RESUMO
The Atg8 family protein LC3 is indispensible for autophagy and plays critical roles in multiple steps of the process. Despite this functional significance, the regulation of LC3 activity at the posttranslational level remains poorly understood. In a recent study, we report that the conserved Ste20 kinases STK3 and STK4, the mammalian orthologs of Hippo kinase, are essential for autophagy in diverse organisms, and both can phosphorylate LC3 on amino acid Thr50. STK3/STK4-mediated phosphorylation is critical for fusion of autophagosomes with lysosomes, as well as the ability of cells to clear intracellular bacteria, an established cargo for autophagy. Our discovery of a novel mode of autophagy regulation involving direct phosphorylation of LC3 by STK3/STK4 significantly enhances our molecular understanding of the autophagy process. Moreover, our findings raise the exciting possibility that STK3/STK4's known roles in immunity are exerted through their ability to regulate autophagy via LC3 phosphorylation.
Assuntos
Autofagia/genética , Fibroblastos/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Serina-Treonina Quinases/genética , Animais , HumanosRESUMO
The protein LC3 is indispensible for the cellular recycling process of autophagy and plays critical roles during cargo recruitment, autophagosome biogenesis, and completion. Here, we report that LC3 is phosphorylated at threonine 50 (Thr(50)) by the mammalian Sterile-20 kinases STK3 and STK4. Loss of phosphorylation at this site blocks autophagy by impairing fusion of autophagosomes with lysosomes, and compromises the ability of cells to clear intracellular bacteria, an established cargo for autophagy. Strikingly, mutation of LC3 mimicking constitutive phosphorylation at Thr(50) reverses the autophagy block in STK3/STK4-deficient cells and restores their capacity to clear bacteria. Loss of STK3/STK4 impairs autophagy in diverse species, indicating that these kinases are conserved autophagy regulators. We conclude that phosphorylation of LC3 by STK3/STK4 is an essential step in the autophagy process. Since several pathological conditions, including bacterial infections, display aberrant autophagy, we propose that pharmacological agents targeting this regulatory circuit hold therapeutic potential.
Assuntos
Autofagia/genética , Fibroblastos/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Serina-Treonina Quinases/genética , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Células Cultivadas , Embrião de Mamíferos , Fibroblastos/microbiologia , Regulação da Expressão Gênica , Humanos , Lisossomos/metabolismo , Fusão de Membrana , Camundongos , Camundongos Knockout , Proteínas Associadas aos Microtúbulos/genética , Mutação , Fragmentos de Peptídeos/química , Fagossomos/metabolismo , Fosforilação , Proteínas Serina-Treonina Quinases/deficiência , Serina-Treonina Quinase 3 , Transdução de Sinais , Streptococcus pyogenes/patogenicidade , Streptococcus pyogenes/fisiologia , Treonina/metabolismoRESUMO
This chapter is dedicated to the study of aging in Caenorhabditis elegans (C. elegans). The assays are divided into two sections. In the first section, we describe detailed protocols for performing life span analysis in solid and liquid medium. In the second section, we describe various assays for measuring age-related changes. Our laboratory has been involved in several fruitful collaborations with non-C. elegans researchers keen on testing a role for their favorite gene in modulating aging (Carrano et al., 2009; Dong et al., 2007; Raices et al., 2008; Wolff et al., 2006). But even with the guidance of trained worm biologists, this undertaking can be daunting. We hope that this chapter will serve as a worthy compendium for those researchers who may or may not have immediate access to laboratories studying C. elegans.
Assuntos
Envelhecimento/genética , Bioensaio , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Transdução de Sinais/genética , Animais , Animais Geneticamente Modificados , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Restrição Calórica , Complexo de Proteínas da Cadeia de Transporte de Elétrons/genética , Complexo de Proteínas da Cadeia de Transporte de Elétrons/metabolismo , Fertilidade , Insulina/genética , Insulina/metabolismo , Fator de Crescimento Insulin-Like I/genética , Fator de Crescimento Insulin-Like I/metabolismo , Lipofuscina/biossíntese , Locomoção , Longevidade , Mitocôndrias/genética , Mitocôndrias/metabolismo , Interferência de RNA , Taxa de SobrevidaRESUMO
We purified the oncoprotein SnoN and found that it functions as a corepressor of the tumor suppressor p53 in the regulation of the hepatic alpha-fetoprotein (AFP) tumor marker gene. p53 promotes SnoN and histone deacetylase interaction at an overlapping Smad binding, p53 regulatory element (SBE/p53RE) in AFP. Comparison of wild-type and p53-null mouse liver tissue by using chromatin immunoprecipitation (ChIP) reveals that the absence of p53 protein correlates with the disappearance of SnoN at the SBE/p53RE and loss of AFP developmental repression. Treatment of AFP-expressing hepatoma cells with transforming growth factor-beta1 (TGF-beta1) induced SnoN transcription and Smad2 activation, concomitant with AFP repression. ChIP assays show that TGF-beta1 stimulates p53, Smad4, P-Smad2 binding, and histone H3K9 deacetylation and methylation, at the SBE/p53RE. Depletion, by small interfering RNA, of SnoN and/or p53 in hepatoma cells disrupted repression of AFP transcription. These findings support a model of cooperativity between p53 and TGF-beta effectors in chromatin modification and transcription repression of an oncodevelopmental tumor marker gene.
