The Ca(2+)/Calcineurin-Dependent Signaling Pathway in the Gray Mold Botrytis cinerea: The Role of Calcipressin in Modulating Calcineurin Activity

In the gray mold fungus Botrytis cinerea the Gα subunit Bcg1 of a heterotrimeric G protein is an upstream activator of the Ca(2+)/calmodulin-dependent phosphatase calcineurin. In this study we focused on the functional characterization of the catalytic subunit of calcineurin (BcCnA) and its putative regulator calcipressin (BcRcn1). We deleted the genes encoding both proteins to examine their role concerning growth, differentiation and virulence. The ΔbccnA mutant shows a severe growth defect, does not produce conidia and is avirulent, while the loss of BcRcn1 caused retardation of hyphal growth and delayed infection of host plants, but had no impact on conidiation and sclerotia formation. Expression of several calcineurin-dependent genes and bccnA itself is positively affected by BcRcn1. Complementation of the Δbcrcn1 mutant with a GFP-BcRcn1 fusion construct revealed that BcRcn1 is localized in the cytoplasm and accumulates around the nuclei. Furthermore, we showed that BcCnA physically interacts with BcRcn1 and the regulatory subunit of calcineurin, BcCnB. We investigated the impact of several protein domains characteristic for modulation and activation of BcCnA via BcRcn1, such as the phosphorylation sites and the calcineurin-docking site, by physical interaction studies between BcCnA and wild-type and mutated copies of BcRcn1. Based on the observed phenotypes we conclude that BcRcn1 acts as a positive modulator of BcCnA and the Ca(2+)/calcineurin-mediated signal transduction in B. cinerea, and that both proteins regulate fungal development and virulence.     

Ca(2+)/calmodulin-independent activation of calcineurin from Dictyostelium by unsaturated long chain fatty acids

This study describes a novel mode of activation for the Ca(2+)/calmodulin-dependent protein phosphatase calcineurin. Using purified calcineurin from Dictyostelium discoideum we found a reversible, Ca(2+)/calmodulin-independent activation by the long chain unsaturated fatty acids arachidonic acid, linoleic acid, and oleic acid, which was of the same magnitude as activation by Ca(2+)/calmodulin. Half-maximal stimulation of calcineurin occurred at fatty acid concentrations of approximately 10 microM with either p-nitrophenyl phosphate or RII phosphopeptide as substrates. The methyl ester of arachidonic acid and the saturated fatty acids palmitic acid and arachidic acid did not activate calcineurin. The activation was shown to be independent of the regulatory subunit, calcineurin B. Activation by Ca(2+)/calmodulin and fatty acids was not additive. In binding assays with immobilized calmodulin, arachidonic acid inhibited binding of calcineurin to calmodulin. Therefore fatty acids appear to mimic Ca(2+)/calmodulin action by binding to the calmodulin-binding site.     

A cluster of basic amino acid residues in calcineurin b participates in the binding of calcineurin to phosphatidylserine vesicles

Interactions between phospholipid membranes and the acyl chain and specific amino acid residues of myristoylated proteins are necessary for membrane association. In the present study we tested the effects of mutations of calcineurin B subunit amino acid residues K(20)K(21), K(24)R(25), K(27)K(28) to Glu on the interactions between calcineurin and phosphatidylserine vesicles. Calcineurin-phosphatidylserine interactions were measured using binding assays and assays of phosphatidylserine-stimulated calcineurin phosphatase activity. The reverse-charge calcineurin B subunit mutant had a slower mobility in SDS-PAGE relative to wild-type calcineurin B. In addition, the myristoylated calcineurin B reverse-charge mutant had a slower mobility in SDS-PAGE compared to the non-myristoylated form, in contrast to the faster mobility of myristoylated wild-type calcineurin B relative to non-myristoylated calcineurin B. The reverse-charge mutations had no apparent effect on N-terminal myristoylation, Ca(2+)-binding, or calcineurin heterodimer formation and stimulation of Ca(2+)/calmodulin-dependent phosphatase activity. However, in contrast to the results obtained using native calcineurin, phosphatidylserine vesicles did not bind to or activate the phosphatase activity of calcineurin containing the calcineurin B reverse-charge mutant. These results indicate that calcineurin B contains an amino terminal basic residue cluster that is involved in the binding of calcineurin to acidic phospholipids.     

