Calmodulin-dependent Protein Phosphatase Calcineurin Research Articles

Inhibition of Cdc42-Interacting Protein 4-calcineurin complexes attenuate pathological cardiac remodeling after myocardial infarction

Myocardial infarction (MI) is a major cause of mortality and morbidity worldwide. Heart failure due to MI results from both acute ischemia/reperfusion (I/R) injury and subsequent post-MI pathological cardiac remodeling. The Ca2+/calmodulin-dependent protein phosphatase calcineurin (CaN) is a critical mediator of pathological cardiac remodeling. Along with its role in the immune system, the pro-survival function of CaN complicates the targeting of the phosphatase in heart disease. CaNAβ is a CaN isoform uniquely associated with pathological cardiac hypertrophy. We have discovered that pools of cardiomyocyte CaNAβ are anchored via the unique CaNAβ N-terminal polyproline domain (PP) to scaffold proteins such as CIP4 (Cdc42 interacting protein 4, TRIP10), creating discrete Ca2+-CaN signaling compartments. Pathological cardiac remodeling induced by pressure overload was inhibited by CIP4 gene knock-out and by inhibition of CaNAβ binding to CIP4 using an adeno-associated virus (AAV) vector that expresses the CaNAβ PP peptide. It remains unknown whether PP-anchored CaNAβ also participates in CaN-dependent cardioprotective signaling in I/R injury. We now consider whether CIP4-CaNAβ signalosomes are required for myocyte survival in ischemic cardiomyopathy. To test the relevance of CIP4 to ischemic reperfusion (IR) injury, tamoxifen-inducible, cardiac-specific CIP4 knock-out (CIP4 CKO), control Tg(Myh6-cre/Esr1*), and control CIP4f/f mice were subjected to 30 minutes of ischemia by transient left anterior descending coronary artery (LAD) ligation. Cardiac function and structure were evaluated 24 hours after I/R by echocardiography and histology. Left ventricular ejection fraction was significantly preserved, while infarct size was significantly decreased by CIP4 CKO when compared to the two control cohorts. Separate mice subjected to permanent LAD ligation showed that CIP4 CKO resulted in long-term preservation of cardiac function. To disrupt CaNAβ-CIP4 complexes in vivo, the CaNAβ PP domain peptide was expressed again using AAV. In contrast to the expression of the VIVIT pan-CaN inhibitory peptide, CaNAβ PP peptide expression did not exacerbate I/R injury and in fact preserved cardiac function immediately after infarction. Moreover, VIVIT peptide expression worsened, while CaNAβ PP peptide expression improved cardiac function long term after induction of MI by permanent LAD ligation. Together, these results suggest that CIP4-CaNAβ signalosomes do not contribute to cardioprotection, while inducing pathological cardiac remodeling. These results suggest that targeting of CIP4-CaNAβ signalosomes constitute a novel strategy for inhibiting I/R injury and preventing the development of heart failure in response to diverse forms of cardiovascular disease. This work was supported by National Institutes of Health R01 grants HL126950 and HL158052. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Abstract P2021: Targeting Of Cdc42-interacting Protein 4 -calcineurin Signaling Complexes Is Cardioprotective During Ischemia/Reperfusion Injury

