RESUMEN
The Ca2+-independent, but diacylglycerol-regulated, novel protein kinase C (PKC) theta (θ) is highly expressed in hematopoietic cells where it participates in immune signaling and platelet function. Mounting evidence suggests that PKCθ may be involved in cancer, particularly blood cancers, breast cancer, and gastrointestinal stromal tumors, yet how to target this kinase (as an oncogene or as a tumor suppressor) has not been established. Here, we examine the effect of four cancer-associated mutations, R145H/C in the autoinhibitory pseudosubstrate, E161K in the regulatory C1A domain, and R635W in the regulatory C-terminal tail, on the cellular activity and stability of PKCθ. Live-cell imaging studies using the genetically-encoded fluorescence resonance energy transfer-based reporter for PKC activity, C kinase activity reporter 2 (CKAR2), revealed that the pseudosubstrate and C1A domain mutations impaired autoinhibition to increase basal signaling. This impaired autoinhibition resulted in decreased stability of the protein, consistent with the well-characterized behavior of Ca2+-regulated PKC isozymes wherein mutations that impair autoinhibition are paradoxically loss-of-function because the mutant protein is degraded. In marked contrast, the C-terminal tail mutation resulted in enhanced autoinhibition and enhanced stability. Thus, the examined mutations were loss-of-function by different mechanisms: mutations that impaired autoinhibition promoted the degradation of PKC, and those that enhanced autoinhibition stabilized an inactive PKC. Supporting a general loss-of-function of PKCθ in cancer, bioinformatics analysis revealed that protein levels of PKCθ are reduced in diverse cancers, including lung, renal, head and neck, and pancreatic. Our results reveal that PKCθ function is lost in cancer.
Asunto(s)
Neoplasias , Proteína Quinasa C-theta , Humanos , Proteína Quinasa C-theta/genética , Proteína Quinasa C-theta/metabolismo , Proteína Quinasa C-theta/química , Neoplasias/genética , Neoplasias/enzimología , Neoplasias/metabolismo , Mutación con Pérdida de Función , Células HEK293 , Dominios Proteicos , Mutación , Proteína Quinasa C/genética , Proteína Quinasa C/metabolismo , Proteína Quinasa C/químicaRESUMEN
The nuclear pore complex (NPC) is the sole and selective gateway for nuclear transport, and its dysfunction has been associated with many diseases. The metazoan NPC subcomplex RanBP2, which consists of RanBP2 (Nup358), RanGAP1-SUMO1, and Ubc9, regulates the assembly and function of the NPC. The roles of immune signaling in regulation of NPC remain poorly understood. Here, we show that in human and murine T cells, following T-cell receptor (TCR) stimulation, protein kinase C-θ (PKC-θ) directly phosphorylates RanGAP1 to facilitate RanBP2 subcomplex assembly and nuclear import and, thus, the nuclear translocation of AP-1 transcription factor. Mechanistically, TCR stimulation induces the translocation of activated PKC-θ to the NPC, where it interacts with and phosphorylates RanGAP1 on Ser504 and Ser506. RanGAP1 phosphorylation increases its binding affinity for Ubc9, thereby promoting sumoylation of RanGAP1 and, finally, assembly of the RanBP2 subcomplex. Our findings reveal an unexpected role of PKC-θ as a direct regulator of nuclear import and uncover a phosphorylation-dependent sumoylation of RanGAP1, delineating a novel link between TCR signaling and assembly of the RanBP2 NPC subcomplex.