Targets of immunophilin-immunosuppressant complexes are distinct highly conserved regions of calcineurin A.

The immunosuppressive complexes cyclophilin A-cyclosporin A (CsA) and FKBP12-FK506 inhibit calcineurin, a heterodimeric Ca(2+)-calmodulin-dependent protein phosphatase that regulates signal transduction. We have characterized CsA- or FK506-resistant mutants isolated from a CsA-FK506-sensitive Saccharomyces cerevisiae strain. Three mutations that confer dominant CsA resistance are single amino acid substitutions (T350K, T350R, Y377F) in the calcineurin A catalytic subunit CMP1. One mutation that confers dominant FK506 resistance alters a single residue (W430C) in the calcineurin A catalytic subunit CMP2. In vitro and in vivo, the CsA-resistant calcineurin mutants bind FKBP12-FK506 but have reduced affinity for cyclophilin A-CsA. When introduced into the CMP1 subunit, the FK506 resistance mutation (W388C) blocks binding by FKBP12-FK506, but not by cyclophilin A-CsA. Co-expression of CsA-resistant and FK506-resistant calcineurin A subunits confers resistance to CsA and to FK506 but not to CsA plus FK506. Double mutant calcineurin A subunits (Y377F, W388C CMP1 and Y419F, W430C CMP2) confer resistance to CsA, to FK506 and to CsA plus FK506. These studies identify cyclophilin A-CsA and FKBP12-FK506 binding targets as distinct, highly conserved regions of calcineurin A that overlap the binding domain for the calcineurin B regulatory subunit.     

A novel calcineurin splice variant that modifies calcineurin activity.

Calcineurin is a Ca(2+)/calmodulin dependent phosphoprotein phosphatase implicated in a wide range of disorders. Here, we report the cloning of a novel calcineurin A alpha splice variant that lacks both the catalytic and calcineurin B binding domains. Biochemical analysis revealed a stimulating effect on calcineurin activity at low calcium concentrations as well as protein-protein interaction with the catalytic calcineurin holoenzyme. By Western blot analysis, expression of similar short splice variants could be seen in the spinal cord of an animal model of familial amyotrophic lateral sclerosis, suggesting a role of these new variants in motor neuron disease.     

The calcineurin regulator sra plays an essential role in female meiosis in Drosophila

Modulatory calcineurin-interacting proteins (MCIPs)--also termed regulators of calcineurin (RCNs), calcipressins, or DSCR1 (Down's syndrome critical region 1)--are highly conserved regulators of calcineurin, a Ca(2+)/calmodulin-dependent protein phosphatase . Although overexpression experiments in several organisms have revealed that MCIPs inhibit calcineurin activity , their in vivo functions remain unclear. Here, we show that the Drosophila MCIP sarah (sra) is essential for meiotic progression in oocytes. Eggs from sra null mothers are arrested at anaphase of meiosis I. This phenotype was due to loss of function of sra specifically in the female germline. Sra is physically associated with the catalytic subunit of calcineurin, and its overexpression suppresses the phenotypes caused by constitutively activated calcineurin, such as rough eye or loss of wing veins. Hyperactivation of calcineurin signaling in the germline cells resulted in a meiotic-arrest phenotype, which can also be suppressed by overexpression of Sra. All these results support the hypothesis that Sra regulates female meiosis by controlling calcineurin activity in the germline. To our knowledge, this is the first unambiguous demonstration that the regulation of calcineurin signaling by MCIPs plays a critical role in a defined biological process.     

Regulation of calcineurin phosphatase activity by its autoinhibitory domain

The Ca(2+)-dependent activation of calcineurin phosphatase activity is regulated by an autoinhibitory element (residues 457-482) located 43 residues COOH-terminal of the calmodulin-binding domain (residues 390-414). Removal of residues 457-482 does not result in full Ca(2+)/calmodulin-independent activity. Full activity in the absence of Ca(2+) requires the removal of residues 420-457. In the present study the presence of additional autoinhibitory elements within residues 420-457 was tested using two calcineurin A subunit COOH-terminal region constructs containing residues 420-511 (AI(420-511)) or 328-511 (AI(328-511)). Using recombinant, Ca(2+)/calmodulin-independent calcineurin, AI(420-511) and AI(328-511) were three- to fourfold more potent inhibitors of calcineurin phosphatase activity than the synthetic calcineurin autoinhibitory peptide(457-482). Calmodulin reversed the inhibition of calcineurin phosphatase activity by AI(328-511) but not AI(420-511). Kinetic studies indicated that AI(420-511) exhibited mixed-type inhibition and that the enzyme/substrate/inhibitor complex is partially active. These results indicate that (i) additional autoinhibitory elements are present within residues 420-457, (ii) calmodulin-binding to the autoinhibitory domain neutralizes the inhibitory function of the 420-457 autoinhibitory segment, (iii) the full-length autoinhibitory domain is a mixed-type inhibitor of calcineurin phosphatase activity, and (iv) the enzyme/substrate/inhibitor complex is partially catalytically active.     

