RESUMEN
Autophagy is a membrane-mediated degradation process of macromolecule recycling. Although the formation of double-membrane degradation vesicles (autophagosomes) is known to have a central role in autophagy, the mechanism underlying this process remains elusive. The serine/threonine kinase Atg1 has a key role in the induction of autophagy. In this study, we show that overexpression of Drosophila Atg1 promotes the phosphorylation-dependent activation of the actin-associated motor protein myosin II. A novel myosin light chain kinase (MLCK)-like protein, Spaghetti-squash activator (Sqa), was identified as a link between Atg1 and actomyosin activation. Sqa interacts with Atg1 through its kinase domain and is a substrate of Atg1. Significantly, myosin II inhibition or depletion of Sqa compromised the formation of autophagosomes under starvation conditions. In mammalian cells, we found that the Sqa mammalian homologue zipper-interacting protein kinase (ZIPK) and myosin II had a critical role in the regulation of starvation-induced autophagy and mammalian Atg9 (mAtg9) trafficking when cells were deprived of nutrients. Our findings provide evidence of a link between Atg1 and the control of Atg9-mediated autophagosome formation through the myosin II motor protein.
Asunto(s)
Autofagia/fisiología , Proteínas de Drosophila/fisiología , Miosina Tipo II/metabolismo , Fagosomas/metabolismo , Proteínas Serina-Treonina Quinasas/fisiología , Inanición/metabolismo , Animales , Animales Modificados Genéticamente , Autofagia/genética , Homólogo de la Proteína 1 Relacionada con la Autofagia , Proteínas Relacionadas con la Autofagia , Células Cultivadas , Drosophila/genética , Drosophila/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Expresión Génica/fisiología , Humanos , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/fisiología , Quinasa de Cadena Ligera de Miosina/genética , Quinasa de Cadena Ligera de Miosina/metabolismo , Quinasa de Cadena Ligera de Miosina/fisiología , Fosforilación , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Inanición/genética , Distribución TisularRESUMEN
Phostensin, a protein phosphatase 1 F-actin cytoskeleton targeting subunit encoded by KIAA1949, consists of 165 amino acids and is located between HLA-C and HLA-E gene clusters on human chromosome 6. In this current study, we characterized the biochemical functions of phostensin. Actin dynamics assays using gelsolin-actin seeds showed that phostensin decreases the elongation and depolymerization rates of actin filament pointed ends. The feature of phostensin that binds to the pointed ends of actin filaments was observed through fluorescent single filament binding assay. Taken together, our results suggested that phostensin is an actin filament pointed end-capping protein that is capable of modulating actin dynamics.
Asunto(s)
Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Proteína Fosfatasa 1/metabolismo , Humanos , Proteína Fosfatasa 1/genéticaRESUMEN
We have identified a novel protein, protein phosphatase 1 F-actin cytoskeleton targeting subunit (phostensin). This protein is encoded by KIAA1949 and was found to associate with protein phosphatase 1 (PP1) in the yeast two-hybrid assay, co-immunoprecipitation, and GST pull-down assay. Northern blot analysis revealed that phostensin mRNA was predominantly distributed in leukocytes and spleen, and phostensin protein was present in crude extracts of human peripheral leukocytes. Immunofluorescence microscopic analysis revealed that the phostensin/PP1 complex was conspicuously localized with the actin cytoskeleton at the cell periphery in Madin-Darby canine kidney (MDCK) epithelial cells. Taken together, our data shows that phostensin targets PP1 to F-actin cytoskeleton. The phostensin/PP1 complex may play a vital role in modulation of actin rearrangements.