RESUMEN
Calmodulin (CaM) is a Ca2+ sensor protein found in all eukaryotic cells that regulates a large number of target proteins in a Ca2+ concentration-dependent manner. As a transient-type hub protein, it recognizes linear motifs of its targets, though for the Ca2+-dependent binding, no consensus sequence was identified. Its complex with melittin, a major component of bee venom, is often used as a model system of protein-protein complexes. Yet, the structural aspects of the binding are not well understood, as only diverse, low-resolution data are available concerning the association. We present the crystal structure of melittin in complex with Ca2+-saturated CaMs from two, evolutionarily distant species, Homo sapiens and Plasmodium falciparum, representing three binding modes of the peptide. Results-augmented by molecular dynamics simulations-indicate that multiple binding modes can exist for CaM-melittin complexes, as an intrinsic characteristic of the binding. While the helical structure of melittin remains, swapping of its salt bridges and partial unfolding of its C-terminal segment can occur. In contrast to the classical way of target recognition by CaM, we found that different sets of residues can anchor at the hydrophobic pockets of CaM, which were considered as main recognition sites. Finally, the nanomolar binding affinity of the CaM-melittin complex is created by an ensemble of arrangements of similar stability-tight binding is achieved not by optimized specific interactions but by simultaneously satisfying less optimal interaction patterns in co-existing different conformers.
Asunto(s)
Calmodulina , Meliteno , Modelos Moleculares , Secuencia de Aminoácidos , Sitios de Unión , Calcio/metabolismo , Calmodulina/química , Calmodulina/metabolismo , Meliteno/química , Meliteno/metabolismo , Unión Proteica , Humanos , Plasmodium falciparum , Estructura Cuaternaria de Proteína , Simulación del Acoplamiento MolecularRESUMEN
Eukaryotic cells use calcium ions (Ca2+) as second messengers, particularly in response to abiotic and biotic stresses. These signals are detected by Ca2+ sensor proteins, such as calmodulin (CaM), which regulate the downstream target proteins. Plants also possess many CaM-like proteins (CMLs), most of which remain unstudied. We recently demonstrated that Arabidopsis CML13 and CML14 interact with proteins containing isoleucine/glutamine (IQ) domains, including CaM-binding transcriptional activators (CAMTAs). Here, we show that CaM, CML13 and CML14 bind all six members of the Arabidopsis CAMTA family. Using a combination of in planta and in vitro protein-interaction assays, we tested 11 members of the CaM/CML family and demonstrated that only CaM, CML13 and CML14 bind to CAMTA IQ domains. CaM, CML13 and CML14 showed Ca2+-independent binding to the IQ region of CAMTA6 and CAMTA3, and CAMTA6 in vitro exhibited some specificity toward individual IQ domains within CAMTA6 in split-luciferase in planta assays. We show that cml13 mutants exhibited enhanced salinity tolerance during germination compared to wild-type plants, a phenotype similar to camta6 mutants. In contrast, plants overexpressing CML13-GFP or CML14-GFP in the wild-type background showed increased NaCl sensitivity. Under mannitol stress, cml13 mutants were more susceptible than camta6 mutants or wild-type plants. The phenotype of cml13 mutants could be rescued with the wild-type CML13 gene. Several salinity-marker genes under CAMTA6 control were similarly misregulated in both camta6 and cml13 mutants, further supporting a role for CML13 in CAMTA6 function. Collectively, our data suggest that CML13 and CML14 participate in abiotic stress signaling as CAMTA effectors.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/metabolismo , Calmodulina/genética , Calmodulina/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Calcio/metabolismo , Salinidad , Factores de Transcripción/metabolismo , Estrés SalinoRESUMEN
Calcium/calmodulin (CaM)-dependent protein kinase kinase 2 (CaMKK2) regulates bone remodeling through its effects on osteoblasts and osteoclasts. However, its role in osteocytes, the most abundant bone cell type and the master regulator of bone remodeling, remains unknown. Here we report that the conditional deletion of CaMKK2 from osteocytes using Dentine matrix protein 1 (Dmp1)-8kb-Cre mice led to enhanced bone mass only in female mice owing to a suppression of osteoclasts. Conditioned media isolated from female CaMKK2-deficient osteocytes inhibited osteoclast formation and function in in vitro assays, indicating a role for osteocyte-secreted factors. Proteomics analysis revealed significantly higher levels of extracellular calpastatin, a specific inhibitor of calcium-dependent cysteine proteases calpains, in female CaMKK2 null osteocyte conditioned media, compared to media from female control osteocytes. Further, exogenously added non-cell permeable recombinant calpastatin domain I elicited a marked, dose-dependent inhibition of female wild-type osteoclasts and depletion of calpastatin from female CaMKK2-deficient osteocyte conditioned media reversed the inhibition of matrix resorption by osteoclasts. Our findings reveal a novel role for extracellular calpastatin in regulating female osteoclast function and unravel a novel CaMKK2-mediated paracrine mechanism of osteoclast regulation by female osteocytes.
