RESUMEN
Targeting Ca2+/calmodulin-dependent protein kinase γ (CaMKIIγ) in macrophages using RNAi nanotechnology represents an innovative and promising strategy in the diagnosis and treatment of atherosclerosis. Nevertheless, it remains elusive because of the current challenges associated with the systemic delivery of siRNA nanoparticle (NP) to atheromatous plaques and the complexity of atherosclerotic plaques. Here, we demonstrate the potential of a thienothiadiazole-based near-infrared-II (NIR-II) organic aggregation-induced emission (AIE) platform encapsulated with the Camk2g siRNA to effectively target CaMKIIγ in macrophages for dynamic imaging and image-guided gene therapy of atherosclerosis. The nanoparticles effectively decreased CaMKIIγ expression and increased the expression of the efferocytosis receptor MerTK in plaque macrophages, leading to a reduction in the necrotic core area of the lesion in an aortic plaque model. Our theranostic approach highlights the substantial promise of near-infrared II (NIR-II) AIEgens for imaging and image-guided therapy of atherosclerosis.
Asunto(s)
Aterosclerosis , Imagen Óptica , ARN Interferente Pequeño , Animales , Humanos , Ratones , Aterosclerosis/diagnóstico por imagen , Aterosclerosis/terapia , Rayos Infrarrojos , Macrófagos/metabolismo , Ratones Endogámicos C57BL , Nanopartículas/química , Placa Aterosclerótica/diagnóstico por imagen , ARN Interferente Pequeño/química , ARN Interferente Pequeño/uso terapéutico , Tiadiazoles/química , Tiadiazoles/farmacología , Proteínas Quinasas Dependientes de Calcio-Calmodulina/química , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismoRESUMEN
The death-associated protein kinase (DAPK) family is a member of the calcium/calmodulin-regulated serine/threonine protein kinase family, and studies have shown that its role, as its name suggests, is mainly to regulate cell death. The DAPK family comprises five members, including DAPK1, DAPK2, DAPK3, DRAK1 and DRAK2, which show high homology in the common N-terminal kinase domain but differ in the extra-catalytic domain. Notably, previous research has suggested that the DAPK family plays an essential role in both the development and regulation of human diseases. However, only a few small-molecule inhibitors have been reported. In this Perspective, we mainly discuss the structure, biological function, and role of DAPKs in diseases and the currently discovered small-molecule inhibitors, providing valuable information for the development of the DAPK field.
Asunto(s)
Proteínas Quinasas Dependientes de Calcio-Calmodulina , Proteínas Serina-Treonina Quinasas , Humanos , Proteínas Quinasas Asociadas a Muerte Celular/química , Proteínas Quinasas Asociadas a Muerte Celular/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Dominio Catalítico , Proteínas Quinasas Dependientes de Calcio-Calmodulina/químicaRESUMEN
CASK (Ca2+/calmodulin-dependent Ser/Thr kinase) is a member of the MAGUK (membrane-associated guanylate kinase) family that functions as neurexin kinases with roles implicated in neuronal synapses and trafficking. The lack of a canonical DFG motif, which is altered to GFG in CASK, led to the classification as a pseudokinase. However, functional studies revealed that CASK can still phosphorylate substrates in the absence of divalent metals. CASK dysfunction has been linked to many diseases, including colorectal cancer, Parkinson's disease, and X-linked mental retardation, suggesting CASK as a potential drug target. Here, we exploited structure-based design for the development of highly potent and selective CASK inhibitors based on 2,4-diaminopyrimidine-5-carboxamides targeting an unusual pocket created by the GFG motif. The presented inhibitor design offers a more general strategy for the development of pseudokinase ligands that harbor unusual sequence motifs. It also provides a first chemical probe for studying the biological roles of CASK.
