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.     

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.     

Regulation of Ca(2+)/calmodulin-dependent protein kinase kinase alpha by cAMP-dependent protein kinase: I. Biochemical analysis

Ca(2+)/calmodulin-dependent protein kinases (CaM-kinases) I and IV are activated upon phosphorylation of their Thr(177) and Thr(196), respectively, by the upstream Ca(2+)/calmodulin-dependent protein kinases CaM-kinase kinase alpha and beta, and deactivated upon dephosphorylation by protein phosphatases such as CaM-kinase phosphatase. Recent studies demonstrated that the activity of CaM-kinase kinase alpha is decreased upon phosphorylation by cAMP-dependent protein kinase (PKA), and the relationship between the inhibition and phosphorylation of CaM-kinase kinase alpha by PKA has been studied. In the present study, we demonstrate that the activity of CaM-kinase kinase alpha toward PKIV peptide, which contains the sequence surrounding Thr(196) of CaM-kinase IV, is increased by incubation with PKA in the presence of Ca(2+)/calmodulin but decreased in its absence, while the activity toward CaM-kinase IV is decreased by incubation with PKA in both the presence and absence of Ca(2+)/calmodulin. Six phosphorylation sites on CaM-kinase kinase alpha, Ser(24) for autophosphorylation, and Ser(52), Ser(74), Thr(108), Ser(458), and Ser(475) for phosphorylation by PKA, were identified by amino acid sequence analysis of the phosphopeptides purified from the tryptic digest of the phosphorylated enzymes. The presence of Ca(2+)/calmodulin suppresses phosphorylation on Ser(52), Ser(74), Thr(108), and Ser(458) by PKA, but accelerates phosphorylation on Ser(475). The changes in the activity of the enzyme upon phosphorylation appear to occur as a result of conformational changes induced by phosphorylation on several sites.     

Activity-dependent phosphorylation of GABAA receptors regulates receptor insertion and tonic current

The expression of GABA(A) receptors and the efficacy of GABAergic neurotransmission are subject to adaptive compensatory regulation as a result of changes in neuronal activity. Here, we show that activation of L-type voltage-gated Ca(2+) channels (VGCCs) leads to Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) phosphorylation of S383 within the β3 subunit of the GABA(A) receptor. Consequently, this results in rapid insertion of GABA(A) receptors at the cell surface and enhanced tonic current. Furthermore, we demonstrate that acute changes in neuronal activity leads to the rapid modulation of cell surface numbers of GABA(A) receptors and tonic current, which are critically dependent on Ca(2+) influx through L-type VGCCs and CaMKII phosphorylation of β3S383. These data provide a mechanistic link between activity-dependent changes in Ca(2+) influx through L-type channels and the rapid modulation of GABA(A) receptor cell surface numbers and tonic current, suggesting a homeostatic pathway involved in regulating neuronal intrinsic excitability in response to changes in activity.     

Calcium-stimulated autophosphorylation site of plant chimeric calcium/calmodulin-dependent protein kinase

The existence of two molecular switches regulating plant chimeric Ca(2+)/calmodulin-dependent protein kinase (CCaMK), namely the C-terminal visinin-like domain acting as Ca(2+)-sensitive molecular switch and calmodulin binding domain acting as Ca(2+)-stimulated autophosphorylation-sensitive molecular switch, has been described (Sathyanarayanan, P. V., Cremo, C. R., and Poovaiah, B. W. (2000) J. Biol. Chem. 275, 30417-30422). Here we report the identification of Ca(2+)-stimulated autophosphorylation site of CCaMK by matrix-assisted laser desorption ionization time of flight-mass spectrometry. Thr(267) was confirmed as the Ca(2+)-stimulated autophosphorylation site by post-source decay experiments and by site-directed mutagenesis. The purified T267A mutant form of CCaMK did not show Ca(2+)-stimulated autophosphorylation, autophosphorylation-dependent variable calmodulin affinity, or Ca(2+)/calmodulin stimulation of kinase activity. Sequence comparison of CCaMK from monocotyledonous plant (lily) and dicotyledonous plant (tobacco) suggests that the autophosphorylation site is conserved. This is the first identification of a phosphorylation site specifically responding to activation by second messenger system (Ca(2+) messenger system) in plants. Homology modeling of the kinase and calmodulin binding domain of CCaMK with the crystal structure of calcium/calmodulin-dependent protein kinase 1 suggests that the Ca(2+)-stimulated autophosphorylation site is located on the surface of the kinase and far from the catalytic site. Analysis of Ca(2+)-stimulated autophosphorylation with increasing concentration of CCaMK indicates the possibility that the Ca(2+)-stimulated phosphorylation occurs by an intermolecular mechanism.     