Asunto(s)
Osteoclastos , Osteocitos , Animales , Femenino , Ratones , Calcio/metabolismo , Quinasa de la Proteína Quinasa Dependiente de Calcio-Calmodulina/metabolismo , Medios de Cultivo Condicionados/farmacología , Osteoclastos/metabolismo , Osteocitos/metabolismo , Caracteres SexualesRESUMEN
Ca2+/calmodulin-dependent inactivation (CDI) of CaV channels is a critical regulatory process that tunes the kinetics of Ca2+ entry for different cell types and physiologic responses. CDI is mediated by calmodulin (CaM), which is bound to the IQ domain of the CaV carboxy tail. This modulatory process is tailored by alternative splicing such that select splice variants of CaV1.3 and CaV1.4 contain a long distal carboxy tail (DCT). The DCT harbors an inhibitor of CDI (ICDI) module that competitively displaces CaM from the IQ domain, thereby diminishing CDI. While this overall mechanism is now well described, the detailed interactions required for ICDI binding to the IQ domain are yet to be elucidated. Here, we perform alanine-scanning mutagenesis of the IQ and ICDI domains and evaluate the contribution of neighboring regions to CDI inhibition. Through FRET binding analysis, we identify functionally relevant residues within the CaV1.3 IQ domain and the CaV1.4 ICDI and nearby A region, which are required for high-affinity IQ/ICDI binding. Importantly, patch-clamp recordings demonstrate that disruption of this interaction commensurately diminishes ICDI function resulting in the re-emergence of CDI in mutant channels. Furthermore, CaV1.2 channels harbor a homologous DCT; however, the ICDI region of this channel does not appear to appreciably modulate CaV1.2 CDI. Yet coexpression of CaV1.2 ICDI with select CaV1.3 splice variants significantly disrupts CDI, implicating a cross-channel modulatory scheme in cells expressing both channel subtypes. In all, these findings provide new insights into a molecular rheostat that fine-tunes Ca2+-entry and supports normal neuronal and cardiac function.
Asunto(s)
Calcio/metabolismo , Calmodulina/metabolismo , Caveolina 1/metabolismo , Activación del Canal Iónico , Mutación , Caveolina 1/antagonistas & inhibidores , Caveolina 1/genética , Células Cultivadas , Humanos , Cinética , Unión Proteica , Relación Estructura-ActividadRESUMEN
The family of K+-dependent Na+/Ca2+-exchangers, NCKX, are important mediators of cellular Ca2+ efflux, particularly in neurons associated with sensory transduction. The NCKX family comprises five proteins, NCKX1-5, each being the product of a different SLC24 gene. NCKX4 (SLC24A4) has been found to have a critical role in termination and adaptation of visual and olfactory signals, melanocortin-dependent satiety signaling, and the maturation of dental enamel. To explore mechanisms that might influence the temporal control of NCKX4 activity, a yeast two-hybrid system was used to search for protein interaction partners. We identified calmodulin as a partner for NCKX4 and confirmed the interaction using glutathione-S-transferase fusion pull-down. Calmodulin binding to NCKX4 was demonstrated in extracts from mouse brain and in transfected HEK293 cells. Calmodulin bound in a Ca2+-dependent manner to a motif present in the central cytosolic loop of NCKX4 and was abolished by the double-mutant I328D/F334D. When cotransfected in HEK293 cells, calmodulin bound to NCKX4 under basal conditions and induced a â¼2.5-fold increase in NCKX4 abundance, but did not influence either cellular location or basal activity. When purinergic stimulation of NCKX4 was examined in these cells, coexpression of wild-type calmodulin, but not a Ca2+ binding-deficient calmodulin mutant, suppressed NCKX4 activation in a time-dependent manner. We propose that Ca2+ binding to calmodulin prepositioned on NCKX4 induces a slow conformational rearrangement that interferes with purinergic stimulation of the exchanger, possibly by obscuring T331, a previously identified potential protein kinase C site.