Myocardial infarction (MI) is a major cause of mortality and morbidity worldwide. Heart failure due to MI results from both acute ischemic/reperfusion (I/R) injury and subsequent post-MI pathological cardiac remodeling. The Ca 2+ /calmodulin-dependent protein phosphatase calcineurin (CaN), which promotes pathological cardiac remodeling, has been problematic as a therapeutic target due to its role in cardioprotection, including during I/R injury. We have shown that a select pool of CaNAβ holoenzyme is localized within the cardiomyocyte by scaffold protein Cdc42-Interacting Protein 4 (CIP4/TRIP10), such that the CaNAβ N-terminal polyproline (PP) domain binds directly to the CIP4 C-terminal SH3 domain. Pathological cardiac remodeling induced by pressure overload was inhibited by CIP4 gene knock-out and by inhibition of CaNAβ binding to CIP4 using an adeno-associated virus (AAV) vector that expresses the CaNAβ PP peptide. We now consider whether targeting CIP4-CaNAβ signalosomes is beneficial in ischemic cardiomyopathy. To test the relevance of CIP4 in vivo to ischemic reperfusion (IR) injury, tamoxifen-inducible, cardiac-specific CIP4 knock-out (CIP4 CKO), control Tg(Myh6-cre/Esr1*), and control CIP4 f/f mice were subjected to 30 minutes of ischemia by transient left anterior descending coronary artery (LAD) ligation. Cardiac function and structure were evaluated 24 hours after reperfusion by echocardiography and histology. Left ventricular ejection fraction was significantly preserved, while infarct size was significantly decreased by CIP4 CKO when compared to the two control cohorts. Separate mice subjected to permanent LAD ligation showed that CIP4 CKO resulted in long-term preservation of cardiac function. To disrupt CaNAβ-CIP4 complexes in vivo, AAV has been used to express the CaNAβ PP domain peptide. In contrast to the expression of the VIVIT pan-CaN inhibitory peptide, CaNAβ PP peptide expression did not worsen I/R injury. Together, these results suggest that PP-anchored CaNAβ does not contribute to cardioprotection. Targeting CIP4 signalosomes may constitute a novel strategy for inhibiting I/R injury and preventing the development of heart failure in response to diverse forms of cardiovascular disease.

Calmodulin Regulates Transient Receptor Potential TRPM3 and TRPM8-Induced Gene Transcription.

Calmodulin is a small protein that binds Ca2+ ions via four EF-hand motifs. The Ca2+/calmodulin complex as well as Ca2+-free calmodulin regulate the activities of numerous enzymes and ion channels. Here, we used genetic and pharmacological tools to study the functional role of calmodulin in regulating signal transduction of TRPM3 and TRPM8 channels. Both TRPM3 and TRPM8 are important regulators of thermosensation. Gene transcription triggered by stimulation of TRPM3 or TRPM8 channels was significantly impaired in cells expressing a calmodulin mutant with mutations in all four EF-hand Ca2+ binding motifs. Similarly, incubation of cells with the calmodulin inhibitor ophiobolin A reduced TRPM3 and TRPM8-induced signaling. The Ca2+/calmodulin-dependent protein phosphatase calcineurin was shown to negatively regulate TRPM3-induced gene transcription. Here, we show that TRPM8-induced transcription is also regulated by calcineurin. We propose that calmodulin plays a dual role in regulating TRPM3 and TRPM8 functions. Calmodulin is required for the activation of TRPM3 and TRPM8-induced intracellular signaling, most likely through a direct interaction with the channels. Ca2+ influx through TRPM3 and TRPM8 feeds back to TRPM3 and TRPM8-induced signaling by activation of the calmodulin-regulated enzyme calcineurin, which acts as a negative feedback loop for both TRPM3 and TRPM8 channel signaling.

Nuanced Interactions between AKAP79 and STIM1 with Orai1 Ca2+ Channels at Endoplasmic Reticulum-Plasma Membrane Junctions Sustain NFAT Activation.

A-kinase anchoring protein 79 (AKAP79) is a human scaffolding protein that organizes Ca2+/calmodulin-dependent protein phosphatase calcineurin, calmodulin, cAMP-dependent protein kinase, protein kinase C, and the transcription factor nuclear factor of activated T cells (NFAT1) into a signalosome at the plasma membrane. Upon Ca2+ store depletion, AKAP79 interacts with the N-terminus of STIM1-gated Orai1 Ca2+ channels, enabling Ca2+ nanodomains to stimulate calcineurin. Calcineurin then dephosphorylates and activates NFAT1, which then translocates to the nucleus. A fundamental question is how signalosomes maintain long-term signaling when key effectors are released and therefore removed beyond the reach of the activating signal. Here, we show that the AKAP79-Orai1 interaction is considerably more transient than that of STIM1-Orai1. Free AKAP79, with calcineurin and NFAT1 in tow, is able to replace rapidly AKAP79 devoid of NFAT1 on Orai1, in the presence of continuous Ca2+ entry. We also show that Ca2+ nanodomains near Orai1 channels activate almost the entire cytosolic pool of NFAT1. Recycling of inactive NFAT1 from the cytoplasm to AKAP79 in the plasma membrane, coupled with the relatively weak interaction between AKAP79 and Orai1, maintain excitation-transcription coupling. By measuring rates for AKAP79-NFAT interaction, we formulate a mathematical model that simulates NFAT dynamics at the plasma membrane.

