Journal articles: 'Calmodulin mediated activation'

1

Moretó, Jemina, Anna Lladó, Maite Vidal-Quadras, Maria Calvo, Albert Pol, Carlos Enrich, and Francesc Tebar. "Calmodulin modulates H-Ras mediated Raf-1 activation." Cellular Signalling 20, no. 6 (June 2008): 1092–103. http://dx.doi.org/10.1016/j.cellsig.2008.01.022.

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Schmitt, John M., Gary A. Wayman, Naohito Nozaki, and Thomas R. Soderling. "Calcium Activation of ERK Mediated by Calmodulin Kinase I." Journal of Biological Chemistry 279, no. 23 (March 29, 2004): 24064–72. http://dx.doi.org/10.1074/jbc.m401501200.

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Miao, Bei, Xiao-Hui Yin, Dong-Sheng Pei, Quan-Guang Zhang, and Guang-Yi Zhang. "Neuroprotective Effects of Preconditioning Ischemia on Ischemic Brain Injury through Down-regulating Activation of JNK1/2 via N-Methyl-D-aspartate Receptor-mediated Akt1 Activation." Journal of Biological Chemistry 280, no. 23 (March 29, 2005): 21693–99. http://dx.doi.org/10.1074/jbc.m500003200.

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Our previous studies have demonstrated that the JNK signaling pathway plays an important role in ischemic brain injury and is mediated via glutamate receptor 6. Others studies have shown that N-methyl-d-aspartate (NMDA) receptor is involved in the neuroprotection of ischemic preconditioning. Here we examined whether ischemic preconditioning down-regulates activation of the mixed lineage kinase-JNK signaling pathway via NMDA receptor-mediated Akt1 activation. In our present results, ischemic preconditioning could not only inhibit activations of mixed lineage kinase 3, JNK1/2, and c-Jun but also enhanced activation of Akt1. In addition, both NMDA (an agonist of NMDA receptor) and preconditioning showed neuroprotective effects. In contrast, ketamine, an antagonist of NMDA receptor, prevented the above effects of preconditioning. Further studies indicated that LY294002, an inhibitor of phosphoinositide 3-kinase that is an upstream signaling protein of Akt1, could block neuroprotection of preconditioning, and KN62, an inhibitor of calmodulin-dependent protein kinase, also achieved the same effects as LY294002. Therefore, both phosphoinositide 3-kinase and calmodulin-dependent protein kinase are involved in the activation of Akt1 in ischemic tolerance. Taken together, our results indicate that preconditioning can inhibit activation of JNK signaling pathway via NMDA receptor-mediated Akt1 activation and induce neuroprotection in hippocampal CA1 region.

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Matthews, R. P., C. R. Guthrie, L. M. Wailes, X. Zhao, A. R. Means, and G. S. McKnight. "Calcium/calmodulin-dependent protein kinase types II and IV differentially regulate CREB-dependent gene expression." Molecular and Cellular Biology 14, no. 9 (September 1994): 6107–16. http://dx.doi.org/10.1128/mcb.14.9.6107-6116.1994.

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Phosphorylation of CREB (cyclic AMP [cAMP]- response element [CRE]-binding protein) by cAMP-dependent protein kinase (PKA) leads to the activation of many promoters containing CREs. In neurons and other cell types, CREB phosphorylation and activation of CRE-containing promoters can occur in response to elevated intracellular Ca2+. In cultured cells that normally lack this Ca2+ responsiveness, we confer Ca(2+)-mediated activation of a CRE-containing promoter by introducing an expression vector for Ca2+/calmodulin-dependent protein kinase type IV (CaMKIV). Activation could also be mediated directly by a constitutively active form of CaMKIV which is Ca2+ independent. The CaMKIV-mediated gene induction requires the activity of CREB/ATF family members but is independent of PKA activity. In contrast, transient expression of either a constitutively active or wild-type Ca2+/calmodulin-dependent protein kinase type II (CaMKII) fails to mediate the transactivation of the same CRE-containing reporter gene. Examination of the subcellular distribution of transiently expressed CaMKIV and CaMKII reveals that only CaMKIV enters the nucleus. Our results demonstrate that CaMKIV, which is expressed in neuronal, reproductive, and lymphoid tissues, may act as a mediator of Ca(2+)-dependent gene induction.

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Matthews, R. P., C. R. Guthrie, L. M. Wailes, X. Zhao, A. R. Means, and G. S. McKnight. "Calcium/calmodulin-dependent protein kinase types II and IV differentially regulate CREB-dependent gene expression." Molecular and Cellular Biology 14, no. 9 (September 1994): 6107–16. http://dx.doi.org/10.1128/mcb.14.9.6107.

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Phosphorylation of CREB (cyclic AMP [cAMP]- response element [CRE]-binding protein) by cAMP-dependent protein kinase (PKA) leads to the activation of many promoters containing CREs. In neurons and other cell types, CREB phosphorylation and activation of CRE-containing promoters can occur in response to elevated intracellular Ca2+. In cultured cells that normally lack this Ca2+ responsiveness, we confer Ca(2+)-mediated activation of a CRE-containing promoter by introducing an expression vector for Ca2+/calmodulin-dependent protein kinase type IV (CaMKIV). Activation could also be mediated directly by a constitutively active form of CaMKIV which is Ca2+ independent. The CaMKIV-mediated gene induction requires the activity of CREB/ATF family members but is independent of PKA activity. In contrast, transient expression of either a constitutively active or wild-type Ca2+/calmodulin-dependent protein kinase type II (CaMKII) fails to mediate the transactivation of the same CRE-containing reporter gene. Examination of the subcellular distribution of transiently expressed CaMKIV and CaMKII reveals that only CaMKIV enters the nucleus. Our results demonstrate that CaMKIV, which is expressed in neuronal, reproductive, and lymphoid tissues, may act as a mediator of Ca(2+)-dependent gene induction.

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ROMERO, Irma, Ana M. MALDONADO, and Pilar ERASO. "Glucose-independent inhibition of yeast plasma-membrane H+-ATPase by calmodulin antagonists." Biochemical Journal 322, no. 3 (March 15, 1997): 823–28. http://dx.doi.org/10.1042/bj3220823.

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Glucose metabolism causes activation of the yeast plasma-membrane H+-ATPase. The molecular mechanism of this regulation is not known, but it is probably mediated by phosphorylation of the enzyme. The involvement in this process of several kinases has been suggested but their actual role has not been proved. The physiological role of a calmodulin-dependent protein kinase in glucose-induced activation was investigated by studying the effect of specific calmodulin antagonists on the glucose-induced ATPase kinetic changes in wild-type and two mutant strains affected in the glucose regulation of the enzyme. Preincubation of the cells with calmidazolium or compound 48/80 impeded the increase in ATPase activity by reducing the Vmax of the enzyme without modifying the apparent affinity for ATP in the three strains. In one mutant, pma1-T912A, the putative calmodulin-dependent protein kinase-phosphorylatable Thr-912 was eliminated, and in the other, pma1-P536L, H+-ATPase was constitutively activated, suggesting that the antagonistic effect was not mediated by a calmodulin-dependent protein kinase and not related to glucose regulation. This was corroborated when the in vitroeffect of the calmodulin antagonists on H+-ATPase activity was tested. Purified plasma membranes from glucose-starved or glucose-fermenting cells from both pma1-P890X, another constitutively activated ATPase mutant, and wild-type strains were preincubated with calmidazolium or melittin. In all cases, ATP hydrolysis was inhibited with an IC50 of ≈1 μM. This inhibition was reversed by calmodulin. Analysis of the calmodulin-binding protein pattern in the plasma-membrane fraction eliminates ATPase as the calmodulin target protein. We conclude that H+-ATPase inhibition by calmodulin antagonists is mediated by an as yet unidentified calmodulin-dependent membrane protein.

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Mangels, L. A., R. R. Neubig, H. E. Hamm, and M. E. Gnegy. "Calmodulin binding distinguishes between βγ subunits of activated G proteins and transducin." Biochemical Journal 283, no. 3 (May 1, 1992): 683–90. http://dx.doi.org/10.1042/bj2830683.

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The interactions between guanine nucleotide regulatory proteins and the Ca(2+)-binding protein calmodulin were studied using calmodulin-Sepharose affinity chromatography. Purified bovine brain beta gamma subunits bound to calmodulin-Sepharose in a Ca(2+)-dependent manner. On the contrary, beta gamma subunits produced in an activated Go/Gi preparation did not bind to calmodulin-Sepharose. The effect was independent of the type of bovine brain G protein (Go/Gi, Gs), method of activation and the presence of magnesium. To distinguish whether the binding of purified beta gamma subunits to calmodulin was unique to brain beta gamma or to the method of purification, similar experiments were performed using transducin. In contrast to bovine brain G proteins, both purified transducin beta gamma subunits and beta gamma released from rhodopsin-activated transducin bound to calmodulin-Sepharose in a Ca(2+)-dependent manner. To assess the functional significance of the binding of bovine brain beta gamma subunits to calmodulin, the ability of purified beta gamma and of beta gamma in unactivated and activated Go/Gi to inhibit partially purified calmodulin-sensitive adenylate cyclase was determined. Purified beta gamma was highly effective in inhibiting calmodulin-stimulated adenylate cyclase activity. However, unactivated Go/Gi and preactivated Go/Gi inhibited calmodulin-stimulated adenylate cyclase activity to the same extent. This Go/Gi-mediated inhibition also occurred in the presence of a 500-fold molar excess of calmodulin over added G protein. These results demonstrate: (1) that beta gamma subunits may not be completely released upon G protein activation, and (2) that inhibition of calmodulin-stimulated adenylate cyclase by beta gamma subunits does not appear to be mediated by a direct beta gamma-calmodulin interaction. Differences in the binding properties of activated bovine brain G proteins versus those of transducin could be explained by differences in the gamma subunit between the proteins, or by differences in affinities of the alpha and beta gamma subunits for each other and for calmodulin. The different functional properties of purified beta gamma subunits and beta gamma subunits produced in situ by activation of G proteins indicates that extrapolation from the effects of purified subunits to events occurring in membranes should be done with caution.

