RESUMO
Persistent activation of p42 mitogen-activated protein kinase (p42 MAPK) during mitosis induces a "cytostatic factor" arrest, the arrest responsible for preventing the parthenogenetic activation of unfertilized eggs. The protein kinase p90 Rsk is a substrate of p42 MAPK; thus, the role of p90 Rsk in p42 MAPK-induced mitotic arrest was examined. Xenopus laevis egg extracts immunodepleted of Rsk lost their capacity to undergo mitotic arrest in response to activation of the Mos-MEK-1-p42 MAPK cascade of protein kinases. Replenishing Rsk-depleted extracts with catalytically competent Rsk protein restored the ability of the extracts to undergo mitotic arrest. Rsk appears to be essential for cytostatic factor arrest.
Assuntos
Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Mitose , Óvulo/citologia , Proteínas Serina-Treonina Quinases , Proteínas Proto-Oncogênicas c-mos/metabolismo , Proteínas Quinases S6 Ribossômicas/metabolismo , Animais , Proteína Quinase CDC2/metabolismo , Extratos Celulares , Ativação Enzimática , MAP Quinase Quinase 1 , Sistema de Sinalização das MAP Quinases , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Dados de Sequência Molecular , Óvulo/enzimologia , Proteínas Proto-Oncogênicas c-mos/farmacologia , Xenopus laevisRESUMO
The 90-kDa ribosomal S6 kinases, the p90 Rsks, are a family of intracellular serine/threonine protein kinases distinguished by two distinct kinase domains. Rsks are activated downstream of the ERK1 (p44) and ERK2 (p42) mitogen-activated protein (MAP) kinases in diverse biological contexts, including progression through meiotic and mitotic M phases in Xenopus oocytes and cycling Xenopus egg extracts, and are critical for the M phase functions of Xenopus p42 MAPK. Here we report the cloning and biochemical characterization of Xenopus Rsk2. Xenopus Rsk1 and Rsk2 are specifically recognized by commercially available RSK1 and RSK2 antisera on immunoblots, but both Rsk1 and Rsk2 are immunoprecipitated by RSK1, RSK2, and RSK3 sera. Rsk2 is about 20-fold more abundant than the previously described Xenopus Rsk1 protein; their concentrations are approximately 120 and 5 nm, respectively. Rsk2, like Rsk1, forms a heteromeric complex with p42 MAP kinase. This interaction depends on sequences at the extreme C terminus of Rsk2 and can be disrupted by a synthetic peptide derived from the C-terminal 20 amino acids of Rsk2. Finally, we demonstrate that p42 MAP kinase can activate recombinant Rsk2 in vitro to a specific activity comparable to that found in Rsk2 that has been activated maximally in vivo. These findings underscore the importance of the Rsk2 isozyme in the M phase functions of p42 MAP kinase and provide tools for further examining Rsk2 function.
Assuntos
Oócitos/enzimologia , Óvulo/enzimologia , Proteínas Quinases S6 Ribossômicas/genética , Proteínas Quinases S6 Ribossômicas/metabolismo , Sequência de Aminoácidos , Animais , Clonagem Molecular , Feminino , Humanos , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Filogenia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas Quinases S6 Ribossômicas/química , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , XenopusRESUMO
Previous work on the responses of mitogen-activated protein (MAP) kinase cascade components in a Xenopus oocyte extract system demonstrated that p42 MAP kinase (MAPK) exhibits a sharp, sigmoidal stimulus/response curve, rather than a more typical hyperbolic curve. One plausible explanation for this behavior requires the assumption that MAP kinase kinase (MAPKK) carries out its dual phosphorylation of p42 MAPK by a distributive mechanism, where MAPKK dissociates from MAPK between the first and second phosphorylations, rather than a processive mechanism, where MAPKK carries out both phosphorylations before dissociating. Here we have investigated the mechanism through which a constitutively active form of human MAPKK-1 (denoted MAPKK-1 R4F or MAPKK-1*) phosphorylates Xenopus p42 MAPK in vitro. We found that the amount of monophosphorylated MAPK formed during the phosphorylation reaction exceeded the amount of MAPKK-1* present, which would not be possible if the phosphorylation occurred exclusively by a processive mechanism. The monophosphorylated MAPK was phosphorylated predominantly on tyrosine, but a small proportion was phosphorylated on threonine, indicating that the first phosphorylation is usually, but not invariably, the tyrosine phosphorylation. We also found that the rate at which pulse-labeled monophosphorylated MAPK became bisphosphorylated depended on the MAPKK-1* concentration, behavior that is predicted by the distributive model but incompatible with the processive model. These findings indicate that MAPKK-1* phosphorylates p42 MAPK by a two-collision, distributive mechanism rather than a single-collision, processive mechanism, and provide a mechanistic basis for understanding how MAP kinase can convert graded inputs into switch-like outputs.
Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Animais , Humanos , Proteína Quinase 1 Ativada por Mitógeno , Quinases de Proteína Quinase Ativadas por Mitógeno , Modelos Químicos , Mapeamento de Peptídeos , Fosforilação , Proteínas Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Treonina/metabolismo , Tripsina/metabolismo , XenopusRESUMO
We have used an in vitro protein synthesis system to construct a very large library of peptides displayed on polysomes. A pool of DNA sequences encoding 10(12) random decapeptides was incubated in an Escherichia coli S30 coupled transcription/translation system. Polysomes were isolated and screened by affinity selection of the nascent peptides on an immobilized monoclonal antibody specific for the peptide dynorphin B. The mRNA from the enriched pool of polysomes was recovered, copied into cDNA, and amplified by the polymerase chain reaction (PCR) to produce template for the next round of in vitro synthesis and selection. A portion of the amplified template from each round was cloned into a filamentous phagemid vector to determine the specificity of peptide binding by phage ELISA and to sequence the DNA. After four rounds of affinity selection, the majority of clones encoded peptides that bound specifically to the antibody and contained a consensus sequence that is similar to the known epitope for the antibody. Synthetic peptides corresponding to several of these sequences have binding affinities ranging from 7 to 140 nM. The in vitro system described here has the potential to screen peptide libraries that are three to six orders of magnitude larger than current biological peptide display systems.
Assuntos
Dinorfinas/análogos & derivados , Endorfinas/química , Peptídeos/química , Polirribossomos/metabolismo , Sequência de Aminoácidos , Anticorpos Monoclonais , Sequência de Bases , Primers do DNA/química , Dinorfinas/química , Epitopos , Escherichia coli , Técnicas In Vitro , Ligantes , Dados de Sequência Molecular , Ligação Proteica , Proteínas Recombinantes de Fusão/química , Relação Estrutura-AtividadeRESUMO
Peptide ligands for the antigen binding site of the surface immunoglobulin receptor of a human B-cell lymphoma cell line were identified with the use of filamentous phage libraries displaying random 8- and 12-amino acid peptides. Corresponding synthetic peptides bound specifically to the antigen binding site of this immunoglobulin receptor and blocked the binding of an anti-idiotype antibody. The ligands, when conjugated to form dimers or tetramers, induced cell death by apoptosis in vitro with an IC50 between 40 and 200 nM. This effect was associated with specific stimulation of intracellular protein tyrosine phosphorylation.