Ca2+ Microdomains, Calcineurin and the Regulation of Gene Transcription.

Ca2+ ions function as second messengers regulating many intracellular events, including neurotransmitter release, exocytosis, muscle contraction, metabolism and gene transcription. Cells of a multicellular organism express a variety of cell-surface receptors and channels that trigger an increase of the intracellular Ca2+ concentration upon stimulation. The elevated Ca2+ concentration is not uniformly distributed within the cytoplasm but is organized in subcellular microdomains with high and low concentrations of Ca2+ at different locations in the cell. Ca2+ ions are stored and released by intracellular organelles that change the concentration and distribution of Ca2+ ions. A major function of the rise in intracellular Ca2+ is the change of the genetic expression pattern of the cell via the activation of Ca2+-responsive transcription factors. It has been proposed that Ca2+-responsive transcription factors are differently affected by a rise in cytoplasmic versus nuclear Ca2+. Moreover, it has been suggested that the mode of entry determines whether an influx of Ca2+ leads to the stimulation of gene transcription. A rise in cytoplasmic Ca2+ induces an intracellular signaling cascade, involving the activation of the Ca2+/calmodulin-dependent protein phosphatase calcineurin and various protein kinases (protein kinase C, extracellular signal-regulated protein kinase, Ca2+/calmodulin-dependent protein kinases). In this review article, we discuss the concept of gene regulation via elevated Ca2+ concentration in the cytoplasm and the nucleus, the role of Ca2+ entry and the role of enzymes as signal transducers. We give particular emphasis to the regulation of gene transcription by calcineurin, linking protein dephosphorylation with Ca2+ signaling and gene expression.

Muscle A-kinase–anchoring protein-β–bound calcineurin toggles active and repressive transcriptional complexes of myocyte enhancer factor 2D

Myocyte enhancer factor 2 (MEF2) transcription factors are key regulators of the development and adult phenotype of diverse tissues, including skeletal and cardiac muscles. Controlled by multiple post-translational modifications, MEF2D is an effector for the Ca2+/calmodulin-dependent protein phosphatase calcineurin (CaN, PP2B, and PPP3). CaN-catalyzed dephosphorylation promotes the desumoylation and acetylation of MEF2D, increasing its transcriptional activity. Both MEF2D and CaN bind the scaffold protein muscle A-kinase-anchoring protein β (mAKAPβ), which is localized to the nuclear envelope, such that C2C12 skeletal myoblast differentiation and neonatal rat ventricular myocyte hypertrophy are inhibited by mAKAPβ signalosome targeting. Using immunoprecipitation and DNA-binding assays, we now show that the formation of mAKAPβ signalosomes is required for MEF2D dephosphorylation, desumoylation, and acetylation in C2C12 cells. Reduced MEF2D phosphorylation was coupled to a switch from type IIa histone deacetylase to p300 histone acetylase binding that correlated with increased MEF2D-dependent gene expression and ventricular myocyte hypertrophy. Together, these results highlight the importance of mAKAPβ signalosomes for regulating MEF2D activity in striated muscle, affirming mAKAPβ as a nodal regulator in the myocyte intracellular signaling network.

Stimulation of TRPM3 channels increases the transcriptional activation potential of Elk-1 involving cytosolic Ca2+, extracellular signal-regulated protein kinase, and calcineurin