Asunto(s)
Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Sondas Moleculares/química , Inhibidores de Proteínas Quinasas/química , Serina/química , Treonina/química , Proteínas Quinasas Dependientes de Calcio-Calmodulina/química , Diseño de Fármacos , Humanos , Sondas Moleculares/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
Calcium/calmodulin-dependent protein kinase (CCaMK) has been shown to play important roles in brassinosteroid (BR)-induced antioxidant defense and enhancing the tolerance of plants to drought stress. The autophosphorylation of CCaMK is a key step for the activation of CCaMK, thus promoting substrate phosphorylation. However, how CCaMK autophosphorylation function in BR-induced antioxidant defense is not known yet. Here, seven potential autophosphorylation sites of ZmCCaMK were identified using mass spectroscopy (liquid chromatography-tandem mass spectrometry [LC-MS/MS]) analysis. The transient gene expression analysis in maize protoplasts showed that Thr420 and Ser454 of ZmCCaMK were important for BR-induced antioxidant defense. Furthermore, Thr420 and Ser454 of ZmCCaMK were crucial for improving drought tolerance and alleviating drought induced oxidative damage of plants via overexpressing various mutant versions of ZmCCaMK in tobacco (Nicotiana tabacum). Mutations of Thr420 and Ser454 in ZmCCaMK substantially blocked the autophosphorylation and substrate phosphorylation of ZmCCaMK in vitro. Taken together, our results demonstrate that Thr420 and Ser454 of ZmCCaMK are crucial for BR-induced antioxidant defense and drought tolerance through modulating the autophosphorylation and substrate phosphorylation activities of ZmCCaMK.
Asunto(s)
Antioxidantes/metabolismo , Brasinoesteroides/farmacología , Proteínas Quinasas Dependientes de Calcio-Calmodulina/química , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Serina/metabolismo , Treonina/metabolismo , Zea mays/enzimología , Adaptación Fisiológica/efectos de los fármacos , Sequías , Proteínas Mutantes/metabolismo , Fosforilación/efectos de los fármacos , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Plantas Modificadas Genéticamente , Relación Estructura-Actividad , Especificidad por Sustrato/efectos de los fármacos , Nicotiana/genética , Zea mays/efectos de los fármacosRESUMEN
Calmodulin binding receptor like cytoplasmic kinase 2 (CRCK2) belongs to the family of receptor like kinases (RLKs) which is mainly implicated in pathways associated with the stress responses in plants. The protein from the stem of Oroxylum indicum was isolated and purified using anion-exchange followed by gel filtration chromatography. The purity of protein was checked using SDS-PAGE, which showed a single band of 50â¯kDa. The purified protein was identified as CRCK2 using MALDI-TOF. Using I-TASSER, a bioinformatics tools, the model of protein was constructed and its secondary structure was predicted using VADAR. The secondary structure content was also determined by far-UV CD, which indicated that the CRCK2 is mainly ß-sheet dominating protein (43% ß-sheet). The secondary structural content predication from computational method is in close agreement with the result obtained by CD spectropolarimeter. This study validates I-TASSER model for determination of structure of a protein. Moreover, stability of CRCK2 was monitored against heat- and guanidinium chloride (GdmCl)-induced denaturation by using circular dichroism (CD) and fluorescence spectroscopy. Denaturation curve analysis gave values of 2.88⯱â¯0.12â¯kcalâ¯mol-1and 4.11⯱â¯0.09â¯M for ∆°GD (Gibbs free energy change at 25⯰C) and Cm (midpoint of denaturation), respectively. It has been observed that purified CRCK2 is quite stable protein against both heat-induced as well as GdmCl-induced denaturation. This is very first report of purification and biophysical characterization of CRCK2 protein from medicinal plant O. indicum.
Asunto(s)
Bignoniaceae/enzimología , Proteínas Quinasas Dependientes de Calcio-Calmodulina/química , Conformación Proteica en Lámina beta , Estructura Secundaria de Proteína , Bignoniaceae/química , Fenómenos Biofísicos , Proteínas Quinasas Dependientes de Calcio-Calmodulina/genética , Dicroismo Circular , Modelos Químicos , Unión Proteica , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
We surveyed genome sequences from the basidiomycetous mushroom Coprinopsis cinerea and isolated a cDNA homologous to CMKA, a calmodulin-dependent protein kinase (CaMK) in Aspergillus nidulans. We designated this sequence, encoding 580 amino acids with a molecular weight of 63,987, as CoPK02. CoPK02 possessed twelve subdomains specific to protein kinases and exhibited 43, 35, 40% identity with rat CaMKI, CaMKII, CaMKIV, respectively, and 40% identity with CoPK12, one of the CaMK orthologs in C. cinerea. CoPK02 showed significant autophosphorylation activity and phosphorylated exogenous proteins in the presence of Ca2+/CaM. By the CaM-overlay assay we confirmed that the C-terminal sequence (Trp346-Arg358) was the calmodulin-binding site, and that the binding of Ca2+/CaM to CoPK02 was reduced by the autophosphorylation of CoPK02. Since CoPK02 evolved in a different clade from CoPK12, and showed different gene expression compared to that of CoPK32, which is homologous to mitogen-activated protein kinase-activated protein kinase, CoPK02 and CoPK12 might cooperatively regulate Ca2+-signaling in C. cinerea.