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Gardiner, Elizabeth E., Denuja Karunakaran, Jane F. Arthur, Fi-Tjen Mu, Maree S. Powell, Ross I. Baker, P. Mark Hogarth, Mark L. Kahn, Robert K. Andrews, and Michael C. Berndt. "Dual ITAM-mediated proteolytic pathways for irreversible inactivation of platelet receptors: de-ITAM-izing FcγRIIa." Blood 111, no. 1 (January 1, 2008): 165–74. http://dx.doi.org/10.1182/blood-2007-04-086983.

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Collagen binding to glycoprotein VI (GPVI) induces signals critical for platelet activation in thrombosis. Both ligand-induced GPVI signaling through its coassociated Fc-receptor γ-chain (FcRγ) immunoreceptor tyrosine-activation motif (ITAM) and the calmodulin inhibitor, W7, dissociate calmodulin from GPVI and induce metalloproteinase-mediated GPVI ectodomain shedding. We investigated whether signaling by another ITAM-bearing receptor on platelets, FcγRIIa, also down-regulates GPVI expression. Agonists that signal through FcγRIIa, the mAbs VM58 or 14A2, potently induced GPVI shedding, inhibitable by the metalloproteinase inhibitor, GM6001. Unexpectedly, FcγRIIa also underwent rapid proteolysis in platelets treated with agonists for FcγRIIa (VM58/14A2) or GPVI/FcRγ (the snake toxin, convulxin), generating an approximate 30-kDa fragment. Immunoprecipitation/pull-down experiments showed that FcγRIIa also bound calmodulin and W7 induced FcγRIIa cleavage. However, unlike GPVI, the approximate 30-kDa FcγRIIa fragment remained platelet associated, and proteolysis was unaffected by GM6001 but was inhibited by a membrane-permeable calpain inhibitor, E64d; consistent with this, μ-calpain cleaved an FcγRIIa tail-fusion protein at 222Lys/223Ala and 230Gly/231Arg, upstream of the ITAM domain. These findings suggest simultaneous activation of distinct extracellular (metalloproteinase-mediated) and intracellular (calpain-mediated) proteolytic pathways irreversibly inactivating platelet GPVI/FcRγ and FcγRIIa, respectively. Activation of both pathways was observed with immunoglobulin from patients with heparin-induced thrombocytopenia (HIT), suggesting novel mechanisms for platelet dysfunction by FcγRIIa after immunologic insult.

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Gray, Michael, Daniel H. Daudelin, and Jorge Golowasch. "Activation mechanism of a neuromodulator-gated pacemaker ionic current." Journal of Neurophysiology 118, no. 1 (July 1, 2017): 595–609. http://dx.doi.org/10.1152/jn.00743.2016.

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The neuromodulator-gated current ( IMI) found in the crab stomatogastric ganglion is activated by neuromodulators that are essential to induce the rhythmic activity of the pyloric network in this system. One of these neuromodulators is also known to control the correlated expression of voltage-gated ionic currents in pyloric neurons, as well as synaptic plasticity and strength. Thus understanding the mechanism by which neuromodulator receptors activate IMI should provide insights not only into how oscillations are initiated but also into how other processes, and currents not directly activated by them, are regulated. To determine what specific signaling molecules are implicated in this process, we used a battery of agonists and antagonists of common signal transduction pathways. We found that the G protein inhibitor GDPβS and the G protein activator GTPγS significantly affect IMI amplitude, suggesting that its activation is mediated by G proteins. Interestingly, when using the more specific G protein blocker pertussis toxin, we observed the expected inhibition of IMI amplitude but, unexpectedly, in a calcium-dependent fashion. We also found that antagonists of calcium- and calmodulin-associated signaling significantly reduce IMI amplitude. In contrast, we found little evidence for the role of cyclic nucleotide signaling, phospholipase C (PLC), or kinases and phosphatases, except two calmodulin-dependent kinases. In sum, these results suggest that proctolin-induced IMI is mediated by a G protein whose pertussis toxin sensitivity is altered by external calcium concentration and appears to depend on intracellular calcium, calmodulin, and calmodulin-activated kinases. In contrast, we found no support for IMI being mediated by PLC signaling or cyclic nucleotides. NEW & NOTEWORTHY Neuronal rhythmic activity is generated by either network-based or cell-autonomous mechanisms. In the pyloric network of decapod crustaceans, the activation of a neuromodulator-gated pacemaker current is crucial for the generation of rhythmic activity. This current is activated by several neuromodulators, including peptides and acetylcholine, presumably via metabotropic receptors. We have previously demonstrated a novel extracellular calcium-sensitive voltage-dependence mechanism of this current. We presently report that the activation mechanism depends on intracellular and extracellular calcium-sensitive components.

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Tokumitsu, Hiroshi, and Hiroyuki Sakagami. "Molecular Mechanisms Underlying Ca2+/Calmodulin-Dependent Protein Kinase Kinase Signal Transduction." International Journal of Molecular Sciences 23, no. 19 (September 20, 2022): 11025. http://dx.doi.org/10.3390/ijms231911025.

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Ca2+/calmodulin-dependent protein kinase kinase (CaMKK) is the activating kinase for multiple downstream kinases, including CaM-kinase I (CaMKI), CaM-kinase IV (CaMKIV), protein kinase B (PKB/Akt), and 5′AMP-kinase (AMPK), through the phosphorylation of their activation-loop Thr residues in response to increasing the intracellular Ca2+ concentration, as CaMKK itself is a Ca2+/CaM-dependent enzyme. The CaMKK-mediated kinase cascade plays important roles in a number of Ca2+-dependent pathways, such as neuronal morphogenesis and plasticity, transcriptional activation, autophagy, and metabolic regulation, as well as in pathophysiological pathways, including cancer progression, metabolic syndrome, and mental disorders. This review focuses on the molecular mechanism underlying CaMKK-mediated signal transduction in normal and pathophysiological conditions. We summarize the current knowledge of the structural, functional, and physiological properties of the regulatory kinase, CaMKK, and the development and application of its pharmacological inhibitors.

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Choi, S. H., B. H. Lee, S. H. Hwang, H. J. Kim, S. M. Lee, H. C. Kim, H. W. Rhim, and S. Y. Nah. "Molecular Mechanisms of Large-Conductance Ca2+-Activated Potassium Channel Activation by Ginseng Gintonin." Evidence-Based Complementary and Alternative Medicine 2013 (2013): 1–14. http://dx.doi.org/10.1155/2013/323709.

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Gintonin is a unique lysophosphatidic acid (LPA) receptor ligand found inPanax ginseng. Gintonin induces transient [Ca2+]ithrough G protein-coupled LPA receptors. Large-conductance Ca2+-activated K+(BKCa) channels are expressed in blood vessels and neurons and play important roles in blood vessel relaxation and attenuation of neuronal excitability. BKCachannels are activated by transient [Ca2+]iand are regulated by various Ca2+-dependent kinases. We investigated the molecular mechanisms of BKCachannel activation by gintonin. BKCachannels are heterologously expressed inXenopus oocytes. Gintonin treatment induced BKCachannel activation in oocytes expressing the BKCachannelαsubunit in a concentration-dependent manner (EC50= 0.71 ± 0.08 µg/mL). Gintonin-mediated BKCachannel activation was blocked by a PKC inhibitor, calphostin, and by the calmodulin inhibitor, calmidazolium. Site-directed mutations in BKCachannels targeting CaM kinase II or PKC phosphorylation sites but not PKA phosphorylation sites attenuated gintonin action. Mutations in the Ca2+bowl and the regulator of K+conductance (RCK) site also blocked gintonin action. These results indicate that gintonin-mediated BKCachannel activations are achieved through LPA1 receptor-phospholipase C-IP3-Ca2+-PKC-calmodulin-CaM kinase II pathways and calcium binding to the Ca2+bowl and RCK domain. Gintonin could be a novel contributor against blood vessel constriction and over-excitation of neurons.

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Trenn, G., R. Taffs, R. Hohman, R. Kincaid, E. M. Shevach, and M. Sitkovsky. "Biochemical characterization of the inhibitory effect of CsA on cytolytic T lymphocyte effector functions." Journal of Immunology 142, no. 11 (June 1, 1989): 3796–802. http://dx.doi.org/10.4049/jimmunol.142.11.3796.

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Abstract We have examined the effects of cyclosporine A (CsA) on a number of CTL effector functions. CsA partially inhibited the CTL-mediated lysis of Ag-bearing target cells. Both target cell- and anti-TCR mAb-induced granule exocytosis were markedly inhibited by CsA. In addition, marked inhibition of PMA and calcium ionophore (A23187) induced granule exocytosis was produced by CsA suggesting that the inhibitory effects of CsA on granule exocytosis involve biochemical events after protein kinase C activation and increases in intracellular free Ca2+. CsA had no inhibitory effects on TCR-mediated phosphatidylinositol metabolism. The inhibitory effects of CsA were not mediated by the cAMP-dependent protein kinase inhibitory pathway and no effect of CsA on the Ca2+-induced binding of calmodulin to calmodulin-binding proteins could be demonstrated. CsA was also a potent inhibitor of IgE receptor-mediated exocytosis in rat basophil leukemia cells. CsA had no effect on receptor-mediated phosphatidylinositol hydrolysis; 400 ng/ml CsA resulted in a 90% inhibition of serotonin release but had no effect on phosphatidylinositol hydrolysis. These results indicate that CsA may inhibit some common event in Ca2+-dependent secretory cells. Taken together, these results suggest that CsA does not inhibit signal transduction but rather interferes with the biochemical events in the later stages of Ca2+-dependent reactions that follow the binding of calmodulin to cytoskeletal or cytoplasmic calmodulin binding proteins.

