Dependent Calmodulin Research Articles

Neurogranin modulates the rate of association between calmodulin and target peptides

The best-known mode of action of calmodulin (CaM) is binding of Ca2+ to its N- and C-domains, followed by binding to target proteins. An underappreciated facet of this process is that CaM is typically bound to proteins at basal levels of free Ca2+, including the small, intrinsically disordered, neuronal IQ-motif proteins called PEP-19 and neurogranin (Ng). PEP-19 and Ng would not be effective competitive inhibitors of high-affinity Ca2+-dependent CaM targets at equilibrium because they bind to CaM with relatively low affinity, but they could influence the time course of CaM signaling by affecting the rate of association of CaM with high-affinity Ca2+-dependent targets. This mode of regulation may be domain specific because PEP-19 binds to the C-domain of CaM, whereas Ng binds to both N- and C-domains. In this report, we used a model CaM binding peptide (CKIIp) to characterize the preferred pathway of complex formation with Ca2+-CaM at low levels of free Ca2+ (0.25–1.5 μM), and how PEP-19 and Ng affect this process. We show that the dominant encounter complex involves association of CKIIp with the N-domain of CaM, even though the C-domain has a greater affinity for Ca2+. We also show that Ng greatly decreases the rate of association of Ca2+-CaM with CKIIp due to the relatively slow dissociation of Ng from CaM, and to interactions between the Gly-rich C-terminal region of Ng with the N-domain of CaM, which inhibits formation of the preferred encounter complex with CKIIp. These results provide the general mechanistic paradigms that binding CaM to targets can be driven by its N-domain, and that low-affinity regulators of CaM signaling have the potential to influence the rate of activation of high-affinity CaM targets and potentially affect the distribution of limited CaM among multiple targets during Ca2+ oscillations.

The control of bionanomachine using calmodulin based ionochromic nanodevice.

A small G-protein (RAS) which leads to the stimulation of downstream pathways, playing an important role in cell proliferation, and differentiation among others may also promote tumorigenesis. Knowing that CaM undergoes a conformational change upon binding to Ca2+, we used Ca2+ dependent Calmodulin as bionanodevice that we successfully incorporate into our M13-Inhibitor-based bionanomachine, to obtain the desired RAS inhibition. A promising inhibition was observed and confirmed by a GTPase essay, along with Ca2+ dependent reversible binding of CAM-I-M13 was monitored by size-exclusion-HPLC. Continuing with the idea of controlling the Ras cycle, some promising bionanodevices were obtained after chemical incorporation of calmodulin target peptide into the functional region of a mutated GEF on one hand and a biological modification of Ras by incorporation of Ras fusion protein with M13 peptide on the HVR region to control by Calmodulin done and succeeded, then a future GEF modification by biological replacement described in this research, showing, as a result, a significant control of the Ras cycle. The mechanism is explained here in this research, guiding us to a potential solution to the enigmatic Ras/GAP/GEF interaction mechanism.

Caveolin-3 regulates the activity of Ca2+/calmodulin-dependent protein kinase II in C2C12 cells.

Caveolins, encoded by the Cav gene family, are the main components of caveolae. Caveolin-3 (Cav3) is specifically expressed in muscle cells. Mutations in Cav3 are responsible for a group of muscle diseases called caveolinopathies, and Cav3 deficiency is associated with sarcolemmal membrane alterations, disorganization of T-tubules, and disruption of specific cell-signaling pathways. However, Cav3 overexpression increases the number of sarcolemmal caveolae and muscular dystrophy-like regenerating muscle fibers with central nuclei, suggesting that the alteration of Cav3 expression levels or localization influences muscle cell functions. Here, we used mouse C2C12 myoblasts in which Cav3 expression was suppressed with short hairpin RNA and found that Cav3 suppression impaired myotube differentiation without affecting the expression of MyoD and Myog. We also observed an increase of intracellular Ca2+ levels, total calpain activity, and Ca2+-dependent calmodulin kinase II (CaMKII) levels in Cav3-depleted myoblasts. Importantly, those phenotypes due to Cav3 suppression were caused by the ryanodine receptor activation. Furthermore, pharmacological inhibition of CaMKII rescued the impairment of myoblast differentiation due to Cav3 knockdown. Our results suggest that Cav3 regulates intracellular Ca2+ concentrations by modulating ryanodine receptor activity in muscle cells and that CaMKII suppression in muscle could be a novel therapy for caveolinopathies.

