Characterization of actin- and lipid-binding domains in severin, a Ca(2+)-dependent F-actin fragmenting protein.

Severin is a Ca(2+)-activated actin-binding protein that nucleates actin assembly and severs and caps the fast growing ends of actin filaments. It consists of three highly conserved domains. To investigate the domain structure of severin, we constructed genetically the N-terminal domain 1, the middle domain 2, and the tandem domains 2 + 3. Their interaction with actin, Ca2+, and lipids was characterized. Domain 1 contains the F-actin capping and a Ca(2+)-binding site [Eichinger, L., Noegel, A. A., & Schleicher, M. (1991) J. Cell Biol. 112, 665-676]. Binding of domain 2 to actin filaments was Ca(2+)-dependent and saturated at a 1:1 molar ratio. In the presence of Ca2+, about 1.5 mol of domains 2 + 3 bound per mole of F-actin subunit. Scatchard analysis gave a Kd of 18 microM for the interaction of domain 2 with F-actin subunits and a Kd of 1.6 microM for domains 2 + 3. Low-shear viscometry, electron microscopy, and low-speed sedimentation assays showed that domains 2 + 3 induced bundling of actin filaments. The influence of PIP2 micelles on the different activities of severin was assayed using native severin and N- and C-terminally truncated fragments. Severin contains at least two PIP2-binding sites since the activities of the two nonoverlapping severin fragments domain 1 and domains 2 + 3 were inhibited by PIP2. The specificity of severin-phospholipid interaction was investigated by studying the regulation of native severin by PIP2 and other pure or mixed phospholipids.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ca(2+) signalling is not required for chemotaxis in Dictyostelium

Dictyostelium cells can move rapidly towards a source of cyclic-AMP (cAMP). This chemoattractant is detected by G-protein-linked receptors, which trigger a signalling cascade including a rapid influx of Ca(2+). We have disrupted an inositol 1,4,5-trisphosphate (InsP(3)) receptor-like gene, iplA, to produce null cells in which Ca(2+) entry in response to chemoattractants is abolished, as is the normal increase in free cytosolic Ca(2+) ([Ca(2+)](c)) that follows chemotactic stimulation. However, the resting [Ca(2+)](c) is similar to wild type. This mutant provides a test for the role of Ca(2+) influx in both chemotaxis and the signalling cascade that controls it. The production of cyclic-GMP and cAMP, and the activation of the MAP kinase, DdERK2, triggered from the cAMP receptor, are little perturbed in the mutant; mobilization of actin into the cytoskeleton also follows similar kinetics to wild type. Mutant cells chemotax efficiently towards cAMP or folic acid and their sensitivity to cAMP is similar to wild type. Finally, they move at similar speeds to wild-type cells, with or without chemoattractant. We conclude that Ca(2+) signalling is not necessary for chemotaxis to cAMP.     

IP(3)-dependent intracellular Ca(2+) release is required for cAMP-induced c-fos expression in hippocampal neurons

Endothelial argininosuccinate synthetase 1 (ASS1) regulates the provision of l-arginine to nitric oxide synthase 3 (NOS3). Previous studies demonstrated that endothelial ASS1 expression was induced by laminar shear stress (LSS) and that this enzyme plays a role in maintaining anti-inflammatory microenvironments through enhancing NO production. However, differently from the case of NOS3, the regulatory mechanism for the endothelial ASS1 expression in response to LSS is not well understood. This study addressed a specific issue whether endothelial ASS1 expression is regulated by Kruppel-like factors (KLFs) that are presumed to coordinate endothelial gene expressions in response to LSS. The cDNA microarray data indicated that LSS stimulated the expression of numerous KLFs in human umbilical vein endothelial cells. KLF4 showed the highest fold increase and LSS-dependent increases of KLF4 and most other KLFs were similar in young versus senescent endothelial cells. LSS-induced KLF4 expression was verified by RT-PCR and Western blotting. LSS-induced ASS1 expression and NO production were suppressed by a small interfering RNA for KLF4. The ectopic expression of KLF4 led to the increase of ASS1 expression and NO production. The present study demonstrated a key regulatory role of KLF4 in the endothelial ASS1 expression and NO production in response to LSS.     