Asunto(s)
Proteínas Activadoras de GTPasa , Chaperonas Moleculares , Proteínas de Complejo Poro Nuclear , Receptores de Antígenos de Linfocitos T/metabolismo , Proteína SUMO-1 , Enzimas Ubiquitina-Conjugadoras , Animales , Proteínas Activadoras de GTPasa/química , Proteínas Activadoras de GTPasa/metabolismo , Humanos , Ratones , Chaperonas Moleculares/química , Chaperonas Moleculares/metabolismo , Poro Nuclear/química , Poro Nuclear/metabolismo , Proteínas de Complejo Poro Nuclear/química , Proteínas de Complejo Poro Nuclear/metabolismo , Fosforilación , Proteína Quinasa C-theta/química , Proteína Quinasa C-theta/metabolismo , Proteína SUMO-1/química , Proteína SUMO-1/metabolismo , Transducción de Señal/fisiología , Enzimas Ubiquitina-Conjugadoras/química , Enzimas Ubiquitina-Conjugadoras/metabolismoRESUMEN
Protein Kinase C theta (PKCθ) is a serine/threonine kinase that belongs to the novel PKC subfamily. In normal tissue, its expression is restricted to skeletal muscle cells, platelets and T lymphocytes in which PKCθ controls several essential cellular processes such as survival, proliferation and differentiation. Particularly, PKCθ has been extensively studied for its role in the immune system where its translocation to the immunological synapse plays a critical role in T cell activation. Beyond its physiological role in immune responses, increasing evidence implicates PKCθ in the pathology of various diseases, especially autoimmune disorders and cancers. In this review, we discuss the implication of PKCθ in various types of cancers and the PKCθ-mediated signaling events controlling cancer initiation and progression. In these types of cancers, the high PKCθ expression leads to aberrant cell proliferation, migration and invasion resulting in malignant phenotype. The recent development and application of PKCθ inhibitors in the context of autoimmune diseases could benefit the emergence of treatment for cancers in which PKCθ has been implicated.
Asunto(s)
Enfermedades Autoinmunes/genética , Regulación Neoplásica de la Expresión Génica , Neoplasias/genética , Proteína Quinasa C-theta/genética , Subunidades de Proteína/genética , Transducción de Señal/genética , Animales , Enfermedades Autoinmunes/enzimología , Enfermedades Autoinmunes/inmunología , Enfermedades Autoinmunes/patología , Movimiento Celular , Proliferación Celular , Humanos , FN-kappa B/genética , FN-kappa B/metabolismo , Invasividad Neoplásica , Metástasis de la Neoplasia , Neoplasias/enzimología , Neoplasias/inmunología , Neoplasias/patología , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Proteína Quinasa C-theta/química , Proteína Quinasa C-theta/inmunología , Subunidades de Proteína/química , Subunidades de Proteína/inmunología , Células Th17/inmunología , Células Th17/patología , Células Th2/inmunología , Células Th2/patologíaRESUMEN
Haemorrhagic disease caused by grass carp reovirus (GCRV) can result in large-scale death of young grass carp, leading to irreparable economic losses that seriously affect large-scale breeding. Protein kinase C (PKC, also known as PRKC) represents a family of serine/threonine protein kinases that includes multiple isozymes in many species. Among these, PKC-θ (PKC theta, also written as PRKCQ) is a novel isoform, mainly expressed in T cells, that is known to be involved in immune system function in mammals. To date, no research on immunological functions of fish Pkc-θ has been reported. To address this issue, we cloned the grass carp pkc-θ gene. Phylogenetic and syntenic analysis showed that this gene is the most evolutionarily conserved relative to zebrafish. Real-time quantitative PCR (RT-qPCR) indicated that pkc-θ was expressed at high levels in the gills and spleen of healthy grass carp. Infection with GCRV down regulated pkc-θ expression in the gills and spleen. Gene products that function upstream and downstream of pkc-θ were up regulated in the gill, but were down-regulated in the spleen. These results suggest that direct or indirect targeting of pkc-θ by GCRV may help the virus evade host immune defences in the spleen. Phorbol ester (PMA) treatment of Jurkat T cells induced translocation of grass carp Pkc-θ from the cytoplasm to the plasma membrane. This response to PMA suggests evolutionary conservation of an immune response function in fish Pkc-θ, as well as conservation of its sequence and structural domains. This study expanded our knowledge of the fish PKC gene family, and explored the role of pkc-θ in function of the grass carp immune system, providing new insights which may facilitate further studies of its biological functions.
