Calcineurin is part of a negative feedback loop in the InsP3/Ca signalling pathway in blowfly salivary glands

The ubiquitous InsP₃/Ca²⁺ signalling pathway is modulated by diverse mechanisms, i.e. feedback of Ca²⁺ and interactions with other signalling pathways. In the salivary glands of the blowfly Calliphora vicina, the hormone serotonin (5-HT) causes a parallel rise in intracellular [Ca²⁺] and [cAMP] via two types of 5-HT receptors. We have shown recently that cAMP/protein kinase A (PKA) sensitizes InsP₃-induced Ca²⁺ release. We have now identified the protein phosphatase that counteracts the effect of PKA on 5-HT-induced InsP₃/Ca²⁺ signalling. We demonstrate that (1) tautomycin and okadaic acid, inhibitors of protein phosphatases PP1 and PP2A, have no effect on 5-HT-induced Ca²⁺ signals; (2) cyclosporin A and FK506, inhibitors of Ca²⁺/calmodulin-activated protein phosphatase calcineurin, cause an increase in the frequency of 5-HT-induced Ca²⁺ oscillations; (3) the sensitizing effect of cyclosporin A on 5-HT-induced Ca²⁺ responses does not involve Ca²⁺ entry into the cells; (4) cyclosporin A increases InsP₃-dependent Ca²⁺ release; (5) inhibition of PKA abolishes the effect of cyclosporin A on the 5-HT-induced Ca²⁺ responses, indicating that PKA and calcineurin act antagonistically on the InsP₃/Ca²⁺ signalling pathway. These findings suggest that calcineurin provides a negative feedback on InsP₃/Ca²⁺ signalling in blowfly salivary glands, counteracting the effect of PKA and desensitizing the signalling cascade at higher 5-HT concentrations.     

The serine/threonine kinase Cmk2 is required for oxidative stress response in fission yeast

Cmk2, a fission yeast Ser/Thr protein kinase homologous to mammalian calmodulin kinases, is essential for oxidative stress response. Cells lacking cmk2 gene were specifically sensitive to oxidative stress conditions. Upon stress, Cmk2 was phosphorylated in vivo, and this phosphorylation was dependent on the stress-activated MAPK Sty1/Spc1. Co-precipitation assays demonstrated that Cmk2 binds Sty1. Furthermore, in vivo or in vitro activated Sty1 was able to phosphorylate Cmk2, and the phosphorylation occurred at the C-terminal regulatory domain at Thr-411. Cell lethality caused by overexpression of Wis1 MAPK kinase was abolished by deletion of cmk2 or by mutation of Thr-411 of Cmk2. Taken together, our data suggest that Cmk2 acts downstream of Sty1 and is an essential kinase for oxidative stress responses.     

Regulatory Subunit Myristoylation Antagonizes Calcineurin Phosphatase Activation in Yeast

The Ca²⁺/calmodulin-stimulated protein phosphatase calcineurin is a critical component of Ca²⁺ signaling cascades in eukaryotic cells. Myristoylation of the regulatory subunit of calcineurin (CNB) is conserved from yeast to humans. Here, we show that CNB myristoylation antagonizes phosphatase activation in yeast. Disruption of CNB myristoylation by mutation of the myristoylated glycine triggered constitutive expression of a calcineurin-dependent reporter gene and enhanced calcineurin-dependent phenotypes. Basal phosphatase activity was also increased in nmt1–181 yeast with reduced N-myristoyltransferase activity. Our findings are the first demonstration of a functional role for CNB myristoylation and reveal the importance of Nmt1 in modulating cellular calcineurin activation.     

Calcineurin is a calmodulin-dependent protein phosphatase

Calcineurin, one of the major calmodulin-binding proteins in the brain, dephosphorylates a phosphorylated protein termed inhibitor-1, a potent inhibitor of protein phosphatase 1. The phosphatase activity was Ca²⁺- and calmodulin-dependent and was reversed by EGTA or trifluoperazine, an antagonist of calmodulin. Using a radioimmunoassay of calcineurin and a phosphatase activity assay, we found that the two activities coincided in a sucrose density gradient and in a non-denaturing polyacrylamide gel. These results demonstrate that calcineurin is a calmodulin-dependent protein phosphatase.     