Stimulation of transient receptor potential M3 (TRPM3) channels with the steroid pregnenolone sulfate increases the transcriptional activation potential of Elk-1, a transcription factor that regulates serum response element-mediated transcription. Here, we show that an influx of Ca2+ ions into the cells is essential for the activation of Elk-1 following stimulation of TRPM3. Using genetically encoded Ca2+ buffers, we show that a rise in cytoplasmic Ca2+ is required for the upregulation of the transcriptional activation potential of Elk-1, while buffering of Ca2+ in the nucleus had no inhibitory effect on the transcriptional activity of Elk-1. Pharmacological and genetic experiments showed that extracellular signal-regulated protein kinase (ERK1/2) functions as signal transducer connecting TRPM3 channels with the Elk-1 transcription factor. Accordingly, dephosphorylation of ERK1/2 in the nucleus by MAP kinase phosphatase attenuated TRPM3-mediated Elk-1 activation. Moreover, we show that the Ca2+/calmodulin-dependent protein phosphatase calcineurin is part of a shut-off-device for the signaling cascade connecting TRPM3 channels with the activation of Elk-1. The fact that TRPM3 channel stimulation activates Elk-1 connects TRPM3 with the biological functions of Elk-1, including the regulation of proliferation, differentiation, survival, transcription, and cell migration.

Dissecting the Roles of the Calcineurin Pathway in Unisexual Reproduction, Stress Responses, and Virulence in Cryptococcus deneoformans.

The Ca2+/calmodulin-dependent protein phosphatase calcineurin orchestrates sexual reproduction, stress responses, and virulence via branched downstream pathways in the opportunistic human fungal pathogen Cryptococcus neoformans The calcineurin-binding protein Cbp1, the calcineurin temperature suppressor Cts1, the calcineurin-responsive zinc finger transcription factor Crz1, and the calcineurin targets Pbp1, Tif3, and Puf4, all function downstream of calcineurin to orchestrate distinct cellular processes. To elucidate how the calcineurin pathway regulatory network governs unisexual reproduction, stress responses, and virulence, we have analyzed the self-filamentous C. deneoformans strain, XL280α, and generated double mutants of these calcineurin downstream genes. We demonstrated that calcineurin governs unisexual reproduction at different sexual developmental stages, in which the initiation of the yeast-hyphal morphological transition is independent of Crz1, whereas the sporulation process is dependent on Crz1. Calcineurin-dependent unisexual reproduction is independent of the pheromone response pathway. Crz1 synergistically interacts with different calcineurin downstream targets in responding to ER, high-calcium, and cell wall stresses. We observed a widespread synergy suggesting that these proteins function in complex branched pathways downstream of calcineurin with some functional redundancy, which may allow efficient signaling network rewiring within the pathway for prompt adaptation to changing environments. Finally, we showed that deletion of PBP1 or TIF3 in the cna1∆ mutant background conferred a modest level of growth tolerance at 37°, but that the cna1∆ pbp1∆ and cna1∆ tif3∆ double mutants were both avirulent, suggesting that calcineurin may control virulence via mechanisms beyond thermotolerance.

Stimulation of B-Raf increases c-Jun and c-Fos expression and upregulates AP-1-regulated gene transcription in insulinoma cells.

Stimulation of pancreatic β-cells with glucose activates the protein kinases B-Raf and extracellular signal-regulated protein kinase that participate in glucose sensing. Inhibition of both kinases results in impairment of glucose-regulated gene transcription. To analyze the signaling pathway controlled by B-Raf, we expressed a conditionally active form of B-Raf in INS-1 insulinoma cells. Here, we show that stimulation of B-Raf strongly activated the transcription factor AP-1 which is accompanied by increased c-Jun and c-Fos promoter activities, an upregulation of c-Jun and c-Fos biosynthesis, and elevated transcriptional activation potentials of c-Jun and c-Fos. Mutational analysis identified the AP-1 sites within the c-Jun promoter and the serum response element (SRE) within the c-Fos promoter as the essential genetic elements connecting B-Raf stimulation with AP-1 activation. In line with this, the transcriptional activation potential of the SRE-binding protein Elk-1 was increased following B-Raf activation. The signal pathway from B-Raf to AP-1 required the activation of c-Jun. We identified the cyclin D1 gene as a delayed response gene for AP-1 following stimulation of B-Raf in insulinoma cells. Moreover, MAP kinase phosphatase-1 and the Ca2+/calmodulin-dependent protein phosphatase calcineurin were identified to function as shut-off-devices for the signaling cascade connecting B-Raf stimulation with the activation of AP-1. The fact that stimulation with glucose, activation of L-type voltage-gated Ca2+ channels, and stimulation of B-Raf all trigger an activation of AP-1 indicates that AP-1 is a point of convergence of signaling pathways in β-cell.