Flotillin-Dependent Membrane Microdomains Are Required for Functional Phagolysosomes against Fungal Infections

Lipid rafts form signaling platforms on biological membranes with incompletely characterized role in immune response to infection. Here we report that lipid-raft microdomains are essential components of phagolysosomal membranes of macrophages and depend on flotillins. Genetic deletion of flotillins demonstrates that the assembly of both major defense complexes vATPase and NADPH oxidase requires membrane microdomains. Furthermore, we describe a virulence mechanism leading to dysregulation of membrane microdomains by melanized wild-type conidia of the important human-pathogenic fungus Aspergillus fumigatus resulting in reduced phagolysosomal acidification. We show that phagolysosomes with ingested melanized conidia contain a reduced amount of free Ca2+ ions and that inhibition of Ca2+-dependent calmodulin activity led to reduced lipid-raft formation. We identify a single-nucleotide polymorphism in the human FLOT1 gene resulting in heightened susceptibility for invasive aspergillosis in hematopoietic stem cell transplant recipients. Collectively, flotillin-dependent microdomains on the phagolysosomal membrane play an essential role in protective antifungal immunity.

Grb7-derived calmodulin-binding peptides inhibit proliferation, migration and invasiveness of tumor cells while they enhance attachment to the substrate

The growth factor receptor bound protein 7 (Grb7) is a Ca2+-dependent calmodulin (CaM)-binding adaptor protein implicated, among other functions, in cell proliferation, migration and tumor-associated angiogenesis. The goal of this study was to determine whether a peptide based on the CaM binding site of Grb7 disrupts cellular processes, relevant for the malignancy of tumor cells, in which this adaptor protein is implicated. We designed synthetic myristoylated and non-myristoylated peptides corresponding to the CaM-binding domain of human Grb7 with the sequence 243RKLWKRFFCFLRRS256 and a variant peptide with the mutated sequence RKLERFFCFLRRE (W246E-ΔK247-S256E). The two non-myristoylated peptides bind dansyl-CaM with higher efficiency in the presence than in the absence of Ca2+ and they enter into the cell, as tested with 5(6)-carboxytetramethylrhodamine (TAMRA)-labeled peptides. The myristoylated and non-myristoylated peptides inhibit the proliferation, migration and invasiveness of A431 tumor cells while they enhance their adhesion to the substrate. The myristoylated peptides have stronger inhibitory effect than the non-myristoylated counterparts, in agreement with their expected higher cell-permeant capacity. The myristoylated and non-myristoylated W246E-ΔK247-S256E mutant peptide has a lesser inhibitory effect on cell proliferation as compared to the wild-type peptide. We also demonstrated that the myristoylated peptides were more efficient than the CaM antagonist N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) inhibiting cell migration and equally efficient inhibiting cell proliferation.

Deficiency of nuclear receptor interaction protein leads to cardiomyopathy by disrupting sarcomere structure and mitochondrial respiration