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Sarria, Ignacio, Jennifer Ling, Michael X. Zhu, and Jianguo G. Gu. "TRPM8 acute desensitization is mediated by calmodulin and requires PIP2: distinction from tachyphylaxis." Journal of Neurophysiology 106, no. 6 (December 2011): 3056–66. http://dx.doi.org/10.1152/jn.00544.2011.

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The cold-sensing channel transient receptor potential melastatin 8 (TRPM8) features Ca2+-dependent downregulation, a cellular process underlying somatosensory accommodation in cold environments. The Ca2+-dependent functional downregulation of TRPM8 is manifested with two distinctive phases, acute desensitization and tachyphylaxis. Here we show in rat dorsal root ganglion neurons that TRPM8 acute desensitization critically depends on phosphatidylinositol 4,5-bisphosphate (PIP2) availability rather than PIP2 hydrolysis and is triggered by calmodulin activation. Tachyphylaxis, on the other hand, is mediated by phospholipase hydrolysis of PIP2 and protein kinase C/phosphatase 1,2A. We further demonstrate that PIP2 switches TRPM8 channel gating to a high-open probability state with short closed times. Ca2+-calmodulin reverses the effect of PIP2, switching channel gating to a low-open probability state with long closed times. Thus, through gating modulation, Ca2+-calmodulin provides a mechanism to rapidly regulate TRPM8 functions in the somatosensory system.

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Talbi, Khaoula, Jiraporn Ousingsawat, Raquel Centeio, Rainer Schreiber, and Karl Kunzelmann. "Calmodulin-Dependent Regulation of Overexpressed but Not Endogenous TMEM16A Expressed in Airway Epithelial Cells." Membranes 11, no. 9 (September 21, 2021): 723. http://dx.doi.org/10.3390/membranes11090723.

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Regulation of the Ca2+-activated Cl− channel TMEM16A by Ca2+/calmodulin (CAM) is discussed controversially. In the present study, we compared regulation of TMEM16A by Ca2+/calmodulin (holo-CAM), CAM-dependent kinase (CAMKII), and CAM-dependent phosphatase calcineurin in TMEM16A-overexpressing HEK293 cells and TMEM16A expressed endogenously in airway and colonic epithelial cells. The activator of the Ca2+/CAM-regulated K+ channel KCNN4, 1-EBIO, activated TMEM16A in overexpressing cells, but not in cells with endogenous expression of TMEM16A. Evidence is provided that CAM-interaction with TMEM16A modulates the Ca2+ sensitivity of the Cl− channel. Enhanced Ca2+ sensitivity of overexpressed TMEM16A explains its activity at basal (non-elevated) intracellular Ca2+ levels. The present results correspond well to a recent report that demonstrates a Ca2+-unbound form of CAM (apo-CAM) that is pre-associated with TMEM16A and mediates a Ca2+-dependent sensitization of activation (and inactivation). However, when using activators or inhibitors for holo-CAM, CAMKII, or calcineurin, we were unable to detect a significant impact of CAM, and limit evidence for regulation by CAM-dependent regulatory proteins on receptor-mediated activation of endogenous TMEM16A in airway or colonic epithelial cells. We propose that regulatory properties of TMEM16A and and other members of the TMEM16 family as detected in overexpression studies, should be validated for endogenous TMEM16A and physiological stimuli such as activation of phospholipase C (PLC)-coupled receptors.

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Lee, Hon Cheung, Robert Aarhus, Richard Graeff, Mary E. Gurnack, and Timothy F. Walseth. "Cyclic ADP ribose activation of the ryanodine receptor is mediated by calmodulin." Nature 370, no. 6487 (July 1994): 307–9. http://dx.doi.org/10.1038/370307a0.

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Della Rocca, Gregory J., Yurii V. Mukhin, Maria N. Garnovskaya, Yehia Daaka, Geoffrey J. Clark, Louis M. Luttrell, Robert J. Lefkowitz, and John R. Raymond. "Serotonin 5-HT1AReceptor-mediated Erk Activation Requires Calcium/Calmodulin-dependent Receptor Endocytosis." Journal of Biological Chemistry 274, no. 8 (February 19, 1999): 4749–53. http://dx.doi.org/10.1074/jbc.274.8.4749.

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Lam, E., M. Benedyk, and N. H. Chua. "Characterization of phytochrome-regulated gene expression in a photoautotrophic cell suspension: possible role for calmodulin." Molecular and Cellular Biology 9, no. 11 (November 1989): 4819–23. http://dx.doi.org/10.1128/mcb.9.11.4819-4823.1989.

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A photoautotrophic suspension culture of soybeans was found to exhibit light-dependent expression of the genes encoding the major chlorophyll a- and b-binding protein (CAB). The expression was mediated by phytochrome, since it was induced by red light and reversed by far-red light. The maximal level as well as the kinetics of the induction were comparable between the suspension culture and soybean seedlings. Using this cell culture, we addressed the question of whether a calcium- and/or calmodulin-mediated step is involved in the signal transduction process between phytochrome and CAB expression. We found that W-7, a potent calmodulin antagonist, severely attenuated the induction of CAB mRNA by light, whereas W-5, a weak calmodulin antagonist, had little effect. Control experiments demonstrated that W-7 treatment did not block the induction of hsp-75 by heat shock. The addition of ionomycin, a calcium ionophore, induced a low level of CAB mRNA accumulation in the dark which could be further enhanced by light treatment. We propose that calmodulin activation by light is necessary but not sufficient to induce maximal CAB expression.

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Wang, Xu, and Abdel A. Abdel-Rahman. "Estrogen modulation of eNOS activity and its association with caveolin-3 and calmodulin in rat hearts." American Journal of Physiology-Heart and Circulatory Physiology 282, no. 6 (June 1, 2002): H2309—H2315. http://dx.doi.org/10.1152/ajpheart.00772.2001.

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Previous studies have shown that estrogen modulation of endothelial nitric oxide (NO) synthase (eNOS) may confer protection against heart disease. Here, we demonstrate an association between reductions in baroreflex-mediated bradycardia and in cardiac NOS activity in ovariectomized (Ovx) rats compared with controls. The latter resulted, at least in part, from a reduction in cardiac eNOS protein. eNOS-derived NO and its biological effects are determined by the levels of eNOS protein and by eNOS catalytic activity; the latter is regulated partly through the dynamic interaction with an inhibitory protein (caveolin) and a stimulatory protein (calmodulin). The association of eNOS immunoprecipitated with caveolin-3 and calmodulin was examined. Caveolin-3 and calmodulin binding with eNOS was increased and decreased, respectively, in Ovx rats. 17β-Estradiol replacement restored, to within normal levels, the baroreflex-mediated bradycardic responses along with eNOS activity, eNOS expression, and the association of eNOS with caveolin-3 and calmodulin. Our findings may help to elucidate the molecular mechanism underlying the favorable effects of estrogen on cardiac responses to baroreflex activation.

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Lam, E., M. Benedyk, and N. H. Chua. "Characterization of phytochrome-regulated gene expression in a photoautotrophic cell suspension: possible role for calmodulin." Molecular and Cellular Biology 9, no. 11 (November 1989): 4819–23. http://dx.doi.org/10.1128/mcb.9.11.4819.

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A photoautotrophic suspension culture of soybeans was found to exhibit light-dependent expression of the genes encoding the major chlorophyll a- and b-binding protein (CAB). The expression was mediated by phytochrome, since it was induced by red light and reversed by far-red light. The maximal level as well as the kinetics of the induction were comparable between the suspension culture and soybean seedlings. Using this cell culture, we addressed the question of whether a calcium- and/or calmodulin-mediated step is involved in the signal transduction process between phytochrome and CAB expression. We found that W-7, a potent calmodulin antagonist, severely attenuated the induction of CAB mRNA by light, whereas W-5, a weak calmodulin antagonist, had little effect. Control experiments demonstrated that W-7 treatment did not block the induction of hsp-75 by heat shock. The addition of ionomycin, a calcium ionophore, induced a low level of CAB mRNA accumulation in the dark which could be further enhanced by light treatment. We propose that calmodulin activation by light is necessary but not sufficient to induce maximal CAB expression.

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20

Rodríguez-Vilarrupla, Aina, Montserrat Jaumot, Neus Abella, Núria Canela, Sonia Brun, Carmen Díaz, Josep M. Estanyol, Oriol Bachs, and Neus Agell. "Binding of Calmodulin to the Carboxy-Terminal Region of p21 Induces Nuclear Accumulation via Inhibition of Protein Kinase C-Mediated Phosphorylation of Ser153." Molecular and Cellular Biology 25, no. 16 (August 15, 2005): 7364–74. http://dx.doi.org/10.1128/mcb.25.16.7364-7374.2005.