Asunto(s)
Basidiomycota/enzimología , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Secuencia de Aminoácidos , Animales , Basidiomycota/genética , Basidiomycota/crecimiento & desarrollo , Sitios de Unión , Señalización del Calcio , Proteínas Quinasas Dependientes de Calcio-Calmodulina/química , Proteínas Quinasas Dependientes de Calcio-Calmodulina/genética , Calmodulina/metabolismo , Catálisis , Clonación Molecular , Electroforesis en Gel de Poliacrilamida , Perfilación de la Expresión Génica , Genes Fúngicos , Fosforilación , Filogenia , Ratas , Homología de Secuencia de AminoácidoRESUMEN
Calmodulin-dependent protein kinase (CAMK) is physiologically activated in fertilized human oocytes and is involved in the Ca2+ response pathways that link the fertilization calmodulin signal to meiosis resumption and cortical granule exocytosis. The kinase has an unstructured C-terminal tail that can be recognized and bound by the PDZ5 domain of its cognate partner, the multi-PDZ domain protein (MUP). In the current study, we reported a rational biomolecular design of halogen-bonding system at the complex interface of CAMK's C-terminal peptide with MUP PDZ5 domain by using high-level computational approaches. Four organic halogens were employed as atom probes to explore the structural geometry and energetic property of designed halogen bonds in the PDZ5-peptide complex. It was found that the heavier halogen elements such as bromine Br and iodine I can confer stronger halogen bond but would cause bad atomic contacts and overlaps at the complex interface, while fluorine F cannot form effective halogen bond in the complex. In addition, the halogen substitution at different positions of peptide's aromatic ring would result in distinct effects on the halogen-bonding system. The computational findings were then verified by using fluorescence analysis; it is indicated that the halogen type and substitution position play critical role in the interaction strength of halogen bonds, and thus the PDZ5-peptide binding affinity can be improved considerably by optimizing their combination.
Asunto(s)
Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Proteínas Portadoras/metabolismo , Secuencia de Aminoácidos , Proteínas Quinasas Dependientes de Calcio-Calmodulina/química , Proteínas Portadoras/química , Halogenación , Humanos , Enlace de Hidrógeno , Proteínas de la Membrana , Estructura Molecular , Unión Proteica , Dominios Proteicos , Teoría CuánticaRESUMEN
Arabidopsis inositol polyphosphate kinase 2ß (AtIpk2ß) has multiple functions in plant development and in responding to abiotic stress. Although some related clues suggested a potential role of AtIpk2ß in ABA signaling, the defined evidence was still lack. Here we discovered that a key ABA signaling component calcium-dependent protein kinase 4 (CPK4) can interact with AtIpk2ß under ABA treated conditions through affinity purification and mass spectrometry detection. The interaction between CPK4 and AtIpk2ß were further confirmed by yeast two hybrid and bimolecular fluorescence complementation assays. Expression of AtIpk2ß also can be rapidly induced by ABA. In addition, we found that CPK4 can phosphorylate AtIpk2ß in vitro and identified five novel phosphorylation sites of AtIpk2ß by CPK4 kinase, including Tyr46, Ser48, Ser51, Thr128, Ser147. Overexpression of AtIpk2ß in Arabidopsis was more sensitive to ABA in seed germination, primary root inhibition, ABA-responsive gene expression than wild type plants, whereas knockout mutant atipk2ß exhibited no significant difference. The AtIpk2ß variants containing Tyr46, Thr128, Ser147 mutated to Ala cannot complement the yeast mutant ipk2 growth in high temperature, suggesting that those three amino acid residues are critical for AtIpk2ß. These findings provide insight into the modulation of ABA signaling by AtIpk2ß.