Asunto(s)
Antiportadores/metabolismo , Calcio/metabolismo , Calmodulina/metabolismo , Intercambiador de Sodio-Calcio/metabolismo , Antiportadores/genética , Señalización del Calcio/fisiología , Membrana Celular/metabolismo , Células HEK293 , Humanos , Unión Proteica , Intercambiador de Sodio-Calcio/química , Técnicas del Sistema de Dos HíbridosRESUMEN
AMP-activated protein kinase (AMPK) is a fundamental component of a protein kinase cascade that is an energy sensor. AMPK maintains energy homeostasis in the cell by promoting catabolic and inhibiting anabolic pathways. Activation of AMPK requires phosphorylation by the liver kinase B1 or by the Ca2+/calmodulin-dependent protein kinase 2 (CaMKK2). The scaffold protein IQGAP1 regulates intracellular signaling pathways, such as the mitogen-activated protein kinase and AKT signaling cascades. Recent work implicates the participation of IQGAP1 in metabolic function, but the molecular mechanisms underlying these effects are poorly understood. Here, using several approaches including binding analysis with fusion proteins, siRNA-mediated gene silencing, RT-PCR, and knockout mice, we investigated whether IQGAP1 modulates AMPK signaling. In vitro analysis reveals that IQGAP1 binds directly to the α1 subunit of AMPK. In addition, we observed a direct interaction between IQGAP1 and CaMKK2, which is mediated by the IQ domain of IQGAP1. Both CaMKK2 and AMPK associate with IQGAP1 in cells. The ability of metformin and increased intracellular free Ca2+ concentrations to activate AMPK is reduced in cells lacking IQGAP1. Importantly, Ca2+-stimulated AMPK phosphorylation was rescued by re-expression of IQGAP1 in IQGAP1-null cell lines. Comparison of the fasting response in wild-type and IQGAP1-null mice revealed that transcriptional regulation of the gluconeogenesis genes PCK1 and G6PC and the fatty acid synthesis genes FASN and ACC1 is impaired in IQGAP1-null mice. Our data disclose a previously unidentified functional interaction between IQGAP1 and AMPK and suggest that IQGAP1 modulates AMPK signaling.
Asunto(s)
Adenilato Quinasa/metabolismo , Proteínas Activadoras de ras GTPasa/metabolismo , Animales , Calcio/metabolismo , Quinasa de la Proteína Quinasa Dependiente de Calcio-Calmodulina/metabolismo , Activación Enzimática , Técnicas de Silenciamiento del Gen , Células HeLa , Células Hep G2 , Humanos , Inmunoprecipitación , Ratones , Unión Proteica , Dominios Proteicos , Proteínas Activadoras de ras GTPasa/genéticaRESUMEN
Calmodulin (CaM) regulation of voltage-gated calcium (CaV1-2) channels is a powerful Ca2+-feedback mechanism to adjust channel activity in response to Ca2+ influx. Despite progress in resolving mechanisms of CaM-CaV feedback, the stoichiometry of CaM interaction with CaV channels remains ambiguous. Functional studies that tethered CaM to CaV1.2 suggested that a single CaM sufficed for Ca2+ feedback, yet biochemical, FRET, and structural studies showed that multiple CaM molecules interact with distinct interfaces within channel cytosolic segments, suggesting that functional Ca2+ regulation may be more nuanced. Resolving this ambiguity is critical as CaM is enriched in subcellular domains where CaV channels reside, such as the cardiac dyad. We here localized multiple CaMs to the CaV nanodomain by tethering either WT or mutant CaM that lack Ca2+-binding capacity to the pore-forming α-subunit of CaV1.2, CaV1.3, and CaV2.1 and/or the auxiliary ß2A subunit. We observed that a single CaM tethered to either the α or ß2A subunit tunes Ca2+ regulation of CaV channels. However, when multiple CaMs are localized concurrently, CaV channels preferentially respond to signaling from the α-subunit-tethered CaM. Mechanistically, the introduction of a second IQ domain to the CaV1.3 carboxyl tail switched the apparent functional stoichiometry, permitting two CaMs to mediate functional regulation. In all, Ca2+ feedback of CaV channels depends exquisitely on a single CaM preassociated with the α-subunit carboxyl tail. Additional CaMs that colocalize with the channel complex are unable to trigger Ca2+-dependent feedback of channel gating but may support alternate regulatory functions.
Asunto(s)
Canales de Calcio Tipo L/fisiología , Calcio/metabolismo , Calmodulina/metabolismo , Transducción de Señal/fisiología , Canales de Calcio Tipo L/química , Células HEK293 , Humanos , Activación del Canal Iónico/fisiología , Unión ProteicaRESUMEN
Myosin-IC (Myo1c) has been proposed to function in delivery of glucose transporter type 4 (GLUT4)-containing vesicles to the plasma membrane in response to insulin stimulation. Current evidence suggests that, upon insulin stimulation, Myo1c is phosphorylated at Ser701, leading to binding of the signaling protein 14-3-3ß. Biochemical and functional details of the Myo1c-14-3-3ß interaction have yet to be described. Using recombinantly expressed proteins and mass spectrometry-based analyses to monitor Myo1c phosphorylation, along with pulldown, fluorescence binding, and additional biochemical assays, we show here that 14-3-3ß is a dimer and, consistent with previous work, that it binds to Myo1c in the presence of calcium. This interaction was associated with dissociation of calmodulin (CaM) from the IQ motif in Myo1c. Surprisingly, we found that 14-3-3ß binds to Myo1c independent of Ser701 phosphorylation in vitro Additionally, in contrast to previous reports, we did not observe Myo1c Ser701 phosphorylation by Ca2+/CaM-dependent protein kinase II (CaMKII), although CaMKII phosphorylated four other Myo1c sites. The presence of 14-3-3ß had little effect on the actin-activated ATPase or motile activities of Myo1c. Given these results, it is unlikely that 14-3-3ß acts as a cargo adaptor for Myo1c-powered transport; rather, we propose that 14-3-3ß binds Myo1c in the presence of calcium and stabilizes the calmodulin-dissociated, nonmotile myosin.
