RESUMEN
The central nervous system regulates neuronal excitability by macromolecular signalling complexes that consist of functionally related proteins, including neurotransmitter receptors, enzymes and scaffolds. The composition of these signal complexes is regulated by post-translational modifications, such as phosphorylation and SUMOylation (SUMO is small ubiquitin-related modifier). In the present study, we searched for proteins interacting with the intracellular C-termini of the metabotropic glutamate receptors mGluR8a and mGluR8b and identified proteins of the SUMOylation and NEDDylation machinery. The SUMO E3 ligases Pias1 [Pias is protein inhibitor of activated STAT (signal transducer and activator of transcription)] and Pias3L interacted strongly with mGluR8b, and were co-localized with the E2-conjugating Ubc9, SUMO1 and mGluR8b in cell bodies present in the ganglion cell layer of the mammalian retina. SUMO1 conjugation of Lys882, present in a bona fide consensus sequence for SUMOylation (VKSE) in the mGluR8b C-terminus, was enhanced by addition of Pias1, consistent with an interaction between both proteins. Mutation of Lys882 to arginine reduced, but did not abolish, mGluR8b SUMOylation. Co-mutating a second lysine residue (Lys903) located in the mGluR8b isoform-specific C-terminus largely prevented SUMO1 conjugation by Ubc9. Modelling studies suggested that Lys903 contacts Ubc9 and thus is part of the non-canonical SUMOylation site VKSG. In summary, the results of the present study show in vivo SUMOylation of the complete mGluR8b and co-localize proteins of the SUMOylation machinery in the retina.
Asunto(s)
Proteínas Inhibidoras de STAT Activados/genética , Proteínas Inhibidoras de STAT Activados/fisiología , Receptores de Glutamato Metabotrópico/metabolismo , Retina/metabolismo , Sumoilación/genética , Secuencia de Aminoácidos , Animales , Dominio Catalítico/genética , Células Cultivadas , Regulación Enzimológica de la Expresión Génica , Humanos , Ratones , Modelos Biológicos , Modelos Moleculares , Datos de Secuencia Molecular , Proteínas Inhibidoras de STAT Activados/metabolismo , Dominios y Motivos de Interacción de Proteínas/genética , Isoformas de Proteínas/metabolismo , Procesamiento Proteico-Postraduccional/genética , Estructura Cuaternaria de Proteína , Ratas , Receptores de Glutamato Metabotrópico/química , Receptores de Glutamato Metabotrópico/genética , Retina/enzimología , Proteína SUMO-1/química , Proteína SUMO-1/genética , Proteína SUMO-1/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Defects of ciliogenesis have been implicated in a wide range of human phenotypes and play a crucial role in signal transduction and cell-cycle coordination. We used homozygosity mapping in two families with autosomal-recessive short-rib polydactyly syndrome Majewski type to identify mutations in NEK1 as an underlying cause of this lethal osteochondrodysplasia. NEK1 encodes a serine/threonine kinase with proposed function in DNA double-strand repair, neuronal development, and coordination of cell-cycle-associated ciliogenesis. We found that absence of functional full-length NEK1 severely reduces cilia number and alters ciliar morphology in vivo. We further substantiate a proposed digenic diallelic inheritance of ciliopathies by the identification of heterozygous mutations in NEK1 and DYNC2H1 in an additional family. Notably, these findings not only increase the broad spectrum of ciliar disorders, but suggest a correlation between the degree of defective microtubule or centriole elongation and organization and the severity of the resulting phenotype.