Asunto(s)
Carpas/genética , Carpas/inmunología , Enfermedades de los Peces/inmunología , Regulación de la Expresión Génica/inmunología , Inmunidad Innata/genética , Proteína Quinasa C-theta/genética , Proteína Quinasa C-theta/inmunología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Proteínas de Peces/química , Proteínas de Peces/genética , Proteínas de Peces/inmunología , Perfilación de la Expresión Génica/veterinaria , Proteína Quinasa C-theta/química , Distribución Aleatoria , Reoviridae/fisiología , Infecciones por Reoviridae/inmunología , Infecciones por Reoviridae/veterinaria , Alineación de Secuencia/veterinariaRESUMEN
A primitive adaptive immune system has recently been suggested to be present in a basal chordate amphioxus (Branchiostoma belcheri, Bb), making it an ideal model for studying the origin of adaptive immune. The novel protein kinase C isoform PKC-θ, but not its closest isoform PKC-δ, plays a critical role for mammalian T-cell activation via translocation to immunological synapse (IS) mediated by a unique PKC-θ V3 domain containing one PxxP motif. To understand the evolution of this unique PKC-θ V3 domain and the primitive adaptive immune system in amphioxus, we comparatively studied the orthologs of PKC-δ and -θ from amphioxus and other species. Phylogenetic analysis showed BbPKC-δ/θ to be the common ancestor of vertebrate PKC-δ and PKC-θ, with a V3 domain containing two PxxP motifs. One motif is conserved in both zebrafish and mammalian PKC-θ but is absent in PKC-δ V3 domain of these species, and has already emerged in drosophila PKC-δ. The other non-conserved motif emerged in BbPKC-δ/θ, and only retained in Danio rerio PKC-δ (DrPKC-δ) but lost in mammalian PKC-δ and -θ. Comparative analyses of the sequence and function of BbPKC-δ/θ, DrPKC-δ, DrPKC-θ and Homo sapiens PKC-θ (HsPKC-θ) in IS translocation and T-cell receptor (TCR)-induced NF-κB activation revealed that retention of the conserved PxxP motif and loss of the non-conserved PxxP motif in mammalian PKC-θ and loss of both PxxP motifs in mammalian PKC-δ accomplish the unique function of PKC-θ in T cells. Together, this study suggests an evolutionary mechanism for PKC-θ unique V3 and reveals BbPKC-δ/θ is the common ancestor of PKC-δ and -θ with a functional proto-V3 domain, supplying new evidence for the existence of primitive adaptive immune system in amphioxus.
Asunto(s)
Inmunidad Adaptativa/genética , Enfermedades de los Peces/inmunología , Regulación de la Expresión Génica/inmunología , Anfioxos/genética , Anfioxos/inmunología , Proteína Quinasa C-delta/genética , Proteína Quinasa C-delta/inmunología , Proteína Quinasa C-theta/genética , Proteína Quinasa C-theta/inmunología , Secuencia de Aminoácidos , Animales , Perfilación de la Expresión Génica/veterinaria , Anfioxos/enzimología , Filogenia , Proteína Quinasa C-delta/química , Proteína Quinasa C-theta/química , Alineación de Secuencia/veterinariaRESUMEN
The PKC isozymes represent the most prominent family of signaling proteins mediating response to the ubiquitous second messenger diacylglycerol. Among them, PKCθ is critically involved in T-cell activation. Whereas all the other conventional and novel PKC isoforms have twin C1 domains with potent binding activity for phorbol esters, in PKCθ only the C1b domain possesses potent binding activity, with little or no activity reported for the C1a domain. In order to better understand the structural basis accounting for the very weak ligand binding of the PKCθ C1a domain, we assessed the effect on ligand binding of twelve amino acid residues which differed between the C1a and C1b domains of PKCθ. Mutation of Pro9 of the C1a domain of PKCθ to the corresponding Lys9 found in C1b restored in vitro binding activity for [3H]phorbol 12,13-dibutyrate to 3.6â¯nM, whereas none of the other residues had substantial effect. Interestingly, the converse mutation in the C1b domain of Lys9 to Pro9 only diminished binding affinity to 11.7â¯nM, compared to 254â¯nM in the unmutated C1a. In confocal experiments, deletion of the C1b domain from full length PKCθ diminished, whereas deletion of the C1a domain enhanced 5-fold (at 100â¯nM PMA) the translocation to the plasma membrane. We conclude that the Pro168 residue in the C1a domain of full length PKCθ plays a critical role in the ligand and membrane binding, while exchanging the residue (Lys240) at the same position in C1b domain of full length PKCθ only modestly reduced the membrane interaction.