Activation of brain calcineurin phosphatase towards nonprotein phosphoesters by Ca2+, calmodulin, and Mg2+

Calcineurin purified from bovine brain was found to be active towards beta-naphthyl phosphate greater than p-nitrophenyl phosphate greater than alpha-naphthyl phosphate much greater than phosphotyrosine. In its native state, calcineurin shows little activity. It requires the synergistic action of Ca2+, calmodulin, and Mg2+ for maximum activation. Ca2+ and Ca2+ X calmodulin exert their activating effects by transforming the enzyme into a potentially active form which requires Mg2+ to express the full activity. Ni2+, Mn2+, and Co2+, but not Ca2+ or Zn2+, can substitute for Mg2+. The pH optimum, and the Vm and Km values of the phosphatase reaction are characteristics of the divalent cation cofactor. Ca2+ plus calmodulin increases the Vm in the presence of a given divalent cation, but has little effect on the Km for p-nitrophenyl phosphate. The activating effects of Mg2+ are different from those of the transition metal ions in terms of effects on Km, Vm, pH optimum of the phosphatase reaction and their affinity for calcineurin. Based on the Vm values determined in their respective optimum conditions, the order of effectiveness is: Mg2+ greater than or equal to Ni2+ greater than Mn2+ much greater than Co2+. The catalytic properties of calcineurin are markedly similar to those of p-nitrophenyl phosphatase activity associated with protein phosphatase 3C and with its catalytic subunit of Mr = 35,000, suggesting that there are common features in the catalytic sites of these two different classes of phosphatase.     

Calcineurin regulatory subunit is essential for virulence and mediates interactions with FKBP12-FK506 in Cryptococcus neoformans

Calcineurin is a Ca2+-calmodulin-regulated protein phosphatase that is the target of the immunosuppressive drugs cyclosporin A and FK506. Calcineurin is a heterodimer composed of a catalytic A and a regulatory B subunit. In previous studies, the calcineurin A homologue was identified and shown to be required for growth at 37 degrees C and hence for virulence of the pathogenic fungus Cryptococcus neoformans. Here, we identify the gene encoding the calcineurin B regulatory subunit and demonstrate that calcineurin B is also required for growth at elevated temperature and virulence. We show that the FKR1-1 mutation, which confers dominant FK506 resistance, results from a 6 bp duplication generating a two-amino-acid insertion in the latch region of calcineurin B. This mutation was found to reduce FKBP12-FK506 binding to calcineurin both in vivo and in vitro. Molecular modelling based on the FKBP12-FK506-calcineurin crystal structure illustrates how this mutation perturbs drug interactions with the phosphatase target. In summary, our studies reveal a central role for calcineurin B in virulence and antifungal drug action in the human fungal pathogen C. neoformans.     

Cch1 mediates calcium entry in Cryptococcus neoformans and is essential in low-calcium environments

The ability of Cryptococcus neoformans to grow at the mammalian body temperature (37 degrees C to 39 degrees C) is a well-established virulence factor. Growth of C. neoformans at this physiological temperature requires calcineurin, a Ca(2+)/calmodulin-dependent protein phosphatase. When cytosolic calcium concentrations are low ( approximately 50 to 100 nM), calcineurin is inactive and becomes active only when cytosolic calcium concentrations rise ( approximately 1 to 10 microM) through the activation of calcium channels. In this study we analyzed the function of Cch1 in C. neoformans and found that Cch1 is a Ca(2+)-permeable channel that mediates calcium entry in C. neoformans. Analysis of the Cch1 protein sequence revealed differences in the voltage sensor (S4 regions), suggesting that Cch1 may have diminished voltage sensitivity or possibly an alternative gating mechanism. The inability of the cch1 mutant to grow under conditions of limited extracellular calcium concentrations ([Ca(2+)](extracellular), approximately 100 nM) suggested that Cch1 was required for calcium uptake in low-calcium environments. These results are consistent with the role of ScCch1 in mediating high-affinity calcium uptake in Saccharomyces cerevisiae. Although the growth defect of the cch1 mutant under conditions of limited [Ca(2+)](extracellular) ( approximately 100 nM) became more severe with increasing temperature (25 degrees C to 38.5 degrees ), this temperature sensitivity was not observed when the cch1 mutant was grown on rich medium ([Ca(2+)](extracellular), approximately 0.140 mM). Accordingly, the cch1 mutant strain displayed only attenuated virulence when tested in the mouse inhalation model of cryptococcosis, further suggesting that C. neoformans may have a limited requirement for Cch1 and that this requirement appears to include ion stress tolerance.     

