Annexin5 Plays a Vital Role in Arabidopsis Pollen Development via Ca2+-Dependent Membrane Trafficking

The regulation of pollen development and pollen tube growth is a complicated biological process that is crucial for sexual reproduction in flowering plants. Annexins are widely distributed from protists to higher eukaryotes and play multiple roles in numerous cellular events by acting as a putative “linker” between Ca²⁺ signaling, the actin cytoskeleton and the membrane, which are required for pollen development and pollen tube growth. Our recent report suggested that downregulation of the function of Arabidopsis annexin 5 (Ann5) in transgenic Ann5-RNAi lines caused severely sterile pollen grains. However, little is known about the underlying mechanisms of the function of Ann5 in pollen. This study demonstrated that Ann5 associates with phospholipid membrane and this association is stimulated by Ca²⁺ in vitro. Brefeldin A (BFA) interferes with endomembrane trafficking and inhibits pollen germination and pollen tube growth. Both pollen germination and pollen tube growth of Ann5-overexpressing plants showed increased resistance to BFA treatment, and this effect was regulated by calcium. Overexpression of Ann5 promoted Ca²⁺-dependent cytoplasmic streaming in pollen tubes in vivo in response to BFA. Lactrunculin (LatB) significantly prohibited pollen germination and tube growth by binding with high affinity to monomeric actin and preferentially targeting dynamic actin filament arrays and preventing actin polymerization. Overexpression of Ann5 did not affect pollen germination or pollen tube growth in response to LatB compared with wild-type, although Ann5 interacts with actin filaments in a manner similar to some animal annexins. In addition, the sterile pollen phenotype could be only partially rescued by Ann5 mutants at Ca²⁺-binding sites when compared to the complete recovery by wild-type Ann5. These data demonstrated that Ann5 is involved in pollen development, germination and pollen tube growth through the promotion of endomembrane trafficking modulated by calcium. Our results provide reliable molecular mechanisms that underlie the function of Ann5 in pollen.     

Characterization of a Maize Ca2+-Dependent Protein Kinase Phosphorylating Phosphoenolpyruvate Carboxylase

Phosphoenolpyruvate carboxylase (PEPC) [EC 4.1.1.31 [EC] ] of plantsundergoes regulatory phosphorylation in response to light ornutritional conditions. However, the nature of protein kinase(s)for this phosphorylation has not yet been fully elucidated.We separated a Ca²⁺-requiring protein kinase from Ca²⁺-independentone, both of which can phosphorylate maize leaf PEPC and characterizedthe former kinase after partial purification. Several linesof evidence indicated that the kinase is one of the characteristicCa²⁺-dependent but calmodulin-independent protein kinase (CDPK).Although the M_r, of native CDPK was estimated to be about 100kDa by gel permeation chromatography, in situ phosphorylationassay of CDPK in a SDS-polyacrylamide gel revealed that thesubunit has an M_r of about 50 kDa suggesting dimer formationor association with other protein(s). Several kinetic parameterswere also obtained using PEPC as a substrate. Although the CDPKshowed an ability of regulatory phosphorylation (Ser-15 in maizePEPC), no significant desensitization to feedback inhibitor,malate, could be observed presumably due to low extent of phosphorylation.The kinase was not specific to PEPC but phosphorylated a varietyof synthetic peptides. The possible physiological role of thiskinase was discussed. ¹Present address: NEOS Central Research Laboratory, 1-1 Ohike-machi,Kosei-cho, Shiga, 520-3213 Japan. ²Present address: Chugai Pharmaceutical Co., Ltd., 1-135 Komakado,Gotemba, 412-0038 Japan. ⁴N.O. and N.Y. contributed equally to this work.     

The EF-Hand Ca2+ Binding Protein MICU Choreographs Mitochondrial Ca2+ Dynamics in Arabidopsis

Plant organelle function must constantly adjust to environmental conditions, which requires dynamic coordination. Ca²⁺ signaling may play a central role in this process. Free Ca²⁺ dynamics are tightly regulated and differ markedly between the cytosol, plastid stroma, and mitochondrial matrix. The mechanistic basis of compartment-specific Ca²⁺ dynamics is poorly understood. Here, we studied the function of At-MICU, an EF-hand protein of Arabidopsis thaliana with homology to constituents of the mitochondrial Ca²⁺ uniporter machinery in mammals. MICU binds Ca²⁺ and localizes to the mitochondria in Arabidopsis. In vivo imaging of roots expressing a genetically encoded Ca²⁺ sensor in the mitochondrial matrix revealed that lack of MICU increased resting concentrations of free Ca²⁺ in the matrix. Furthermore, Ca²⁺ elevations triggered by auxin and extracellular ATP occurred more rapidly and reached higher maximal concentrations in the mitochondria of micu mutants, whereas cytosolic Ca²⁺ signatures remained unchanged. These findings support the idea that a conserved uniporter system, with composition and regulation distinct from the mammalian machinery, mediates mitochondrial Ca²⁺ uptake in plants under in vivo conditions. They further suggest that MICU acts as a throttle that controls Ca²⁺ uptake by moderating influx, thereby shaping Ca²⁺ signatures in the matrix and preserving mitochondrial homeostasis. Our results open the door to genetic dissection of mitochondrial Ca²⁺ signaling in plants.     