Protein synthesis is required for the transition to Ca(2+)-dependent regulated secretion in progesterone-matured Xenopus oocytes

Calcium (Ca) ionophores trigger cortical granule exocytosis in progesterone-matured Xenopus oocytes (eggs), but not in immature oocytes. Prior work suggested that this secretory transition involved a Ca-dependent isoform of protein kinase C (PKC). To address this possibility, we treated eggs with several different inhibitors of Ca-dependent PKCs. Although these agents (eg., staurosporine, Ro31-8220) completely blocked cortical granule exocytosis that is triggered in eggs by phorbol esters, they had no impact on ionomycin-evoked secretion of cortical granule lectin. These data suggest that Ca-dependent PKCs do not mediate secretory triggering in eggs. Instead, further investigation revealed that protein synthesis (but not RNA synthesis) was required for eggs to secrete in response to ionomycin. Moreover, we observed that when oocytes were matured by injection of maturation promoting factor (MPF), they failed to secrete in response to ionomycin. Collectively, these results suggest that the progesterone-dependent maturation pathway induces these cells either to synthesize de novo, a protein that mediates Ca-dependent secretory triggering, or that intrinsic Ca-sensing machinery is modified in a protein-synthesis-dependent fashion. Initial efforts to distinguish between these possibilities (using Ca overlay, pharmacological and immunoblot strategies) revealed that such Ca-binding proteins as calmodulin, synaptotagmin1, CAPS, rabphilin-3A and calcineurin were unlikely to transduce the secretory effects of ionomycin in eggs. Thus, the cortical reaction in these cells may rely on a novel mechanism for initiating Ca-dependent exocytosis.     

A Xestospongin C-sensitive Ca(2+) store is required for cAMP-induced Ca(2+) influx and cAMP oscillations in Dictyostelium

Xestospongin C (XeC) is known to bind to the inositol 1,4, 5-trisphosphate (IP(3))-sensitive store in mammalian cells and to inhibit IP(3)- and thapsigargin-induced Ca(2+) release. In this study we show that this is also true for Dictyostelium. In addition, XeC inhibited Ca(2+) uptake into purified vesicle fractions and induced Ca(2+) release. This suggests that, in the case of Dictyostelium, XeC opens rather than plugs the IP(3) receptor channel as was proposed for mammalian cells (Gafni, J., Munsch, J. A. , Lam, T. H., Catlin, M. C., Costa, L. G., Molinski, T. F., and Pessah, I. N. (1997) Neuron 19, 723-733). In order to elucidate the function of the XeC-sensitive Ca(2+) store in Dictyostelium during differentiation, we applied XeC to the cells and found that it caused a time-dependent increase of basal [Ca(2+)](i) and inhibited cAMP-induced Ca(2+) influx in single cells as well as in cell suspensions. Moreover, XeC blocked light scattering spikes and pulsatile cAMP signaling.     

Minimal model for Ca(2+)-dependent oscillations in excitable cells.

A minimal model for calcium controlled oscillations is presented. The model considers only an exchange of potassium and calcium ions over the plasma membrane. Calcium ions leak into the cell through a potential dependent channel and is extruded by a pump. Potassium leaks out through a calcium dependent, but voltage independent, channel. The cytosolic calcium concentration is buffered, so a fixed fraction is free. Inactivation, membrane capacity, and time delays for the conductance changes are not included, so the time dependence is solely introduced through the temporal changes of the intracellular Ca(2+)-concentration. With continuous parameter changes the model can switch between five states: (1) a non-excitable, stable state; (2) single-spike excitability; (3) slow, spontaneous oscillations; (4) reverse-spike excitability; and (5) another non-excitable, stable state. One of the key parameters for this switching behavior is the rate constant for the calcium pump.     

A C-terminal proline is required for bioluminescence of the Ca(2+)-binding photoprotein, aequorin.

The requirement for a proline residue at the C-terminus of the Ca(2+)-binding photoprotein, aequorin, was investigated by measuring luminescence activities of a series of C-terminal deletion mutants, substitution mutants and an addition mutant. CD spectral measurements of apoaequorin with the C-terminal proline deleted showed a small change in secondary structure. In all cases studied, the C-terminal proline was required for bioluminescence activity.     