The calmodulin-dependent protein phosphatase catalytic subunit (calcineurin A) is an essential gene in Aspergillus nidulans.

The gene encoding the homologue of the catalytic subunit of the Ca2+/calmodulin-regulated protein phosphatase 2B (calcineurin A) has been isolated from Aspergillus nidulans. This gene, cnaA+, is essential in this fungal system. Analysis of growth-arrested cells following gene disruption by homologous recombination reveals that they are blocked early in the cell cycle. The cnaA+ gene encodes a 2.5 kb mRNA and the deduced protein sequence is highly homologous to the calcineurin A subunit of other species. The mRNA varies in a cell cycle-dependent manner with maximal levels found early in G1 and considerably before the G1/S boundary. As calmodulin is also essential for A.nidulans cell cycle progression and levels rise before the G1/S boundary, our data suggest that calcineurin may represent a primary target for calmodulin at this cell cycle transition point.     

The calmodulin-dependent protein phosphatase catalytic subunit (calcineurin A) is an essential gene in Aspergillus nidulans.

The gene encoding the homologue of the catalytic subunit of the Ca2+/calmodulin-regulated protein phosphatase 2B (calcineurin A) has been isolated from Aspergillus nidulans. This gene, cnaA+, is essential in this fungal system. Analysis of growth-arrested cells following gene disruption by homologous recombination reveals that they are blocked early in the cell cycle. The cnaA+ gene encodes a 2.5 kb mRNA and the deduced protein sequence is highly homologous to the calcineurin A subunit of other species. The mRNA varies in a cell cycle-dependent manner with maximal levels found early in G1 and considerably before the G1/S boundary. As calmodulin is also essential for A. nidulans cell cycle progression and levels rise before the G1/S boundary, our data suggest that calcineurin may represent a primary target for calmodulin at this cell cycle transition point.     

Intra-subunit and inter-subunit electron transfer in neuronal nitric-oxide synthase: effect of calmodulin on heterodimer catalysis

In neuronal nitric-oxide synthase (nNOS), calmodulin (CaM) binding is thought to trigger electron transfer from the reductase domain to the heme domain, which is essential for O(2) activation and NO formation. To elucidate the electron-transfer mechanism, we characterized a series of heterodimers consisting of one full-length nNOS subunit and one oxygenase-domain subunit. The results support an inter-subunit electron-transfer mechanism for the wild type nNOS, in that electrons for catalysis transfer in a Ca(2+)/CaM-dependent way from the reductase domain of one subunit to the heme of the other subunit, as proposed for inducible NOS. This suggests that the two different isoforms form similar dimeric complexes. In a series of heterodimers containing a Ca(2+)/CaM-insensitive mutant (delta40), electrons transferred from the reductase domain to both hemes in a Ca(2+)/CaM-independent way. Thus, in the delta40 mutant electron transfer from the reductase domains to the heme domains can occur via both inter-subunit and intra-subunit mechanisms. However, NO formation activity was exclusively linked to inter-subunit electron transfer and was observed only in the presence of Ca(2+)/CaM. This suggests that the mechanism of activation of nNOS by CaM is not solely dependent on the activation of electron transfer to the nNOS hemes but may involve additional structural factors linked to the catalytic action of the heme domain.     

Ca2+-calmodulin promotes survival of pheromone-induced growth arrest by activation of calcineurin and Ca2+-calmodulin-dependent protein kinase.