Ca2+-Dependent Protein Kinase11 and 24 Modulate the Activity of the Inward Rectifying K+ Channels in Arabidopsis Pollen Tubes

Potassium (K⁺) influx into pollen tubes via K⁺ transporters is essential for pollen tube growth; however, the mechanism by which K⁺ transporters are regulated in pollen tubes remains unknown. Here, we report that Arabidopsis thaliana Ca²⁺-dependent protein kinase11 (CPK11) and CPK24 are involved in Ca²⁺-dependent regulation of the inward K⁺ (K+_in) channels in pollen tubes. Using patch-clamp analysis, we demonstrated that K+_in currents of pollen tube protoplasts were inhibited by elevated [Ca²⁺]_cyt. However, disruption of CPK11 or CPK24 completely impaired the Ca²⁺-dependent inhibition of K+_in currents and enhanced pollen tube growth. Moreover, the cpk11 cpk24 double mutant exhibited similar phenotypes as the corresponding single mutants, suggesting that these two CDPKs function in the same signaling pathway. Bimolecular fluorescence complementation and coimmunoprecipitation experiments showed that CPK11 could interact with CPK24 in vivo. Furthermore, CPK11 phosphorylated the N terminus of CPK24 in vitro, suggesting that these two CDPKs work together as part of a kinase cascade. Electrophysiological assays demonstrated that the Shaker pollen K+_in channel is the main contributor to pollen tube K+_in currents and acts as the downstream target of the CPK11-CPK24 pathway. We conclude that CPK11 and CPK24 together mediate the Ca²⁺-dependent inhibition of K+_in channels and participate in the regulation of pollen tube growth in Arabidopsis.     