Identification of three signaling molecules required for calcineurin-dependent monopolar growth induced by the DNA replication checkpoint in fission yeast

Cell polarity is coordinately regulated with the cell cycle. Growth polarity of the fission yeast Schizosaccharomyces pombe transits from monopolar to bipolar during G2 phase, termed NETO (new end take off). Upon perturbation of DNA replication, the checkpoint kinase Cds1/CHK2 induces NETO delay through activation of Ca2+/calmodulin-dependent protein phosphatase calcineurin (CN). CN in turn regulates its downstream targets including the microtubule (MT) plus-end tracking CLIP170 homologue Tip1 and the Casein kinase 1γ Cki3. However, whether and which Ca2+ signaling molecules are involved in the NETO delay remains elusive. Here we show that 3 genes (trp1322, vcx1 and SPAC6c3.06c encoding TRP channel, antiporter and P-type ATPase, respectively) play vital roles in the NETO delay. Upon perturbation of DNA replication, these 3 genes are required for not only the NETO delay but also for the maintenance of cell viability. Trp1322 and Vcx1 act downstream of Cds1 and upstream of CN for the NETO delay, whereas SPAC6c3.06c acts downstream of CN. Consistently, Trp1322 and Vcx1, but not SPAC6c3.06c, are essential for activation of CN. Interestingly, we have found that elevated extracellular Ca2+ per se induces a NETO delay, which depends on CN and its downstream target genes. These findings imply that Ca2+-CN signaling plays a central role in cell polarity control by checkpoint activation.

Extracellular Signal-Regulated Protein Kinase, c-Jun N-Terminal Protein Kinase, and Calcineurin Regulate Transient Receptor Potential M3 (TRPM3) Induced Activation of AP-1

Stimulation of transient receptor potential M3 (TRPM3) cation channels with pregnenolone sulfate induces an influx of Ca2+ ions into the cells and a rise in the intracellular Ca2+ concentration, leading to the activation of the activator protein-1 (AP-1) transcription factor. Here, we show that expression of a constitutively active mutant of the Ca2+ /calmodulin-dependent protein phosphatase calcineurin attenuated pregnenolone sulfate-induced AP-1 activation in TRPM3-expressing cells. Likewise, expression of the regulatory B subunit of calcineurin reduced AP-1 activity in the cells following stimulation of TRPM3 channels. MAP kinase phosphatase-1 has been shown to attenuate TRPM3-mediated AP-1 activation. Here, we show that pregnenolone sulfate-induced stimulation of TRPM3 triggers the phosphorylation and activation of the MAP kinase extracellular signal-regulated protein kinase (ERK1/2). Pharmacological and genetic experiments revealed that stimulation of ERK1/2 is essential for the activation of AP-1 in cells expressing stimulated TRPM3 channels. ERK1/2 is required for the activation of the transcription factor c-Jun, a key component of the AP-1 transcription factor, and regulates c-Fos promoter activity. In addition, we identified c-Jun N-terminal protein kinase (JNK1/2) as a second signal transducer of activated TRPM3 channels. Together, the data show that calcineurin and the protein kinases ERK1/2 and JNK1/2 are important regulators within the signaling cascade connecting TRPM3 channel stimulation with increased AP-1-regulated transcription. J. Cell. Biochem. 118: 2409-2419, 2017. © 2017 Wiley Periodicals, Inc.

Abstract 306: Targeting of Cdc42-Interacting Protein 4 - Calcineurin Aβ Complexes Ameliorates Pressure Overload-induced Cardiac Remodeling