BackgroundCardiomyopathy is a common and lethal complication in patients with limb-girdle muscular dystrophy (LGMD), one of the most prevalent forms of muscular dystrophy. The pathogenesis underlying LGMD-related cardiomyopathy remains unclear. NRIP (gene name DCAF6), a Ca2+-dependent calmodulin binding protein, was reduced in dystrophic muscles from LGMD patients. Mice lacking NRIP exhibit a myopathic phenotype resembling that in LGMD patients, making NRIP deficiency a potential culprit leading to cardiomyopathy. This study aimed to determine if NRIP deficiency leads to cardiomyopathy and to explore the underlying molecular mechanisms. Methods and resultsNRIP expression was reduced in both human and mouse failing hearts. Muscle-specific NRIP knockout (MCK-Cre::Dcaf6flox/flox) mouse heart and isolated cardiomyocytes exhibited markedly reduced contractility. Transmission electron microscopy revealed abnormal sarcomere structures and mitochondrial morphology in MCK-Cre::Dcaf6flox/flox hearts. Protein co-immunoprecipitation and confocal imaging revealed that NRIP interacts with α-actinin 2 (ACTN2) at the Z-disc. We found that NRIP facilitated ACTN2-mediated F-actin bundling, and that NRIP deficiency resulted in reduced binding between Z-disc proteins ACTN2 and Cap-Z. In addition, NRIP-deficiency led to increased mitochondrial ROS and impaired mitochondrial respiration/ATP production owing to elevated cellular NADH/NAD+ ratios. Treatment with mitochondria-directed antioxidant mitoTEMPO or NAD+ precursor nicotinic acid restored mitochondrial function and cardiac contractility in MCK-Cre::Dcaf6flox/flox mice. ConclusionsNRIP is essential to maintain sarcomere structure and mitochondrial/contractile function in cardiomyocytes. Our results revealed a novel role for NRIP deficiency in the pathogenesis of LGMD and heart failure. Targeting NRIP, therefore, could be a powerful new approach to treat myocardial dysfunction in LGMD and heart failure patients.

Ca2+-dependent regulation of sodium channels NaV1.4 and NaV1.5 is controlled by the post-IQ motif

Skeletal muscle voltage-gated Na+ channel (NaV1.4) activity is subject to calmodulin (CaM) mediated Ca2+-dependent inactivation; no such inactivation is observed in the cardiac Na+ channel (NaV1.5). Taken together, the crystal structures of the NaV1.4 C-terminal domain relevant complexes and thermodynamic binding data presented here provide a rationale for this isoform difference. A Ca2+-dependent CaM N-lobe binding site previously identified in NaV1.5 is not present in NaV1.4 allowing the N-lobe to signal other regions of the NaV1.4 channel. Consistent with this mechanism, removing this binding site in NaV1.5 unveils robust Ca2+-dependent inactivation in the previously insensitive isoform. These findings suggest that Ca2+-dependent inactivation is effected by CaM’s N-lobe binding outside the NaV C-terminal while CaM’s C-lobe remains bound to the NaV C-terminal. As the N-lobe binding motif of NaV1.5 is a mutational hotspot for inherited arrhythmias, the contributions of mutation-induced changes in CDI to arrhythmia generation is an intriguing possibility.

Inhibition of Autophagy in Cardiomyocytes by Calmodulin Antagonists

Macroautophagy (hereafter referred to as autophagy) is a catabolic process that regulates important aspects of myocardial metabolism. While numerous studies have indicated involvement of signaling via Ca2+ in autophagic flux, the precise mechanisms remain unknown. Calmodulin (CaM) is a ubiquitous transducer of intracellular Ca2+ signals by interacting with an estimated network of 300 cellular proteins. We have begun to examine the role of CaM in autophagic flux in cardiomyocytes. A basal level of autophagy is required for normal myocardial function. Chloroquine dose‐dependently reduces basal autophagic flux in H9c2 cardiomyocytes, as indicated by LC3 immunoblotting, p62 immunofluorescence, and autophagic flux assays. Competitive binding assays using purified CaM and FRET‐based CaM biosensors demonstrate direct Ca2+‐dependent CaM binding to chloroquine. The structurally distinct CaM antagonists W‐7, trifluoperazine, and CGS 9343B also dose‐dependently inhibit basal autophagic flux by reducing autophagosome degradation. Most CaM‐dependent processes depend at the front end on the amplitude and dynamics of intracellular Ca2+ signals. Interestingly, chloroquine, W‐7, trifluoperazine, and CGS 9343b all mobilize intracellular Ca2+. The effects of these pharmacological agents to inhibit autophagic flux are thus associated with their inhibition of CaM and not of initial Ca2+ signals produced in cells. These data indicate a role for CaM in the control of autophagic flux in cardiomyocytes.Support or Funding InformationAmerican Heart Association (15SDG25090279) and Iowa Osteopathic Education and Research Funds (Eric Wauson); NIH HL112184 and DMU IOER‐090717 (Quang‐Kim Tran)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Calcium/calmodulin regulates signaling at the α1A adrenoceptor