Ca(2+)-ATPase function is required for intracellular trafficking of the Notch receptor in Drosophila.

Maintaining high Ca(2+) concentrations in the lumen of the endoplasmic reticulum is important for protein synthesis and transport. We identified a lethal complementation group recovered in a screen for mutations that reduce Notch activity as loss-of-function alleles of the Drosophila Ca(2+)-ATPase gene Ca-P60A. Analysis of Ca-P60A mutants indicates that Ca(2+)-ATPase is essential for cell viability and tissue morphogenesis during development. Cultured cells treated with Ca(2+)-ATPase inhibitors exhibit impaired Notch cleavage and receptor trafficking to the cell surface, explaining the genetic interaction between Ca(2+)-ATPase and Notch. Notch and several other transmembrane proteins are mislocalized in tissue clones homozygous for Ca-P60A mutations, demonstrating a general effect on membrane protein trafficking caused by a deficiency in Ca(2+)-ATPase.     

A sustained increase in intracellular Ca(2+) is required for the acrosome reaction in sea urchin sperm.

The acrosome reaction (AR), necessary for fertilization in many species, requires an increase in intracellular Ca(2+) ([Ca(2+)](i)). In sea urchin sperm, the AR is triggered by an egg-jelly factor: the associated [Ca(2+)](i) elevation lasts minutes and involves two Ca(2+) permeable channels. Both the opening of the second channel and the onset of the AR occur approximately 5 s after treatment with egg factor, suggesting that these events are linked. In agreement, removal of Ca(2+) from sea water or addition of Ca(2+) channel blockers at the time when opening of the second channel is first detected inhibits AR and causes a "rapid" (t(1/2) = 3--15 s) decrease in [Ca(2+)](i) and partial inhibition of the intracellular pH change associated with the AR. Simultaneous addition of NH(4)Cl and either EGTA, Co(2+), or Ni(2+) 5 s after egg factor prevents the partial inhibition of the evoked pH(i) change observed but does not reverse AR inhibition. Therefore, the sustained increase in [Ca(2+)](i) caused by the second Ca(2+) channel is needed for the sperm AR. Experiments with agents that induce capacitative Ca(2+) uptake (thapsigargin and cyclopiazonic acid) suggest that the second channel opened during the AR could be a store-operated Ca(2+) channel.     

Ca(2+)-dependent in vivo protein phosphorylation and encystment induction in the ciliated protozoan Colpoda cucullus

Encystment induction of Colpoda cucullus is promoted by an increase in external Ca²⁺ and overpopulation of Colpoda vegetative cells. Using phos-tag detection assays, the present study revealed that the in vivo phosphorylation level in several proteins [33 kDa, 37 kDa, 37.5 kDa, 43 kDa, 47 kDa, 49 kDa, etc.] was raised when the vegetative cells were stimulated by overpopulation to encyst in a medium containing 0.1 mM Ca²⁺ or without the addition of Ca²⁺. Both overpopulation-mediated encystment induction and protein phosphorylation were suppressed by the addition of EGTA. Ca²⁺/overpopulation-stimulated encystment induction and protein phosphorylation were also suppressed by the addition of BAPTA-AM. These results suggest that the Ca²⁺ inflow promoted by cell-to-cell stimulation due to overpopulation may activate signaling pathways involving protein phosphorylation and encystment induction. In the presence of cAMP-AM, the phosphorylation levels of 33 kDa, 37 kDa, 37.5 kDa, 43 kDa, 47 kDa and 49 kDa proteins were enhanced, and encystment induction was promoted. Enzyme immunoassays (EIAs) showed that intracellular cAMP concentration was raised prior to encystment when the cells were stimulated by overpopulation. These results suggest that cAMP/PKA-dependent protein phosphorylation, which is an event on Ca²⁺-triggered signaling pathways, may be involved in encystment induction.     

