CBL)等,进而引起胞内一系列生理生化变化,最终对胁迫做出响应。钙信号在植物响应干旱胁迫信号系统中起枢纽作用,主要通过调节气孔运动,水通道蛋白(aquaporin,AQP)和抗氧化酶活性来减少水分流失,提高水分利用率,最终降低干旱对植物细胞的伤害,并具有一定的生态学功能。该文对近年来国内外有关植物体内钙信号的研究进展以及在干旱逆境中的调节作用进行综述,并对今后的研究做了展望。     

FKBP12.6 protects heart from AngII‐induced hypertrophy through inhibiting Ca2+/calmodulin‐mediated signalling pathways in vivo and in vitro

We previously observed that disruption of FK506‐binding protein 12.6 (FKBP12.6) gene resulted in cardiac hypertrophy in male mice. Studies showed that overexpression of FKBP12.6 attenuated thoracic aortic constriction (TAC)‐induced cardiac hypertrophy in mice, whereas the adenovirus‐mediated overexpression of FKBP12.6 induced hypertrophy and apoptosis in cultured neonatal cardiomyocytes, indicating that the role of FKBP12.6 in cardiac hypertrophy is still controversial. In this study, we aimed to investigate the roles and mechanisms of FKBP12.6 in angiotensin II (AngII)‐induced cardiac hypertrophy using various transgenic mouse models in vivo and in vitro. FKBP12.6 knockout (FKBP12.6^?/?) mice and cardiac‐specific FKBP12.6 overexpressing (FKBP12.6 TG) mice were infused with AngII (1500 ng/kg/min) for 14 days subcutaneously by implantation of an osmotic mini‐pump. The results showed that FKBP12.6 deficiency aggravated AngII‐induced cardiac hypertrophy, while cardiac‐specific overexpression of FKBP12.6 prevented hearts from the hypertrophic response to AngII stimulation in mice. Consistent with the results in vivo, overexpression of FKBP12.6 in H9c2 cells significantly repressed the AngII‐induced cardiomyocyte hypertrophy, seen as reductions in the cell sizes and the expressions of hypertrophic genes. Furthermore, we demonstrated that the protection of FKBP12.6 on AngII‐induced cardiac hypertrophy was involved in reducing the concentration of intracellular Ca²⁺ ([Ca²⁺]i), in which the protein significantly inhibited the key Ca²⁺/calmodulin‐dependent signalling pathways such as calcineurin/cardiac form of nuclear factor of activated T cells 4 (NFATc4), calmodulin kinaseII (CaMKII)/MEF‐2, AKT/Glycogen synthase kinase 3β (GSK3β)/NFATc4 and AKT/mTOR signalling pathways. Our study demonstrated that FKBP12.6 protects heart from AngII‐induced cardiac hypertrophy through inhibiting Ca²⁺/calmodulin‐mediated signalling pathways.     

Retracted: Elevated expression of CaMKIIγ during osteoclastogenesis and its functional implications

Ca²⁺/calmodulin signaling has been recognized recently as a major regulator in osteoclastogenesis. Efforts have ensued to identify the downstream targets of this signaling pathway in the context of regulating osteoclastogenesis. The calcineurin‐NFAT pathway has thus been identified as one such target. In this article, we describe the discovery of another novel downstream target, CaMKIIγ. We also demonstrate that CaMKIIγ is the sole known CaMK expressed in significant amounts in osteoclasts and their precursors. Other known CaMKs such as CaMKIV and CaMKIIα, β, δ, were not detectable, and CaMKI was only expressed at a negligible level. Furthermore, the expression of CaMKIIγ was tightly correlated with the osteoclastogenic process, with a peak level on Day 3 of cell culturing. Osteoclastogenesis is halted by treatment with the CaMKIIγ inhibitor, KN93, independently from apoptosis, with the IC₅₀ for osteoclastogenesis matching that for blocking CaMKIIγ function. Collectively, these data indicate that CaMKIIγ may be a significant regulator of osteoclastogenesis. J. Cell. Biochem. 101: 1038–1045, 2007. © 2006 Wiley‐Liss, Inc.     

The presence and binding characteristics of calmodulin in microsomal preparations enriched in sarcoplasmic reticulum from rabbit skeletal muscle

ATP-dependent oxalate facilitated calcium transport in sarcoplasmic reticulum (SR) preparations obtained from rabbit vastus lateralis muscle (fast skeletal muscle; F_sr) and soleus (slow skeletal muscle; S_sr) was determined. Addition of exogenous calmodulin did not stimulate calcium transport in either F_sr or S_sr preparations. F_sr and S_sr previously washed in 1 mM EGTA demonstrated a reduced capacity to transport Ca²⁺; the exogenous addition of calmodulin (0.24 μM) under these conditions, did not restore uptake activity but significantly decreased the steady-state level of Ca²⁺ uptake. Extracts of skeletal SR prepared by treatment with 0.2 mM EDTA and boiling produced significantly more stimulation of red cell Ca²⁺ATPase activity than extracts prepared by boiling alone. This stimulation of red cell Ca²⁺-ATPase was inhibited to a significant extent by $\text{48}\text{80}$, a known anti-calmodulin agent. Radioimmunoassay revealed that extracts prepared by boiling or EDTA-treatment followed by boiling contained considerable amounts of calmodulin. Washing with 1 mM EGTA, though, did not release any calmodulin from SR. These studies reveal that calmodulin is present in both F_sr and S_sr and can only be removed by harsh treatments. The role of calmodulin in skeletal muscle Ca²⁺-transport remains to be determined.     

