RESUMEN

Mouse embryonic stem (ES) cells remain "pluripotent" in vitro in the continuous presence of leukemia inhibitory factor (LIF). In the absence of LIF, ES cells are irreversibly committed to differentiate into various lineages. In this study we have set up an in vitro assay based on the anti-apoptotic activity of LIF to distinguish pluripotent from "differentiation-committed" ES cells. We have examined the phosphorylation profiles of known (STAT3 and ERKs) and identified new (ribosomal S6 kinases (RSKs) and cAMP-responsive element-binding protein (CREB)) LIF-regulated targets in ES and in ES-derived neuronal cells. We have demonstrated that although STAT3, a crucial player in the maintenance of ES cell pluripotency, is induced by LIF in all cell types tested, the LIF-dependent activation of RSKs is restricted to ES cells. We have shown that LIF-induced phosphorylation of RSKs in ES cells is dependent on ERKs, whereas STAT3 phosphorylation is not mediated by any known MAPK activities. Our results also demonstrate that the LIF-dependent phosphorylation of CREB is partially under the control of the RSK2 kinase.

Asunto(s)

Proteínas de Unión al ADN/metabolismo , Embrión de Mamíferos/citología , Inhibidores de Crecimiento/metabolismo , Interleucina-6 , Linfocinas/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Quinasas S6 Ribosómicas/metabolismo , Transducción de Señal , Células Madre/metabolismo , Transactivadores/metabolismo , Animales , Apoptosis , Proteína de Unión a CREB , Diferenciación Celular , Factor Inhibidor de Leucemia , Ratones , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Proteínas Nucleares/química , Fosforilación , Factor de Transcripción STAT3 , Células Madre/citología , Transactivadores/química

RESUMEN

The protein kinase ribosomal S6 kinase 2 (RSK2) has been implicated in phosphorylation of transcription factor CREB and histone H3 in response to mitogenic stimulation by epidermal growth factor. Binding of phospho-CREB to the coactivator CBP allows gene activation through recruitment of the basal transcriptional machinery. Acetylation of H3 by histone acetyltransferase (HAT) activities, such as the one carried by CBP, has been functionally coupled to H3 phosphorylation. While various lines of evidence indicate that coupled histone acetylation and phosphorylation may act in concert to induce chromatin remodeling events facilitating gene activation, little is known about the coupling of the two processes at the signaling level. Here we show that CBP and RSK2 are associated in a complex in quiescent cells and that they dissociate within a few minutes upon mitogenic stimulus. CBP preferentially interacts with unphosphorylated RSK2 in a complex where both RSK2 kinase activity and CBP acetylase activity are inhibited. Dissociation is dependent on phosphorylation of RSK2 on Ser227 and results in stimulation of both kinase and HAT activities. We propose a model in which dynamic formation and dissociation of the CBP-RSK2 complex in response to mitogenic stimulation allow regulated phosphorylation and acetylation of specific substrates, leading to coordinated modulation of gene expression.

Asunto(s)

Acetilesterasa/metabolismo , Regulación Enzimológica de la Expresión Génica , Mitógenos/metabolismo , Proteínas Nucleares/metabolismo , Fosfotransferasas/metabolismo , Proteínas Quinasas S6 Ribosómicas/metabolismo , Proteínas de Saccharomyces cerevisiae , Transactivadores/metabolismo , Acetiltransferasas/metabolismo , Animales , Western Blotting , Células COS , Proteína de Unión a CREB , Factor de Crecimiento Epidérmico/farmacología , Glutatión Transferasa/metabolismo , Histona Acetiltransferasas , Humanos , Modelos Biológicos , Ésteres del Forbol/farmacología , Fosforilación , Pruebas de Precipitina , Unión Proteica , Proteínas Recombinantes de Fusión/metabolismo , Transcripción Genética , Activación Transcripcional , Transfección , Rayos Ultravioleta

RESUMEN

RSK2 is a growth factor-regulated serine-threonine protein kinase, acting in the Ras-Mitogen-Activated Protein Kinase (MAPK) signaling pathway. Mutations in the RSK2 gene (RPS6KA3) on chromosome Xp22.2, have been found to cause Coffin-Lowry syndrome (CLS), an X-linked disorder characterized by psychomotor retardation, characteristic facial and digital abnormalities, and progressive skeletal deformations. By screening of 250 patients with clinical features suggestive of Coffin-Lowry syndrome, 71 distinct disease-associated RSK2 mutations have been identified in 86 unrelated families. Thirty-eight percent of mutations are missense mutations, 20% are nonsense mutations, 18% are splicing errors, and 21% are short deletion or insertion events. About 57% of mutations result in premature translation termination, and the vast majority are predicted to cause loss of function of the mutant allele. These changes are distributed throughout the RSK2 gene and show no obvious clustering or phenotypic association. However, some missense mutations are associated with milder phenotypes. In one family, one such mutation was associated solely with mild mental retardation. It is noteworthy that nine mutations were found in female probands, with no affected male relatives, ascertained through learning disability and mild but suggestive facial and digital dysmorphisms.