The cmd1-6 allele contains three mutations that block Ca2+ binding to calmodulin from Saccharomyces cerevisiae. We find that strains containing cmd1-6 lose viability during cell cycle arrest induced by the mating pheromone alpha-factor. The 50% lethal dose (LD50) of alpha-factor for the calmodulin mutant is almost fivefold below the LD50 for a wild-type strain. The calmodulin mutants are not more sensitive to alpha-factor, as measured by activation of a pheromone-responsive reporter gene. Two observations indicate that activation of the Ca2+-calmodulin-dependent protein phosphatase calcineurin contributes to survival of pheromone-induced arrest. First, deletion of the gene encoding the calcineurin regulatory B subunit, CNB1, from a wild-type strain decreases the LD50 of alpha-factor but has no further effect on a cmd1-6 strain. Second, a dominant constitutive calcineurin mutant partially restores the ability of the cmd1-6 strain to survive exposure to alpha-factor. Activation of the Ca2+-calmodulin-dependent protein kinase (CaMK) also contributes to survival, thus revealing a new function for this enzyme. Deletion of the CMK1 and CMK2 genes, which encode CaMK, decreases the LD50 of pheromone compared with that for a wild-type strain but again has no effect in a cmd1-6 strain. Furthermore, the LD50 of alpha-factor for a mutant in which the calcineurin and CaMK genes have been deleted is the same as that for the calmodulin mutant. Finally, the CaMK and calcineurin pathways appear to be independent since the ability of constitutive calcineurin to rescue a cmd1-6 strain is not blocked by deletion of the CaMK genes.     

Calcineurin-dependent growth control in Saccharomyces cerevisiae mutants lacking PMC1, a homolog of plasma membrane Ca2+ ATPases

Ca2+ ATPases deplete the cytosol of Ca2+ ions and are crucial to cellular Ca2+ homeostasis. The PMC1 gene of Saccharomyces cerevisiae encodes a vacuole membrane protein that is 40% identical to the plasma membrane Ca2+ ATPases (PMCAs) of mammalian cells. Mutants lacking PMC1 grow well in standard media, but sequester Ca2+ into the vacuole at 20% of the wild-type levels. pmc1 null mutants fail to grow in media containing high levels of Ca2+, suggesting a role of PMC1 in Ca2+ tolerance. The growth inhibitory effect of added Ca2+ requires activation of calcineurin, a Ca2+ and calmodulin-dependent protein phosphatase. Mutations in calcineurin A or B subunits or the inhibitory compounds FK506 and cyclosporin A restore growth of pmc1 mutants in high Ca2+ media. Also, growth is restored by recessive mutations that inactivate the high-affinity Ca(2+)-binding sites in calmodulin. This mutant calmodulin has apparently lost the ability to activate calcineurin in vivo. These results suggest that activation of calcineurin by Ca2+ and calmodulin can negatively affect yeast growth. A second Ca2+ ATPase homolog encoded by the PMR1 gene acts together with PMC1 to prevent lethal activation of calcineurin even in standard (low Ca2+) conditions. We propose that these Ca2+ ATPase homologs are essential in yeast to deplete the cytosol of Ca2+ ions which, at elevated concentrations, inhibits yeast growth through inappropriate activation of calcineurin.     

Regulation of aflatoxin production by Ca(2+)/calmodulin-dependent protein phosphorylation and dephosphorylation

To elucidate Ca(2+)-mediated regulation of aflatoxin production, the status of Ca(2+)/calmodulin-dependent protein phosphorylation and dephosphorylation was investigated employing toxigenic and non-toxigenic strains of Aspergillus parasiticus. Incubation of cytoplasmic extracts with [gamma-(32)P]ATP followed by SDS-PAGE and autoradiography revealed total absence of protein phosphorylation during periods corresponding to aflatoxin production in the toxigenic strain (NRRL 2999). In contrast, protein phosphorylation was unaffected in the non-toxigenic strain (SRRC 255). Aflatoxin production in the toxigenic strain was also accompanied by enhanced (26-fold) activity of calcineurin (calmodulin-dependent protein phosphatase 2B) concomitant with a lowered (6-fold) activity of calmodulin-dependent protein kinase. In addition, the in vitro activity of Ca(2+)/calmodulin-dependent protein kinase was susceptible to dose-dependent inhibition by aflatoxin. Since calcineurin remains active in the absence of phosphorylation by calmodulin-dependent protein kinase, it is suggested that calcineurin-mediated dephosphorylation of regulatory enzymes ensures continued production of aflatoxins.