Asunto(s)
Proteínas 14-3-3/metabolismo , Miosina Tipo I/metabolismo , Proteínas 14-3-3/química , Proteínas 14-3-3/genética , Secuencias de Aminoácidos , Calcio/química , Calcio/metabolismo , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Calmodulina/metabolismo , Dimerización , Ácido Egtácico/química , Humanos , Espectrometría de Masas , Miosina Tipo I/química , Miosina Tipo I/genética , Fosforilación , Unión Proteica , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , UltracentrifugaciónRESUMEN
Production of reactive oxygen species caused by dysregulated endothelial nitric-oxide synthase (eNOS) activity is linked to vascular dysfunction. eNOS is a major target protein of the primary calcium-sensing protein calmodulin. Calmodulin is often modified by the main biomarker of nitroxidative stress, 3-nitrotyrosine (nitroTyr). Despite nitroTyr being an abundant post-translational modification on calmodulin, the mechanistic role of this modification in altering calmodulin function and eNOS activation has not been investigated. Here, using genetic code expansion to site-specifically nitrate calmodulin at its two tyrosine residues, we assessed the effects of these alterations on calcium binding by calmodulin and on binding and activation of eNOS. We found that nitroTyr-calmodulin retains affinity for eNOS under resting physiological calcium concentrations. Results from in vitro eNOS assays with calmodulin nitrated at Tyr-99 revealed that this nitration reduces nitric-oxide production and increases eNOS decoupling compared with WT calmodulin. In contrast, calmodulin nitrated at Tyr-138 produced more nitric oxide and did so more efficiently than WT calmodulin. These results indicate that the nitroTyr post-translational modification, like tyrosine phosphorylation, can impact calmodulin sensitivity for calcium and reveal Tyr site-specific gain or loss of functions for calmodulin-induced eNOS activation.
Asunto(s)
Calcio/metabolismo , Calmodulina/metabolismo , Óxido Nítrico Sintasa de Tipo III/metabolismo , Tirosina/metabolismo , Animales , Bovinos , Extractos Celulares , Fluorescencia , Células HEK293 , Humanos , Interferometría , Nitrosación , Unión ProteicaRESUMEN
Specialized transporting and sensory epithelial cells employ homologous protocadherin-based adhesion complexes to remodel their apical membrane protrusions into organized functional arrays. Within the intestine, the nutrient-transporting enterocytes utilize the intermicrovillar adhesion complex (IMAC) to assemble their apical microvilli into an ordered brush border. The IMAC bears remarkable homology to the Usher complex, whose disruption results in the sensory disorder type 1 Usher syndrome (USH1). However, the entire complement of proteins that comprise both the IMAC and Usher complex are not yet fully elucidated. Using a protein isolation strategy to recover the IMAC, we have identified the small EF-hand protein calmodulin-like protein 4 (CALML4) as an IMAC component. Consistent with this finding, we show that CALML4 exhibits marked enrichment at the distal tips of enterocyte microvilli, the site of IMAC function, and is a direct binding partner of the IMAC component myosin-7b. Moreover, distal tip enrichment of CALML4 is strictly dependent upon its association with myosin-7b, with CALML4 acting as a light chain for this myosin. We further show that genetic disruption of CALML4 within enterocytes results in brush border assembly defects that mirror the loss of other IMAC components and that CALML4 can also associate with the Usher complex component myosin-7a. Our study further defines the molecular composition and protein-protein interaction network of the IMAC and Usher complex and may also shed light on the etiology of the sensory disorder USH1H.