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ABSTRACT Intracellular localization plays an important role in the functional regulation of the cell cycle inhibitor p21. We have previously shown that calmodulin binds to p21 and that calmodulin is essential for the nuclear accumulation of p21. Here, we analyze the mechanism of this regulation. We show that calmodulin inhibits in vitro phosphorylation of p21 by protein kinase C (PKC) and that this inhibition is dependent upon calmodulin binding to p21. Two-dimensional electrophoresis analysis of cells expressing the p21 wild type or p21S153A, a nonphosphorylatable mutant of p21 at position 153, indicates that Ser153 of p21 is a phosphorylable residue in vivo. Furthermore, Western blot analysis using phospho-Ser153-specific antibodies indicates that Ser153 phosphorylation in vivo is induced when PKC is activated and calmodulin is inhibited. The mutation of Ser153 to aspartate, a pseudophosphorylated residue, inhibits the nuclear accumulation of p21. Finally, whereas wild-type p21 translocates to the cytoplasm after PKC activation in the presence of calmodulin inhibitors, p21 carrying a nonphosphorylatable residue at position 153 remains in the nucleus. We propose that calmodulin binding to p21 prevents its phosphorylation by PKC at Ser153 and consequently allows its nuclear localization. When phosphorylated at Ser153, p21 is located at the cytoplasm and disrupts stress fibers.

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Park, C. S., S. H. Chang, H. S. Lee, S. H. Kim, J. W. Chang, and C. D. Hong. "Inhibition of renin secretion by Ca2+ through activation of myosin light chain kinase." American Journal of Physiology-Cell Physiology 271, no. 1 (July 1, 1996): C242—C247. http://dx.doi.org/10.1152/ajpcell.1996.271.1.c242.

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This study sought to identify specific enzyme(s) involved in the biochemical cascade of inhibition of renin secretion through Ca(2+)-calmodulin mediation with the use of inhibitors of protein kinase and phosphatases. Inhibition of renin secretion mediated by Ca(2+)-calmodulin was induced by incubating rat renal cortical slices in K(+)-rich depolarizing medium, producing > 50% inhibition. This inhibition was completely blocked by the calmodulin antagonist calmidazolium. The inhibitor of protein kinase with broad specificity, K-252a, blocked the inhibition of renin secretion. Neither KN-62, a specific inhibitor of Ca(2+)-calmodulin-dependent protein kinase II (CaMK II), nor specific inhibitors of protein phosphatase 2B (PP2B), cyclosporin A and FK-506, blocked the inhibition. On the other hand, all four known inhibitors specific for myosin light chain kinase (MLCK), with different chemical structures and mechanisms of inhibition (ML-9, ML-7, KT-5926 and wortmannin), almost completely protected renin secretion against the inhibition by Ca2+. In particular, ML-9 reversively protected > 77% secretion against the inhibition both in K(+)-rich medium alone and in combination with the calcium ionophore A-23187 in a concentration-dependent manner. Together, these findings from our present study provide the first evidence, albeit indirect in nature, for the possibility that activation of Ca(2+)-calmodulin-dependent MLCK at the downstream of Ca2+ influx into juxtaglomerular (JG) cells leads to phosphorylation of 20-kDa regulatory myosin light chain (MLC20). Through interaction with actin, the phosphorylated MLC20 may play an important role in the inhibitory stimulus-secretion coupling of renin.

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Comte, M., A. Malnoë, and J. A. Cox. "Affinity purification of seminalplasmin and characterization of its interaction with calmodulin." Biochemical Journal 240, no. 2 (December 1, 1986): 567–73. http://dx.doi.org/10.1042/bj2400567.

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Bull seminalplasmin antagonizes with high potency and selectivity the activating effect of calmodulin on target enzymes [Gietzen & Galla (1985) Biochem. J. 230, 277-280]. In the present paper we establish that seminalplasmin forms a 1:1, Ca2+-dependent and urea-resistant complex with calmodulin. The dissociation constant equals 1.6 nM. In the absence of Ca2+ a low-affinity complex is formed that is disrupted by 4 M-urea. On the basis of these properties, a fast affinity purification of seminalplasmin was developed. The high specificity of seminalplasmin as a calmodulin antagonist was demonstrated for the multipathway-regulated adenylate cyclase of bovine cerebellum. Far-u.v. c.d. properties are consistent with a random form of seminalplasmin in aqueous solution; 23% alpha-helix is induced on interaction with calmodulin. The fluorescence properties of the single tryptophan residue of seminalplasmin are markedly changed on formation of the complex. These studies allowed us to locate tentatively the peptide segment that interacts with calmodulin, and to ascertain the structural homology between seminalplasmin and other calmodulin-binding peptides. Additional material, showing the inhibition of calmodulin-mediated activation of bovine brain phosphodiesterase by melittin and seminalplasmin and also the near-u.v. spectrum of affinity-purified seminalplasmin, has been deposited as supplement SUP 50135 (4 pages) at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies may be obtained on the terms indicated in Biochem. J. (1986) 233, 5.

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Hoshino, Mitsunobu, and Shun Nakamura. "Small GTPase Rin induces neurite outgrowth through Rac/Cdc42 and calmodulin in PC12 cells." Journal of Cell Biology 163, no. 5 (December 8, 2003): 1067–76. http://dx.doi.org/10.1083/jcb.200308070.

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The novel Ras-like small GTPase Rin is expressed prominently in adult neurons, and binds calmodulin (CaM) through its COOH-terminal–binding motif. It might be involved in calcium/CaM-mediated neuronal signaling, but Rin-mediated signal transduction pathways have not yet been elucidated. Here, we show that expression of Rin induces neurite outgrowth without nerve growth factor or mitogen-activated protein kinase activation in rat pheochromocytoma PC12 cells. Rin-induced neurite outgrowth was markedly inhibited by coexpression with dominant negative Rac/Cdc42 protein or CaM inhibitor treatment. We also found that expression of Rin elevated the endogenous Rac/Cdc42 activity. Rin mutant proteins, in which the mutation disrupted association with CaM, failed to induce neurite outgrowth irrespective of Rac/Cdc42 activation. Disruption of endogenous Rin function inhibited the neurite outgrowth stimulated by forskolin and extracellular calcium entry through voltage-dependent calcium channel evoked by KCl. These findings suggest that Rin-mediated neurite outgrowth signaling requires not only endogenous Rac/Cdc42 activation but also Rin–CaM association, and that endogenous Rin is involved in calcium/CaM-mediated neuronal signaling pathways.

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Murthy, K. S., K. M. Zhang, J. G. Jin, J. R. Grider, and G. M. Makhlouf. "VIP-mediated G protein-coupled Ca2+ influx activates a constitutive NOS in dispersed gastric muscle cells." American Journal of Physiology-Gastrointestinal and Liver Physiology 265, no. 4 (October 1, 1993): G660—G671. http://dx.doi.org/10.1152/ajpgi.1993.265.4.g660.

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Vasoactive intestinal peptide (VIP) and peptide histidine-isoleucine (PHI) receptors and the signaling pathways to which they are coupled were characterized in dispersed gastric smooth muscle cells. Radioligand binding using 125I-labeled VIP and PHI identified 4 classes of receptors: VIP-preferring and PHI-preferring receptors recognized by both ligands and readily desensitized by the preferred ligand, and VIP-specific and PHI-specific receptors recognized by only 1 ligand and resistant to desensitization. All except VIP-specific receptors were coupled to adenylate cyclase. VIP-specific receptors mediated a G protein-coupled Ca2+ influx that led to activation of NO synthase (NOS), NO-dependent activation of soluble guanylate cyclase, and activation of guanosine 3',5'-cyclic monophosphate (cGMP) kinase resulting in muscle relaxation. The entire cascade was blocked by Ca2+ channel and/or calmodulin antagonists. The NOS inhibitor NG-nitro-L-arginine abolished L-[3H]citrulline (coproduct of NO synthesis) and cGMP generation and partly inhibited (52 +/- 4%) relaxation. The components of response mediated by VIP-specific receptors (increase in [Ca2+]i, L-[3H]citrulline, and cGMP) were preserved after desensitization. Insertion of guanosine 5'-O-(beta-thio)diphosphate into reversibly permeabilized muscle cells abolished responses mediated by VIP-preferring and VIP-specific receptors. VIP stimulated both adenosine 3',5'-cyclic monophosphate (cAMP)-kinase and cGMP-kinase activities consistent with stimulation of cAMP and cGMP. Both kinases contributed to relaxation that was partly inhibited by cAMP-kinase [H-89 and (R)-p-adenosine 3',5'-cyclic monophosphorothioate] and cGMP-kinase (KT-5823) inhibitors and abolished by a combination of the 2 types of inhibitors. We conclude that VIP-specific receptors mediate a G protein-coupled Ca2+ influx leading to activation of a constitutive Ca2+/calmodulin-dependent NOS and generation of NO, which is partly responsible for relaxation in smooth muscle.

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Bozoky, Zoltan, Saumel Ahmadi, Tal Milman, Tae Hun Kim, Kai Du, Michelle Di Paola, Stan Pasyk, et al. "Synergy of cAMP and calcium signaling pathways in CFTR regulation." Proceedings of the National Academy of Sciences 114, no. 11 (February 27, 2017): E2086—E2095. http://dx.doi.org/10.1073/pnas.1613546114.

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Cystic fibrosis results from mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel, leading to defective apical chloride transport. Patients also experience overactivation of inflammatory processes, including increased calcium signaling. Many investigations have described indirect effects of calcium signaling on CFTR or other calcium-activated chloride channels; here, we investigate the direct response of CFTR to calmodulin-mediated calcium signaling. We characterize an interaction between the regulatory region of CFTR and calmodulin, the major calcium signaling molecule, and report protein kinase A (PKA)-independent CFTR activation by calmodulin. We describe the competition between calmodulin binding and PKA phosphorylation and the differential effects of this competition for wild-type CFTR and the major F508del mutant, hinting at potential therapeutic strategies. Evidence of CFTR binding to isolated calmodulin domains/lobes suggests a mechanism for the role of CFTR as a molecular hub. Together, these data provide insights into how loss of active CFTR at the membrane can have additional consequences besides impaired chloride transport.