Calmodulin has the Potential to Function as a Ca2+-Dependent Adaptor Protein

Calmodulin (CaM) is a versatile Ca²⁺-binding protein that regulates the activity of numerous effector proteins in response to Ca²⁺ signals. Several CaM-dependent regulatory mechanisms have been identified, including autoinhibitory domain displacement, sequestration of a ligand-binding site, active site reorganization, and target protein dimerization. We recently showed that the N- and C-lobes of animal and plant CaM isoforms could independently and sequentially bind to target peptides derived from the CaM-binding domain of Nicotiana tabacum mitogen-activated protein kinase phosphatase (NtMKP1), to form a 2:1 peptide:CaM complex. This suggests that CaM might facilitate the dimerization of NtMKP1, although the dimerization mechanism is distinct from the previously described simultaneous binding of other target peptides to CaM. The independent and sequential binding of the NtMKP1 peptides to CaM also suggests an alternative plausible scenario in which the C-lobe of CaM remains tethered to NtMKP1, and the N-lobe is free to recruit a second target protein to the complex, such as an NtMKP1 target. Thus, we hypothesize that CaM may be capable of functioning as a Ca²⁺-dependent adaptor or recruiter protein.Key Words: calmodulin, calcium, EF-hand, adaptor protein, mitogen-activated protein kinase phosphataseCalcium (Ca²⁺) is a dynamic secondary messenger that regulates many signaling events in both plant and animal cells. Intracellular Ca²⁺ transients and oscillations (Ca²⁺ signals) are decoded by a large superfamily of calcium-binding proteins, the most important of which is calmodulin (CaM).^1–3 The prototypical CaM protein consists of four tandem helix-loop-helix “EF-hand” Ca²⁺-binding motifs that are divided into distinct N- and C-terminal globular lobes connected by a flexible linker. CaM proteins from all species including the single mammalian CaM and the many different plant CaM isoforms each undergo similar Ca²⁺-induced conformational changes involving a rearrangement of the position of its α-helices that opens distinct hydrophobic target protein-binding patches on the surface of each lobe; known as the “open” conformation (Fig. 1B). These hydrophobic patches can interact with numerous different target proteins including protein kinases, protein phosphatases, cytoskeletal proteins and other cell signaling enzymes, to regulate their activity. The closed or semi-open conformations adopted by the N- and C-lobes of Ca²⁺-free CaM (apo-CaM) (Fig. 1A) can also interact with another subset of proteins, to target CaM to certain cellular locations or facilitate Ca²⁺-independent regulatory events.^1–3Open in a separate windowFigure 1Structures of CaM and CaM-target complexes. (A) apo-CaM (PDB:1DMo), (B) Ca²⁺-CaM (PDB:1CLL). Complexes of CaM bound to (C) CaMBD of smooth muscle myosin light chain kinase (PDB:1CDL), (D) partial CaMBD of plasma membrane Ca²⁺-pump C20W (PDB:1CFF), (E) the adenylyl cyclase protein from Bacillus anthracis (PDB:1K93), (F) 2 glutamate decarboxylase CaMBD''s (PDB:1NWD), (G) 2 CaM proteins bound to 2 small conductance Ca²⁺-activated potassium channel (SK channel) CaMBD''s (PDB:1G4Y), (H) 2 apo-CaM proteins bound to 2 tandem IQ motifs from murine myosin V (PDB:2IX7). In each panel CaM is shown in ivory, the target molecule is shown in blue and the Ca²⁺ ions bound to the N- and/or C-lobes of CaM are represented by red spheres.The CaM-dependent regulation of target proteins can occur through numerous different mechanisms. For example, Ca²⁺-CaM can relieve autoinhibition by binding to a short (20–25 residue) calmodulin-binding domain (CaMBD) sequence that is adjacent to or within an autoinhibitory region of the enzyme (Fig. 2A).³ Numerous structures of these Ca²⁺-CaM-CaMBD complexes have been reported, which reveal a characteristic “wrap-around” binding mode (Fig. 1C). Typically the CaM C-lobe binds with high affinity to a Trp residue within the N-terminal part of the target sequence, and the flexible central linker allows the N-lobe to pivot and bind to a second bulky hydrophobic “anchor” residue within the C-terminal part of the target sequence.³ Truncation of this second anchor residue can lead to binding of only one CaM domain and an extended CaM conformation (Fig. 1D).^4,5 Studies with plant CaM isoforms having mutations to non-CaMBD-coordinating residues have also suggested that a secondary binding interface exists on the opposite surface of the CaM protein which also contributes to the activation of some of these target enzymes.^6,7Open in a separate windowFigure 2Schematic model for the various mechanisms of CaM-dependent target regulation. (A) autoinhibitory domain displacement, (B) sequestering of a ligand binding site, (C) active-site reorganization, (D) CaM-induced target protein dimerization (1:2 complex), (E) CaM-induced target protein dimerization (2:2 complex), (F) hypothesized model for CaM acting as an adaptor/recruiter protein. In each panel CaM is shown as a red dumbbell shaped molecule with Ca²⁺ ions represented by yellow circles, and the target proteins are shown in various colors. See the text for details on each model.Another regulatory mechanism involving Ca²⁺-CaM-binding to a single contiguous CaMBD sequence may occur with the potato kinesin-like CaM-binding protein (KCBP)⁸ as well as some plant cyclic-nucleotide gated channels (CNGC''s).⁹ In both cases the Ca²⁺-CaM binding site on the target protein overlaps with the respective ligand binding site, and thus the binding of KCBP to microtubules or the binding of cyclic nucleotide monophosphates to CNGC''s may be prevented by interaction with Ca²⁺-CaM (Fig. 2B). In a variation on this mechanism, CaM can bind to the cytoplasmic juxtamembrane region of the human epidermal growth factor receptor and sequester a threonine residue which is a specific phosphorylation target of protein kinase C (PKC). CaM-binding inhibits PKC phosphorylation of this threonine, and PKC phosphorylation inhibits CaM-binding.