Asunto(s)
Calmodulina/metabolismo , Membrana Celular/metabolismo , Enterocitos/metabolismo , Cadenas Ligeras de Miosina/metabolismo , Síndromes de Usher/metabolismo , Animales , Células COS , Células CACO-2 , Calmodulina/genética , Membrana Celular/genética , Membrana Celular/patología , Chlorocebus aethiops , Enterocitos/patología , Células HEK293 , Humanos , Ratones , Ratones Noqueados , Cadenas Pesadas de Miosina/metabolismo , Cadenas Ligeras de Miosina/genética , Miosina Tipo II/metabolismo , Síndromes de Usher/genética , Síndromes de Usher/patologíaRESUMEN
Mutations in the genes encoding the highly conserved Ca2+-sensing protein calmodulin (CaM) cause severe cardiac arrhythmias, including catecholaminergic polymorphic ventricular tachycardia or long QT syndrome and sudden cardiac death. Most of the identified arrhythmogenic mutations reside in the C-terminal domain of CaM and mostly affect Ca2+-coordinating residues. One exception is the catecholaminergic polymorphic ventricular tachycardia-causing N53I substitution, which resides in the N-terminal domain (N-domain). It does not affect Ca2+ coordination and has only a minor impact on binding affinity toward Ca2+ and on other biophysical properties. Nevertheless, the N53I substitution dramatically affects CaM's ability to reduce the open probability of the cardiac ryanodine receptor (RyR2) while having no effect on the regulation of the plasmalemmal voltage-gated Ca2+ channel, Cav1.2. To gain more insight into the molecular disease mechanism of this mutant, we used NMR to investigate the structures and dynamics of both apo- and Ca2+-bound CaM-N53I in solution. We also solved the crystal structures of WT and N53I CaM in complex with the primary calmodulin-binding domain (CaMBD2) from RyR2 at 1.84-2.13 Å resolutions. We found that all structures of the arrhythmogenic CaM-N53I variant are highly similar to those of WT CaM. However, we noted that the N53I substitution exposes an additional hydrophobic surface and that the intramolecular dynamics of the protein are significantly altered such that they destabilize the CaM N-domain. We conclude that the N53I-induced changes alter the interaction of the CaM N-domain with RyR2 and thereby likely cause the arrhythmogenic phenotype of this mutation.
Asunto(s)
Arritmias Cardíacas , Calcio/química , Calmodulina/química , Calmodulina/genética , Mutación Missense , Canal Liberador de Calcio Receptor de Rianodina/química , Sustitución de Aminoácidos , Calcio/metabolismo , Calmodulina/metabolismo , Humanos , Resonancia Magnética Nuclear Biomolecular , Dominios Proteicos , Canal Liberador de Calcio Receptor de Rianodina/genética , Canal Liberador de Calcio Receptor de Rianodina/metabolismoRESUMEN
The calcium-calmodulin-dependent protein kinase kinase-2 (CaMKK2) is a key regulator of cellular and whole-body energy metabolism. It is known to be activated by increases in intracellular Ca2+, but the mechanisms by which it is inactivated are less clear. CaMKK2 inhibition protects against prostate cancer, hepatocellular carcinoma, and metabolic derangements induced by a high-fat diet; therefore, elucidating the intracellular mechanisms that inactivate CaMKK2 has important therapeutic implications. Here we show that stimulation of cAMP-dependent protein kinase A (PKA) signaling in cells inactivates CaMKK2 by phosphorylation of three conserved serine residues. PKA-dependent phosphorylation of Ser495 directly impairs calcium-calmodulin activation, whereas phosphorylation of Ser100 and Ser511 mediate recruitment of 14-3-3 adaptor proteins that hold CaMKK2 in the inactivated state by preventing dephosphorylation of phospho-Ser495 We also report the crystal structure of 14-3-3ζ bound to a synthetic diphosphorylated peptide that reveals how the canonical (Ser511) and noncanonical (Ser100) 14-3-3 consensus sites on CaMKK2 cooperate to bind 14-3-3 proteins. Our findings provide detailed molecular insights into how cAMP-PKA signaling inactivates CaMKK2 and reveals a pathway to inhibit CaMKK2 with potential for treating human diseases.
Asunto(s)
Proteínas 14-3-3/metabolismo , Quinasa de la Proteína Quinasa Dependiente de Calcio-Calmodulina/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Transducción de Señal , Proteínas 14-3-3/genética , Animales , Células COS , Quinasa de la Proteína Quinasa Dependiente de Calcio-Calmodulina/genética , Línea Celular Tumoral , Chlorocebus aethiops , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Activación Enzimática , HumanosRESUMEN
In plants, rapid and reversible biological responses to environmental cues may require complex cellular reprograming. This is enabled by signaling molecules such as the cyclic nucleotide monophosphates (cNMPs) cAMP and cGMP, as well as Ca2+. While the roles and synthesis of cAMP and cGMP in plants are increasingly well-characterized, the "off signal" afforded by cNMP-degrading enzymes, the phosphodiesterases (PDEs), is, however, poorly understood, particularly so in monocots. Here, we identified a candidate PDE from the monocot Brachypodium distachyon (BDPDE1) and showed that it can hydrolyze cNMPs to 5'NMPs but with a preference for cAMP over cGMP in vitro. Notably, the PDE activity was significantly enhanced by Ca2+ only in the presence of calmodulin (CaM), which interacts with BDPDE1, most likely at a predicted CaM-binding site. Finally, based on our biochemical, mutagenesis and structural analyses, we constructed a comprehensive amino acid consensus sequence extracted from the catalytic centers of annotated and/or experimentally validated PDEs across species to enable a broad application of this search motif for the identification of similar active sites in eukaryotes and prokaryotes.