Asunto(s)
Ácido Abscísico/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Transducción de Señal , Secuencia de Aminoácidos , Arabidopsis/química , Arabidopsis/genética , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Proteínas Quinasas Dependientes de Calcio-Calmodulina/química , Regulación de la Expresión Génica de las Plantas , Fosforilación , Fosfotransferasas (Aceptor de Grupo Alcohol)/química , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Mapas de Interacción de Proteínas , Regulación hacia ArribaRESUMEN
Nutrient signalling integrates and coordinates gene expression, metabolism and growth. However, its primary molecular mechanisms remain incompletely understood in plants and animals. Here we report unique Ca2+ signalling triggered by nitrate with live imaging of an ultrasensitive biosensor in Arabidopsis leaves and roots. A nitrate-sensitized and targeted functional genomic screen identifies subgroup III Ca2+-sensor protein kinases (CPKs) as master regulators that orchestrate primary nitrate responses. A chemical switch with the engineered mutant CPK10(M141G) circumvents embryo lethality and enables conditional analyses of cpk10 cpk30 cpk32 triple mutants to define comprehensive nitrate-associated regulatory and developmental programs. Nitrate-coupled CPK signalling phosphorylates conserved NIN-LIKE PROTEIN (NLP) transcription factors to specify the reprogramming of gene sets for downstream transcription factors, transporters, nitrogen assimilation, carbon/nitrogen metabolism, redox, signalling, hormones and proliferation. Conditional cpk10 cpk30 cpk32 and nlp7 mutants similarly impair nitrate-stimulated system-wide shoot growth and root establishment. The nutrient-coupled Ca2+ signalling network integrates transcriptome and cellular metabolism with shoot-root coordination and developmental plasticity in shaping organ biomass and architecture.
Asunto(s)
Amidohidrolasas/metabolismo , Arabidopsis/crecimiento & desarrollo , Arabidopsis/metabolismo , Calcio/metabolismo , Nitratos/metabolismo , Proteínas Quinasas/metabolismo , Transducción de Señal , Amidohidrolasas/genética , Secuencia de Aminoácidos , Arabidopsis/genética , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Biomasa , Señalización del Calcio , Proteínas de Unión al Calcio/química , Proteínas de Unión al Calcio/genética , Proteínas de Unión al Calcio/metabolismo , Proteínas Quinasas Dependientes de Calcio-Calmodulina/química , Proteínas Quinasas Dependientes de Calcio-Calmodulina/genética , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Carbono/metabolismo , Reprogramación Celular , Alimentos , Regulación de la Expresión Génica de las Plantas , Nitrógeno/metabolismo , Oxidación-Reducción , Fosforilación , Raíces de Plantas/crecimiento & desarrollo , Raíces de Plantas/metabolismo , Brotes de la Planta/crecimiento & desarrollo , Brotes de la Planta/metabolismo , Plantas Modificadas Genéticamente , Proteínas Quinasas/química , Proteínas Quinasas/genética , Transcripción Genética , TranscriptomaRESUMEN
The α-kinases are a family of a typical protein kinases present in organisms ranging from protozoa to mammals. Here we report an autoinhibited conformation for the α-kinase domain of Dictyostelium myosin-II heavy chain kinase A (MHCK-A) in which nucleotide binding to the catalytic cleft, located at the interface between an N-terminal and C-terminal lobe, is sterically blocked by the side chain of a conserved arginine residue (Arg592). Previous α-kinase structures have shown that an invariant catalytic aspartic acid residue (Asp766) is phosphorylated. Unexpectedly, in the autoinhibited conformation the phosphoryl group is transferred to the adjacent Asp663, creating an interaction network that stabilizes the autoinhibited state. The results suggest that Asp766 phosphorylation may play both catalytic and regulatory roles. The autoinhibited structure also provides the first view of a phosphothreonine residue docked into the phospho-specific allosteric binding site (Pi-pocket) in the C-lobe of the α-kinase domain.