Pathological cardiac remodeling is the primary mechanism underlying the development of heart failure. The Ca 2+ /calmodulin-dependent protein phosphatase calcineurin is a key regulator of pathological myocyte hypertrophy. Previously, it was shown that the β-isoform of the calcineurin catalytic A-subunit (CaNAβ) was uniquely required for hypertrophy. In order to determine the mechanism of CaN A-subunit specificity, we performed a yeast two-hybrid screen using the CaNAβ-specific N-terminal polyproline domain. We now show that a new binding partner for CaNAβ is the F-Bar (Fes-CIP4 homology [ F CH] - B in/ A mphyphysin/ R vs) scaffold protein Cdc42-interacting protein 4 (CIP4/TRIP10). The CaNAβ polyproline domains binds the SH3 domain of CIP4. Using a new CIP4 conditional knock-out mouse, we show that CIP4 contributes to the hypertrophy induced in vivo by pressure overload. After induction of conditional gene deletion using tamoxifen, CIP4 fl/fl ;Tg(Myh6-cre/Esr1*) mice exhibited decreased hypertrophy and improved cardiac function over a period of 16 weeks following transverse aortic constriction survival surgery when compared to control tamoxifen-treated Tg(Myh6-cre/Esr1*) and CIP4 fl/fl mice. In order to show the relevance of CaNAβ-CIP4 binding, we also developed an adeno-associated virus (AAV) gene therapy vector that expresses a CaNAβ polyproline domain - GFP fusion protein under the control of the cardiac myocyte-specific troponin T promoter. Disruption of CIP4-CaNAβ binding in vivo by the AAV vector attenuates cardiac hypertrophy following two weeks of pressure overload and improved systolic function. Together these results both demonstrate the role of a F-Bar scaffold protein in cardiac remodeling and reveal a novel mechanism for specificity in calcineurin signaling. Identification of this new signaling pathway also suggests a new therapeutic approach to heart failure with reduced ejection fraction.

Cooperative autoinhibition and multi-level activation mechanisms of calcineurin.

The Ca(2+)/calmodulin-dependent protein phosphatase calcineurin (CN), a heterodimer composed of a catalytic subunit A and an essential regulatory subunit B, plays critical functions in various cellular processes such as cardiac hypertrophy and T cell activation. It is the target of the most widely used immunosuppressants for transplantation, tacrolimus (FK506) and cyclosporin A. However, the structure of a large part of the CNA regulatory region remains to be determined, and there has been considerable debate concerning the regulation of CN activity. Here, we report the crystal structure of full-length CN (β isoform), which revealed a novel autoinhibitory segment (AIS) in addition to the well-known autoinhibitory domain (AID). The AIS nestles in a hydrophobic intersubunit groove, which overlaps the recognition site for substrates and immunosuppressant-immunophilin complexes. Indeed, disruption of this AIS interaction results in partial stimulation of CN activity. More importantly, our biochemical studies demonstrate that calmodulin does not remove AID from the active site, but only regulates the orientation of AID with respect to the catalytic core, causing incomplete activation of CN. Our findings challenge the current model for CN activation, and provide a better understanding of molecular mechanisms of CN activity regulation.

Spatial control of calcineurin in response to heat shock in fission yeast.

In fission yeast, Ppb1, the Ca2+/calmodulin-dependent protein phosphatase calcineurin regulates multiple biological processes, such as cytokinesis, Ca2+-homeostasis, membrane trafficking and cell wall integrity. Calcineurin dephosphorylates the Prz1 transcription factor, leading to its nuclear translocation and gene expression under the control of CDRE (calcineurin-dependent response element). Although the calcineurin-mediated spatial control of downstream transcription factors has been intensively studied in many organisms, less is known about the spatial regulation of calcineurin on stresses. Here, we show that heat shock stimulates calcineurin-dependent nuclear translocation of Prz1 and CDRE-dependent gene expression. Notably, calcineurin exhibited a dramatic change in subcellular localization, translocating from diffuse cytoplasmic to dot-like structures on heat shock. The calcineurin dots colocalized with Dcp2 or Pabp, the constituent of P-bodies or stress granules, respectively, thus suggesting that calcineurin is a component of RNA granules under heat shock. Importantly, the calcineurin inhibitor FK506 markedly inhibited the accumulation of calcineurin granules, whereas the constitutively active calcineurin strongly accumulated in the granules on heat shock, suggesting that phosphatase activity is important for calcineurin localization. Notably, the depletion of calcineurin induced a rapid appearance of Nrd1- and Pabp-positive RNA granules. The possible roles of calcineurin in response to heat shock will be discussed.