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Coroneos, E. J., M. Kester, J. Maclouf, P. Thomas, and M. J. Dunn. "Calcium-regulated protein tyrosine phosphorylation is required for endothelin-1 to induce prostaglandin endoperoxide synthase-2 mRNA expression and protein synthesis in mesangial cells." Journal of the American Society of Nephrology 8, no. 7 (July 1997): 1080–90. http://dx.doi.org/10.1681/asn.v871080.

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The role of endothelin (ET)-1-mediated cytosolic calcium ([Ca2+]i) elevation in regulating ET-1-induced prostaglandin endoperoxide synthase, prostaglandin G/H synthase (PGHS)-2 mRNA expression and protein synthesis was investigated in mesangial cells (MC). Ionomycin, a calcium ionophore, and thapsigargin, an inhibitor of calcium ATPase, mimicked the ET-1-stimulated PGHS-2 mRNA and protein induction. Inhibition of [Ca2+]i increases with (2-¿C2-bis-(carboxymethyl)-amino-5 methylphenoxy]methyl¿-6-methoxy-8-bis-(carboxymethyl)-aminoquinoline tetra-(acetoxymethyl)ester (Quin/AM), a calcium chelator, or with the combined presence of [8-(diethylamino)-octyl-3,4,5-trimethoxybenzoate, HCl] (TMB), an inhibitor of intracellular calcium stores release, and ethyleneglycol-bis-(beta-aminoethyl)- N,N,N',N'-tetra-acetic acid (EGTA) suppressed ET-1, as well as ionomycin and thapsigargin-mediated PGHS-2 mRNA and protein formation. Also, the ET-1-, ionomycin-, and thapsigargin-induced PGHS-2 mRNA expression and protein formation was inhibited in MC pretreated with inhibitors of calcium calmodulin kinase. In contrast, these conditions did not inhibit interleukin (IL)-1-induced PGHS-2 mRNA expression and protein synthesis. Pretreatment with tyrosine kinase inhibitors abolished the ET-1-, ionomycin-, thapsigargin-, and IL-1-mediated PGHS-2 mRNA and protein induction. ET-1-, ionomycin-, and thapsigargin- induced protein tyrosine phosphorylation, but not IL-1-induced protein tyrosine phosphorylation, was suppressed by inhibiting either [Ca2+]i elevation or calcium calmodulin kinase activation. It was concluded that elevation of [Ca2+]i and activation of calcium calmodulin kinases are upstream mediators of ET-1-induced PGHS-2 gene expression through activation of non-receptor-linked protein tyrosine kinase in MC.

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Ishiguro, Kazuhiro, Todd Green, Joseph Rapley, Heather Wachtel, Cosmas Giallourakis, Aimee Landry, Zhifang Cao, et al. "Ca2+/Calmodulin-Dependent Protein Kinase II Is a Modulator of CARMA1-Mediated NF-κB Activation." Molecular and Cellular Biology 26, no. 14 (July 15, 2006): 5497–508. http://dx.doi.org/10.1128/mcb.02469-05.

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ABSTRACT CARMA1 is a central regulator of NF-κB activation in lymphocytes. CARMA1 and Bcl10 functionally interact and control NF-κB signaling downstream of the T-cell receptor (TCR). Computational analysis of expression neighborhoods of CARMA1-Bcl10MALT 1 for enrichment in kinases identified calmodulin-dependent protein kinase II (CaMKII) as an important component of this pathway. Here we report that Ca2+/CaMKII is redistributed to the immune synapse following T-cell activation and that CaMKII is critical for NF-κB activation induced by TCR stimulation. Furthermore, CaMKII enhances CARMA1-induced NF-κB activation. Moreover, we have shown that CaMKII phosphorylates CARMA1 on Ser109 and that the phosphorylation facilitates the interaction between CARMA1 and Bcl10. These results provide a novel function for CaMKII in TCR signaling and CARMA1-induced NF-κB activation.

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Daibata, M., R. E. Humphreys, K. Takada, and T. Sairenji. "Activation of latent EBV via anti-IgG-triggered, second messenger pathways in the Burkitt's lymphoma cell line Akata." Journal of Immunology 144, no. 12 (June 15, 1990): 4788–93. http://dx.doi.org/10.4049/jimmunol.144.12.4788.

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Abstract Anti-IgG treatment activated latent EBV genomes in 50 to 70% of the cells of the Burkitt's lymphoma cell line Akata. The EBV-activating role of intracellular Ca2+, as potentiated by diacylglycerol (DAG) and suppressed by cAMP, was analyzed in the cells through effects of agonists and antagonists of these second messenger pathways. Early Ag (EA) was induced in 10% of cells with the calcium ionophore A23187 (A23187). EA induction with anti-IgG or A23187 was blocked by a calmodulin antagonist, trifluoperazine. The DAG pathway had a potentiating but not direct effect on EBV activation because: 1) the DAG analog, dioctanoylglycerol (diC8), an agonist for protein kinase C, alone induced only 2% EA-positive cells, 2) diC8 synergized with A23187 for EA induction, and 3) the protein kinase C antagonist, staurosporine, almost completely inhibited EA induction by anti-IgG. When cells were reincubated in medium with fresh diC8 and A23187 at 3, 6, 9, and 12 h, EA induction at 24 h reached the levels seen with anti-IgG stimulation. A cAMP-mediated pathway suppressed EBV activation because dibutyryl cAMP or 8-bromo-cAMP, plus blockage of phosphodiesterase by theophylline, or use of forskolin, inhibited EA induction with anti-IgG. Although the principal stimulatory role in EBV activation of a Ca2(+)-mediated, second messenger pathway, as synergized by DAG and inhibited by cAMP, was established, we did not explain the significant lag in EA induction by A23187 and diC8 as compared with anti-IgG induction of EA. We conclude that EBV genome activation with anti-IgG is mediated by Ca2+/calmodulin and DAG pathways in Akata cells, that the cAMP pathway suppresses EA induction by anti-IgG, and that a mechanism regulating the speed of EA induction remains unexplained.

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Tian, Xixi, Yijun Tang, Yingjie Liu, Ruiwu Wang, and S. R. Wayne Chen. "Calmodulin modulates the termination threshold for cardiac ryanodine receptor-mediated Ca2+ release." Biochemical Journal 455, no. 3 (October 10, 2013): 367–75. http://dx.doi.org/10.1042/bj20130805.

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CaM modulates the termination threshold for RyR2-mediated Ca2+ release, and that the CaM-binding domain of RyR2 is an important determinant of Ca2+ release termination and activation. Defective CaM-RyR2 interaction may lead to cardiomyopathies and cardiac arrhythmias.

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Amemiya, Yuna, Nao Nakamura, Nao Ikeda, Risa Sugiyama, Chiaki Ishii, Masatoshi Maki, Hideki Shibata, and Terunao Takahara. "Amino Acid-Mediated Intracellular Ca2+ Rise Modulates mTORC1 by Regulating the TSC2-Rheb Axis through Ca2+/Calmodulin." International Journal of Molecular Sciences 22, no. 13 (June 27, 2021): 6897. http://dx.doi.org/10.3390/ijms22136897.

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Mechanistic target of rapamycin complex 1 (mTORC1) is a master growth regulator by controlling protein synthesis and autophagy in response to environmental cues. Amino acids, especially leucine and arginine, are known to be important activators of mTORC1 and to promote lysosomal translocation of mTORC1, where mTORC1 is thought to make contact with its activator Rheb GTPase. Although amino acids are believed to exclusively regulate lysosomal translocation of mTORC1 by Rag GTPases, how amino acids increase mTORC1 activity besides regulation of mTORC1 subcellular localization remains largely unclear. Here we report that amino acids also converge on regulation of the TSC2-Rheb GTPase axis via Ca2+/calmodulin (CaM). We showed that the amino acid-mediated increase of intracellular Ca2+ is important for mTORC1 activation and thereby contributes to the promotion of nascent protein synthesis. We found that Ca2+/CaM interacted with TSC2 at its GTPase activating protein (GAP) domain and that a CaM inhibitor reduced binding of CaM with TSC2. The inhibitory effect of a CaM inhibitor on mTORC1 activity was prevented by loss of TSC2 or by an active mutant of Rheb GTPase, suggesting that a CaM inhibitor acts through the TSC2-Rheb axis to inhibit mTORC1 activity. Taken together, in response to amino acids, Ca2+/CaM-mediated regulation of the TSC2-Rheb axis contributes to proper mTORC1 activation, in addition to the well-known lysosomal translocation of mTORC1 by Rag GTPases.

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Praveen Rao, J., and C. Subramanyam. "Calmodulin mediated activation of acetyl-CoA carboxylase during aflatoxin production by Aspergillus parasiticus." Letters in Applied Microbiology 30, no. 4 (April 2000): 277–81. http://dx.doi.org/10.1046/j.1472-765x.2000.00717.x.

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Kane, Christopher D., and Anthony R. Means. "Activation of orphan receptor-mediated transcription by Ca2+/calmodulin-dependent protein kinase IV." EMBO Journal 19, no. 4 (February 15, 2000): 691–701. http://dx.doi.org/10.1093/emboj/19.4.691.

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Murakami, Itsuo, Sakae Takeuchi, Toshiyuki Kudo, Shizuyo Sutou, and Sumio Takahashi. "Corticotropin-releasing hormone or dexamethasone regulates rat proopiomelanocortin transcription through Tpit/Pitx-responsive element in its promoter." Journal of Endocrinology 193, no. 2 (May 2007): 279–90. http://dx.doi.org/10.1677/joe-06-0143.