The KSR2-calcineurin complex regulates STIM1-ORAI1 dynamics and store-operated calcium entry (SOCE)

Store-operated calcium entry (SOCE) is the predominant Ca²⁺ entry mechanism in nonexcitable cells and controls a variety of physiological and pathological processes. Although significant progress has been made in identifying the components required for SOCE, the molecular mechanisms underlying it are elusive. The present study provides evidence for a direct involvement of kinase suppressor of Ras 2 (KSR2) in SOCE. Using lymphocytes and fibroblasts from ksr2^−/− mice and shKSR2-depleted cells, we find that KSR2 is critical for the elevation of cytosolic Ca²⁺ concentration. Specifically, our results show that although it is dispensable for Ca²⁺-store depletion, KSR2 is required for optimal calcium entry. We observe that KSR2 deficiency affects stromal interaction molecule 1 (STIM1)/ORAI1 puncta formation, which is correlated with cytoskeleton disorganization. Of interest, we find that KSR2-associated calcineurin is crucial for SOCE. Blocking calcineurin activity impairs STIM1/ORAI1 puncta-like formation and cytoskeleton organization. In addition, we observe that calcineurin activity and its role in SOCE are both KSR2 dependent.     

Signal tranduction: regulation of cAMP concentration in cardiac muscle by calmodulin-dependent cyclic nucleotide phosphodiesterase

The bovine heart calmodulin-dependent phosphodiesterase can be phosphorylated by cAMP-dependent protein kinase, resulting in a decrease in the enzyme's affinity for calmodulin. The phosphorylation of calmodulin-dependent phosphodiesterase is blocked by Ca²⁺ and calmodulin and reversed by the calmodulin-dependent phosphatase. The dephosphorylation is accompanied by an increase in the affinity of the phosphodiesterase for calmodulin. The CaM-dependent phosphodiesterase isozymes of heart and brain are regulated by calmodulin, but the affinity for calmodulin are different. Furthermore, the bovine heart CaM-dependent phosphodiesterase isozyme in stimulated at much lower Ca²⁺ concentration than the bovine brain isozymes. Results from this study suggest that the activity of this phosphodiesterase is precisely regulated by cross-talk between Ca²⁺ and cAMP signalling pathways.     

Heart-specific Deletion of CnB1 Reveals Multiple Mechanisms Whereby Calcineurin Regulates Cardiac Growth and Function

Calcineurin is a protein phosphatase that is uniquely regulated by sustained increases in intracellular Ca²⁺ following signal transduction events. Calcineurin controls cellular proliferation, differentiation, apoptosis, and inducible gene expression following stress and neuroendocrine stimulation. In the adult heart, calcineurin regulates hypertrophic growth of cardiomyocytes in response to pathologic insults that are associated with altered Ca²⁺ handling. Here we determined that calcineurin signaling is directly linked to the proper control of cardiac contractility, rhythm, and the expression of Ca²⁺-handling genes in the heart. Our approach involved a cardiomyocyte-specific deletion using a CnB1-LoxP-targeted allele in mice and three different cardiac-expressing Cre alleles/transgenes. Deletion of calcineurin with the Nkx2.5-Cre knock-in allele resulted in lethality at 1 day after birth due to altered right ventricular morphogenesis, reduced ventricular trabeculation, septal defects, and valvular overgrowth. Slightly later deletion of calcineurin with the α-myosin heavy chain Cre transgene resulted in lethality in early mid adulthood that was characterized by substantial reductions in cardiac contractility, severe arrhythmia, and reduced myocyte content in the heart. Young calcineurin heart-deleted mice died suddenly after pressure overload stimulation or neuroendocrine agonist infusion, and telemetric monitoring of older mice showed arrhythmia leading to sudden death. Mechanistically, loss of calcineurin reduced expression of key Ca²⁺-handling genes that likely lead to arrhythmia and reduced contractility. Loss of calcineurin also directly impacted cellular proliferation in the postnatal developing heart. These results reveal multiple mechanisms whereby calcineurin regulates cardiac development and myocyte contractility.     

Cmk2, a novel serine/threonine kinase in fission yeast

The cmk2 gene of Schizosaccharomyces pombe encodes a 504 amino acid protein kinase with sequence homology with the calmodulin-dependent protein kinase family. The cmk2(+) gene is not essential for cell viability but overexpression of cmk2(+) blocks the cell cycle at G2 phase and this inhibition is cdc2-dependent. The Cmk2 is a cytoplasmic protein expressed in a cell cycle-dependent manner, peaking at the G1/S boundary. Overexpression of Cmk2 suppresses fission yeast DNA replication checkpoint defects but not DNA damage checkpoint defects, suggesting that the G2 cell cycle arrest mediated by high levels of Cmk2 provides sufficient time to correct DNA replication alterations.     