Asunto(s)
Brachypodium/enzimología , Calcio/metabolismo , Calmodulina/metabolismo , Hidrolasas Diéster Fosfóricas/metabolismo , Proteínas de Plantas/metabolismo , Adenosina Monofosfato/metabolismo , Secuencia de Aminoácidos , Sitios de Unión/genética , Brachypodium/genética , Dominio Catalítico , AMP Cíclico , GMP Cíclico/metabolismo , Guanosina Monofosfato/metabolismo , Hidrólisis , Cinética , Hidrolasas Diéster Fosfóricas/química , Hidrolasas Diéster Fosfóricas/genética , Proteínas de Plantas/química , Proteínas de Plantas/genética , Unión Proteica , Homología de Secuencia de Aminoácido , Especificidad por Sustrato , TemperaturaRESUMEN
Cell compartmentalization is an essential process by which eukaryotic cells separate and control biological processes. Although calmodulins are well-known to regulate catalytic properties of their targets, we show here their involvement in the subcellular location of two plant proteins. Both proteins exhibit a dual location, namely in the cytosol in addition to their association to plastids (where they are known to fulfil their role). One of these proteins, ceQORH, a long-chain fatty acid reductase, was analyzed in more detail, and its calmodulin-binding site was identified by specific mutations. Such a mutated form is predominantly targeted to plastids at the expense of its cytosolic location. The second protein, TIC32, was also shown to be dependent on its calmodulin-binding site for retention in the cytosol. Complementary approaches (bimolecular fluorescence complementation and reverse genetics) demonstrated that the calmodulin isoform CAM5 is specifically involved in the retention of ceQORH in the cytosol. This study identifies a new role for calmodulin and sheds new light on the intriguing CaM-binding properties of hundreds of plastid proteins, despite the fact that no CaM or CaM-like proteins were identified in plastids.
Asunto(s)
Proteínas de Arabidopsis/genética , Calmodulina/genética , Compartimento Celular/genética , Proteínas de Cloroplastos/genética , Proteínas de la Membrana/genética , Arabidopsis/química , Arabidopsis/genética , Proteínas de Arabidopsis/química , Sitios de Unión/genética , Señalización del Calcio/genética , Calmodulina/química , Proteínas de Cloroplastos/química , Cloroplastos/química , Cloroplastos/genética , Citosol/química , Proteínas de la Membrana/química , Plastidios/química , Plastidios/genética , Unión Proteica/genéticaRESUMEN
In plants, strict regulation of stomatal pores is critical for modulation of CO2 fixation and transpiration. Under certain abiotic and biotic stressors, pore closure is initiated through anionic flux, with calcium (Ca2+) playing a central role. The aluminum-activated malate transporter 12 (ALMT12) is a malate-activated, voltage-dependent member of the aluminum-activated malate transporter family that has been implicated in anionic flux from guard cells controlling the stomatal aperture. Herein, we report the characterization of the regulatory mechanisms mediating channel activities of an ALMT from the grass Brachypodium distachyon (BdALMT12) that has the highest sequence identity to Arabidopsis thaliana ALMT12. Electrophysiological studies in a heterologous cell system confirmed that this channel is malate- and voltage-dependent. However, this was shown to be true only in the presence of Ca2+ Although a general kinase inhibitor increased the current density of BdALMT12, a calmodulin (CaM) inhibitor reduced the Ca2+-dependent channel activation. We investigated the physiological relevance of the CaM-based regulation in planta, where stomatal closure, induced by exogenous Ca2+ ionophore and malate, was shown to be inhibited by exogenous application of a CaM inhibitor. Subsequent analyses revealed that the double substitutions R335A/R338A and R335A/K342A, within a predicted BdALMT12 CaM-binding domain (CBD), also decreased the channels' ability to activate. Using isothermal titration calorimetry and CBD-mimetic peptides, as well as CaM-agarose affinity pulldown of full-length recombinant BdALMT12, we confirmed the physical interaction between the CBD and CaM. Together, these findings support a co-regulatory mechanism of BdALMT12 activation by malate, and Ca2+/CaM, emphasizing that a complex regulatory network modulates BdALMT12 activity.