Asunto(s)

Anomalías Múltiples/genética , Discapacidad Intelectual/genética , Proteínas Quinasas S6 Ribosómicas/genética , Cromosoma X/genética , Anomalías Múltiples/patología , Ligamiento Genético , Genotipo , Humanos , Mutación , Fenotipo , Literatura de Revisión como Asunto , Síndrome

RESUMEN

Ribosomal S6 kinases (RSKs) are serine/threonine kinases activated by mitogenic signals through the Mitogen-Activated Protein Kinases/Extracellular Signal-Regulated Kinases (MAPK/ERK). RSKs contain two heterologous complete protein kinase domains. Phosphorylation by ERK of the C-terminal kinase domain allows activation of the N-terminal kinase domain, which mediates substrate phosphorylation. In human, there are three isoforms of RSK (RSK1, RSK2, RSK3), whose functional specificity remains undefined. Importantly, we have shown that mutations in the RSK2 gene lead to the Coffin-Lowry syndrome (CLS). In this study, we characterize two monoclonal antibodies raised against phosphorylated forms of the N- and C-terminal domain of RSK2 (P-S227 and P-T577, respectively). Using these two antibodies, we show that stress signals, such as UV light, induce phosphorylation and activation of the three RSKs to an extent which is comparable to Epidermal Growth Factor (EGF)-mediated activation. The use of specific kinase inhibitors indicates that UV-induced phosphorylation and activation of RSK2 is mediated by the MAPK/ERK pathway, but that the Stress-Activated Protein Kinase 2 (SAPK2)/p38 pathway is also involved. These results modify the view of RSKs as kinases restricted to the mitogenic response and reveal a previously unappreciated role of MAPKs in stress induced signaling. Oncogene (2000) 19, 4221 - 4229

Asunto(s)

Isoenzimas/efectos de la radiación , Sistema de Señalización de MAP Quinasas/efectos de la radiación , Procesamiento Proteico-Postraduccional/efectos de la radiación , Proteínas Quinasas S6 Ribosómicas/efectos de la radiación , Estrés Fisiológico/fisiopatología , Rayos Ultravioleta , Células 3T3/enzimología , Células 3T3/efectos de la radiación , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/inmunología , Células COS/enzimología , Células COS/efectos de la radiación , Células Cultivadas , Inhibidores Enzimáticos/farmacología , Factor de Crecimiento Epidérmico/farmacología , Fibroblastos/enzimología , Fibroblastos/efectos de la radiación , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Sistema de Señalización de MAP Quinasas/fisiología , Ratones , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Proteínas Quinasas Activadas por Mitógenos/fisiología , Datos de Secuencia Molecular , Fosforilación/efectos de la radiación , Estructura Terciaria de Proteína , Proteínas Quinasas S6 Ribosómicas/antagonistas & inhibidores , Proteínas Quinasas S6 Ribosómicas/genética , Proteínas Quinasas S6 Ribosómicas/inmunología , Proteínas Quinasas S6 Ribosómicas/metabolismo , Acetato de Tetradecanoilforbol/farmacología , Proteínas Quinasas p38 Activadas por Mitógenos

RESUMEN

The 90 kDa ribosomal S6 kinase-2 (RSK2) is a growth factor-stimulated protein kinase with two kinase domains. The C-terminal kinase of RSK2 is activated by ERK-type MAP kinases, leading to autophosphorylation of RSK2 at Ser386 in a hydrophobic motif. The N-terminal kinase is activated by 3-phosphoinositide-dependent protein kinase-1 (PDK1) through phosphorylation of Ser227, and phosphorylates the substrates of RSK. Here, we identify Ser386 in the hydrophobic motif of RSK2 as a phosphorylation-dependent docking site and activator of PDK1. Treatment of cells with growth factor induced recruitment of PDK1 to the Ser386-phosphorylated hydrophobic motif and phosphorylation of RSK2 at Ser227. A RSK2-S386K mutant showed no interaction with PDK1 or phosphorylation at Ser227. Interaction with Ser386-phosphorylated RSK2 induced autophosphorylation of PDK1. Addition of a synthetic phosphoSer386 peptide (RSK2(373-396)) increased PDK1 activity 6-fold in vitro. Finally, mutants of RSK2 and MSK1, a RSK-related kinase, with increased affinity for PDK1, were constitutively active in vivo and phosphorylated histone H3. Our results suggest a novel regulatory mechanism based on phosphoserine-mediated recruitment of PDK1 to RSK2, leading to coordinated phosphorylation and activation of PDK1 and RSK2.