Cardiovascular functions are mediated by multiple 7-pass transmembrane receptors whose activation promotes contraction or relaxation of the tissues. The α1 adrenoceptor type 1A plays important roles in the control of vascular tone and myocardial contractility via Ca2+-dependent actions. Here, using novel FRET-based biosensors, we identified a novel Ca2+-dependent interaction between calmodulin (CaM) and the human α1A adrenoceptor at the juxtamembranous region of its 4th submembrane domain (SMD4JM, a.a. 333–361). SMD4JM houses the known nuclear localization signal of α1A adrenoceptor (NLS, a.a. 334–349). We found that NLS itself also interacts with CaM, but with lower affinity and Ca2+ sensitivity, indicating that full interaction between CaM and α1A receptor in this region requires segment a.a. 333–361. Combined K353Q/L356A substitutions in the non-NLS segment of SMD4JM cause a 3.5-fold reduction in the affinity of CaM-SMD4JM interaction. Overexpression of wild-type α1A adrenoceptor in cells enhances phosphorylation of the extracellular signal-regulated kinases 1/2 (ERK1/2) stimulated by A61603, while overexpression of the K353Q/L356A α1A receptor mutant significantly reduces this signal. Norepinephrine stimulates intracellular Ca2+ signals that are higher in cells overexpressing wild-type receptor but lower in cells overexpressing the K353Q/L356A receptor compared to non-transfected cells in the same microscopic environments. These data support a novel and important role for Ca2+-dependent CaM interaction at SMD4JM in α1A adrenoceptor-mediated signaling.

Ranolazine prevents pressure overload-induced cardiac hypertrophy and heart failure by restoring aberrant Na+ and Ca2+ handling.

Cardiac hypertrophy and heart failure are characterized by increased late sodium current and abnormal Ca2+ handling. Ranolazine, a selective inhibitor of the late sodium current, can reduce sodium accumulation and Ca 2+ overload. In this study, we investigated the effects of ranolazine on pressure overload-induced cardiac hypertrophy and heart failure in mice. Inhibition of late sodium current with the selective inhibitor ranolazine suppressed cardiac hypertrophy and fibrosis and improved heart function assessed by echocardiography, hemodynamics, and histological analysis in mice exposed to chronic pressure overload induced by transverse aortic constriction (TAC). Ca2+ imaging of ventricular myocytes from TAC mice revealed both abnormal SR Ca 2+ release and increased SR Ca 2+ leak. Ranolazine restored aberrant SR Ca 2+ handling induced by pressure overload. Ranolazine also suppressed Na + overload induced in the failing heart, and restored Na + -induced Ca 2+ overload in an sodium-calcium exchanger (NCX)-dependent manner. Ranolazine suppressed the Ca 2+ -dependent calmodulin (CaM)/CaMKII/myocyte enhancer factor-2(MEF2) and CaM/CaMKII/calcineurin/nuclear factor of activated T-cells (NFAT) hypertrophy signaling pathways triggered by pressure overload. Pressure overload also prolonged endoplasmic reticulum (ER) stress leading to ER-initiated apoptosis, while inhibition of late sodium current or NCX relieved ER stress and ER-initiated cardiomyocyte apoptosis. Our study demonstrates that inhibition of late sodium current with ranolazine improves pressure overload-induced cardiac hypertrophy and systolic and diastolic function by restoring Na+ and Ca 2+ handling, inhibiting the downstream hypertrophic pathways and ER stress. Inhibition of late sodium current may provide a new treatment strategy for cardiac hypertrophy and heart failure.

Identification and characterization of a novel calmodulin binding site in Drosophila TRP C-terminus