A TRPV Channel in Drosophila Motor Neurons Regulates Presynaptic Resting Ca2+ Levels, Synapse Growth, and Synaptic Transmission

Presynaptic resting Ca(2+) influences synaptic vesicle (SV) release probability. Here, we report that a TRPV channel, Inactive (Iav), maintains presynaptic resting [Ca(2+)] by promoting Ca(2+) release from the endoplasmic reticulum in Drosophila motor neurons, and is required for both synapse development and neurotransmission. We find that Iav activates the Ca(2+)/calmodulin-dependent protein phosphatase calcineurin, which is essential for presynaptic microtubule stabilization at the neuromuscular junction. Thus, loss of Iav induces destabilization of presynaptic microtubules, resulting in diminished synaptic growth. Interestingly, expression of human TRPV1 in Iav-deficient motor neurons rescues these defects. We also show that the absence of Iav causes lower SV release probability and diminished synaptic transmission, whereas Iav overexpression elevates these synaptic parameters. Together, our findings indicate that Iav acts as a key regulator of synaptic development and function by influencing presynaptic resting [Ca(2+)].

Calcineurin/nuclear factor of activated T cells-coupled vanilliod transient receptor potential channel 4 ca2+ sparklets stimulate airway smooth muscle cell proliferation.

Proliferation of airway smooth muscle cells (ASMCs) contributes to the remodeling and irreversible obstruction of airways during severe asthma, but the mechanisms underlying this disease process are poorly understood. Here we tested the hypothesis that Ca(2+) influx through the vanilliod transient receptor potential channel (TRPV) 4 stimulates ASMC proliferation. We found that synthetic and endogenous TRPV4 agonists increase proliferation of primary ASMCs. Furthermore, we demonstrate that Ca(2+) influx through individual TRPV4 channels produces Ca(2+) microdomains in ASMCs, called "TRPV4 Ca(2+) sparklets." We also show that TRPV4 channels colocalize with the Ca(2+)/calmodulin-dependent protein phosphatase calcineurin in ASMCs. Activated calcineurin dephosphorylates nuclear factor of activated T cells (NFAT) transcription factors cytosolic (c) to allow nuclear translocation and activation of synthetic transcriptional pathways. We show that ASMC proliferation in response to TRPV4 activity is associated with calcineurin-dependent nuclear translocation of the NFATc3 isoform tagged with green florescent protein. Our findings suggest that Ca(2+) microdomains created by TRPV4 Ca(2+) sparklets activate calcineurin to stimulate nuclear translocation of NFAT and ASMC proliferation. These findings further suggest that inhibition of TRPV4 could diminish asthma-induced airway remodeling.

Calcineurin-Crz1 Signaling in Lower Eukaryotes

Calcium ions are ubiquitous intracellular messengers. An increase in the cytosolic Ca(2+) concentration activates many proteins, including calmodulin and the Ca(2+)/calmodulin-dependent protein phosphatase calcineurin. The phosphatase is conserved from yeast to humans (except in plants), and many target proteins of calcineurin have been identified. The most prominent and best-investigated targets, however, are the transcription factors NFAT (nuclear factor of activated T cells) in mammals and Crz1 (calcineurin-responsive zinc finger 1) in yeast. In recent years, many orthologues of Crz1 have been identified and characterized in various species of fungi, amoebae, and other lower eukaryotes. It has been shown that the functions of calcineurin-Crz1 signaling, ranging from ion homeostasis through cell wall biogenesis to the building of filamentous structures, are conserved in the different organisms. Furthermore, frequency-modulated gene expression through Crz1 has been discovered as a striking new mechanism by which cells can coordinate their response to a signal. In this review, I focus on the latest findings concerning calcineurin-Crz1 signaling in fungi, amoebae and other lower eukaryotes. I discuss the potential of Crz1 and its orthologues as putative drug targets, and I also discuss possible parallels with calcineurin-NFAT signaling in mammals.

Regulatory role of regucalcin in heart calcium signaling: Insight into cardiac failure (Review).