¹⁰There are also several examples of CaM-target interactions where the N- and C-lobes bind to noncontiguous target protein regions, and play distinct roles in target regulation. The structures of a CaM-activated adenylyl cyclase from Bacillus anthracis with and without bound CaM shows how the N- and C-lobes of CaM can bind two distant regions of the adenylyl cyclase enzyme and induce a conformation reorganization that creates the enzyme''s active site (Figs. 1E and ​and2C2C).¹¹ An interesting feature of this interaction is that the CaM N-lobe remains Ca²⁺-free and in a closed conformation, while the C-lobe is in a canonical Ca²⁺-bound open conformation. Indeed, Ca²⁺-binding to the C-lobe but not N-lobe is required for activation of the adenylyl cyclase.¹²The N- and C-lobes of Ca²⁺-CaM can also each simultaneously bind to identical peptides derived from the petunia glutamate decarboxylase (GAD) enzyme to form a 1:2 Ca²⁺-CaM:GAD complex (Fig. 1F).^13,14 This suggests that Ca²⁺-CaM-induced target protein dimerization may be another way in which CaM can regulate target proteins (Fig. 2D). CaM-dependent dimerization has also been shown to regulate the activity of small conductance Ca²⁺-activated K⁺ channels (SK channel), although in this case a novel 2:2 CaM:SK channel complex is formed (Figs. 1G and ​and2E2E).¹⁵ This structure is also unique because Ca²⁺ is bound to the “lower affinity” N-lobe EF-hands, but not to the “higher affinity” C-lobe EF-hands of CaM.In addition to the SK channel, CaM can regulate voltage-gated sodium channels, voltage-gated calcium channels, as well as ryanodine-sensitive calcium release channels.¹⁶ With these channels CaM typically binds in complex Ca²⁺-dependent and Ca²⁺-independent ways to several noncontiguous target sequences in the same protein, and often to so-called IQ motifs (IQXXXRGXXXR). IQ motifs are generally thought to be constitutive apo-CaM binding sites which retain CaM under resting (low [Ca²⁺]) cellular conditions to ensure a rapid response to Ca²⁺-stimuli.¹⁷ However many IQ motifs can also bind specifically to Ca²⁺-CaM or to both apo-CaM and Ca²⁺-CaM. Structures of some Ca²⁺-CaM-IQ domain complexes have revealed wrap-around binding modes, albeit with differences in lobe and peptide orientation compared to other complexes.^18–20 For a discussion about the mechanisms of CaM-dependent ion channel regulation (see ref. ¹⁶). A very recent crystal structure of apo-CaM bound to an IQ domain from myosin V (Fig. 1H) has also revealed yet another variation on the wrap-around binding mode, where the apo-C-lobe of CaM adopts a semi-open conformation and forms numerous interactions with the target sequence, while the apo-N-lobe adopts a closed conformation and forms weaker interactions with the IQ domain.²¹Using several biophysical techniques we recently characterized the interaction between CaM isoforms (mammalian CaM, soybean CaM isoforms SCaM-1 and SCaM-4) and a novel CaMBD derived from the Nicotiana tabacum mitogen-activated protein kinase phosphatase (NtMKP1).²² The NtMKP1 protein was initially identified as a CaM-binding protein by Ohashi and coworkers,²³ and the same group recently showed that CaM-binding NtMKP1 homologs are also present in other plant species as well.²⁴ We found that each CaM isoform was capable of binding to the NtMKP1 CaMBD in the absence of Ca²⁺ using only the apo-C-lobe, with the primary binding site consisting of NtMKP1 residues N438 - S449, and additional C-terminal residues G450 - K460 enhancing the overall binding affinity (K_d ∼10⁻⁵ M). In the presence of Ca²⁺, a 1:1 complex could be formed with the CaM C-lobe having significantly increased affinity for the N438 - S449 region of NtMKP1 (K_d 10⁻⁷ − 10⁻¹⁰ M). However, the Ca²⁺-loaded CaM N-lobe interacted only very weakly with the C-terminal NtMKP1 sequence in this 1:1 complex, despite an abundance of seemingly suitable hydrophobic “anchor” residues in this region. Interestingly, the addition of more peptide triggered the independent binding of a second NtMKP1 peptide to the Ca²⁺-CaM N-lobe (Kd 10⁻⁵ − 10⁻⁶ M) to form a 1:2 Ca²⁺-CaM:NtMKP1 complex. As with GAD, these results suggest that CaM is capable of facilitating the dimerization of NtMKP1, although the independent and sequential NtMKP1 peptide binding to the C- and N-lobes markedly distinguishes the CaM-NtMKP1 interaction from the simultaneous high-affinity binding of 2 GAD CaMBD''s to CaM.While our NtMKP1 study was ongoing, Ohashi and coworkers reported that CaM is incapable of stimulating the phosphatase activity of the NtMKP1 enzyme, thereby implying that the CaM-NtMKP1 interaction is necessary for something other than direct enzyme regulation.²⁵ The independent and sequential binding of the NtMKP1 fragments to the Ca²⁺-saturated C- and then N-lobes of CaM observed in our study suggests a plausible situation in which the C-lobe of CaM is tightly bound to NtMKP1, leaving the N-lobe free to recruit a different target protein to the complex, for example, a NtMKP1 protein substrate. Therefore, CaM may be capable of acting as an adaptor or recruiter protein, which would add yet another mechanism of target regulation to CaM''s repertoire (Fig. 2F). In addition to NtMKP1 peptides, the isolated N-lobe of CaM is capable of binding to other CaMBD peptides^26,27 as well as intact target proteins,²⁸ increasing the likelihood that the N-lobe could serve as a recruiter domain. The pre-association of the apo-C-lobe of CaM with NtMKP1 under resting conditions would also ensure a rapid response response to Ca²⁺-stimuli, since CaM would only need to recruit one rather than both protein targets.Although the ability of CaM to act as an adaptor protein in vivo has not yet been demonstrated, there are examples of related EF-hand proteins acting as adaptor proteins, including centrin²⁹ and calcium- and integrin-binding protein 1.³⁰ With the abundance of poorly characterized CaM-binding proteins in plants, many of which have CaMBD''s with little sequence resemblance to the better characterized motifs in animals¹ it seems likely that sequences will be identified which bind preferentially to the CaM N-lobe. Considering the incredible assortment of known CaM interaction modes and regulatory mechanisms, many of which have only been identified within the last decade, it is likely only a matter of time before CaM is proven to function as an adaptor protein in vivo.     