Transient receptor potential (TRP) channels are a group of essential cation channels involved in many important sensory signal transduction processes, such as light, temperature, tastes and pressure sensing. Drosophila TRP channel is the first discovered family member and plays important roles in photo-transduction in Drosophila. Calmodulin (CaM), an important downstream effector of Ca2+ signal, was considered as a vital regulator of TRP activities. In this study, we discovered a novel Ca2+ dependent CaM binding site (TRP 783–862) in between the previously reported two calmodulin binding sites (CBSs). The isothermal titration calorimetry (ITC) and the size exclusion chromatography coupled with multi-angle static light scattering (SEC-MALS) results showed that the dissociation constant (Kd) between TRP 783–862 and Ca2+-CaM is 0.10 ± 0.04 μM and their binding stoichiometry is 1:1. In addition, the shortest Ca2+-CaM interaction region and core CaM binding sequences in TRP 783–862 were dissected by the boundary mapping and mutagenesis experiments. More interestingly, by comparing the circular dichroism (CD) spectra before and after Ca2+-CaM binding, the TRP 783–862 fragment showed Ca2+-CaM binding dependent secondary structure changes, indicating that the interaction between CaM and Drosophila TRP channel may have a conformational impact on TRP structure. In summary, by identifying and characterizing a novel CaM binding site in TRP C-terminus, our findings provided a biochemical and structural basis for further in vivo functional studies of Ca2+-mediated TRP channel regulation through CaM/TRP interaction.

AMP-activated protein kinase–mediated feedback phosphorylation controls the Ca2+/calmodulin (CaM) dependence of Ca2+/CaM-dependent protein kinase kinase β

The Ca2+/calmodulin-dependent protein kinase kinase β (CaMKKβ)/5'-AMP-activated protein kinase (AMPK) phosphorylation cascade affects various Ca2+-dependent metabolic pathways and cancer growth. Unlike recombinant CaMKKβ that exhibits higher basal activity (autonomous activity), activation of the CaMKKβ/AMPK signaling pathway requires increased intracellular Ca2+ concentrations. Moreover, the Ca2+/CaM dependence of CaMKKβ appears to arise from multiple phosphorylation events, including autophosphorylation and activities furnished by other protein kinases. However, the effects of proximal downstream kinases on CaMKKβ activity have not yet been evaluated. Here, we demonstrate feedback phosphorylation of CaMKKβ at multiple residues by CaMKKβ-activated AMPK in addition to autophosphorylation in vitro, leading to reduced autonomous, but not Ca2+/CaM-activated, CaMKKβ activity. MS analysis and site-directed mutagenesis of AMPK phosphorylation sites in CaMKKβ indicated that Thr144 phosphorylation by activated AMPK converts CaMKKβ into a Ca2+/CaM-dependent enzyme as shown by completely Ca2+/CaM-dependent CaMKK activity of a phosphomimetic T144E CaMKKβ mutant. CaMKKβ mutant analysis indicated that the C-terminal domain (residues 471-587), including the autoinhibitory region, plays an important role in stabilizing an inactive conformation in a Thr144 phosphorylation-dependent manner. Furthermore, immunoblot analysis with anti-phospho-Thr144 antibody revealed phosphorylation of Thr144 in CaMKKβ in transfected COS-7 cells that was further enhanced by exogenous expression of AMPKα. These results indicate that AMPK-mediated feedback phosphorylation of CaMKKβ regulates the CaMKKβ/AMPK signaling cascade and may be physiologically important for intracellular maintenance of Ca2+-dependent AMPK activation by CaMKKβ.

Estrogen Enhances Linkage in the Vascular Endothelial Calmodulin Network via a Feedforward Mechanism at the G Protein-coupled Estrogen Receptor 1

Estrogen exerts many effects on the vascular endothelium. Calmodulin (CaM) is the transducer of Ca(2+) signals and is a limiting factor in cardiovascular tissues. It is unknown whether and how estrogen modifies endothelial functions via the network of CaM-dependent proteins. Here we show that 17β-estradiol (E2) up-regulates total CaM level in endothelial cells. Concurrent measurement of Ca(2+) and Ca(2+)-CaM indicated that E2 also increases free Ca(2+)-CaM. Pharmacological studies, gene silencing, and receptor expression-specific cell studies indicated that the G protein-coupled estrogen receptor 1 (GPER/GPR30) mediates these effects via transactivation of EGFR and subsequent MAPK activation. The outcomes were then examined on four distinct members of the intracellular CaM target network, including GPER/GPR30 itself and estrogen receptor α, the plasma membrane Ca(2+)-ATPase (PMCA), and endothelial nitric-oxide synthase (eNOS). E2 substantially increases CaM binding to estrogen receptor α and GPER/GPR30. Mutations that reduced CaM binding to GPER/GPR30 in separate binding domains do not affect GPER/GPR30-Gβγ preassociation but decrease GPER/GPR30-mediated ERK1/2 phosphorylation. E2 increases CaM-PMCA association, but the expected stimulation of Ca(2+) efflux is reversed by E2-stimulated tyrosine phosphorylation of PMCA. These effects sustain Ca(2+) signals and promote Ca(2+)-dependent CaM interactions with other CaM targets. Consequently, E2 doubles CaM-eNOS interaction and also promotes dual phosphorylation of eNOS at Ser-617 and Ser-1179. Calculations using in-cell and in vitro data revealed substantial individual and combined contribution of these effects to total eNOS activity. Taken together, E2 generates a feedforward loop via GPER/GPR30, which enhances Ca(2+)/CaM signals and functional linkage in the endothelial CaM target network.

