RESUMEN
BACKGROUND: Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common genetic cause of Parkinson disease (PD). LRRK2 contains an "enzymatic core" composed of GTPase and kinase domains that is flanked by leucine-rich repeat (LRR) and WD40 protein-protein interaction domains. While kinase activity and GTP-binding have both been implicated in LRRK2 neurotoxicity, the potential role of other LRRK2 domains has not been as extensively explored. PRINCIPAL FINDINGS: We demonstrate that LRRK2 normally exists in a dimeric complex, and that removing the WD40 domain prevents complex formation and autophosphorylation. Moreover, loss of the WD40 domain completely blocks the neurotoxicity of multiple LRRK2 PD mutations. CONCLUSION: These findings suggest that LRRK2 dimerization and autophosphorylation may be required for the neurotoxicity of LRRK2 PD mutations and highlight a potential role for the WD40 domain in the mechanism of LRRK2-mediated cell death.
Asunto(s)
Neurotoxinas/química , Neurotoxinas/toxicidad , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/toxicidad , Animales , Línea Celular , Humanos , Ratones , Modelos Moleculares , Peso Molecular , Fosforilación/efectos de los fármacos , Multimerización de Proteína/efectos de los fármacos , Estructura Terciaria de Proteína , Eliminación de Secuencia , Homología Estructural de Proteína , Relación Estructura-ActividadRESUMEN
Spinocerebellar ataxia type 1 (SCA1) is one of nine inherited neurodegenerative disorders caused by a mutant protein with an expanded polyglutamine tract. Phosphorylation of ataxin-1 (ATXN1) at serine 776 is implicated in SCA1 pathogenesis. Previous studies, utilizing transfected cell lines and a Drosophila photoreceptor model of SCA1, suggest that phosphorylating ATXN1 at S776 renders it less susceptible to degradation. This work also indicated that oncogene from AKR mouse thymoma (Akt) promotes the phosphorylation of ATXN1 at S776 and severity of neurodegeneration. Here, we examined the phosphorylation of ATXN1 at S776 in cerebellar Purkinje cells, a prominent site of pathology in SCA1. We found that while phosphorylation of S776 is associated with a stabilization of ATXN1 in Purkinje cells, inhibition of Akt either in vivo or in a cerebellar extract-based phosphorylation assay did not decrease the phosphorylation of ATXN1-S776. In contrast, immunodepletion and inhibition of cyclic AMP-dependent protein kinase decreased phosphorylation of ATXN1-S776. These results argue against Akt as the in vivo kinase that phosphorylates S776 of ATXN1 and suggest that cyclic AMP-dependent protein kinase is the active ATXN1-S776 kinase in the cerebellum.
Asunto(s)
Cerebelo/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Serina/metabolismo , Alanina/genética , Alanina/metabolismo , Secuencia de Aminoácidos , Animales , Ataxina-1 , Ataxinas , Cerebelo/enzimología , Estabilidad de Enzimas/genética , Humanos , Ratones , Ratones Endogámicos AKR , Ratones Endogámicos C57BL , Ratones Transgénicos , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/genética , Proteínas Nucleares/genética , Fosforilación , Mutación Puntual , Proteínas Proto-Oncogénicas c-akt/genética , Células de Purkinje/enzimología , Células de Purkinje/metabolismo , Serina/genéticaRESUMEN
Spinocerebellar ataxia type 1 (SCA1) is an inherited neurodegenerative disorder. The mutation causing SCA1 is an expansion in the polyglutamine tract of the ATXN1 protein. Previous work demonstrated that phosphorylation of mutant ATXN1 at serine 776 (S776), a putative Akt phosphorylation site, is critical for pathogenesis. To examine this pathway further, we utilized a cell-transfection system that allowed the targeting of Akt to either the cytoplasm or the nucleus. In contrast to HeLa cells, we found that Akt targeted to the cytoplasm increased the degradation of ATXN1 in Chinese hamster ovary cells. However, Akt targeted to the cytoplasm failed to destabilize ATXN1 if Hsp70/Hsc70 was present. Thus, Hsp70/Hsc70 can regulate ATXN1 levels in concert with phosphorylation of ATXN1 at S776.