Homer2 Protein Regulates Plasma Membrane Ca2+-ATPase-mediated Ca2+ Signaling in Mouse Parotid Gland Acinar Cells

Homer proteins are scaffold molecules with a domain structure consisting of an N-terminal Ena/VASP homology 1 protein-binding domain and a C-terminal leucine zipper/coiled-coil domain. The Ena/VASP homology 1 domain recognizes proline-rich motifs and binds multiple Ca²⁺-signaling proteins, including G protein-coupled receptors, inositol 1,4,5-triphosphate receptors, ryanodine receptors, and transient receptor potential channels. However, their role in Ca²⁺ signaling in nonexcitable cells is not well understood. In this study, we investigated the role of Homer2 on Ca²⁺ signaling in parotid gland acinar cells using Homer2-deficient (Homer2^−/−) mice. Homer2 is localized at the apical pole in acinar cells. Deletion of Homer2 did not affect inositol 1,4,5-triphosphate receptor localization or channel activity and did not affect the expression and activity of sarco/endoplasmic reticulum Ca²⁺-ATPase pumps. In contrast, Homer2 deletion markedly increased expression of plasma membrane Ca²⁺-ATPase (PMCA) pumps, in particular PMCA4, at the apical pole. Accordingly, Homer2 deficiency increased Ca²⁺ extrusion by acinar cells. These findings were supported by co-immunoprecipitation of Homer2 and PMCA in wild-type parotid cells and transfected human embryonic kidney 293 (HEK293) cells. We identified a Homer-binding PPXXF-like motif in the N terminus of PMCA that is required for interaction with Homer2. Mutation of the PPXXF-like motif did not affect the interaction of PMCA with Homer1 but inhibited its interaction with Homer2 and increased Ca²⁺ clearance by PMCA. These findings reveal an important regulation of PMCA by Homer2 that has a central role on PMCA-mediated Ca²⁺ signaling in parotid acinar cells.     

Partial Purification and Characterization of a Ca2+-Dependent Protein Kinase from Pea Nuclei

Almost all the Ca²⁺-dependent protein kinase activity in nuclei purified from etiolated pea (Pisum sativum, L.) plumules is present in a single enzyme that can be extracted from chromatin by 0.3 molar NaCl. This protein kinase can be further purified 80,000-fold by salt fractionation and high performance liquid chromatography, after which it has a high specific activity of about 100 picomoles per minute per microgram in the presence of Ca²⁺ and reaches half-maximal activation at about 3 ×10⁻⁷ molar free Ca²⁺, without calmodulin. It is a monomer with a molecular weight near 90,000. It can efficiently use histone III-S, ribosomal S6 protein, and casein as artificial substrates, but it phosphorylates phosvitin only weakly. Its Ca²⁺-dependent kinase activity is half-maximally inhibited by 0.1 millimolar chlorpromazine, by 35 nanomolar K-252a and by 7 nanomolar staurosporine. It is insensitive to sphingosine, an inhibitor of protein kinase C, and to basic polypeptides that block other Ca²⁺-dependent protein kinases. It is not stimulated by exogenous phospholipids or fatty acids. In intact isolated pea nuclei it preferentially phosphorylates several chromatin-associated proteins, with the most phosphorylated protein band being near the same molecular weight (43,000) as a nuclear protein substrate whose phosphorylation has been reported to be stimulated by phytochrome in a calcium-dependent fashion.     

Ca2+ Activated Protein Kinases in Hypocotyl of Soybean (Glycine max L.)