Asunto(s)

Proteínas Quinasas Activadas por Mitógenos/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Quinasas S6 Ribosómicas 90-kDa , Serina , Proteínas Quinasas Dependientes de 3-Fosfoinosítido , Animales , Sitios de Unión , Catálisis , Activación Enzimática , Humanos , Ratones , Proteínas Quinasas Activadas por Mitógenos/genética , Modelos Biológicos , Mutación , Fosforilación , Unión Proteica , Estructura Terciaria de Proteína , Proteínas Quinasas S6 Ribosómicas

RESUMEN

The antagonist activity of short-chain toxins from snake venoms toward the nicotinic acetylcholine receptor (nAChR) is neutralized upon binding to a toxin-specific monoclonal antibody called Malpha2-3 (1). To establish the molecular basis of this specificity, we predicted from both mutational analyses and docking procedures the structure of the Malpha2-3-toxin complex. From knowledge of the functional paratope and epitope, and using a double-mutation cycle procedure, we gathered evidence that Asp(31) in complementarity determining region 1H is close to, and perhaps interacts with, Arg(33) in the antigen. The use of this pair of proximate residues during the selection procedure yielded three models based on docking calculations. The selected models predicted the proximity of Tyr(49) and/or Tyr(50) in the antibody to Lys(47) in the toxin. This was experimentally confirmed using another round of double-mutation cycles. The two models finally selected were submitted to energy minimization in a CHARMM22 force field, and were characterized by a root mean square deviation of 7.0 +/- 2.9 A. Both models display most features of antibody-antigen structures. Since Malpha2-3 also partially mimics some binding properties of nAChR, these structural features not only explain its fine specificity of recognition, but may also further clarify how toxins bind to nAChR.

Asunto(s)

Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/inmunología , Receptores Nicotínicos/inmunología , Venenos de Serpiente/química , Sustitución de Aminoácidos , Animales , Ácido Aspártico , Sitios de Unión , Ensayo de Inmunoadsorción Enzimática , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Antagonistas Nicotínicos/química , Antagonistas Nicotínicos/farmacología , Conformación Proteica , Receptores Nicotínicos/efectos de los fármacos , Receptores Nicotínicos/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/inmunología , Venenos de Serpiente/farmacocinética , Venenos de Serpiente/farmacología , Serpientes , Termodinámica

RESUMEN

An unreported missense mutation of the ribosomal S6 kinase 2 (RSK2) gene has been identified in two male sibs with a mild form of Coffin-Lowry syndrome (CLS) inherited from their healthy mother. They exhibit transient severe hypotonia, macrocephaly, delay in closure of the fontanelles, normal gait, and mild mental retardation, associated in the first sib with transient autistic behaviour. Some dysmorphic features of CLS (in particular forearm fullness and tapering fingers) and many atypical findings (some of which were reminiscent of FG syndrome) were observed as well. The moderate phenotypic expression of this mutation extends the CLS phenotype to include less severe mental retardation and minor, hitherto unreported signs. The missense mutation identified may be less deleterious than those previously described. As this mutation occurs in a protein domain with no predicted function, it could be responsible for a conformational change affecting the protein catalytic function, since a non-polar amino acid is replaced by a charged residue.