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Tpit/Pitx-responsive element (Tpit/PitxRE), which binds transcription factors Tpit and Pitx1, confers cell-type specific expression of proopiomelanocortin (POMC) gene in pituitary corticotrops where the gene expression is mainly regulated by corticotropin-releasing hormone (CRH) and glucocorticoids (Gcs). CRH stimulates POMC gene expression, which is mediated by the accumulation of intracellular cAMP and requires binding of Nur factors to Nur-responsive element (NurRE). Gcs antagonize NurRE-dependent POMC gene expression through direct interaction between glucocorticoid receptors and Nur factors. We examined whether Tpit/PitxRE and NurRE are involved in CRH/cAMP-induced activation and Gc-induced repression of POMC gene expression by reporter assay in AtT-20 corticotropic cells. Deletion and mutation of Tpit/PitxRE markedly reduced basal activity of the promoter, and those of NurRE decreased the levels of the CRH/cAMP-induced activation. Nifedipine, KN-62, and W-7, specific inhibitors of the L-type calcium channel, calmodulin-dependent protein kinase II, and calmodulin respectively, attenuated CRH/cAMP-induced activation of promoters with three copies of either Tpit/PitxRE or NurRE, indicating that both Tpit/PitxRE and NurRE mediate CRH-induced activation of POMC gene expression in a calcium-dependent manner. Deletion and mutation of Tpit/PitxRE abolished dexamethasone (DEX)-induced repression of POMC gene expression, while those of NurRE did not, indicating that Tpit/PitxRE predominantly mediates Gc-induced repression of POMC transcription. However, DEX treatment attenuated activities of promoters with three copies of either Tpit/PitxRE or NurRE, suggesting that Gcs act at NurRE as well as Tpit/PitxRE to repress POMC gene expression. We conclude that Tpit/PitxRE is an important element by which CRH and Gcs regulate the POMC gene expression.

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Yin, Jing-Xiang, Rui-Fang Yang, Shumin Li, Alex O. Renshaw, Yu-Long Li, Harold D. Schultz, and Matthew C. Zimmerman. "Mitochondria-produced superoxide mediates angiotensin II-induced inhibition of neuronal potassium current." American Journal of Physiology-Cell Physiology 298, no. 4 (April 2010): C857—C865. http://dx.doi.org/10.1152/ajpcell.00313.2009.

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Reactive oxygen species (ROS), particularly superoxide (O2·−), have been identified as key signaling intermediates in ANG II-induced neuronal activation and sympathoexcitation associated with cardiovascular diseases, such as hypertension and heart failure. Studies of the central nervous system have identified NADPH oxidase as a primary source of O2·− in ANG II-stimulated neurons; however, additional sources of O2·−, including mitochondria, have been mostly overlooked. Here, we tested the hypothesis that ANG II increases mitochondria-produced O2·− in neurons and that increased scavenging of mitochondria-produced O2·− attenuates ANG II-dependent intraneuronal signaling. Stimulation of catecholaminergic (CATH.a) neurons with ANG II (100 nM) increased mitochondria-localized O2·− levels, as measured by MitoSOX Red fluorescence. This response was significantly attenuated in neurons overexpressing the mitochondria-targeted O2·−-scavenging enzyme Mn-SOD. To examine the biological significance of the ANG II-mediated increase in mitochondria-produced O2·−, we used the whole cell configuration of the patch-clamp technique to record the well-characterized ANG II-induced inhibition of voltage-gated K+ current ( IKv) in neurons. Adenovirus-mediated Mn-SOD overexpression or pretreatment with the cell-permeable antioxidant tempol (1 mM) significantly attenuated ANG II-induced inhibition of IKv. In contrast, pretreatment with extracellular SOD protein (400 U/ml) had no effect. Mn-SOD overexpression also inhibited ANG II-induced activation of Ca2+/calmodulin kinase II, a redox-sensitive protein known to modulate IKv. These data indicate that ANG II increases mitochondrial O2·−, which mediates, at least in part, ANG II-induced activation of Ca2+/calmodulin kinase II and inhibition of IKv in neurons.

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Cao, Youjia, Shiyang Li, Ming-Juan Du, Ya-Juan Wan, Bei Lan, Yao-Hui Liu, Yin Yang, and Cui-zhu Zhang. "The N-terminal region of the guanine nucleotide exchange factor Vav1 plays a distinguished role in T cell receptor mediated calcium signaling (P1158)." Journal of Immunology 190, no. 1_Supplement (May 1, 2013): 190.4. http://dx.doi.org/10.4049/jimmunol.190.supp.190.4.

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Abstract Vav1 is a guanine nucleotide exchange factor specifically expressed in hematopoietic cells. It consists of multiple structural domains and plays important roles in T cell activation. The other highly conserved isoforms of Vav family, Vav2 and Vav3, are ubiquitously expressed in human tissues including lymphocytes. All the three Vav proteins activate Rho family small GTPases, which are involved in a variety of biological processes during T cell activation. Intensive studies have demonstrated that Vav1 is indispensable for T cell receptor mediated signal transduction. T cells lacking Vav1 exhibited severe defect in TCR-mediated calcium elevation, indicating that the co-existing Vav2 and Vav3 did not compensate Vav1 in calcium signaling. What is the functional particularity of Vav1 in lymphocytes? In this study, we identified the N-terminal region of Vav1 in calponin homology domain to be essential for its interaction with Calmodulin, that leads to TCR-induced calcium mobilization. Substitution of the region of Vav1 with the homologous region of Vav2 or Vav3 abolished the association with CaM, and the N-terminal mutations of Vav1 failed to potentiate normal TCR-induced calcium mobilization, that in turn, suspended nuclear factor of activated T cells activation and IL-2 production. This study highlights the importance of the N-terminal region of Vav1 for calmodulin binding, and that attributes irreplaceable role of Vav1 in T cell activation and signal transduction.

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Blystone, Scott D., Suzanne E. Slater, Matthew P. Williams, Michael T. Crow, and Eric J. Brown. "A Molecular Mechanism of Integrin Crosstalk: αvβ3 Suppression of Calcium/Calmodulin-dependent Protein Kinase II Regulates α5β1 Function." Journal of Cell Biology 145, no. 4 (May 17, 1999): 889–97. http://dx.doi.org/10.1083/jcb.145.4.889.

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Many cells express more than one integrin receptor for extracellular matrix, and in vivo these receptors may be simultaneously engaged. Ligation of one integrin may influence the behavior of others on the cell, a phenomenon we have called integrin crosstalk. Ligation of the integrin αvβ3 inhibits both phagocytosis and migration mediated by α5β1 on the same cell, and the β3 cytoplasmic tail is necessary and sufficient for this regulation of α5β1. Ligation of α5β1 activates the calcium- and calmodulin-dependent protein kinase II (CamKII). This activation is required for α5β1-mediated phagocytosis and migration. Simultaneous ligation of αvβ3 or expression of a chimeric molecule with a free β3 cytoplasmic tail prevents α5β1-mediated activation of CamKII. Expression of a constitutively active CamKII restores α5β1 functions blocked by αvβ3-initiated integrin crosstalk. Thus, αvβ3 inhibition of α5β1 activation of CamKII is required for its role in integrin crosstalk. Structure-function analysis of the β3 cytoplasmic tail demonstrates a requirement for Ser752 in β3-mediated suppression of CamKII activation, while crosstalk is independent of Tyr747 and Tyr759, implicating Ser752, but not β3 tyrosine phosphorylation in initiation of the αvβ3 signal for integrin crosstalk.

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Du, Jianyang, Jia Xie, and Lixia Yue. "Intracellular calcium activates TRPM2 and its alternative spliced isoforms." Proceedings of the National Academy of Sciences 106, no. 17 (April 16, 2009): 7239–44. http://dx.doi.org/10.1073/pnas.0811725106.

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Melastatin-related transient receptor potential channel 2 (TRPM2) is a Ca2+-permeable, nonselective cation channel that is involved in oxidative stress-induced cell death and inflammation processes. Although TRPM2 can be activated by ADP-ribose (ADPR) in vitro, it was unknown how TRPM2 is gated in vivo. Moreover, several alternative spliced isoforms of TRPM2 identified recently are insensitive to ADPR, and their gating mechanisms remain unclear. Here, we report that intracellular Ca2+ ([Ca2+]i) can activate TRPM2 as well as its spliced isoforms. We demonstrate that TRPM2 mutants with disrupted ADPR-binding sites can be activated readily by [Ca2+]i, indicating that [Ca2+]i gating of TRPM2 is independent of ADPR. The mechanism by which [Ca2+]i activates TRPM2 is via a calmodulin (CaM)-binding domain in the N terminus of TRPM2. Whereas Ca2+-mediated TRPM2 activation is independent of ADPR and ADPR-binding sites, both [Ca2+]i and the CaM-binding motif are required for ADPR-mediated TRPM2 gating. Importantly, we demonstrate that intracellular Ca2+ release activates both recombinant and endogenous TRPM2 in intact cells. Moreover, receptor activation-induced Ca2+ release is capable of activating TRPM2. These results indicate that [Ca2+]i is a key activator of TRPM2 and the only known activator of the spliced isoforms of TRPM2. Our findings suggest that [Ca2+]i-mediated activation of TRPM2 and its alternative spliced isoforms may represent a major gating mechanism in vivo, therefore conferring important physiological and pathological functions of TRPM2 and its spliced isoforms in response to elevation of [Ca2+]i.