Asunto(s)
Proteínas Quinasas Dependientes de Calcio-Calmodulina/química , Dictyostelium/enzimología , Proteínas Protozoarias/química , Apoenzimas/química , Dominios ProteicosRESUMEN
The α-kinases are a widely expressed family of serine/threonine protein kinases that exhibit no sequence identity with conventional eukaryotic protein kinases. In this report, we provide new information on the catalytic properties of the α-kinase domain of Dictyostelium myosin-II heavy chain kinase-A (termed A-CAT). Crystallization of A-CAT in the presence of MgATP yielded structures with AMP or adenosine in the catalytic cleft together with a phosphorylated Asp-766 residue. The results show that the ß- and α-phosphoryl groups are transferred either directly or indirectly to the catalytically essential Asp-766. Biochemical assays confirmed that A-CAT hydrolyzed ATP, ADP, and AMP with kcat values of 1.9, 0.6, and 0.32 min(-1), respectively, and showed that A-CAT can use ADP to phosphorylate peptides and proteins. Binding assays using fluorescent 2'/3'-O-(N-methylanthraniloyl) analogs of ATP and ADP yielded Kd values for ATP, ADP, AMP, and adenosine of 20 ± 3, 60 ± 20, 160 ± 60, and 45 ± 15 µM, respectively. Site-directed mutagenesis showed that Glu-713, Leu-716, and Lys-645, all of which interact with the adenine base, were critical for nucleotide binding. Mutation of the highly conserved Gln-758, which chelates a nucleotide-associated Mg(2+) ion, eliminated catalytic activity, whereas loss of the highly conserved Lys-722 and Arg-592 decreased kcat values for kinase and ATPase activities by 3-6-fold. Mutation of Asp-663 impaired kinase activity to a much greater extent than ATPase, indicating a specific role in peptide substrate binding, whereas mutation of Gln-768 doubled ATPase activity, suggesting that it may act to exclude water from the active site.
Asunto(s)
Nucleótidos de Adenina/química , Proteínas Quinasas Dependientes de Calcio-Calmodulina/química , Dictyostelium/enzimología , Proteínas Protozoarias/química , Nucleótidos de Adenina/genética , Nucleótidos de Adenina/metabolismo , Proteínas Quinasas Dependientes de Calcio-Calmodulina/genética , Cristalografía por Rayos X , Dictyostelium/genética , Mutagénesis Sitio-Dirigida , Estructura Terciaria de Proteína , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismoRESUMEN
Many human protein kinases are regulated by the calcium-sensor protein calmodulin, which binds to a short flexible segment C-terminal to the enzyme's catalytic kinase domain. Our understanding of the molecular mechanism of kinase activity regulation by calcium/calmodulin has been advanced by the structures of two protein kinases-calmodulin kinase II and death-associated protein kinase 1-bound to calcium/calmodulin. Comparison of these two structures reveals a surprising level of diversity in the overall kinase-calcium/calmodulin arrangement and functional readout of activity, as well as complementary mechanisms of kinase regulation such as phosphorylation.
Asunto(s)
Proteínas Quinasas Dependientes de Calcio-Calmodulina/química , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Secuencia de Aminoácidos , Animales , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/química , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Proteínas Quinasas Asociadas a Muerte Celular/química , Proteínas Quinasas Asociadas a Muerte Celular/metabolismo , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Conformación Proteica , Alineación de SecuenciaRESUMEN
Eukaryotic elongation factor 2 kinase (eEF2K) is the best-characterized member of the α-kinase family. Within this group, only eEF2K and myosin heavy chain kinases (MHCKs) have known substrates. Here we have studied the roles of specific residues, selected on the basis of structural data for MHCK A and TRPM7, in the function of eEF2K. Our data provide the first information regarding the basis of the substrate specificity of α-kinases, in particular the roles of residues in the so-called N/D loop, which appears to occupy a position in the structure of α-kinases similar to that of the activation loop in other kinases. Several mutations in the EEF2K gene occur in tumors, one of which (Arg303Cys) is at a highly conserved residue in the N/D loop. This mutation greatly enhances eEF2K activity and may be cytoprotective. Our data support the concept that the major autophosphorylation site (Thr348 in eEF2K) docks into a binding pocket to help create the kinase-competent conformation. This is similar to the situation for MHCK A and is consistent with this being a common feature of α-kinases.