Asunto(s)

Anomalías Múltiples/genética , Discapacidad Intelectual/genética , Mutación Missense , Proteínas Quinasas S6 Ribosómicas/genética , Niño , Ligamiento Genético , Humanos , Masculino , Fenotipo , Síndrome , Cromosoma X

Asunto(s)

Anomalías Múltiples/genética , Discapacidad Intelectual/genética , Análisis Mutacional de ADN , Salud de la Familia , Femenino , Humanos , Masculino , Mutación , Linaje , Polimorfismo Conformacional Retorcido-Simple , Proteínas Quinasas S6 Ribosómicas/genética , Síndrome

Asunto(s)

Discapacidad Intelectual/genética , Proteínas Quinasas/genética , Proteínas Quinasas S6 Ribosómicas 90-kDa , Secuencia de Aminoácidos , Secuencia de Bases , Femenino , Ligamiento Genético , Humanos , Discapacidad Intelectual/enzimología , Masculino , Mutación Missense , Linaje , Polimorfismo Conformacional Retorcido-Simple , Proteínas Quinasas/metabolismo , Cromosoma X/genética

RESUMEN

Coffin-Lowry syndrome (CLS) is a syndromal form of X linked mental retardation, in which some associated facial, hand, and skeletal abnormalities are diagnostic features. Accurate diagnosis, critical for genetic counselling, is often difficult, especially in early childhood. We have recently shown that Coffin-Lowry syndrome is caused by mutations in the gene encoding RSK2, a growth factor regulated protein kinase. RSK2 mutations are very heterogeneous and most of them lead to premature termination of translation or to loss of phosphotransferase activity or both. In the present study, we have evaluated immunoblot and RSK2 kinase assays as a rapid and simple diagnostic test for CLS, using cultured lymphoblastoid or fibroblast cell lines. Western blot analysis failed to detect RSK2 in six patients, suggesting the presence of truncated proteins in these patients. This conclusion was confirmed in four patients, in whom the causative mutations, all leading to premature termination of translation, were identified. Of four patients showing a normal amount of RSK2 protein on western blot and tested for RSK2 phosphotransferase activity, one had a dramatically impaired activity. Analysis of the RSK2 cDNA sequence in this patient showed a mutation of a putative phosphorylation site that would be critical for RSK2 activity. Preliminary results show that, at least, the western blot protocol can be successfully applied to lymphocyte protein extracts prepared directly from blood samples. These assays promise to become important diagnostic tools for CLS, particularly with regard to very young patients with no family history of the condition.

Asunto(s)

Anomalías Múltiples/enzimología , Discapacidad Intelectual/enzimología , Proteínas Quinasas S6 Ribosómicas/análisis , Cromosoma X , Adolescente , Adulto , Western Blotting , Niño , Preescolar , Pruebas Enzimáticas Clínicas , Humanos , Masculino , Síndrome , Factores de Tiempo

RESUMEN

Coffin-Lowry syndrome (CLS) is an X-linked disorder characterized by severe psychomotor retardation, facial and digital dysmorphisms, and progressive skeletal deformations. By using a positional cloning approach, we have recently shown that mutations in the gene coding for the RSK2 serine-threonine protein kinase are responsible for this syndrome. To facilitate mutational analysis, we have now determined the genomic structure of the human RSK2 gene. The open reading frame of the RSK2 coding region is split into 22 exons. Primers were designed for PCR amplification of single exons from genomic DNA and subsequent single-strand conformation polymorphism analysis. We screened 37 patients with clinical features suggestive of CLS. Twenty-five nucleotide changes predicted to be disease-causing mutations were identified, including eight splice-site alterations, seven nonsense mutations, five frameshift mutations, and five missense mutations. Twenty-three of them were novel mutations. Coupled with previously reported mutations, these findings bring the total of different RSK2 mutations to 34. These are distributed throughout the RSK2 gene, with no clustering, and all but two, which have been found in two independent patients, are unique. A very high (68%) rate of de novo mutations was observed. It is noteworthy also that three mutations were found in female probands, with no affected male relatives, ascertained through learning disability and mild but suggestive facial and digital dysmorphisms. No obvious correlation was observed between the position or type of the RSK2 mutations and the severity or particular clinical features of CLS.

Asunto(s)

Anomalías Múltiples/genética , Heterogeneidad Genética , Discapacidad Intelectual/genética , Mutación , Proteínas Quinasas S6 Ribosómicas/genética , Anomalías Múltiples/enzimología , Adolescente , Adulto , Alelos , Secuencia de Bases , Niño , Preescolar , Análisis Mutacional de ADN , Exones/genética , Femenino , Ligamiento Genético , Humanos , Discapacidad Intelectual/enzimología , Intrones/genética , Masculino , Sistemas de Lectura Abierta/genética , Fenotipo , Polimorfismo Conformacional Retorcido-Simple , Síndrome , Cromosoma X

RESUMEN

We have identified a Coffin-Lowry syndrome pedigree where the disorder is associated with a novel splice site mutation in the RSK2 gene, leading to in-phase skipping of exon 5. Western blot analysis, using an antibody directed against the C-terminus of RSK2, failed to reveal RSK2 in this patient, suggesting strongly that the resulting internally deleted protein is unstable. The mutation was present in the DNA of one affected son and one manifesting daughter but was absent in two asymptomatic daughters, who carry the at-risk haplotype, and in the mother's somatic cell (lymphocyte) DNA. The results are consistent with the mutation arising as a postzygotic event in the mother, who therefore is a germinal mosaic. The application of linked markers to identify the disease allele for conventional genetic counselling would have been misleading in this family. This observation again highlights the importance of precise identification of the disease-causing mutation.