Calcium/calmodulin-dependent kinases are involved in growth,thermotolerance, oxidative stress survival,and fertility in Neurospora crassa

Calcium/calmodulin-dependent kinases (Ca²⁺/CaMKs) are Ser/Thr protein kinases that respond to change in cytosolic free Ca²⁺ ([Ca²⁺]_c) and play multiple cellular roles in organisms ranging from fungi to humans. In the filamentous fungus Neurospora crassa, four Ca²⁺/CaM-dependent kinases, Ca²⁺/CaMK-1 to 4, are encoded by the genes NCU09123, NCU02283, NCU06177, and NCU09212, respectively. We found that camk-1 and camk-2 are essential for full fertility in N. crassa. The survival of ?camk-2 mutant was increased in induced thermotolerance and oxidative stress conditions. In addition, the ?camk-1 ?camk-2, ?camk-4 ?camk-2, and ?camk-3 ?camk-2 double mutants display slow growth phenotype, reduced aerial hyphae, decreased thermotolerance, and increased sensitivity to oxidative stress, revealing the genetic interactions among these kinases. Therefore, Ca²⁺/CaMKs are involved in growth, thermotolerance, oxidative stress tolerance, and fertility in N. crassa.     

Myosin light chain kinase and the role of myosin light chain phosphorylation in skeletal muscle

Skeletal muscle myosin light chain kinase (skMLCK) is a dedicated Ca²⁺/calmodulin-dependent serine–threonine protein kinase that phosphorylates the regulatory light chain (RLC) of sarcomeric myosin. It is expressed from the MYLK2 gene specifically in skeletal muscle fibers with most abundance in fast contracting muscles. Biochemically, activation occurs with Ca²⁺ binding to calmodulin forming a (Ca²⁺)₄•calmodulin complex sufficient for activation with a diffusion limited, stoichiometric binding and displacement of a regulatory segment from skMLCK catalytic core. The N-terminal sequence of RLC then extends through the exposed catalytic cleft for Ser15 phosphorylation. Removal of Ca²⁺ results in the slow dissociation of calmodulin and inactivation of skMLCK. Combined biochemical properties provide unique features for the physiological responsiveness of RLC phosphorylation, including (1) rapid activation of MLCK by Ca²⁺/calmodulin, (2) limiting kinase activity so phosphorylation is slower than contraction, (3) slow MLCK inactivation after relaxation and (4) much greater kinase activity relative to myosin light chain phosphatase (MLCP). SkMLCK phosphorylation of myosin RLC modulates mechanical aspects of vertebrate skeletal muscle function. In permeabilized skeletal muscle fibers, phosphorylation-mediated alterations in myosin structure increase the rate of force-generation by myosin cross bridges to increase Ca²⁺-sensitivity of the contractile apparatus. Stimulation-induced increases in RLC phosphorylation in intact muscle produces isometric and concentric force potentiation to enhance dynamic aspects of muscle work and power in unfatigued or fatigued muscle. Moreover, RLC phosphorylation-mediated enhancements may interact with neural strategies for human skeletal muscle activation to ameliorate either central or peripheral aspects of fatigue.     

Intermedilysin induces EGR-1 expression through calcineurin/NFAT pathway in human cholangiocellular carcinoma cells

Intermedilysin (ILY) is a cholesterol-dependent cytolysin produced by Streptococcus intermedius, which is associated with human brain and liver abscesses. Although intrahepatic bile duct cells play a valuable role in the pathogenesis of liver abscess, the molecular mechanism of ILY-treated intrahepatic bile duct cells remains unknown. In this study, we report that ILY induced a nuclear accumulation of intracellular calcium ([Ca²⁺]i) in human cholangiocellular cells HuCCT1. We also demonstrate that 10 ng/ml ILY induced NFAT1 dephosphorylation and its nuclear translocation in HuCCT1 cells. In contrast to the result that ILY induced NF-κB translocation in human hepatic HepG2 cells, ILY did not affect NF-κB localization in HuCCT1 cells. Dephosphorylation and nuclear translocation of NFAT1 caused by ILY were prevented by [Ca²⁺]i calcium chelator, BAPTA/AM, and calcineurin inhibitors, cyclosporine A and tacrolimus. ILY induced early growth response-1 (EGR-1) expression and it was inhibited by the pre-treatment with cyclosporine A, indicating that the calcineurin/NFAT pathway was involved in EGR-1 expression in response to ILY. ILY-induced calcineurin/NFAT1 activation and sequential EGR-1 expression might be related to the pathogenesis of S. intermedius in human bile duct cells.     

Calcineurin and its regulation by Sra/RCAN is required for completion of meiosis in Drosophila

Ca²⁺ signaling pathways play important roles to complete meiosis from metaphase II arrest in vertebrate oocytes. However, less is known about the molecular mechanism of completion of meiosis in Drosophila females. Here, we provide direct evidence that calcineurin, a Ca²⁺/calmodulin (CaM)-dependent phosphatase, is essential for meiotic progression beyond metaphase I in Drosophila oocytes. Oocytes from germline clones lacking CanB2, a calcineurin regulatory subunit B, failed to complete meiosis after egg activation, and laid eggs exhibited a meiotic arrested anaphase I chromosome configuration. Genetic analyses suggest that calcineurin activity is regulated by Sarah (Sra), a family member of regulators of calcineurin (RCANs), through a Sra phosphorylation-dependent mechanism. Our results support a view in which the phosphorylation of Sra not only acts to relieve the inhibitory effects of Sra, but also acts to activate calcineurin, thus explaining the role of RCAN proteins as positive regulators of calcineurin.