RESUMEN
The growth factor/insulin-stimulated AGC kinases share an activation mechanism based on three phosphorylation sites. Of these, only the role of the activation loop phosphate in the kinase domain and the hydrophobic motif (HM) phosphate in a C-terminal tail region are well characterized. We investigated the role of the third, so-called turn motif phosphate, also located in the tail, in the AGC kinases PKB, S6K, RSK, MSK, PRK and PKC. We report cooperative action of the HM phosphate and the turn motif phosphate, because it binds a phosphoSer/Thr-binding site above the glycine-rich loop within the kinase domain, promoting zipper-like association of the tail with the kinase domain, serving to stabilize the HM in its kinase-activating binding site. We present a molecular model for allosteric activation of AGC kinases by the turn motif phosphate via HM-mediated stabilization of the alphaC helix. In S6K and MSK, the turn motif phosphate thereby also protects the HM from dephosphorylation. Our results suggest that the mechanism described is a key feature in activation of upto 26 human AGC kinases.
Asunto(s)
Péptidos y Proteínas de Señalización Intercelular/fisiología , Modelos Moleculares , Proteínas Serina-Treonina Quinasas/química , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Sitios de Unión , Activación Enzimática , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Datos de Secuencia Molecular , Fosforilación , Proteínas Serina-Treonina Quinasas/fisiología , Estructura Secundaria de Proteína , Transducción de SeñalRESUMEN
The gamma-actin gene (ACTG1) encodes a major cytoskeletal protein of the sensory hair cells of the cochlea. Recently, mutations in ACTG1 were found to cause autosomal dominant, progressive, sensorineural hearing impairment linked to the DFNA20/26 locus on chromosome 17q25.3 in four American families and in one Dutch family. We report here the linkage of autosomal dominant, progressive, sensorineural hearing impairment in a large Norwegian family to the DFNA20/26 locus. Sequencing of ACTG1 identified a novel missense mutation (c.1109T>C; p.V370A) segregating with the hearing loss. Functional analysis in yeast showed that the p.V370A mutation restricts cell growth at elevated temperature or under hyperosmolar stress. Molecular modelling suggested that the p.V370A mutation modestly alters a site for protein-protein interaction in gamma-actin and thereby modestly alters gamma-actin-based cytoskeletal structures. Nineteen Norwegian and Danish families with autosomal, dominant hearing impairment were analyzed for mutations in ACTG1 by sequencing, but no disease-associated mutations were identified. Finally, a long-term follow-up of the hearing loss progression associated with the p.V370A mutation in ACTG1 is provided. The present study expands our understanding of the genotype-phenotype relationship of this deafness gene and provides a sensitive and simple functional assay for missense mutations in this gene, which may assist future molecular diagnosis of autosomal-dominant hearing impairment. Finally, the present results do not indicate that mutations in ACTG1 are a frequent cause of autosomal-dominant postlingual sensorineural hearing impairment in Norway nor Denmark.
Asunto(s)
Actinas/genética , Pérdida Auditiva Sensorineural/genética , Mutación Missense/genética , Análisis Mutacional de ADN , Estudios de Seguimiento , Genes Dominantes , Noruega , Linaje , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crecimiento & desarrolloRESUMEN
Methylation of lysine and arginine residues on histone tails affects chromatin structure and gene transcription. Tri- and dimethylation of lysine 9 on histone H3 (H3K9me3/me2) is required for the binding of the repressive protein HP1 and is associated with heterochromatin formation and transcriptional repression in a variety of species. H3K9me3 has long been regarded as a 'permanent' epigenetic mark. In a search for proteins and complexes interacting with H3K9me3, we identified the protein GASC1 (gene amplified in squamous cell carcinoma 1), which belongs to the JMJD2 (jumonji domain containing 2) subfamily of the jumonji family, and is also known as JMJD2C. Here we show that three members of this subfamily of proteins demethylate H3K9me3/me2 in vitro through a hydroxylation reaction requiring iron and alpha-ketoglutarate as cofactors. Furthermore, we demonstrate that ectopic expression of GASC1 or other JMJD2 members markedly decreases H3K9me3/me2 levels, increases H3K9me1 levels, delocalizes HP1 and reduces heterochromatin in vivo. Previously, GASC1 was found to be amplified in several cell lines derived from oesophageal squamous carcinomas, and in agreement with a contribution of GASC1 to tumour development, inhibition of GASC1 expression decreases cell proliferation. Thus, in addition to identifying GASC1 as a histone trimethyl demethylase, we suggest a model for how this enzyme might be involved in cancer development, and propose it as a target for anti-cancer therapy.