Asunto(s)
Dominio Catalítico , Secuencia Conservada , Quinasa del Factor 2 de Elongación/química , Quinasa del Factor 2 de Elongación/metabolismo , Secuencia de Aminoácidos , Aminoácidos/genética , Sitios de Unión , Proteínas Quinasas Dependientes de Calcio-Calmodulina/química , Células HEK293 , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Mutación/genética , Neoplasias/genética , Neoplasias/patología , Fosforilación , Fosfotreonina/metabolismo , Unión Proteica , Estructura Secundaria de Proteína , Proteínas Protozoarias/química , Homología Estructural de Proteína , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
Ca(2+)/calmodulin-dependent protein kinase phosphatase (CaMKP/PPM1F) and its nuclear homolog CaMKP-N (PPM1E) are Ser/Thr protein phosphatases that belong to the PPM family. CaMKP-N is expressed in the brain and undergoes proteolytic processing to yield a C-terminally truncated form. The physiological significance of this processing, however, is not fully understood. Using a wheat-embryo cell-free protein expression system, we prepared human CaMKP-N (hCaMKP-N(WT)) and the truncated form, hCaMKP-N(1-559), to compare their enzymatic properties using a phosphopeptide substrate. The hCaMKP-N(1-559) exhibited a much higher V(max) value than the hCaMKP-N(WT) did, suggesting that the processing may be a regulatory mechanism to generate a more active species. The active form, hCaMKP-N(1-559), showed Mn(2+) or Mg(2+)-dependent phosphatase activity with a strong preference for phospho-Thr residues and was severely inhibited by NaF, but not by okadaic acid, calyculin A, or 1-amino-8-naphthol-2,4-disulfonic acid, a specific inhibitor of CaMKP. It could bind to postsynaptic density and dephosphorylate the autophosphorylated Ca(2+)/calmodulin-dependent protein kinase II. Furthermore, it was inactivated by H2O2 treatment, and the inactivation was completely reversed by treatment with DTT, implying that this process is reversibly regulated by oxidation/reduction. The truncated CaMKP-N may play an important physiological role in neuronal cells.
Asunto(s)
Encéfalo/enzimología , Proteínas Quinasas Dependientes de Calcio-Calmodulina/química , Fosfopéptidos/química , Fosfoproteínas Fosfatasas/química , Animales , Activación Enzimática , Estabilidad de Enzimas , Ratas , Relación Estructura-ActividadRESUMEN
The TNF-IL-6-STAT3 pathway plays a crucial role in promoting ulcerative colitis-associated carcinoma (UCC). To date, the negative regulation of STAT3 is poorly understood. Interestingly, intestinal epithelial cells of UCC in comparison to ulcerative colitis show high expression levels of anti-inflammatory death-associated protein kinase (DAPK) and low levels of pSTAT3. Accordingly, epithelial DAPK expression was enhanced in STAT3(IEC-KO) mice. To unravel a possible regulatory mechanism, we used an in vitro TNF-treated intestinal epithelial cell model. We identified a new function of DAPK in suppressing TNF-induced STAT3 activation as DAPK siRNA knockdown and treatment with a DAPK inhibitor potentiated STAT3 activation, IL-6 mRNA expression, and secretion. DAPK attenuated STAT3 activity directly by physical interaction shown in three-dimensional structural modeling. This model suggests that DAPK-induced conformational changes in the STAT3 dimer masked its nuclear localization signal. Alternatively, pharmacological inactivation of STAT3 led to an increase in DAPK mRNA and protein levels. Chromatin immunoprecipitation showed that STAT3 restricted DAPK expression by promoter binding, thereby reinforcing its own activation by inducing IL-6. This novel negative regulation principle might balance TNF-induced inflammation and seems to play an important role in the inflammation-associated transformation process as confirmed in an AOM+DSS colon carcinogenesis mouse model. DAPK as a negative regulator of STAT3 emerges as therapeutic option in the treatment of ulcerative colitis and UCC.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/metabolismo , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Células Epiteliales/enzimología , Intestinos/patología , Factor de Transcripción STAT3/metabolismo , Animales , Proteínas Reguladoras de la Apoptosis/química , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Quinasas Dependientes de Calcio-Calmodulina/química , Proteínas Quinasas Dependientes de Calcio-Calmodulina/genética , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Transformación Celular Neoplásica/efectos de los fármacos , Transformación Celular Neoplásica/patología , Colitis Ulcerosa/enzimología , Colitis Ulcerosa/patología , Neoplasias del Colon/enzimología , Neoplasias del Colon/patología , Proteínas Quinasas Asociadas a Muerte Celular , Activación Enzimática/efectos de los fármacos , Células Epiteliales/efectos de los fármacos , Células Epiteliales/patología , Humanos , Enlace de Hidrógeno/efectos de los fármacos , Interacciones Hidrofóbicas e Hidrofílicas/efectos de los fármacos , Inflamación/patología , Interleucina-6/metabolismo , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/enzimología , Mucosa Intestinal/patología , Ratones , Ratones Endogámicos C57BL , Proteínas Mutantes/metabolismo , Fosforilación/efectos de los fármacos , Regiones Promotoras Genéticas/genética , Unión Proteica/efectos de los fármacos , Multimerización de Proteína/efectos de los fármacos , Factor de Transcripción STAT3/química , Transducción de Señal/efectos de los fármacos , Factor de Necrosis Tumoral alfa/metabolismo , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