The soluble protein extract of soybean hypocotyl was autophosphorylated, the labeling products were analyzed by SDS-PAGE. A 18 kD protein band was intensely labeled when a relatively high concentration of calcium was present, meanwhile a weakly labeled 67 kD protein band was also observed. When the reaction time was prolonged to 15 or 30 min, the labeling intensity of them was weakened gradually and the labeled bands disappeared eventual ly from the autoradiograph. If the calcium chelater EGTA was added into the reaction sys tem, only 67 kD was phosphorylated with high intensity. When non-labeled ATP was added during the reaction process, 32p in the labeled proteins could be substituted gradually by Pi. This indicated that the reaction system was in a dynamic equilibrium of phosphorylation-de- phosphorylation. There were also data inferred that it was a calcium dependent process. Histon H1 could speed up the phosphorylation, suggesting that it was a suitable substrate for protein kinases in the extract. Findings support that 18 kD and 67 kD protein may be Ca2+ sensitive protein kinases that can be autophosphorylated. Their different responses to Ca2+ may make the calcium signal transduction controllable.     

Loss of Arabidopsis thaliana Dynamin-Related Protein 2B Reveals Separation of Innate Immune Signaling Pathways

Vesicular trafficking has emerged as an important means by which eukaryotes modulate responses to microbial pathogens, likely by contributing to the correct localization and levels of host components necessary for effective immunity. However, considering the complexity of membrane trafficking in plants, relatively few vesicular trafficking components with functions in plant immunity are known. Here we demonstrate that Arabidopsis thaliana Dynamin-Related Protein 2B (DRP2B), which has been previously implicated in constitutive clathrin-mediated endocytosis (CME), functions in responses to flg22 (the active peptide derivative of bacterial flagellin) and immunity against flagellated bacteria Pseudomonas syringae pv. tomato (Pto) DC3000. Consistent with a role of DRP2B in Pattern-Triggered Immunity (PTI), drp2b null mutant plants also showed increased susceptibility to Pto DC3000 hrcC ⁻, which lacks a functional Type 3 Secretion System, thus is unable to deliver effectors into host cells to suppress PTI. Importantly, analysis of drp2b mutant plants revealed three distinct branches of the flg22-signaling network that differed in their requirement for RESPIRATORY BURST OXIDASE HOMOLOGUE D (RBOHD), the NADPH oxidase responsible for flg22-induced apoplastic reactive oxygen species production. Furthermore, in drp2b, normal MAPK signaling and increased immune responses via the RbohD/Ca²⁺-branch were not sufficient for promoting robust PR1 mRNA expression nor immunity against Pto DC3000 and Pto DC3000 hrcC⁻. Based on live-cell imaging studies, flg22-elicited internalization of the plant flagellin-receptor, FLAGELLIN SENSING 2 (FLS2), was found to be partially dependent on DRP2B, but not the closely related protein DRP2A, thus providing genetic evidence for a component, implicated in CME, in ligand-induced endocytosis of FLS2. Reduced trafficking of FLS2 in response to flg22 may contribute in part to the non-canonical combination of immune signaling defects observed in drp2b. In conclusion, this study adds DRP2B to the relatively short list of known vesicular trafficking proteins with roles in flg22-signaling and PTI in plants.     

Signaling diversity of PKA achieved via a Ca2+-cAMP-PKA oscillatory circuit

Many protein kinases are key nodal signaling molecules that regulate a wide range of cellular functions. These functions may require complex spatiotemporal regulation of kinase activities. Here, we show that protein kinase A (PKA), Ca(2+) and cyclic AMP (cAMP) oscillate in sync in insulin-secreting MIN6 beta cells, forming a highly integrated oscillatory circuit. We found that PKA activity was essential for this oscillatory circuit and was capable of not only initiating the signaling oscillations but also modulating their frequency, thereby diversifying the spatiotemporal control of downstream signaling. Our findings suggest that exquisite temporal control of kinase activity, mediated via signaling circuits resulting from cross-regulation of signaling pathways, can encode diverse inputs into temporal parameters such as oscillation frequency, which in turn contribute to proper regulation of complex cellular functions in a context-dependent manner.     

Passive Ca2+ Transport and Ca2+-Dependent K+ Transport in Plasmodium falciparum-Infected Red Cells

Previous reports have indicated that Plasmodium falciparum-infected red cells (pRBC) have an increased Ca²⁺ permeability. The magnitude of the increase is greater than that normally required to activate the Ca²⁺-dependent K⁺ channel (K _Ca channel) of the red cell membrane. However, there is evidence that this channel remains inactive in pRBC. To clarify this discrepancy, we have reassessed both the functional status of the K _Ca channel and the Ca²⁺ permeability properties of pRBC. For pRBC suspended in media containing Ca²⁺, K _Ca channel activation was elicited by treatment with the Ca²⁺ ionophore A23187. In the absence of ionophore the channel remained inactive. In contrast to previous claims, the unidirectional influx of Ca²⁺ into pRBC in which the Ca²⁺ pump was inhibited by vanadate was found to be within the normal range (30–55 μmol (10¹³ cells · hr)⁻¹), provided the cells were suspended in glucose-containing media. However, for pRBC in glucose-free media the Ca²⁺ influx increased to over 1 mmol (10¹³ cells · hr)⁻¹, almost an order of magnitude higher than that seen in uninfected erythrocytes under equivalent conditions. The pathway responsible for the enhanced influx of Ca²⁺ into glucose-deprived pRBC was expressed at approximately 30 hr post-invasion, and was inhibited by Ni²⁺. Possible roles for this pathway in pRBC are considered. Received: 12 May 1999/Revised: 8 July 1999     