Asunto(s)
Brachypodium , Calcio , Calmodulina , Transportadores de Anión Orgánico , Proteínas de Plantas , Estomas de Plantas , Sustitución de Aminoácidos , Brachypodium/química , Brachypodium/genética , Brachypodium/metabolismo , Calcio/química , Calcio/metabolismo , Calmodulina/química , Calmodulina/genética , Calmodulina/metabolismo , Activación del Canal Iónico/fisiología , Malatos/química , Malatos/metabolismo , Mutación Missense , Transportadores de Anión Orgánico/química , Transportadores de Anión Orgánico/genética , Transportadores de Anión Orgánico/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Estomas de Plantas/química , Estomas de Plantas/genética , Estomas de Plantas/metabolismoRESUMEN
Calmodulin (CaM) conveys intracellular Ca2+ signals to KCNQ (Kv7, "M-type") K+ channels and many other ion channels. Whether this "calmodulation" involves a dramatic structural rearrangement or only slight perturbations of the CaM/KCNQ complex is as yet unclear. A consensus structural model of conformational shifts occurring between low nanomolar and physiologically high intracellular [Ca2+] is still under debate. Here, we used various techniques of biophysical chemical analyses to investigate the interactions between CaM and synthetic peptides corresponding to the A and B domains of the KCNQ4 subtype. We found that in the absence of CaM, the peptides are disordered, whereas Ca2+/CaM imposed helical structure on both KCNQ A and B domains. Isothermal titration calorimetry revealed that Ca2+/CaM has higher affinity for the B domain than for the A domain of KCNQ2-4 and much higher affinity for the B domain when prebound with the A domain. X-ray crystallography confirmed that these discrete peptides spontaneously form a complex with Ca2+/CaM, similar to previous reports of CaM binding KCNQ-AB domains that are linked together. Microscale thermophoresis and heteronuclear single-quantum coherence NMR spectroscopy indicated the C-lobe of Ca2+-free CaM to interact with the KCNQ4 B domain (Kd â¼10-20 µm), with increasing Ca2+ molar ratios shifting the CaM-B domain interactions via only the CaM C-lobe to also include the N-lobe. Our findings suggest that in response to increased Ca2+, CaM undergoes lobe switching that imposes a dramatic mutually induced conformational fit to both the proximal C terminus of KCNQ4 channels and CaM, likely underlying Ca2+-dependent regulation of KCNQ gating.
Asunto(s)
Calcio/química , Calmodulina/química , Canales de Potasio KCNQ/química , Animales , Células CHO , Calcio/metabolismo , Calmodulina/genética , Calmodulina/metabolismo , Cricetulus , Cristalografía por Rayos X , Humanos , Activación del Canal Iónico , Canales de Potasio KCNQ/genética , Canales de Potasio KCNQ/metabolismo , Dominios Proteicos , Estructura Secundaria de ProteínaRESUMEN
Isocitrate dehydrogenase (IDH) mutations are found in low-grade gliomas, and the product of the IDH mutant (MT), 2-hydroxyglutarate (2-HG), is the first known oncometabolite. However, the roles of the IDH wild type (WT) in high-grade glioblastoma, which rarely has the IDH mutation, are still unknown. To investigate possible pathways related to IDH WT in gliomas, we carried out bioinformatics analysis, and found that IDH1 has several putative calmodulin (CaM) binding sites. Pull-down and quantitative dissociation constant (Kd) measurements using recombinant proteins showed that IDH1 WT indeed binds to CaM with a higher affinity than IDH1 R132H MT. This biochemical interaction was demonstrated also in the cellular environment by immunoprecipitation with glioblastoma cell extracts. A synthetic peptide for the suggested binding region interfered with the interaction between CaM and IDH1, confirming the specificity of the binding. Direct binding between the synthetic peptide and CaM was observed in an NMR binding experiment, which additionally revealed that the peptide initially binds to the C-lobe of CaM. The physiological meaning of the CaM-IDH1 WT binding was shown with trifluoperazine (TFP), a CaM antagonist, which disrupted the binding and inhibited survival and migration of glioblastoma cells with IDH1 WT. As CaM signaling is activated in glioblastoma, our results suggest that IDH1 WT may be involved in the CaM-signaling pathway in the tumorigenesis of high-grade gliomas.