Asunto(s)
Histonas/química , Histonas/metabolismo , Lisina/química , Lisina/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas Oncogénicas/metabolismo , Factores de Transcripción/metabolismo , Proliferación Celular , Células HeLa , Humanos , Hidroxilación , Histona Demetilasas con Dominio de Jumonji , Metilación , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/clasificación , Proteínas de Neoplasias/genética , Proteínas Oncogénicas/antagonistas & inhibidores , Proteínas Oncogénicas/clasificación , Proteínas Oncogénicas/genética , Oncogenes/genética , Unión Proteica , Especificidad por Sustrato , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/clasificación , Factores de Transcripción/genéticaRESUMEN
Phosphoinositide-dependent kinase-1 (PDK1) mediates activation of many AGC kinases by docking onto a phosphorylated hydrophobic motif located C-terminal of the catalytic domain in the AGC kinase. The interaction shifts PDK1 into a conformation with increased catalytic activity and leads to autophosphorylation of PDK1. We demonstrate here that addition of a hydrophobic motif peptide increases the catalytic activity of PDK1 orthologues from Homo sapiens, Aplysia californica, Arabidopsis thaliana, Schizosaccharomyces pombe (ksg1), and Saccharomyces cerevisiae (Pkh1 and Pkh2) 2- to 12-fold. Furthermore, the hydrophobic motif peptide increases autophosphorylation of PDK1 from Homo sapiens, S. pombe, and S. cerevisiae (Phk2). Our results suggest that PDK1 interaction and activation by the hydrophobic motif of AGC kinases is a central mechanism in PDK1 function, which is conserved during eukaryotic evolution.
Asunto(s)
Proteínas Quinasas/química , Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Quinasas Dependientes de 3-Fosfoinosítido , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Aplysia/enzimología , Aplysia/genética , Arabidopsis/enzimología , Arabidopsis/genética , Dominio Catalítico , Activación Enzimática , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Técnicas In Vitro , Datos de Secuencia Molecular , Filogenia , Conformación Proteica , Proteínas Quinasas/genética , Proteínas Serina-Treonina Quinasas/genética , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Schizosaccharomyces/enzimología , Schizosaccharomyces/genéticaRESUMEN
The flavin enzyme dihydroorotate dehydrogenase (DHOD; EC 1.3.99.11) catalyzes the oxidation of dihydroorotate to orotate, the fourth step in the de novo pyrimidine biosynthesis of UMP. The enzyme is a promising target for drug design in different biological and clinical applications for cancer and arthritis. The first crystal structure of the class 2 dihydroorotate dehydrogenase from rat has been determined in complex with its two inhibitors brequinar and atovaquone. These inhibitors have shown promising results as anti-proliferative, immunosuppressive, and antiparasitic agents. A unique feature of the class 2 DHODs is their N-terminal extension, which folds into a separate domain comprising two alpha-helices. This domain serves as the binding site for the two inhibitors and the respiratory quinones acting as the second substrate for the class 2 DHODs. The orientation of the first N-terminal helix is very different in the two complexes of rat DHOD (DHODR). Binding of atovaquone causes a 12 A movement of the first residue in the first alpha-helix. Based on the information from the two structures of DHODR, a model for binding of the quinone and the residues important for the interactions could be defined. His 56 and Arg 136, which are fully conserved in all class 2 DHODs, seem to play a key role in the interaction with the electron acceptor. The differences between the membrane-bound rat DHOD and membrane-associated class 2 DHODs exemplified by the Escherichia coli DHOD has been investigated by GRID computations of the hydrophobic probes predicted to interact with the membrane.
Asunto(s)
Compuestos de Anilina/metabolismo , Compuestos de Bifenilo/metabolismo , Inhibidores Enzimáticos/metabolismo , Hidroxibutiratos/metabolismo , Inmunosupresores/metabolismo , Naftoquinonas/metabolismo , Ácido Orótico/análogos & derivados , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/metabolismo , Secuencia de Aminoácidos , Compuestos de Anilina/química , Animales , Atovacuona , Compuestos de Bifenilo/química , Catálisis , Crotonatos , Cristalografía por Rayos X , Dihidroorotato Deshidrogenasa , Diseño de Fármacos , Inhibidores Enzimáticos/química , Enlace de Hidrógeno , Hidroxibutiratos/química , Inmunosupresores/química , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Molecular , Naftoquinonas/química , Nitrilos , Ácido Orótico/química , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/química , Unión Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Ratas , Alineación de Secuencia , Especificidad por Sustrato , ToluidinasRESUMEN
The growth factor-activated AGC protein kinases RSK, S6K, PKB, MSK and SGK are activated by serine/threonine phosphorylation in the activation loop and in the hydrophobic motif, C-terminal to the kinase domain. In some of these kinases, phosphorylation of the hydrophobic motif creates a specific docking site that recruits and activates PDK1, which then phosphorylates the activation loop. Here, we discover a pocket in the kinase domain of PDK1 that recognizes the phosphoserine/phosphothreonine in the hydrophobic motif by identifying two oppositely positioned arginine and lysine residues that bind the phosphate. Moreover, we demonstrate that RSK2, S6K1, PKBalpha, MSK1 and SGK1 contain a similar phosphate-binding pocket, which they use for intramolecular interaction with their own phosphorylated hydrophobic motif. Molecular modelling and experimental data provide evidence for a common activation mechanism in which the phosphorylated hydrophobic motif and activation loop act on the alphaC-helix of the kinase structure to induce synergistic stimulation of catalytic activity. Sequence conservation suggests that this mechanism is a key feature in activation of >40 human AGC kinases.