Ca2+/calmodulin-dependent kinase II and calcineurin play critical roles in endothelin-1-induced cardiomyocyte hypertrophy

Endothelin-1 (ET-1) induces cardiac hypertrophy. Because Ca(2+) is a major second messenger of ET-1, the role of Ca(2+) in ET-1-induced hypertrophic responses in cultured cardiac myocytes of neonatal rats was examined. ET-1 activated the promoter of the beta-type myosin heavy chain gene (beta-MHC) (-354 to +34 base pairs) by about 4-fold. This activation was inhibited by chelation of Ca(2+) and the blocking of protein kinase C activity. Similarly, the beta-MHC promoter was activated by Ca(2+) ionophores and a protein kinase C activator. beta-MHC promoter activation induced by ET-1 was suppressed by pretreatment with the calmodulin inhibitor, W7, the Ca(2+)/calmodulin-dependent kinase II (CaMKII) inhibitor, KN62, and the calcineurin inhibitor, cyclosporin A. beta-MHC promoter activation by ET-1 was also attenuated by overexpression of dominant-negative mutants of CaMKII and calcineurin. ET-1 increased the activity of CaMKII and calcineurin in cardiac myocytes. Pretreatment with KN62 and cyclosporin A strongly suppressed ET-1-induced increases in [(3)H]phenylalanine uptake and in cell size. These results suggest that Ca(2+) plays a critical role in ET-1-induced cardiomyocyte hypertrophy by activating CaMKII- and calcineurin-dependent pathways.     

Phosphorylation and characterization of bovine heart calmodulin-dependent phosphodiesterase.

Calmodulin-dependent phosphodiesterase was purified to apparent homogeneity from the total calmodulin-binding fraction of bovine heart in a single step by immunoaffinity chromatography. The isolated enzyme had significantly higher affinity for calmodulin than the bovine brain 60-kDa phosphodiesterase isozyme. The cAMP-dependent protein kinase was found to catalyze the phosphorylation of the purified cardiac calmodulin-dependent phosphodiesterase with the incorporation of 1 mol of phosphate/mol of subunit. The phosphodiesterase phosphorylation rate was increased severalfold by histidine without affecting phosphate incorporation into the enzyme. Phosphorylation of phosphodiesterase lowered its affinity for calmodulin and Ca2+. At constant saturating concentrations of calmodulin (650 nM), the phosphorylated calmodulin-dependent phosphodiesterase required a higher concentration of Ca2+ (20 microM) than the nonphosphorylated phosphodiesterase (0.8 microM) for 50% activity. Phosphorylation could be reversed by the calmodulin-dependent phosphatase (calcineurin), and dephosphorylation was accompanied by an increase in the affinity of phosphodiesterase for calmodulin.     

EPYLRFamide-mediated reduction of acetylcholine-induced inward currents in Helix lucorum-identified neurones: role of NAADP-dependent and IP3-dependent Ca2+ release from internal stores,calmodulin and Ca2+/calmodulin-dependent protein kinase II

The effect of seven compounds intracellularly applied by spontaneous diffusion were investigated on the EPYLRFamide-induced reduction of acetylcholine-induced inward current (ACh-current) recorded from identified neurones from Helix lucorum. Inward currents were recorded from neurones LPa2, LPa3, RPa3 and RPa2 in isolated ganglia preparations using two-electrode voltage clamp technique. ACh was applied ionophoretically. Heparin, an antagonist of IP(3) receptors (IP(3)Rs), and IP(3), the agonist of IP(3)Rs, decreased the effect of EPYLRFamide. Thio-NADP, a blocker of NAADP-induced Ca(2+) release, beta-NAADP, Ca(2+) releaser, R24571, W-7 (both calmodulin antagonists), and KN-62, a selective inhibitor of Ca(2+)/calmodulin-dependent protein kinase II, did not change the modulatory effect of EPYLRFamide. These data suggest that EPYLRFamide decreases ACh-current through elevation of the basal intracellular level of the putative endogenous agonist of IP(3)Rs which activates release of Ca(2+) from intracellular stores. It is concluded that intracellular free Ca(2+) acts on ACh receptor/ionic channel without activation of calmodulin and Ca(2+)/calmodulin-dependent protein kinase II.     