14-3-3 proteins play an important role in the regulation of many cellular processes. The Arabidopsis vacuolar two-pore K(+) channel 1 (TPK1) interacts with the 14-3-3 protein GRF6 (GF14-λ). Upon phosphorylation of the putative binding motif in the N-terminus of TPK1, GRF6 binds to TPK1 and activates the potassium channel. In order to gain a deeper understanding of this 14-3-3-mediated signal transduction, we set out to identify the respective kinases, which regulate the phosphorylation status of the 14-3-3 binding motif in TPK1. Here, we report that the calcium-dependent protein kinases (CDPKs) can phosphorylate and thereby activate the 14-3-3 binding motif in TPK1. Focusing on the stress-activated kinase CPK3, we visualized direct and specific interaction of TPK1 with the kinase at the tonoplast in vivo. In line with its proposed role in K(+) homeostasis, TPK1 phosphorylation was found to be induced by salt stress in planta, and both cpk3 and tpk1 mutants displayed salt-sensitive phenotypes. Molecular modeling of the TPK1-CPK3 interaction domain provided mechanistic insights into TPK1 stress-regulated phosphorylation responses and pinpointed two arginine residues in the N-terminal 14-3-3 binding motif in TPK1 critical for kinase interaction. Taken together, our studies provide evidence for an essential role of the vacuolar potassium channel TPK1 in salt-stress adaptation as a target of calcium-regulated stress signaling pathways involving Ca(2+), Ca(2+)-dependent kinases, and 14-3-3 proteins.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/citología , Arabidopsis/fisiología , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Canales de Potasio/metabolismo , Sales (Química)/farmacología , Estrés Fisiológico/efectos de los fármacos , Vacuolas/metabolismo , Proteínas 14-3-3/metabolismo , Secuencias de Aminoácidos , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Proteínas Quinasas Dependientes de Calcio-Calmodulina/química , Proteínas Quinasas Dependientes de Calcio-Calmodulina/deficiencia , Proteínas Quinasas Dependientes de Calcio-Calmodulina/genética , Citosol/metabolismo , Regulación de la Expresión Génica de las Plantas , Técnicas de Inactivación de Genes , Germinación , Homeostasis , Modelos Moleculares , Mutación , Fosforilación , Potasio/metabolismo , Canales de Potasio/química , Canales de Potasio/genética , Conformación Proteica , Estabilidad Proteica , Transporte de Proteínas , Transducción de SeñalRESUMEN
DAPK (death-associated protein kinase) is a newly recognized member of the mammalian family of ROCO proteins, characterized by common ROC (Ras of complex proteins) and COR (C-terminal of ROC) domains. In the present paper, we review our recent work showing that DAPK is functionally a ROCO protein; its ROC domain binds and hydrolyses GTP. Furthermore, GTP binding regulates DAPK catalytic activity in a novel manner by enhancing autophosphorylation on inhibitory Ser308, thereby promoting the kinase 'off' state. This is a novel mechanism for in cis regulation of kinase activity by the distal ROC domain. The functional similarities between DAPK and the Parkinson's disease-associated protein LRRK2 (leucine-rich repeat protein kinase 2), another member of the ROCO family, are also discussed.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/química , Proteínas Reguladoras de la Apoptosis/metabolismo , Proteínas Quinasas Dependientes de Calcio-Calmodulina/química , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Guanosina Trifosfato/metabolismo , Proteínas Quinasas Asociadas a Muerte Celular , Humanos , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina , Enfermedad de Parkinson/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas p21(ras)/química , Proteínas Proto-Oncogénicas p21(ras)/metabolismoRESUMEN