RYR2 proteins contribute to the formation of Ca(2+) sparks in smooth muscle

Calcium release through ryanodine receptors (RYR) activates calcium-dependent membrane conductances and plays an important role in excitation-contraction coupling in smooth muscle. The specific RYR isoforms associated with this release in smooth muscle, and the role of RYR-associated proteins such as FK506 binding proteins (FKBPs), has not been clearly established, however. FKBP12.6 proteins interact with RYR2 Ca(2+) release channels and the absence of these proteins predictably alters the amplitude and kinetics of RYR2 unitary Ca(2+) release events (Ca(2+) sparks). To evaluate the role of specific RYR2 and FBKP12.6 proteins in Ca(2+) release processes in smooth muscle, we compared spontaneous transient outward currents (STOCs), Ca(2+) sparks, Ca(2+)-induced Ca(2+) release, and Ca(2+) waves in smooth muscle cells freshly isolated from wild-type, FKBP12.6(-/-), and RYR3(-/-) mouse bladders. Consistent with a role of FKBP12.6 and RYR2 proteins in spontaneous Ca(2+) sparks, we show that the frequency, amplitude, and kinetics of spontaneous, transient outward currents (STOCs) and spontaneous Ca(2+) sparks are altered in FKBP12.6 deficient myocytes relative to wild-type and RYR3 null cells, which were not significantly different from each other. Ca(2+) -induced Ca(2+) release was similarly augmented in FKBP12.6(-/-), but not in RYR3 null cells relative to wild-type. Finally, Ca(2+) wave speed evoked by CICR was not different in RYR3 cells relative to control, indicating that these proteins are not necessary for normal Ca(2+) wave propagation. The effect of FKBP12.6 deletion on the frequency, amplitude, and kinetics of spontaneous and evoked Ca(2+) sparks in smooth muscle, and the finding of normal Ca(2+) sparks and CICR in RYR3 null mice, indicate that Ca(2+) release through RYR2 molecules contributes to the formation of spontaneous and evoked Ca(2+) sparks, and associated STOCs, in smooth muscle.     

14-3-3-Regulated Ca2+-dependent protein kinase CPK3 is required for sphingolipid-induced cell death in Arabidopsis

In eukaryotic cells, sphingoid long chain bases (LCBs) such as sphingosine or phytosphingosine (PHS) behave as second messengers involved in various processes including programmed cell death (PCD). In plants, induction of PCD by LCBs has now been described, but the signalling pathway is still enigmatic. Using Arabidopsis, we identify new key steps in this pathway. We demonstrate that PHS induces activation of the calcium-dependent kinase CPK3, which phosphorylates its binding partners, the 14-3-3 proteins. This phosphorylation leads to the disruption of the complex and to CPK3 degradation. Using cpk3 knockout lines, we demonstrate that CPK3 is a positive regulator of LCB-mediated PCD. These findings establish 14-3-3-regulated CPK3 as a key component of the LCB pathway leading to PCD in plants.     

Ca(2+)-dependent and Ca(2+)-independent calmodulin binding sites in erythrocyte protein 4.1. Implications for regulation of protein 4.1 interactions with transmembrane proteins

In vitro protein binding assays identified two distinct calmodulin (CaM) binding sites within the NH(2)-terminal 30-kDa domain of erythrocyte protein 4.1 (4.1R): a Ca(2+)-independent binding site (A(264)KKLWKVCVEHHTFFRL) and a Ca(2+)-dependent binding site (A(181)KKLSMYGVDLHKAKDL). Synthetic peptides corresponding to these sequences bound CaM in vitro; conversely, deletion of these peptides from a 30-kDa construct reduced binding to CaM. Thus, 4.1R is a unique CaM-binding protein in that it has distinct Ca(2+)-dependent and Ca(2+)-independent high affinity CaM binding sites. CaM bound to 4.1R at a stoichiometry of 1:1 both in the presence and absence of Ca(2+), implying that one CaM molecule binds to two distinct sites in the same molecule of 4.1R. Interactions of 4.1R with membrane proteins such as band 3 is regulated by Ca(2+) and CaM. While the intrinsic affinity of the 30-kDa domain for the cytoplasmic tail of erythrocyte membrane band 3 was not altered by elimination of one or both CaM binding sites, the ability of Ca(2+)/CaM to down-regulate 4. 1R-band 3 interaction was abrogated by such deletions. Thus, regulation of protein 4.1 binding to membrane proteins by Ca(2+) and CaM requires binding of CaM to both Ca(2+)-independent and Ca(2+)-dependent sites in protein 4.1.     