Isolation and characterization of a novel calcium/calmodulin-dependent protein kinase, AtCK, from arabidopsis

Protein phosphorylation is one of the major mechanisms by which eukaryotic cells transduce extracellular signals into intracellular responses. Calcium/calmodulin (Ca(2+)/CaM)-dependent protein phosphorylation has been implicated in various cellular processes, yet little is known about Ca(2+)/CaM-dependent protein kinases (CaMKs) in plants. From an Arabidopsis expression library screen using a horseradish peroxidase-conjugated soybean calmodulin isoform (SCaM-1) as a probe, we isolated a full-length cDNA clone that encodes AtCK (Arabidopsis thaliana calcium/calmodulin-dependent protein kinase). The predicted structure of AtCK contains a serine/threonine protein kinase catalytic domain followed by a putative calmodulin-binding domain and a putative Ca(2+)-binding domain. Recombinant AtCK was expressed in E. coli and bound to calmodulin in a Ca(2+)-dependent manner. The ability of CaM to bind to AtCK was confirmed by gel mobility shift and competition assays. AtCK exhibited its highest levels of autophosphorylation in the presence of 3 mM Mn(2+). The phosphorylation of myelin basic protein (MBP) by AtCK was enhanced when AtCK was under the control of calcium-bound CaM, as previously observed for other Ca(2+)/CaM-dependent protein kinases. In contrast to maize and tobacco CCaMKs (calcium and Ca(2+)/CaM-dependent protein kinase), increasing the concentration of calmodulin to more than 3 microgram suppressed the phosphorylation activity of AtCK. Taken together our results indicate that AtCK is a novel Arabidopsis Ca(2+)/CaM-dependent protein kinase which is presumably involved in CaM-mediated signaling.     

A model for molecular mechanisms of synaptic competition for a finite resource

Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) undergoes Ca(2+)/calmodulin-dependent autophosphorylation of threonine-286/287 (Thr(286/287)). Extremely high concentration of CaMKII in the postsynaptic spine indicates that increase in the Ca(2+) concentration in the spine induced by synaptic activation results in Thr(286/287) autophosphorylation of this enzyme. It has recently been shown that the K(d) value of CaMKII for Ca(2+)/calmodulin (Ca(2+)/CaM) drastically decreases after Thr(286/287) autophosphorylation. Therefore, Ca(2+)/CaM associated with CaMKII becomes tightly bound to this kinase after Thr(286/287) autophosphorylation. This has been called 'Ca(2+)/CaM trapping'. We discussed the functional significance of Ca(2+)/CaM trapping in the neuronal system by a mathematical-modelling approach. We considered neighbouring synapses formed on the same dendrite and different increase in the Ca(2+) concentration in each spine. CaMKII undergoing Thr(286/287) autophosphorylation in each spine is eager to recruit nearby calmodulin in the dendrite for Ca(2+)/CaM trapping. Since the amount of calmodulin is limited, recruiting calmodulin to each spine causes competition among synapses for this finite resource. The results of our computer simulation show that this competition leads to 'winner-take-all': almost all calmodulin is taken by a few synapses to which relatively large increases in the Ca(2+) concentration are assigned. These results suggest a novel form of synaptic encoding of information.     

Functional characterization of the Aspergillus fumigatus calcineurin

Aspergillus fumigatus is an aggressive opportunistic pathogen of humans as well as a major allergen. Environmental sensing and retrieving essential nutrients from the environment are general metabolic traits associated with the growth of this saprophytic fungus. Two important mediators of calcium signals in eukaryotic cells are the Ca(2+)-binding protein calmodulin and the Ca(2+)/calmodulin-dependent phosphatase calcineurin. Calcineurin is a heterodimer that consists of a catalytic subunit A and a Ca(2+)/calmodulin binding unit. We deleted the A. fumigatus calA gene, which encodes the calcineurin A catalytic subunit, and demonstrated that this gene is not essential in this fungus. The DeltacalA mutant strain has severe defects in growth extension, branching and conidial architecture. Furthermore, the A. fumigatus DeltacalA mutant strain has decreased fitness in a low dose murine infection and cannot grow in fetal bovine serum (FBS). After potassium phosphate was added to liquid FBS, the DeltacalA mutant strain could grow with the characteristic phenotype of the DeltacalA mutation. When A. fumigatus calcineurin is inhibited by tacrolimus in a phosphate depleted medium, there is a reduction in the inorganic phosphate transport and six putative phosphate transporter genes have altered mRNA levels. However, there is no effect on the acid phosphatase activity. These results suggest that calcineurin is involved in the regulation of the PHO pathway in A. fumigatus. Our work on calcineurin opens new venues for the research on sensing and nutrient acquisition in A. fumigatus.     

The role of calmodulin as a signal integrator for synaptic plasticity

Excitatory synapses in the brain show several forms of synaptic plasticity, including long-term potentiation (LTP) and long-term depression (LTD), which are initiated by increases in intracellular Ca(2+) that are generated through NMDA (N-methyl-D-aspartate) receptors or voltage-sensitive Ca(2+) channels. LTP depends on the coordinated regulation of an ensemble of enzymes, including Ca(2+)/calmodulin-dependent protein kinase II, adenylyl cyclase 1 and 8, and calcineurin, all of which are stimulated by calmodulin, a Ca(2+)-binding protein. In this review, we discuss the hypothesis that calmodulin is a central integrator of synaptic plasticity and that its unique regulatory properties allow the integration of several forms of signal transduction that are required for LTP and LTD.     