Characteristics of magnesium-dependent, Ca2+ -stimulated endogenous protein kinase-catalyzed phosphorylation of basic proteins in myelin isolated from rat brain stem white matter

Purified myelin fraction isolated from rat brain white matter contained Mg²⁺-dependent protein kinase capable of phosphorylation of myelin basic proteins. The Mg²⁺-supported kinase was markedly stimulated (two- to fivefold) by micromolar concentrations of free Ca²⁺ with and without Triton X-100 in the assay, the degree of stimulation being greater with the detergent present. Cyclic AMP, on the other hand, failed to show any effect on phosphorylation of myelin in the absence of Triton X-100 and in the presence of Triton caused only 25–30% stimulation. The phosphorylation reaction was temperature dependent and exhibited a pH optimum at pH 6.5. Apparent affinity toward MgATP^2? was found to be about 70 μm and Ca²⁺ had no effect on this parameter. Dependence on MgCl₂ of myelin phosphorylation indicated the presence of high- and low-affinity sites toward Mg²⁺; Ca²⁺ appeared to influence the low-affinity site. Maximal level of phosphorylation was attained by 10–15 min at 30 °C and it declined at longer incubation times due to phosphatase activity present in the preparation. Stimulatory effect of Ca²⁺ on phosphorylation was not due to inhibition of phosphatase activity. Dephosphorylation experiments showed that neither cyclic AMP nor Ca²⁺ influenced the myelin phosphatase activity. Autoradiographic analysis revealed that phosphorylation of myelin basic proteins accounted for nearly 90% of total myelin phosphorylation. This was supported by the observation that the HCl extract of myelin contained 85% of total activity and comigrated with purified myelin basic proteins. Basal and Ca²⁺-stimulated phosphorylation of basic proteins were due to phosphorylation of serines mainly, although threonine was phosphorylated to a minor extent. Within myelin, Ca²⁺ and cyclic AMP kinases are differentially bound. It appears that the myelin kinase (studied in vitro) is primarily influenced by Ca²⁺ rather than cyclic AMP. Inhibitors (Type I and Type II) of cyclic nucleotide-stimulated protein kinases had no effect on the Ca²⁺-stimulated phosphorylation although basal and cyclic AMP-stimulated phosphorylation was inhibited, indicating that the Ca²⁺ kinase is a separate and distinct enzyme from the cyclic AMP-stimulated and basal kinase(s). Also, leupeptin, a protease inhibitor, did not influence basal, cyclic AMP-stimulated, or Ca²⁺-stimulated myelin phosphorylation, indicating that under the conditions used protease(s) did not alter the myelin kinase activity. The potential significance of phosphorylation of myelin basic proteins and the stimulatory action of Ca²⁺ on this reaction are discussed.     

A Highlights from MBoC Selection: Regulation of the epithelial Ca2+ channel TRPV5 by reversible histidine phosphorylation mediated by NDPK-B and PHPT1

The kidney, together with bone and intestine, plays a crucial role in maintaining whole-body calcium (Ca²⁺) homoeostasis, which is primarily mediated by altering the reabsorption of Ca²⁺ filtered by the glomerulus. The transient receptor potential-vanilloid-5 (TRPV5) channel protein forms a six- transmembrane Ca²⁺-permeable channel that regulates urinary Ca²⁺ excretion by mediating active Ca²⁺ reabsorption in the distal convoluted tubule of the kidney. Here we show that the histidine kinase, nucleoside diphosphate kinase B (NDPK-B), activates TRPV5 channel activity and Ca²⁺ flux, and this activation requires histidine 711 in the carboxy-terminal tail of TRPV5. In addition, the histidine phosphatase, protein histidine phosphatase 1, inhibits NDPK-B–activated TRPV5 in inside/out patch experiments. This is physiologically relevant to Ca²⁺ reabsorption in vivo, as short hairpin RNA knockdown of NDPK-B leads to decreased TRPV5 channel activity, and urinary Ca²⁺ excretion is increased in NDPK-B^−/− mice fed a high-Ca²⁺ diet. Thus these findings identify a novel mechanism by which TRPV5 and Ca²⁺ reabsorption is regulated by the kidney and support the idea that histidine phosphorylation plays other, yet-uncovered roles in mammalian biology.