Inhibitory Ca(2+)-regulation of myosin light chain kinase in the lower eukaryote, Physarum polycephalum: role of a Ca(2+)-dependent inhibitory factor.

ATP-dependent interactions between myosin and actin in the lower eukaryote, Physarum polycephalum, are inhibited by micromolar levels of Ca2+. This inhibition is mediated by the binding of Ca2+ to myosin, the phosphorylation of which is required if Ca2+ is to inhibit the activities of myosin (Kohama, K., Trends Pharmacol. Sci. 11, 433-435 (1990)). As the first step to examine whether Ca2+ also regulates phosphorylation in the actomyosin system, we purified myosin light chain kinase (MLCK) of 55 kDa almost to homogeneity. The MLCK activity was high whether or not Ca2+ was present. However, a Ca(2+)-dependent inhibitory factor (CIF) purified from Physarum (Okagaki et al., Biochem. Biophys. Res. Commun. 176, 564-570 (1991)) was shown to reduce the MLCK activity in a Ca(2+)-dependent manner. Using crude preparations, not only MLCK but also myosin heavy chain kinase and actin kinase were shown to be inhibited by Ca2+ half-maximally at micromolar levels. Since CIF is the only Ca(2+)-binding protein in the preparations, we propose that this inhibitory Ca(2+)-regulation of the kinases for actomyosin is mediated by CIF.     

The yeast Ca(2+)-ATPase homologue, PMR1, is required for normal Golgi function and localizes in a novel Golgi-like distribution.

PMR1, a Ca(2+)-adenosine triphosphatase (ATPase) homologue in the yeast Saccharomyces cerevisiae localizes to a novel Golgi-like organelle. Consistent with a Golgi localization, the bulk of PMR1 comigrates with Golgi markers in subcellular fractionation experiments, and staining of PMR1 by indirect immunofluorescence reveals a punctate pattern resembling Golgi staining in yeast. However, PMR1 shows only partial colocalization with known Golgi markers, KEX2 and SEC7, in double-label immunofluorescence experiments. The effect of PMR1 on Golgi function is indicated by pleiotropic defects in various Golgi processes in pmr1 mutants, including impaired proteolytic processing of pro-alpha factor and incomplete outer chain glycosylation of invertase. Consistent with the proposed role of PMR1 as a Ca2+ pump, these defects are reversed by the addition of millimolar levels of extracellular Ca2+, suggesting that Ca2+ disposition is essential to normal Golgi function. Absence of PMR1 function partially suppresses the temperature-sensitive growth defects of several sec mutants, and overexpression of PMR1 restricts the growth of others. Some of these interactions are modulated by changes in external Ca2+ concentrations. These results imply a global role for Ca2+ in the proper function of components governing transit and processing through the secretory pathway.     

Mitogen-activated protein kinase stimulation of Ca(2+) signaling is required for survival of endoplasmic reticulum stress in yeast

Endoplasmic reticulum (ER) stress in the budding yeast Saccharomyces cerevisiae triggers Ca2+ influx through a plasma membrane channel composed of Cch1 and Mid1. This response activates calcineurin, which helps to prevent cell death during multiple forms of ER stress, including the response to azole-class antifungal drugs. Herein, we show that ER stress activates the cell integrity mitogen-activate protein kinase cascade in yeast and that the activation of Pkc1 and Mpk1 is necessary for stimulation of the Cch1-Mid1 Ca2+ channel independent of many known targets of Mpk1 (Rlm1, Swi4, Swi6, Mih1, Hsl1, and Swe1). ER stress generated in response to miconazole, tunicamycin, or other inhibitors also triggered a transient G2/M arrest that depended upon the Swe1 protein kinase. Calcineurin played little role in the Swe1-dependent cell cycle arrest and Swe1 had little effect on calcineurin-dependent avoidance of cell death. These findings help to clarify the interactions between Mpk1, calcineurin, and Swe1 and suggest that the calcium cell survival pathway promotes drug resistance independent of both the unfolded protein response and the G2/M cell cycle checkpoint.     