Ca2+-Dependent activities of (Na+ + K+)-ATPase

Experiments on the effects of varying concentrations of Ca²⁺ on the Mg²⁺ + Na⁺-dependent ATPase activity of a highly purified preparation of dog kidney (Na⁺ + K⁺)-ATPase showed that Ca²⁺ was a partial inhibitor of this activity. When Ca²⁺ was added to the reaction mixture instead of Mg²⁺, there was a ouabain-sensitive Ca²⁺ + Na⁺-dependent ATPase activity the maximal velocity of which was 30 to 50% of that of Mg²⁺ + Na⁺-dependent activity. The apparent affinities of the enzyme for Ca²⁺ and CaATP seemed to be higher than those for Mg²⁺ and MgATP. Addition of K⁺, along with Ca²⁺ and Na⁺, increased the maximal velocity and the concentration of ATP required to obtain half-maximal velocity. The maximal velocity of the ouabain-sensitive Ca²⁺ + Na⁺ + K⁺-dependent ATPase was about two orders of magnitude smaller than that of Mg²⁺ + Na⁺ + K⁺-dependent activity. In agreement with previous observations, it was shown that in the presence of Ca²⁺, Na⁺, and ATP, an acid-stable phosphoenzyme was formed that was sensitive to either ADP or K⁺. The enzyme also exhibited a Ca²⁺ + Na⁺-dependent ADP-ATP exchange activity. Neither the inhibitory effects of Ca²⁺ on Mg²⁺-dependent activities, nor the Ca²⁺-dependent activities were influenced by the addition of calmodulin. Because of the presence of small quantities of endogenous Mg²⁺ in all reaction mixtures, it could not be determined whether the apparent Ca²⁺-dependent activities involved enzyme-substrate complexes containing Ca²⁺ as the divalent cation or both Ca²⁺ and Mg²⁺.     

Blockade of MCU-Mediated Ca2+ Uptake Perturbs Lipid Metabolism via PP4-Dependent AMPK Dephosphorylation

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Ca2+在茉莉酸甲酯诱导拟南芥气孔关闭中的信号转导作用

以拟南芥叶片下表皮为材料 ,分别用表皮生物分析法和激光扫描共聚焦显微镜成像技术 ,研究茉莉酸甲酯 (JA Me)促进气孔关闭过程中胞质Ca2 浓度的变化及其与气孔关闭的关系。结果表明 ,10 - 7到 10 - 3mol L的JA Me处理能促进拟南芥叶片的气孔关闭 ,其中 ,10 - 5mol L是最适浓度。用 10 - 5mol L的JA Me处理5min ,胞质Ca2 浓度从静息态的 10 5nmol L增加到 332 0nmol L ;质膜Ca2 通道阻断剂LaCl3和Ca2 螯合剂EGTA均能明显地降低JA Me对气孔关闭的促进作用。由此推测 ,胞质Ca2 可能是JA Me促进气孔关闭的重要信号转导因子     

KCa3.1-Dependent Hyperpolarization Enhances Intracellular Ca2+ Signaling Induced by fMLF in Differentiated U937 Cells

Formylated peptides are chemotactic agents generated by pathogens. The most relevant peptide is fMLF (formyl-Met-Leu-Phe) which participates in several immune functions, such as chemotaxis, phagocytosis, cytokine release and generation of reactive oxygen species. In macrophages fMLF-dependent responses are dependent on both, an increase in intracellular calcium concentration and on a hyperpolarization of the membrane potential. However, the molecular entity underlying this hyperpolarization remains unknown and it is not clear whether changes in membrane potential are linked to the increase in intracellular Ca²⁺. In this study, differentiated U937 cells, as a macrophage-like cell model, was used to characterize the fMLF response using electrophysiological and Ca²⁺ imaging techniques. We demonstrate by means of pharmacological and molecular biology tools that fMLF induces a Ca²⁺-dependent hyperpolarization via activation of the K⁺ channel K_Ca3.1 and thus, enhancing fMLF-induced intracellular Ca²⁺ increase through an amplification of the driving force for Ca²⁺ entry. Consequently, enhanced Ca²⁺ influx would in turn lengthen the hyperpolarization, operating as a positive feedback mechanism for fMLF-induced Ca²⁺ signaling.     