Asunto(s)

Anomalías Múltiples/genética , Células Germinativas , Discapacidad Intelectual/genética , Mosaicismo , Secuencia de Aminoácidos , Secuencia de Bases , ADN Complementario , Humanos , Recién Nacido , Masculino , Datos de Secuencia Molecular , Linaje , Polimorfismo Conformacional Retorcido-Simple , Empalme del ARN , Síndrome

RESUMEN

Malpha2-3 is a monoclonal antibody that partially mimics the nicotinic acetylcholine receptor (AChR). Its three-dimensional structure has been previously predicted by molecular modeling, suggesting that 29 complementarity determining region (CDR) residues and 2 framework residues are exposed to solvent. To identify the antibody residues that bind to the antigen, i.e. snake toxin that binds specifically to AChR, we (i) produced the scFv form of Malpha2-3 fused to alkaline phosphatase, in the periplasmic space of Escherichia coli; (ii) submitted approximately 75% of exposed residues of the fused scFv to individual or combined mutations, and (iii) identified the residues whose mutations affect scFv binding to the toxin, using a sensitive enzyme-linked immunosorbent assay. 11 critical residues were identified, including 8 heavy chain residues, 2 framework residues, and 1 light chain residue. They cover a surface of approximately 800 A2, with a subset of most critical residues (VHD31, VHY32, and VHG101) and several aromatic residues. This functional architecture not only constitutes a plausible complementary binding surface for the snake toxin but also offers a structural basis to ultimately understand the capacity of the antibody to partially mimic AChR.

Asunto(s)

Anticuerpos Monoclonales/inmunología , Imitación Molecular , Receptores Colinérgicos/inmunología , Fosfatasa Alcalina/genética , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/genética , Sitios de Unión de Anticuerpos , Ensayo de Inmunoadsorción Enzimática , Escherichia coli/genética , Fragmentos de Inmunoglobulinas/química , Fragmentos de Inmunoglobulinas/genética , Fragmentos de Inmunoglobulinas/inmunología , Ratones , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Receptores Colinérgicos/química , Receptores Colinérgicos/genética , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Homología de Secuencia de Aminoácido , Torpedo

RESUMEN

In several instances, a monoclonal antibody raised against a receptor ligand has been claimed to mimic the ligand receptor. Thus, a specific monoclonal antibody (Malpha2-3) raised against a short-chain toxin from snake was proposed to mimic the nicotinic acetylcholine receptor (AChR) (). Further confirming this mimicry, we show that (i) like AChR, Malpha2-3 elicits anti-AChR antibodies, which in turn elicit anti-toxin antibodies; and (ii) the region 106-122 of the alpha-chain of AChR shares 66% primary structure identity with complementarity-determining regions of Malpha2-3. Also, a mutational analysis of erabutoxin a reveals that the epitope recognized by Malpha2-3 consists of 10 residues, distributed within the three toxin loops. Eight of these residues also belong to the 10-residue epitope recognized by AChR, a result that offers an explanation as to the functional similarities between the receptor and the antibody. Strikingly, however, most of the residues common to the two epitopes contribute differentially to the energetic formation of the antibody-toxin and the receptor-toxin complexes. Together, the data suggest that the mimicry between AChR and Malpha2-3 is partial only.

Asunto(s)

Anticuerpos Monoclonales/metabolismo , Receptores Colinérgicos/metabolismo , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/inmunología , Proteínas Neurotóxicas de Elápidos/inmunología , Proteínas Neurotóxicas de Elápidos/metabolismo , Reacciones Cruzadas , Curare/inmunología , Curare/metabolismo , Epítopos/química , Epítopos/inmunología , Erabutoxinas/inmunología , Erabutoxinas/metabolismo , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Receptores Colinérgicos/inmunología , Alineación de Secuencia , Torpedo