RESUMEN
Phosphopeptides have been used to study phosphorylation and dephosphorylation, which are key events in protein expression. Backbone cyclization has been shown to increase the stability and selectivity of peptides. Backbone cyclic peptides with conformational diversity have produced bioactive peptides with improved pharmaceutical properties, metabolic stability, and enhanced intestinal permeability. We demonstrate a successful methodology for incorporating phospho-amino acids into backbone cyclic peptides. The nuclear factor-kappa B (NF-kappaB) is a latent mammalian protein prototype of dimeric transcription factors that exists in all cell types and plays a pivotal role in a huge number of genes, such as those responsible for chronic and acute inflammatory diseases. To inhibit NF-kappaB, backbone cyclic phosphopeptides were designed and synthesized based on the conserved sequence of the Inhibitor kappa B (IkappaB). The peptides were screened for inhibiting IkappaB ubiquitylation. The best compound showed 90% inhibition at a concentration of 3 microM, and its solution structure showed similarity to a related beta-catenin protein. This general methodology can be use for synthesizing cyclic phosphorylated, as well as backbone cyclic phosphorylated peptides for various biological targets.
Asunto(s)
Proteínas I-kappa B/química , FN-kappa B/química , Péptidos Cíclicos/química , Fosfopéptidos/química , Secuencia de Aminoácidos , Animales , Cromatografía Líquida de Alta Presión , Cromatografía en Capa Delgada , Secuencia Conservada , Drosophila , Proteínas de Drosophila/síntesis química , Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , Quinasa I-kappa B/metabolismo , Espectroscopía de Resonancia Magnética , Espectrometría de Masas , Modelos Moleculares , Inhibidor NF-kappaB alfa , Péptidos Cíclicos/síntesis química , Péptidos Cíclicos/metabolismo , Fosfopéptidos/síntesis química , Fosfopéptidos/metabolismo , FosforilaciónRESUMEN
The Wnt pathway controls numerous developmental processes via the beta-catenin-TCF/LEF transcription complex. Deregulation of the pathway results in the aberrant accumulation of beta-catenin in the nucleus, often leading to cancer. Normally, cytoplasmic beta-catenin associates with APC and axin and is continuously phosphorylated by GSK-3beta, marking it for proteasomal degradation. Wnt signaling is considered to prevent GSK-3beta from phosphorylating beta-catenin, thus causing its stabilization. However, the Wnt mechanism of action has not been resolved. Here we study the regulation of beta-catenin phosphorylation and degradation by the Wnt pathway. Using mass spectrometry and phosphopeptide-specific antibodies, we show that a complex of axin and casein kinase I (CKI) induces beta-catenin phosphorylation at a single site: serine 45 (S45). Immunopurified axin and recombinant CKI phosphorylate beta-catenin in vitro at S45; CKI inhibition suppresses this phosphorylation in vivo. CKI phosphorylation creates a priming site for GSK-3beta and is both necessary and sufficient to initiate the beta-catenin phosphorylation-degradation cascade. Wnt3A signaling and Dvl overexpression suppress S45 phosphorylation, thereby precluding the initiation of the cascade. Thus, a single, CKI-dependent phosphorylation event serves as a molecular switch for the Wnt pathway.
Asunto(s)
Proteínas del Citoesqueleto/metabolismo , Proteínas Quinasas/metabolismo , Proteínas/metabolismo , Proteínas Represoras , Serina/metabolismo , Transactivadores , Proteínas Adaptadoras Transductoras de Señales , Proteína Axina , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Caseína Quinasas , Células Cultivadas , Proteínas del Citoesqueleto/genética , Proteínas Dishevelled , Glucógeno Sintasa Quinasa 3 , Humanos , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Fosforilación , Proteínas Quinasas/genética , Proteínas/inmunología , Transducción de Señal , Proteínas Wnt , Proteína Wnt3 , Proteína Wnt3A , beta CateninaRESUMEN
beta-TrCP/E3RS (E3RS) is the F-box protein that functions as the receptor subunit of the SCF(beta-TrCP) ubiquitin ligase (E3). Surprisingly, although its two recognized substrates, IkappaB(alpha) and beta-catenin, are present in the cytoplasm, we have found that E3RS is located predominantly in the nucleus. Here we report the isolation of the major E3RS-associated protein, hnRNP-U, an abundant nuclear phosphoprotein. This protein occupies E3RS in a specific and stoichiometric manner, stabilizes the E3 component, and is likely responsible for its nuclear localization. hnRNP-U binding was abolished by competition with a pIkappaB(alpha) peptide, or by a specific point mutation in the E3RS WD region, indicating an E3-substrate-type interaction. However, unlike pI(kappa)Balpha, which is targeted by SCF(beta-TrCP) for degradation, the E3-bound hnRNP-U is stable and is, therefore, a pseudosubstrate. Consequently, hnRNP-U engages a highly neddylated active SCF(beta-TrCP), which dissociates in the presence of a high-affinity substrate, resulting in ubiquitination of the latter. Our study points to a novel regulatory mechanism, which secures the localization, stability, substrate binding threshold, and efficacy of a specific protein-ubiquitin ligase.