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Lu, H. K., R. J. Fern, J. J. Nee, and P. Q. Barrett. "Ca(2+)-dependent activation of T-type Ca2+ channels by calmodulin-dependent protein kinase II." American Journal of Physiology-Renal Physiology 267, no. 1 (July 1, 1994): F183—F189. http://dx.doi.org/10.1152/ajprenal.1994.267.1.f183.

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The T-type Ca2+ channel is unique among voltage-dependent Ca2+ channels in its low threshold for opening and its slow kinetics of deactivation. Here, we evaluate the importance of intracellular Ca2+ (Cai2+) in promoting low-threshold gating of T-type channels in adrenal glomerulosa cells. We observe that 390 nM to 1.27 microM Cai2+ enhances T-type current by shifting the voltage dependence of channel activation to more negative potentials. This Ca(2+)-induced shift is mediated by calmodulin-dependent protein kinase II (CaMKII), because it is abolished by inhibitors of CaMKII but not of protein kinase C and is subsequently restored by exogenous calmodulin. This Ca(2+)-induced reduction in gating threshold would render T-type Ca2+ channels uniquely suited to transduce depolarizing stimuli of low amplitude into a Ca2+ signal sufficient to support a physiological response.

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Juvvadi, Praveen R., Alberto Muñoz, Frédéric Lamoth, Erik J. Soderblom, M. Arthur Moseley, Nick D. Read, and William J. Steinbach. "Calcium-Mediated Induction of Paradoxical Growth following Caspofungin Treatment Is Associated with Calcineurin Activation and Phosphorylation in Aspergillus fumigatus." Antimicrobial Agents and Chemotherapy 59, no. 8 (June 8, 2015): 4946–55. http://dx.doi.org/10.1128/aac.00263-15.

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ABSTRACTThe echinocandin antifungal drug caspofungin at high concentrations reverses the growth inhibition ofAspergillus fumigatus, a phenomenon known as the “paradoxical effect,” which is not consistently observed with other echinocandins (micafungin and anidulafungin). Previous studies ofA. fumigatusrevealed the loss of the paradoxical effect following pharmacological or genetic inhibition of calcineurin, yet the underlying mechanism is poorly understood. Here, we utilized a codon-optimized bioluminescent Ca2+reporter aequorin expression system inA. fumigatusand showed that caspofungin elicits a transient increase in cytosolic free Ca2+([Ca2+]c) in the fungus that acts as the initial trigger of the paradoxical effect by activating calmodulin-calcineurin signaling. While the increase in [Ca2+]cwas also observed upon treatment with micafungin, another echinocandin without the paradoxical effect, a higher [Ca2+]cincrease was noted with the paradoxical-growth concentration of caspofungin. Treatments with a Ca2+-selective chelator, BAPTA [1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid], or the L-type Ca2+channel blocker verapamil abolished caspofungin-mediated paradoxical growth in both the wild-type and the echinocandin-resistant (EMFR-S678P) strains. Concomitant with increased [Ca2+]clevels at higher concentrations of caspofungin, calmodulin and calcineurin gene expression was enhanced. Phosphoproteomic analysis revealed that calcineurin is activated through phosphorylation at its serine-proline-rich region (SPRR), a domain previously shown to be essential for regulation of hyphal growth, only at a paradoxical-growth concentration of caspofungin. Our results indicate that as opposed to micafungin, the increased [Ca2+]cat high concentrations of caspofungin activates calmodulin-calcineurin signaling at both a transcriptional and a posttranslational level and ultimately leads to paradoxical fungal growth.

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Chen, Yun, Xinshuai Li, Yuyun Hua, Yue Ding, Guoliang Meng, and Wei Zhang. "RIPK3-Mediated Necroptosis in Diabetic Cardiomyopathy Requires CaMKII Activation." Oxidative Medicine and Cellular Longevity 2021 (June 7, 2021): 1–19. http://dx.doi.org/10.1155/2021/6617816.

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Activation of Ca2+/calmodulin-dependent protein kinase (CaMKII) has been proved to play a vital role in cardiovascular diseases. Receptor-interaction protein kinase 3- (RIPK3-) mediated necroptosis has crucially participated in cardiac dysfunction. The study is aimed at investigating the effect as well as the mechanism of CaMKII activation and necroptosis on diabetic cardiomyopathy (DCM). Wild-type (WT) and the RIPK3 gene knockout (RIPK3-/-) mice were intraperitoneally injected with 60 mg/kg/d streptozotocin (STZ) for 5 consecutive days. After 12 w of feeding, 100 μL recombinant adenovirus solution carrying inhibitor 1 of protein phosphatase 1 (I1PP1) gene was injected into the caudal vein of mice. Echocardiography, myocardial injury, CaMKII activity, necroptosis, RIPK1 expression, mixed lineage kinase domain-like protein (MLKL) phosphorylation, and mitochondrial ultrastructure were measured. The results showed that cardiac dysfunction, CaMKII activation, and necroptosis were aggravated in streptozotocin- (STZ-) stimulated mice, as well as in (Lepr) KO/KO (db/db) mice. RIPK3 deficiency alleviated cardiac dysfunction, CaMKII activation, and necroptosis in DCM. Furthermore, I1PP1 overexpression reversed cardiac dysfunction, myocardial injury and necroptosis augment, and CaMKII activity enhancement in WT mice with DCM but not in RIPK3-/- mice with DCM. The present study demonstrated that CaMKII activation and necroptosis augment in DCM via a RIPK3-dependent manner, which may provide therapeutic strategies for DCM.

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Martin, C., R. Göggel, A. R. Ressmeyer, and S. Uhlig. "Pressor responses to platelet-activating factor and thromboxane are mediated by Rho-kinase." American Journal of Physiology-Lung Cellular and Molecular Physiology 287, no. 1 (July 2004): L250—L257. http://dx.doi.org/10.1152/ajplung.00420.2003.

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Platelet-activating factor (PAF) contracts smooth muscle of airways and vessels primarily via release of thromboxane. Contraction of smooth muscle is thought to be mediated either by calcium and inositol trisphosphate (IP3)-dependent activation of the myosin light chain kinase or, alternatively, via the recently discovered Rho-kinase pathway. Here we investigated the contribution of these two pathways to PAF and thromboxane receptor-mediated broncho- and vasoconstriction in two different rat models: the isolated perfused lung (IPL) and precision-cut lung slices. Inhibition of the IP3 receptor (1–10 μM xestospongin C) or inhibition of phosphatidylinositol-specific PLC (30 μM L-108) did not affect bronchoconstriction but attenuated the sustained vasoconstriction by PAF. Inhibition of myosin light chain kinase (35 μM ML-7) or of calmodulin kinase kinase (26 μM STO609), which regulates the phosphorylation of the myosin light chain, had only a small effect on PAF- or thromboxane-induced pressor responses. Similarly, calmidazolium (10 μM), which inhibits calmodulin-dependent proteins, only weakly reduced the airway responses. In contrast, Y-27632 (10 μM), a Rho-kinase inhibitor, attenuated the thromboxane release triggered by PAF and provided partial or complete inhibition against PAF- and thromboxane-induced pressor responses, respectively. Together, our data indicate that PAF- and thus thromboxane receptor-mediated smooth muscle contraction depends largely on the Rho-kinase pathway.

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Thommesen, Liv, Eva Hofsli, Ruth H. Paulssen, Marit W. Anthonsen, and Astrid Lægreid. "Molecular mechanisms involved in gastrin-mediated regulation of cAMP-responsive promoter elements." American Journal of Physiology-Endocrinology and Metabolism 281, no. 6 (December 1, 2001): E1316—E1325. http://dx.doi.org/10.1152/ajpendo.2001.281.6.e1316.

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In the present study, we explore the role of cAMP-responsive (CRE) promoter elements in gastrin-mediated gene activation. By using the minimal CRE promoter reporter plasmid, pCRELuc, we show that gastrin can activate CRE. This activation is blocked by H-89 and GF 109203x, which inhibit protein kinases A and C, respectively. Moreover, Ca2+-activated pathways seem to be involved, because the calmodulin inhibitor W-7 reduced gastrin-mediated activation of pCRELuc. Deletion of CRE from the c- fos promoter rendered this promoter completely unresponsive to gastrin, indicating that CRE plays a central role in c- fos transactivation. Interestingly, gastrin-induced expression of the inducible cAMP early repressor (ICER), a gene that is known to be regulated by CRE promoter elements, was not reduced by H-89, W-7, or GF 109203x. Furthermore, bandshift analyses indicated that the region of the ICER promoter containing the CRE-like elements CARE 3–4 binds transcription factors that are not members of the CRE-binding protein-CRE modulator protein-activating transcription factor, or CREB/CREM/ATF-1, family. Our results underline the significance of the CRE promoter element in gastrin-mediated gene regulation and indicate that a variety of signaling mechanisms are involved, depending on the CRE promoter context.

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GOPPELT-STRUEBE, Margarete, Angelika HAHN, Martin STROEBEL, and Christian O. A. REISER. "Independent regulation of cyclo-oxygenase 2 expression by p42/44 mitogen-activated protein kinases and Ca2+/calmodulin-dependent kinase." Biochemical Journal 339, no. 2 (April 8, 1999): 329–34. http://dx.doi.org/10.1042/bj3390329.