Novel regulation of equlibrative nucleoside transporter 1 (ENT1) by receptor-stimulated Ca2+-dependent calmodulin binding.

Equilibrative nucleoside transporters (ENTs) facilitate the flux of nucleosides, such as adenosine, and nucleoside analog (NA) drugs across cell membranes. A correlation between adenosine flux and calcium-dependent signaling has been previously reported; however, the mechanistic basis of these observations is not known. Here we report the identification of the calcium signaling transducer calmodulin (CaM) as an ENT1-interacting protein, via a conserved classic 1-5-10 motif in ENT1. Calcium-dependent human ENT1-CaM protein interactions were confirmed in human cell lines (HEK293, RT4, U-87 MG) using biochemical assays (HEK293) and the functional assays (HEK293, RT4), which confirmed modified nucleoside uptake that occurred in the presence of pharmacological manipulations of calcium levels and CaM function. Nucleoside and NA drug uptake was significantly decreased (∼12% and ∼39%, respectively) by chelating calcium (EGTA, 50 μM; BAPTA-AM, 25 μM), whereas increasing intracellular calcium (thapsigargin, 1.5 μM) led to increased nucleoside uptake (∼26%). Activation of N-methyl-d-aspartate (NMDA) receptors (in U-87 MG) by glutamate (1 mM) and glycine (100 μM) significantly increased nucleoside uptake (∼38%) except in the presence of the NMDA receptor antagonist, MK-801 (50 μM), or CaM antagonist, W7 (50 μM). These data support the existence of a previously unidentified novel receptor-dependent regulatory mechanism, whereby intracellular calcium modulates nucleoside and NA drug uptake via CaM-dependent interaction of ENT1. These findings suggest that ENT1 is regulated via receptor-dependent calcium-linked pathways resulting in an alteration of purine flux, which may modulate purinergic signaling and influence NA drug efficacy.

The Key Role of Calmodulin in KRAS-Driven Adenocarcinomas.

KRAS4B is a highly oncogenic splice variant of the KRAS isoform. It is the only isoform associated with initiation of adenocarcinomas. Insight into why and how KRAS4B can mediate ductal adenocarcinomas, particularly of the pancreas, is vastly important for its therapeutics. Here we point out the overlooked critical role of calmodulin (CaM). Calmodulin selectively binds to GTP-bound K-Ras4B; but not to other Ras isoforms. Cell proliferation and growth require the MAPK (Raf/MEK/ERK) and PI3K/Akt pathways. We propose that Ca(2+)/calmodulin promote PI3Kα/Akt signaling, and suggest how. The elevated calcium levels clinically observed in adenocarcinomas may explain calmodulin's involvement in recruiting and stimulating PI3Kα through interaction with its n/cSH2 domains as well as K-Ras4B; importantly, it also explains why K-Ras4B specifically is a key player in ductal carcinomas, such as pancreatic (PDAC), colorectal (CRC), and lung cancers. We hypothesize that calmodulin recruits and helps activate PI3Kα at the membrane, and that this is the likely reason for Ca(2+)/calmodulin dependence in adenocarcinomas. Calmodulin can contribute to initiation/progression of ductal cancers via both PI3Kα/Akt and Raf/MEK/ERK pathways. Blocking the K-Ras4B/MAPK pathway and calmodulin/PI3Kα binding in a K-Ras4B/calmodulin/PI3Kα trimer could be a promising adenocarcinoma-specific therapeutic strategy.