Cadmium Impairs Autophagy Leading to Apoptosis by Ca2+-Dependent Activation of JNK Signaling Pathway in Neuronal Cells

Neurochemical Research - Autophagy, a process for self-degradation of intracellular components and dysfunctional organelles, is closely related with neurodegenerative diseases. It has been shown...     

Dicer-2-Dependent Activation of Culex Vago Occurs via the TRAF-Rel2 Signaling Pathway

Despite their importance as vectors of human and livestock diseases, relatively little is known about innate antiviral immune pathways in mosquitoes and other insects. Previous work has shown that Culex Vago (CxVago), which is induced and secreted from West Nile virus (WNV)-infected mosquito cells, acts as a functional homolog of interferon, by activating Jak-STAT pathway and limiting virus replication in neighbouring cells. Here we describe the Dicer-2-dependent pathway leading to WNV-induced CxVago activation. Using a luciferase reporter assay, we show that a NF-κB-like binding site in CxVago promoter region is conserved in mosquito species and is responsible for induction of CxVago expression following WNV infection. Using dsRNA-based gene knockdown, we show that the NF-κB ortholog, Rel2, plays significant role in the signaling pathway that activates CxVago in mosquito cells in vitro and in vivo. Using similar approaches, we also show that TRAF, but not TRAF-3, is involved in activation of Rel2 after viral infection. Overall the study shows that a conserved signaling pathway, which is similar to mammalian interferon activation pathway, is responsible for the induction and antiviral activity of CxVago.     

Characterization of Ca2+-Dependent Protein-Protein Interactions within the Ca2+ Release Units of Cardiac Sarcoplasmic Reticulum

In the heart, excitation-contraction (E-C) coupling is mediated by Ca²⁺ release from sarcoplasmic reticulum (SR) through the interactions of proteins forming the Ca²⁺ release unit (CRU). Among them, calsequestrin (CSQ) and histidine-rich Ca²⁺ binding protein (HRC) are known to bind the charged luminal region of triadin (TRN) and thus directly or indirectly regulate ryanodine receptor 2 (RyR2) activity. However, the mechanisms of CSQ and HRC mediated regulation of RyR2 activity through TRN have remained unclear. We first examined the minimal KEKE motif of TRN involved in the interactions with CSQ2, HRC and RyR2 using TRN deletion mutants and in vitro binding assays. The results showed that CSQ2, HRC and RyR2 share the same KEKE motif region on the distal part of TRN (aa 202–231). Second, in vitro binding assays were conducted to examine the Ca²⁺ dependence of protein-protein interactions (PPI). The results showed that TRN-HRC interaction had a bell-shaped Ca²⁺ dependence, which peaked at pCa4, whereas TRN-CSQ2 or TRN-RyR2 interaction did not show such Ca²⁺ dependence pattern. Third, competitive binding was conducted to examine whether CSQ2, HRC, or RyR2 affects the TRN-HRC or TRN-CSQ2 binding at pCa4. Among them, only CSQ2 or RyR2 competitively inhibited TRN-HRC binding, suggesting that HRC can confer functional refractoriness to CRU, which could be beneficial for reloading of Ca²⁺ into SR at intermediate Ca²⁺ concentrations.     

K+-Dependent Selectivity and External Ca2+ Block of Shab K+ Channels

Potassium channels allow the selective flux of K⁺ excluding the smaller, and more abundant in the extracellular solution, Na⁺ ions. Here we show that Shab is a typical K⁺ channel that excludes Na⁺ under bi-ionic, Na_o/K_i or Na_o/Rb_i, conditions. However, when internal K⁺ is replaced by Cs⁺ (Na_o/Cs_i), stable inward Na⁺ and outward Cs⁺ currents are observed. These currents show that Shab selectivity is not accounted for by protein structural elements alone, as implicit in the snug-fit model of selectivity. Additionally, here we report the block of Shab channels by external Ca²⁺ ions, and compare the effect that internal K⁺ replacement exerts on both Ca²⁺ and TEA block. Our observations indicate that Ca²⁺ blocks the channels at a site located near the external TEA binding site, and that this pore region changes conformation under conditions that allow Na⁺ permeation. In contrast, the latter ion conditions do not significantly affect the binding of quinidine to the pore central cavity. Based on our observations and the structural information derived from the NaK bacterial channel, we hypothesize that Ca²⁺ is probably coordinated by main chain carbonyls of the pore´s first K⁺-binding site.