Asunto(s)
Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , Calmodulina/metabolismo , Movimiento Celular , Glioblastoma/metabolismo , Glioblastoma/patología , Isocitrato Deshidrogenasa/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Humanos , Isocitrato Deshidrogenasa/química , Modelos Moleculares , Unión Proteica/efectos de los fármacos , Trifluoperazina/farmacologíaRESUMEN
Cortical microtubule (MT) arrays play a critical role in plant cell shape determination by defining the direction of cell expansion. As plants continuously adapt to ever-changing environmental conditions, multiple environmental and developmental inputs need to be translated into changes of the MT cytoskeleton. Here, we identify and functionally characterize an auxin-inducible and MT-localized protein OsIQ67-DOMAIN14 (OsIQD14), which is highly expressed in rice seed hull cells. We show that while deficiency of OsIQD14 results in short and wide seeds and increases overall yield, overexpression leads to narrow and long seeds, caused by changed MT alignment. We further show that OsIQD14-mediated MT reordering is regulated by specifically affecting MT dynamics, and ectopic expression of OsIQD14 in Arabidopsis could change the cell shape both in pavement cells and in hypocotyl cells. Additionally, OsIQD14 activity is tightly controlled by calmodulin proteins, providing an alternative way to modify the OsIQD14 activity. Our results indicate that OsIQD14 acts as a key factor in regulating MT rearrangements in rice hull cells and hence the grain shape, and allows effective local cell shape manipulation to improve the rice yield trait.
Asunto(s)
Proteínas de Arabidopsis , Oryza , Citoesqueleto , Proteínas Asociadas a Microtúbulos , Microtúbulos , Oryza/genéticaRESUMEN
Connexin channels help maintain eye lens homeostasis and transparency. The G143R missense substitution in connexin (Cx) 46 is associated with congenital Coppock cataracts; however, the underlying molecular mechanism is largely unknown. Here, we report that compared with WT Cx46, the G143R substitution abolishes hemichannel conductance in Xenopus oocytes and in HeLa cells. Moreover, this substitution is dominant-negative and inhibits conductance of WT Cx46. CD analysis indicated that the substitution greatly reduces the α-helical structure of the intracellular Cx46 loop domain. Protein pulldown assays and isothermal titration calorimetry revealed that this Cx46 domain directly interacts with calmodulin (CaM) in a Ca2+-dependent fashion, an observation confirmed by immunofluorescent co-localization of Cx46 with CaM. Interestingly, the G143R substitution enhanced the Cx46-CaM interaction and attenuated its abolishment by Ca2+ depletion. Moreover, Cx46 increased dye influx, and the G143R substitution augmented this effect. Inhibition of Ca2+-mediated CaM activation blocked hemichannel permeability. The membrane potential plays a crucial role in Cx46 membrane permeability. We found that the activity of hemichannels is detectable under rest and hyperpolarization conditions but is eliminated with depolarization. These results suggested that the G143R substitution impairs voltage-dependent electrical conductance and alters membrane permeability mediated by Cx46 hemichannels. The latter likely is caused by the substitution-induced structural changes of the intracellular loop domain associated with the increased interaction with CaM and reduced Ca2+ sensitivity. The data suggest that the G143R-induced enhancement of the CaM-Cx46 interaction results in altered hemichannel activities and might be related to cataract formation.
Asunto(s)
Calmodulina/metabolismo , Catarata/genética , Conexinas/genética , Mutación Missense , Animales , Calcio/metabolismo , Calmodulina/química , Calmodulina/genética , Catarata/congénito , Catarata/metabolismo , Conexinas/química , Conexinas/metabolismo , Femenino , Uniones Comunicantes/metabolismo , Células HeLa , Humanos , Cristalino/metabolismo , Potenciales de la Membrana , Oocitos/química , Oocitos/metabolismo , Unión Proteica , Dominios Proteicos , XenopusRESUMEN
Arabidopsis thaliana rapid alkalinization factor 1 (AtRALF1) is a small secreted peptide hormone that inhibits root growth by repressing cell expansion. Although it is known that AtRALF1 binds the plasma membrane receptor FERONIA and conveys its signals via phosphorylation, the AtRALF1 signaling pathway is largely unknown. Here, using a yeast two-hybrid system to search for AtRALF1-interacting proteins in Arabidopsis, we identified calmodulin-like protein 38 (CML38) as an AtRALF1-interacting partner. We also found that CML38 and AtRALF1 are both secreted proteins that physically interact in a Ca2+- and pH-dependent manner. CML38-knockout mutants generated via T-DNA insertion were insensitive to AtRALF1, and simultaneous treatment with both AtRALF1 and CML38 proteins restored sensitivity in these mutants. Hybrid plants lacking CML38 and having high accumulation of the AtRALF1 peptide did not exhibit the characteristic short-root phenotype caused by AtRALF1 overexpression. Although CML38 was essential for AtRALF1-mediated root inhibition, it appeared not to have an effect on the AtRALF1-induced alkalinization response. Moreover, acridinium-labeling of AtRALF1 indicated that the binding of AtRALF1 to intact roots is CML38-dependent. In summary, we describe a new component of the AtRALF1 response pathway. The new component is a calmodulin-like protein that binds AtRALF1, is essential for root growth inhibition, and has no role in AtRALF1 alkalinization.