Abstract 37: Nrip Deficiency Leads to Dilated Cardiomyopathy

Previously, we demonstrate a gene, nuclear receptor interaction protein (NRIP, also named DCAF6 or IQWD1) as a Ca2+- dependent calmodulin binding protein that can activate calcineurin phosphatase activity. Here, we found that α-actinin-2 (ACTN2), is one of NRIP-interacting proteins from the yeast two-hybrid system using NRIP as a prey. We further confirmed the direct bound between NRIP and ACTN2 using in vitro protein-protein interaction and in vivo co-immunoprecipitation assays. To further map the binding domain of each protein, the results showed the IQ domain of NRIP responsible for ACTN2 binding, and EF hand motif of ACTN2 responsible for NRIP bound. Due to ACTN2 is a biomarker of muscular Z-disc complex; we found the co-localization of NRIP and ACTN2 in cardiac tissues by immunofluorescence assays. Taken together, NRIP is a novel ACTN2-interacting protein. To investigate insights into in vivo function of NRIP, we generated conventional NRIP-null mice. The H&E staining results are shown in the hearts of NRIP KO mice are enlarged and dilated and the cell width of NRIP KO cardiomyocyte is increased. The EM of NRIP KO heart muscles reveal the reduction of I-band width and extension length of Z-disc in sarcomere structure; and the echocardiography shows the diminished fractional shortening in heart functions. Additionally, the calcium transient and sarcomere contraction length in cardiomyocytes of NRIP KO is weaker and shorter than wt; respectively. In conclusion, NRIP is a novel Z-disc protein and has function for maintenance of sarcomere integrity structure and function for calcium transient and muscle contraction.

What Currents Underlie Pulmonary Vein Automaticity?

What Currents Underlie Pulmonary Vein Automaticity?

Abstract 2282: Calmodulin binding to DR-5 and the role of CaM-DR-5 binding in DR-5-mediated DISC formation in breast cancer

Abstract Molecular classification of breast cancers into receptor-based subtypes, including estrogen positive or triple negative breast cancers, establish targets that guide individualized treatment for patients (Journal of clinical oncology:27, 1153-1154, 2009). However, drug resistance observed for breast cancer raises the challegens for the breast cancer treatment (CA: a cancer journal for clinicians 59, 303-313, 2009). Targeting the death receptor-5 (DR-5) to induce breast cancer apoptosis is a promising strategy for breast cancer treatment (Immunity 3, 673-682, 1995, Breast Cancer Res Treat 113, 217-230, 2009). TRA-8 is a DR-5 specific agonistic antibody (Nature medicine 7, 954-960, 2001), and TRA-8 can induce apoptosis in breast cancer cells (Clinical cancer research: 9, 3731-3741, 2003). TRA-8 signal DR-5-mediated apoptotic signal via formation of the death inducing signaling complex (DISC) at the DR-5 death domain (Immunity 12, 599-609, 2000). Calmodulin (CaM) is a mediator of calcium signaling and has been shown to play a role in regulating DR-5 mediated apoptosis (Biochem Cell Biol 87, 919-926, 2009). Understanding the mechanism of DR-5-CaM interaction and the role of CaM-DR-5 binding in regulating DR-5 mediated DISC formation, may unveil CaM-DR-5 binding as a potential target to modulate DR-5 mediated apoptosis. In this study we characterize an interaction between CaM and DR-5 and the role of CaM-DR-5 binding in DR-5-mediated DISC formation in MCF-7 and MDA-MB-231 breast cancers. Complex-immunoprecipitation of DR-5 from MCF-7 and MDA-MB-231 lysates demonstrated endogenous CaM and DR-5 form a complex. Native PAGE of recombinant 6xHis-SUMO-CaM incubated with MCF-7 and MDA-MB-231 lysates showed the formation of a CaM and DR-5 complex. CaM pull down of DR-5 from MCF-7 and MDA-MB231 lysates in the presence of 1 mM Ca2+ or 2 mM EGTA showed CaM/DR-5 interaction is calcium dependent. TRA-8 activation of DR-5 resulted in CaM recruitment into the DR-5 mediated DISC. Additionally, Either EGTA or CaM antagonist trifluopherazine (TFP) attenuated DR-5 mediated DISC formation in MCF-7 and MDA-MB-231 breast cancers. We present evidence for the Ca2+ dependent CaM and DR-5 interaction and a role of CaM-DR-5 binding in regulating DR-5 mediated DISC formation in breast cancer cells. Results from this study provide the basis for the further characterization of CaM-DR5 interactions and its important role in cellular activities, which could lead to novel strategies for effectively regulating DR-5-mediated apoptosis in breast cancer. Citation Format: Romone M. Fancy, Hong Wang, Tong Zhou, Yuhua Song. Calmodulin binding to DR-5 and the role of CaM-DR-5 binding in DR-5-mediated DISC formation in breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2282. doi:10.1158/1538-7445.AM2014-2282