The ROCO proteins are a family of large, multidomain proteins characterised by the presence of a Ras of complex proteins (ROC) domain followed by a COR, or C-terminal of ROC, domain. It has previously been shown that the ROC domain of the human ROCO protein Leucine Rich Repeat Kinase 2 (LRRK2) controls its kinase activity. Here, the ability of the ROC domain of another human ROCO protein, Death Associated Protein Kinase 1 (DAPK1), to bind GTP and control its kinase activity has been evaluated. In contrast to LRRK2, loss of GTP binding by DAPK1 does not result in loss of kinase activity, instead acting to modulate this activity. These data highlight the ROC domain of DAPK1 as a target for modifiers of this proteins function, and casts light on the role of ROC domains as intramolecular regulators in complex proteins with implications for a broad range of human diseases.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/química , Proteínas Reguladoras de la Apoptosis/metabolismo , Proteínas Quinasas Dependientes de Calcio-Calmodulina/química , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Guanosina Trifosfato/metabolismo , Dominios y Motivos de Interacción de Proteínas , Animales , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Proteínas Quinasas Dependientes de Calcio-Calmodulina/genética , Línea Celular , Chlorobium/química , Proteínas Quinasas Asociadas a Muerte Celular , Guanosina Trifosfato/química , Células HEK293 , Humanos , Simulación del Acoplamiento Molecular , Mutación , Unión Proteica , Conformación Proteica , Transporte de ProteínasRESUMEN
A calcium/calmodulin-dependent protein kinase (CCaMK) is essential in the interpretation of calcium oscillations in plant root cells for the establishment of symbiotic relationships with rhizobia and mycorrhizal fungi. Some of its properties have been studied in detail, but its calcium ion binding properties and subsequent conformational change have not. A biophysical approach was taken with constructs comprising either the visinin-like domain of Medicago truncatula CCaMK, which contains EF-hand motifs, or this domain together with the autoinhibitory domain. The visinin-like domain binds three calcium ions, leading to a conformational change involving the exposure of hydrophobic surfaces and a change in tertiary but not net secondary or quaternary structure. The affinity for calcium ions of visinin-like domain EF-hands 1 and 2 (K(d) = 200 ± 50 nM) was appropriate for the interpretation of calcium oscillations (~125-850 nM), while that of EF-hand 3 (K(d) ≤ 20 nM) implied occupancy at basal calcium ion levels. Calcium dissociation rate constants were determined for the visinin-like domain of CCaMK, M. truncatula calmodulin 1, and the complex between these two proteins (the slowest of which was 0.123 ± 0.002 s(-1)), suggesting the corresponding calcium association rate constants were at or near the diffusion-limited rate. In addition, the dissociation of calmodulin from the protein complex was shown to be on the same time scale as the dissociation of calcium ions. These observations suggest that the formation and dissociation of the complex between calmodulin and CCaMK would substantially mirror calcium oscillations, which typically have a 90 s periodicity.
Asunto(s)
Proteínas Quinasas Dependientes de Calcio-Calmodulina/química , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Calcio/metabolismo , Medicago truncatula/enzimología , Secuencia de Aminoácidos , Sitios de Unión , Señalización del Calcio , Calmodulina/metabolismo , Motivos EF Hand , Interacciones Hidrofóbicas e Hidrofílicas , Medicago truncatula/química , Medicago truncatula/metabolismo , Datos de Secuencia Molecular , Unión Proteica , Conformación Proteica , Estructura Terciaria de ProteínaRESUMEN
Förster resonance energy transfer (FRET) microscopy is frequently used to study protein interactions and conformational changes in living cells. The utility of FRET is limited by false positive and negative signals. To overcome these limitations we have developed Fluorescence Polarization and Fluctuation Analysis (FPFA), a hybrid single-molecule based method combining time-resolved fluorescence anisotropy (homo-FRET) and fluorescence correlation spectroscopy. Using FPFA, homo-FRET (a 1-10 nm proximity gauge), brightness (a measure of the number of fluorescent subunits in a complex), and correlation time (an attribute sensitive to the mass and shape of a protein complex) can be simultaneously measured. These measurements together rigorously constrain the interpretation of FRET signals. Venus based control-constructs were used to validate FPFA. The utility of FPFA was demonstrated by measuring in living cells the number of subunits in the α-isoform of Venus-tagged calcium-calmodulin dependent protein kinase-II (CaMKIIα) holoenzyme. Brightness analysis revealed that the holoenzyme has, on average, 11.9 ± 1.2 subunit, but values ranged from 10-14 in individual cells. Homo-FRET analysis simultaneously detected that catalytic domains were arranged as dimers in the dodecameric holoenzyme, and this paired organization was confirmed by quantitative hetero-FRET analysis. In freshly prepared cell homogenates FPFA detected only 10.2 ± 1.3 subunits in the holoenzyme with values ranging from 9-12. Despite the reduction in subunit number, catalytic domains were still arranged as pairs in homogenates. Thus, FPFA suggests that while the absolute number of subunits in an auto-inhibited holoenzyme might vary from cell to cell, the organization of catalytic domains into pairs is preserved.