Regucalcin was first identified in 1978 as a regulatory protein of Ca2+ signaling in liver cells. Regucalcin was shown to play a multifunctional role in cell regulation, such as maintainance of intracellular Ca2+ homeostasis and suppression of signal transduction, protein synthesis, nuclear function, cell proliferation and apoptosis in various types of cells and tissues. Cardiac excitation-contraction coupling is based on the regulation of intracellular Ca2+ concentration by the Ca2+ pump in the sarcoplasmic reticulum of heart muscle cells. Regucalcin, which is expressed in the heart, was found to increase rat heart sarcoplasmic reticulum Ca2+-ATPase activity and ATP-dependent Ca2+ uptake and mitochondrial Ca2+-ATPase activity. Regucalcin was also shown to suppress Ca2+-dependent protein tyrosine phosphatase, Ca2+/calmodulin-dependent protein phosphatase (calcineurin) and nitric oxide (NO) synthase activity in the heart cytoplasm. Moreover, regucalcin was found to activate superoxide dismutase (SOD), which plays a significant role in the prevention of cell death and apoptosis in the heart. Regucalcin may be a key molecule in heart muscle cell regulation through Ca2+ signaling. Regucalcin may also play a pathophysiological role in heart failure. The aim of this study was to review the recent findings regarding the role of regucalcin in Ca2+ signaling in the heart.

Calcineurin Selectively Docks with the Dynamin Ixb Splice Variant to Regulate Activity-dependent Bulk Endocytosis

Depolarization of nerve terminals stimulates rapid dephosphorylation of two isoforms of dynamin I (dynI), mediated by the calcium-dependent phosphatase calcineurin (CaN). Dephosphorylation at the major phosphorylation sites Ser-774/778 promotes a dynI-syndapin I interaction for a specific mode of synaptic vesicle endocytosis called activity-dependent bulk endocytosis (ADBE). DynI has two main splice variants at its extreme C terminus, long or short (dynIxa and dynIxb) varying only by 20 (xa) or 7 (xb) residues. Recombinant GST fusion proteins of dynIxa and dynIxb proline-rich domains (PRDs) were used to pull down interacting proteins from rat brain nerve terminals. Both bound equally to syndapin, but dynIxb PRD exclusively bound to the catalytic subunit of CaNA, which recruited CaNB. Binding of CaN was increased in the presence of calcium and was accompanied by further recruitment of calmodulin. Point mutations showed that the entire C terminus of dynIxb is a CaN docking site related to a conserved CaN docking motif (PXIXI(T/S)). This sequence is unique to dynIxb among all other dynamin variants or genes. Peptide mimetics of the dynIxb tail blocked CaN binding in vitro and selectively inhibited depolarization-evoked dynI dephosphorylation in nerve terminals but not of other dephosphins. Therefore, docking to dynIxb is required for the regulation of both dynI splice variants, yet it does not regulate the phosphorylation cycle of other dephosphins. The peptide blocked ADBE, but not clathrin-mediated endocytosis of synaptic vesicles. Our results indicate that Ca(2+) influx regulates assembly of a fully active CaN-calmodulin complex selectively on the tail of dynIxb and that the complex is recruited to sites of ADBE in nerve terminals.

The Novel Calcineurin Inhibitor CN585 Has Potent Immunosuppressive Properties in Stimulated Human T Cells

The Ca2+/calmodulin-dependent protein phosphatase calcineurin is a key mediator in antigen-specific T cell activation. Thus, inhibitors of calcineurin, such as cyclosporin A or FK506, can block T cell activation and are used as immunosuppressive drugs to prevent graft-versus-host reactions and autoimmune diseases. In this study we describe the identification of 2,6- diaryl-substituted pyrimidine derivatives as a new class of calcineurin inhibitors, obtained by screening of a substance library. By rational design of the parent compound we have attained the derivative 6-(3,4-dichloro-phenyl)-4-(N,N-dimethylaminoethylthio)-2-phenyl-pyrimidine (CN585) that noncompetitively and reversibly inhibits calcineurin activity with a K(i) value of 3.8 mum. This derivative specifically inhibits calcineurin without affecting other Ser/Thr protein phosphatases or peptidyl prolyl cis/trans isomerases. CN585 shows potent immunosuppressive effects by inhibiting NFAT nuclear translocation and transactivation, cytokine production, and T cell proliferation. Moreover, the calcineurin inhibitor exhibits no cytotoxicity in the effective concentration range. Therefore, calcineurin inhibition by CN585 may represent a novel promising strategy for immune intervention.