Asunto(s)
Ésteres del Forbol/metabolismo , Dominios y Motivos de Interacción de Proteínas , Proteína Quinasa C-theta/química , Proteína Quinasa C-theta/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Sitios de Unión/genética , Humanos , Modelos Moleculares , Simulación del Acoplamiento Molecular , Mutagénesis Sitio-Dirigida , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Unión Proteica/genética , Dominios y Motivos de Interacción de Proteínas/genética , Proteína Quinasa C-theta/genética , Células Tumorales CultivadasRESUMEN
Protein kinases are ubiquitously expressed as Serine/Threonine kinases, and play a crucial role in cellular activities. Protein kinases have evolved through stringent regulation mechanisms. Protein kinases are also involved in tauopathy, thus are important targets for developing Anti-Alzheimer's disease compounds. Structures with an indole scaffold turned out to be potent new leads. With the aim of developing new inhibitors for human protein kinase C, here we report the generation of four point 3D geometric featured pharmacophore model. In order to identify novel and potent PKCθ inhibitors, the pharmacophore model was screened against 80,000,00 compounds from various chemical databases such as., ZINC, SPEC, ASINEX, which resulted in 127 compound hits, and were taken for molecular docking filters (HTVS, XP docking). After in-depth analysis of binding patterns, induced fit docking (flexible) was employed for six compounds along with the cocrystallized inhibitor. Molecular docking study reveals that compound 6F found to be tight binder at the active site of PKCθ as compared to the cocrystal and has occupancy of 90 percentile. MM-GBSA also confirmed the potency of the compound 6F as better than cocrystal. Molecular dynamics results suggest that compound 6F showed good binding stability of active sites residues similar to cocrystal 7G compound. Present study corroborates the pharmacophore-based virtual screening, and finds the compound 6F as a potent Inhibitor of PKC, having therapeutic potential for Alzheimer's disease. Worldwide, 46.8 million people are believed to be living with Alzheimer's disease. When elderly population increases rapidly and neurodegenerative burden also increases in parallel, we project the findings from this study will be useful for drug developing efforts targeting Alzheimer's disease.
Asunto(s)
Simulación de Dinámica Molecular , Nootrópicos/química , Proteína Quinasa C-theta/química , Inhibidores de Proteínas Quinasas/química , Pirroles/química , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/enzimología , Enfermedad de Alzheimer/fisiopatología , Dominio Catalítico , Bases de Datos de Compuestos Químicos , Diseño de Fármacos , Expresión Génica , Ensayos Analíticos de Alto Rendimiento , Humanos , Enlace de Hidrógeno , Cinética , Ligandos , Simulación del Acoplamiento Molecular , Nootrópicos/farmacología , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Proteína Quinasa C-theta/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Pirroles/farmacología , Relación Estructura-Actividad Cuantitativa , TermodinámicaRESUMEN
AIM: Ligand-based pharmacophore modeling requires long list of inhibitors, while pharmacophores based on single ligand-receptor crystallographic structure can be too restricted or promiscuous. METHODOLOGY: This prompted us to combine simulated annealing molecular dynamics (SAMD) with ligand-receptor contacts analysis as means to construct pharmacophore model(s) from single ligand-receptor complex. Ligand-receptor contacts that survive numerous heating-cooling SAMD cycles are considered critical and are used to guide pharmacophore development. RESULTS: This methodology was implemented to develop pharmacophores for acetylcholinesterase and protein kinase C-θ. The resulting models were validated by receiver-operating characteristic analysis and in vitro bioassay. Assay identified four new protein kinase C-θ inhibitors among captured hits, two of which exhibited nanomolar potencies. CONCLUSION: The results illustrate the ability of the new method to extract valid pharmacophores from single ligand-protein complex.