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5-Hydroxytryptamine (5-HT, ‘serotonin ’) is a potent inducer of the early response gene cyclo-oxygenase 2 (Cox-2; prostaglandin G/H synthase) in mesangial cells. Protein kinase C (PKC), Ca2+-dependent enzymes and mitogen-activated protein kinase (p42/44 MAPK) have previously been shown to be essential modules of the signalling pathway leading from the pertussis-insensitive 5-HT2A receptor to the induction of Cox-2 mRNA expression. In the present study, PKC activation was linked to the 5-HT-mediated phosphorylation and thus the activation of p42/44 MAPK: the inhibition of PKC by the specific inhibitor GF109203x prevented p42/44 MAPK activation. Ca2+/calmodulin-dependent (CaM) kinase II δ2 was detected in mesangial cells by Western blot analysis. The inhibition of CaM kinase by the inhibitors KN62 or KN93 led to a partial inhibition of 5-HT-induced Cox-2 mRNA expression and decreased basal, but not PMA-mediated, Cox-2 expression. The 5-HT-mediated activation of MAPK was not decreased by KN62 or KN93, excluding CaM kinase as a signalling module upstream of p42/44 MAPK. Taken together, these results indicate a modulatory involvement of CaM kinase in the regulation of 5-HT-mediated Cox-2 mRNA expression in addition to the main pathway that consists of the activation of PKC and p42/44 MAPK.

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Burnham, D. B. "Characterization of Ca2+-activated protein phosphatase activity in exocrine pancreas." Biochemical Journal 231, no. 2 (October 15, 1985): 335–41. http://dx.doi.org/10.1042/bj2310335.

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Ca2+-activated protein phosphatase activity was demonstrated in mouse pancreatic acinar cytosol with α-casein and skeletal-muscle phosphorylase kinase as substrates. This phosphatase activity preferentially dephosphorylated the alpha subunit of phosphorylase kinase. After DEAE-cellulose chromatography, the Ca2+-activated phosphatase activity became dependent on exogenous calmodulin for maximal activity. Half-maximal activation was achieved at 0.5 +/- 0.1 microM-Ca2+. Trifluoperazine completely inhibited Ca2+-activated phosphatase activity, with half-maximal inhibition occurring at 8.5 +/- 0.6 microM. Mn2+, but not Mg2+, at 1 mM concentration could substitute for Ca2+ in eliciting full enzyme activation. The apparent Mr of the phosphatase as determined by Sephadex G-150 chromatography was 93000 +/- 1000. Submitting active fractions obtained after Sephadex chromatography to calmodulin affinity chromatography resulted in the resolution of a major protein of Mr 55500 +/- 300. In conclusion, Ca2+-activated protein phosphatase activity has been identified in exocrine pancreas and has several features in common with Ca2+-activated calmodulin-dependent protein phosphatases previously isolated from brain and skeletal muscle. It is possible that this Ca2+-activated phosphatase may utilize as substrates certain acinar-cell phosphoproteins previously shown to undergo dephosphorylation in response to Ca2+-mediated secretagogues.

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Noori, S., Z. M. Hassan, B. Rezaei, A. Rustaiyan, Z. Habibi, and F. Fallahian. "Artemisinin can inhibit the calmodulin-mediated activation of phosphodiesterase in comparison with Cyclosporin A." International Immunopharmacology 8, no. 13-14 (December 2008): 1744–47. http://dx.doi.org/10.1016/j.intimp.2008.08.012.

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Belcheva, Mariana M., Maria Szùcs, Danxin Wang, Wolfgang Sadee, and Carmine J. Coscia. "μ-Opioid Receptor-mediated ERK Activation Involves Calmodulin-dependent Epidermal Growth Factor Receptor Transactivation." Journal of Biological Chemistry 276, no. 36 (July 16, 2001): 33847–53. http://dx.doi.org/10.1074/jbc.m101535200.

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Briggs, Michael W., Zhigang Li, and David B. Sacks. "IQGAP1-mediated Stimulation of Transcriptional Co-activation by β-Catenin Is Modulated by Calmodulin." Journal of Biological Chemistry 277, no. 9 (December 4, 2001): 7453–65. http://dx.doi.org/10.1074/jbc.m104315200.

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Yu, Kuai, Jinqiu Zhu, Zhiqiang Qu, Yuan-Yuan Cui, and H. Criss Hartzell. "Activation of the Ano1 (TMEM16A) chloride channel by calcium is not mediated by calmodulin." Journal of General Physiology 143, no. 2 (January 13, 2014): 253–67. http://dx.doi.org/10.1085/jgp.201311047.

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The Ca2+-activated Cl channel anoctamin-1 (Ano1; Tmem16A) plays a variety of physiological roles, including epithelial fluid secretion. Ano1 is activated by increases in intracellular Ca2+, but there is uncertainty whether Ca2+ binds directly to Ano1 or whether phosphorylation or additional Ca2+-binding subunits like calmodulin (CaM) are required. Here we show that CaM is not necessary for activation of Ano1 by Ca2+ for the following reasons. (a) Exogenous CaM has no effect on Ano1 currents in inside-out excised patches. (b) Overexpression of Ca2+-insensitive mutants of CaM have no effect on Ano1 currents, whereas they eliminate the current mediated by the small-conductance Ca2+-activated K+ (SK2) channel. (c) Ano1 does not coimmunoprecipitate with CaM, whereas SK2 does. Furthermore, Ano1 binds very weakly to CaM in pull-down assays. (d) Ano1 is activated in excised patches by low concentrations of Ba2+, which does not activate CaM. In addition, we conclude that reversible phosphorylation/dephosphorylation is not required for current activation by Ca2+ because the current can be repeatedly activated in excised patches in the absence of ATP or other high-energy compounds. Although Ano1 is blocked by the CaM inhibitor trifluoperazine (TFP), we propose that TFP inhibits the channel in a CaM-independent manner because TFP does not inhibit Ano1 when applied to the cytoplasmic side of excised patches. These experiments lead us to conclude that CaM is not required for activation of Ano1 by Ca2+. Although CaM is not required for channel opening by Ca2+, work of other investigators suggests that CaM may have effects in modulating the biophysical properties of the channel.

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Shivnan, E., L. Clayton, L. Allridge, K. E. Keating, M. Gullberg, and D. R. Alexander. "CD45 monoclonal antibodies inhibit TCR-mediated calcium signals, calmodulin-kinase IV/Gr activation, and oncoprotein 18 phosphorylation." Journal of Immunology 157, no. 1 (July 1, 1996): 101–9. http://dx.doi.org/10.4049/jimmunol.157.1.101.

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Abstract The effects of a pan-CD45 mAb (CD45.2) on TCR-mediated signaling pathways were investigated in Jurkat T cells. The simultaneous addition of CD45 mAb with an activating OKT3 mAb had little effect on TCR-stimulated signals. However, when Jurkat cells were exposed to the CD45 mAb for 10 to 20 min before the addition of OKT3, a partial uncoupling of the TCR from intracellular signals was observed. The maximal increase in intracellular calcium was inhibited 47 +/- 10% (n = 11, range 33-67%), whereas no inhibition of inositol trisphosphate production was detected. The transient TCR-mediated activation of the Ca2+/calmodulin-activated kinase IV/Gr was also inhibited by the CD45 mAb, and this was reflected in a 50 to 60% inhibition in the TCR-stimulated generation of the p21 and p23 phosphoisomers of oncoprotein 18, a Ca2+/calmodulin-activated kinase IV/Gr substrate recently implicated in cell cycle regulatory events. Oncoprotein 18 is also a substrate for mitogen- activated protein kinase, but no inhibition by the CD45 mAb of TCR-triggered mitogen-activated protein kinase activation was observed. The CD45 mAb was therefore selective in causing the uncoupling of the TCR from calcium signals and calcium-regulated events without promoting a general inhibition of all TCR-mediated signals. Confocal microscopy revealed that binding of the CD45 mAb caused patching of CD45 molecules at the cell surface and, unexpectedly, a marked redistribution of intracellular CD45. However, no change was observed in the total level of CD45 expressed at the cell surface. Aggregation of CD45 at the cell surface may result in its sequestration from its tyrosine kinase substrates, with a consequent selective uncoupling of the TCR from intracellular signaling pathways.

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Collins, K., J. R. Sellers, and P. Matsudaira. "Calmodulin dissociation regulates brush border myosin I (110-kD-calmodulin) mechanochemical activity in vitro." Journal of Cell Biology 110, no. 4 (April 1, 1990): 1137–47. http://dx.doi.org/10.1083/jcb.110.4.1137.

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110-kD-calmodulin, when immobilized on nitrocellulose-coated coverslips, translocates actin filaments at a maximal rate of 0.07-0.1 micron/s at 37 degrees C. Actin activates MgATPase activity greater than 40-fold, with a Km of 40 microM and Vmax of 0.86 s-1 (323 nmol/min/mg). The rate of motility mediated by 110-kD-calmodulin is dependent on temperature and concentration of ATP, but independent of time, actin filament length, amount of enzyme, or ionic strength. Tropomyosin inhibits actin binding by 110-kD-calmodulin in MgATP and inhibits motility. Micromolar calcium slightly increases the rate of motility and increases the actin-activated MgATP hydrolysis of the intact complex. In 0.1 mM or higher calcium, motility ceases and actin-dependent MgATPase activity remains at a low rate not activated by increasing actin concentration. Correlated with these inhibitions of activity, a subset of calmodulin is dissociated from the complex. To determine if calmodulin loss is the cause of calcium inhibition, we assayed the ability of calmodulin to rescue the calcium-inactivated enzyme. Readdition of calmodulin to the nitrocellulose-bound, calcium-inactivated enzyme completely restores motility. Addition of calmodulin also restores actin activation to MgATPase activity in high calcium, but does not affect the activity of the enzyme in EGTA. These results demonstrate that in vitro 110-kD-calmodulin functions as a calcium-sensitive mechanoenzyme, a vertebrate myosin I. The properties of this enzyme suggest that despite unique structure and regulation, myosins I and II share a molecular mechanism of motility.

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Journal articles on the topic 'Calmodulin mediated activation'

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