Calcineurin and intracellular Ca2+-release channels: regulation or association?

The Ca(2+)- and calmodulin-dependent phosphatase calcineurin was reported to interact with the inositol 1,4,5-trisphosphate receptor (IP(3)R) and the ryanodine receptor (RyR) and to modulate their phosphorylation status and activity. However, controversial data on the molecular mechanisms involved and on the functional relevance of calcineurin for these channel-complexes have been described. Hence, we will focus on the functional importance of calcineurin for IP(3)R and RyR function and on the different mechanisms by which Ca(2+)-dependent dephosphorylation can affect the gating of those intracellular Ca(2+)-release channels. Since many studies made use of immunosuppressive drugs that are inhibiting calcineurin activity, we will also have to take the different side effects of these drugs into account for the proper interpretation of the effects of calcineurin on intracellular Ca(2+)-release channels. In addition, it became recently known that various other phosphatases and kinases can associate with these channels, thereby forming macromolecular complexes. The relevance of these enzymes for IP(3)R and RyR functioning will be reviewed since in some cases they could interfere with the effects ascribed to calcineurin. Finally, we will discuss the downstream effects of calcineurin on the regulation of the expression levels of intracellular Ca(2+)-release channels as well as the relation between IP(3)R- and RyR-mediated Ca(2+) release and calcineurin-dependent gene expression.     

Invited review: regulation of myosin phosphorylation in smooth muscle.

Phosphorylation of the regulatory light chains of myosin II (rMLC) by the Ca(2+)/calmodulin-dependent myosin light-chain kinase (MLCK) and dephosphorylation by a type 1 phosphatase (MLCP), which is targeted to myosin by a regulatory subunit (MYPT1), are the predominant mechanisms of regulation of smooth muscle tone. The activities of both enzymes are modulated by several protein kinases. MLCK is inhibited by the Ca(2+)/calmodulin-dependent protein kinase II, whereas the activity of MLCP is increased by cGMP and perhaps also cAMP-dependent protein kinases. In either case, this results in a decrease in the Ca(2+) sensitivity of rMLC phosphorylation and force production. The activity of MLCP is inhibited by Rho-associated kinase, one of the effectors of the monomeric GTPase Rho, and protein kinase C, leading to an increase in Ca(2+) sensitivity. Hence, smooth muscle tone appears to be regulated by a network of activating and inactivating intracellular signaling cascades.     

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.     

Differential modulation of Ca2+/calmodulin-dependent protein kinase II activity by regulated interactions with N-methyl-D-aspartate receptor NR2B subunits and alpha-actinin

Neuronal Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) interacts with several prominent dendritic spine proteins, which have been termed CaMKII-associated proteins. The NR2B subunit of N-methyl-d-aspartate (NMDA)-type glutamate receptor, densin-180, and alpha-actinin bind comparable, approximately stoichiometric amounts of Thr(286)-autophosphorylated CaMKIIalpha, forming a ternary complex (Robison, A. J., Bass, M. A., Jiao, Y., Macmillan, L. B., Carmody, L. C., Bartlett, R. K., and Colbran, R. J. (2005) J. Biol. Chem. 280, 35329-35336), but their impacts on CaMKII function are poorly understood. Here we show that these interactions are differentially regulated and exert distinct effects on CaMKII activity. Nonphosphorylated and Thr(286)-autophosphorylated CaMKII bind to alpha-actinin with similar efficacy, but autophosphorylation at Thr(305/306) or Ca(2+)/calmodulin binding significantly reduce this binding. Moreover, alpha-actinin antagonizes CaMKII activation by Ca(2+)/calmodulin, as assessed by autophosphorylation and phosphorylation of a peptide substrate. CaMKII binding to densin (1247-1542) is partially independent of Thr(286) autophosphorylation and is unaffected by Ca(2+)-independent autophosphorylation or Ca(2+)/calmodulin. In addition, the CaMKII binding domain of densin-180 has little effect on CaMKII activity. In contrast, the interaction of CaMKIIalpha with NR2B requires either Thr(286) autophosphorylation or the binding of both Ca(2+)/calmodulin and adenine nucleotides. NR2B inhibits both the Ca(2+)/calmodulin-dependent and autonomous activities of CaMKII by a mechanism that is competitive with autocamtide-2 substrate, non-competitive with syntide-2 substrate, and uncompetitive with respect to ATP. In combination, these data suggest that dynamically regulated interactions with CaMKII-associated proteins could play pleiotropic roles in finetuning CaMKII signaling in defined subcellular compartments.