Two Ca2+-dependent ATPases in rat liver plasma membrane. The previously purified (Ca2+-Mg2+)-ATPase is not a Ca2+-pump but an ecto-ATPase

We have shown that the rat liver plasma membrane has at least two (Ca2+-Mg2+)-ATPases. One of them has the properties of a plasma membrane Ca2+-pump (Lin, S.-H. (1985) J. Biol. Chem. 260, 7850-7856); the other one, which we have purified (Lin, S.-H., and Fain, J.N. (1984) J. Biol. Chem. 259, 3016-3020) and characterized (Lin, S.-H. (1985) J. Biol. Chem. 260, 10976-10980) has no established function. In this study we present evidence that the purified (Ca2+-Mg2+)-ATPase is a plasma membrane ecto-ATPase. In hepatocytes in primary culture, we can detect Ca2+-ATPase and Mg2+-ATPase activities by addition of ATP to the intact cells. The external localization of the active site of the ATPase was confirmed by the observation that the Ca2+-ATPase and Mg2+-ATPase activities were the same for intact cells, saponin-treated cells, and cell homogenates. Less than 14% of total intracellular lactate dehydrogenase, a cytosolic enzyme, was released during a 30-min incubation of the hepatocytes with 2 mM ATP. This indicates that the hepatocytes maintained cytoplasmic membrane integrity during the 30-min incubation with ATP, and the Ca2+-ATPase and Mg2+-ATPase activity measured in the intact cell preparation was due to cell surface ATPase activity. The possibility that the ecto-Ca2+-ATPase and Mg2+-ATPase may be the same protein as the previously purified (Ca2+-Mg2+)-ATPase was tested by comparing the properties of the ecto-ATPase with those of (Ca2+-Mg2+)-ATPase. Both the ecto-ATPase and the (Ca2+-Mg2+)-ATPase have broad nucleotide-hydrolyzing activity, i.e. they both hydrolyze ATP, GTP, UTP, CTP, ADP, and GDP to a similar extent. The effect of Ca2+ and Mg2+ on the ecto-ATPase activity is not additive indicating that both Ca2+- and Mg2+-ATPase activities are part of the same enzyme. The ecto-ATPase activity, like the (Ca2+-Mg2+)-ATPase, is not sensitive to oligomycin, vanadate, N-ethylmaleimide and p-chloromercuribenzoate; and both the ecto-ATPase and purified (Ca2+-Mg2+)-ATPase activities are insensitive to protease treatments. These properties indicate that the previously purified (Ca2+-Mg2+)-ATPase is an ecto-ATPase and may function in regulating the effect of ATP and ADP on hepatocyte Ca2+ mobilization (Charest, R., Blackmore, P.F., and Exton, J.H. (1985) J. Biol. Chem. 260, 15789-15794).     

Dopamine release in PC12 cells is mediated by Ca(2+)-dependent production of ceramide via sphingomyelin pathway

A presynaptic membrane disturbance is an essential process for the release of various neurotransmitters. Ceramide, which is a tumor suppressive lipid, has been shown to act as a channel-forming molecule and serve as a precursor of ceramide-1-phosphate, which can disturb the cellular membrane. This study found that while permeable ceramide increases the rate of dopamine release in the presence of a Ca(2+)-ionophore, A23187, permeable ceramide-1-phosphate provoked its release even without the ionophore. The treatment of PC12 cells with the ionophore at concentrations < 2 microM produced ceramide via the sphingomyelin (SM) pathway with a concomitant release of dopamine, and no cell damage was observed. The addition of a Ca(2+) chelator, EGTA, to the medium inhibited the increase in the release of both the ceramide and dopamine. This suggests that ceramide might be produced by Ca(2+) and is implicated in the membrane disturbance associated with the release of dopamine as a result of its conversion to ceramide-1-phosphate. Consistent with these results, this study detected a membrane-associated and neutral pH optimum sphingomyelinase (SMase) whose activity was increased by Ca(2+). Together, these results demonstrate that ceramide can be produced via the activation of a neutral form of SMase through Ca(2+), and is involved in the dopamine release in concert with Ca(2+).