Role of CA2+/calmodulin on ethanol neurobehavioral effects.

The cAMP-dependent protein kinase A (PKA) signaling transduction pathway has been shown to play an important role in the modulation of several ethanol-induced behaviors. Different studies have demonstrated intracellular calcium (Ca(2+))-dependent activation of the PKA cascade after ethanol administration. Thus, the cAMP cascade mediator Ca(2+)-dependent calmodulin (CaM) has been strongly implicated in the central effects of ethanol. In this study, we assessed the role of the CaM inhibitor W7 on ethanol-induced stimulation, ethanol intake, and ethanol-induced activation of PKA. Swiss mice were pretreated with W7 (0-10mg/kg) 30min before ethanol (0-3.75g/kg) administration. Immediately, animals were placed during 20min in an open-field chamber. Ethanol (10%, v/v) intake in 2h was assessed using a limited access paradigm. Experiments with caffeine (0-15mg/kg), cocaine (0-4mg/kg), and saccharine (0.1%, w/v) were designed to compare their results to those obtained with ethanol. Western blot was assayed 45min after ethanol administration. Results showed that pretreatment with W7, reduced selectively in a dose-dependent fashion ethanol-induced locomotor stimulation and ethanol intake. The ethanol-induced activation of PKA was also prevented by W7 administration. These results demonstrate that CaM inhibition resulted in a selective reduction of ethanol-stimulating effects and ethanol intake. The PKA activation induced by ethanol was blocked after the CaM blockade with W7. These results provide further evidence of the key role of cellular Ca(2+)-dependent pathways on the central effects of ethanol.

Phosphodiesterase 1A modulates cystogenesis in zebrafish.

Substantial evidence indicates the importance of elevated cAMP in polycystic kidney disease (PKD). Accumulation of cAMP in cystic tissues may be, in part, caused by enhanced adenylyl cyclase activity, but inhibition of cAMP degradation by phosphodiesterases (PDE) likely has an important role, because cAMP is inactivated much faster than it is synthesized. PDE1 is the only PDE family activated by Ca(2+), which is reduced in PKD cells. To assess the contribution of the PDE1A subfamily to renal cyst formation, we examined the expression and function of PDE1A in zebrafish. We identified two splice isoforms with alternative starts corresponding to human PDE1A1 and PDE1A4. Expression of the two isoforms varied in embryos and adult tissues, and both isoforms hydrolyzed cAMP with Ca(2+)/calmodulin dependence. Depletion of PDE1A in zebrafish embryos using splice- and translation-blocking morpholinos (MOs) caused pronephric cysts, hydrocephalus, and body curvature. Human PDE1A RNA and the PKA inhibitors, H89 and Rp-cAMPS, partially rescued phenotypes of pde1a morphants. Additionally, MO depletion of PDE1A aggravated phenotypes in pkd2 morphants, causing more severe body curvature, and human PDE1A RNA partially rescued pkd2 morphant phenotypes, pronephric cysts, hydrocephalus, and body curvature. Together, these data indicate the integral role of PDE1A and cAMP signaling in renal development and cystogenesis, imply that PDE1A activity is altered downstream of polycystin-2, and suggest that PDE1A is a viable drug target for PKD.