RESUMEN
Autophagy is a potent cellular degradation pathway, and its activation needs to be tightly controlled. Cargo receptors mediate selectivity during autophagy by bringing cargo to the scaffold protein Atg11 and, in turn, to the autophagic machinery, including the central autophagy kinase Atg1. Here we show how selective autophagy is tightly regulated in space and time to prevent aberrant Atg1 kinase activation and autophagy induction. We established an induced bypass approach (iPass) that combines genetic deletion with chemically induced dimerization to evaluate the roles of Atg13 and cargo receptors in Atg1 kinase activation and selective autophagy progression. We show that Atg1 activation does not require cargo receptors, cargo-bound Atg11, or Atg13 per se. Rather, these proteins function in two independent pathways that converge to activate Atg1 at the vacuole. This pathway architecture underlies the spatiotemporal control of Atg1 kinase activity, thereby preventing inappropriate autophagosome formation.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Relacionadas con la Autofagia/genética , Autofagia/genética , Regulación Fúngica de la Expresión Génica , Proteínas Quinasas/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Transporte Vesicular/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Aminopeptidasas/genética , Aminopeptidasas/metabolismo , Proteínas Relacionadas con la Autofagia/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Fagosomas/metabolismo , Proteínas Quinasas/metabolismo , Multimerización de Proteína , Transporte de Proteínas , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Transducción de Señal , Vacuolas/metabolismo , Proteínas de Transporte Vesicular/metabolismoRESUMEN
Gene regulation by cytokine-activated transcription factors of the signal transducer and activator of transcription (STAT) family requires serine phosphorylation within the transactivation domain (TAD). STAT1 and STAT3 TAD phosphorylation occurs upon promoter binding by an unknown kinase. Here, we show that the cyclin-dependent kinase 8 (CDK8) module of the Mediator complex phosphorylated regulatory sites within the TADs of STAT1, STAT3, and STAT5, including S727 within the STAT1 TAD in the interferon (IFN) signaling pathway. We also observed a CDK8 requirement for IFN-γ-inducible antiviral responses. Microarray analyses revealed that CDK8-mediated STAT1 phosphorylation positively or negatively regulated over 40% of IFN-γ-responsive genes, and RNA polymerase II occupancy correlated with gene expression changes. This divergent regulation occurred despite similar CDK8 occupancy at both S727 phosphorylation-dependent and -independent genes. These data identify CDK8 as a key regulator of STAT1 and antiviral responses and suggest a general role for CDK8 in STAT-mediated transcription. As such, CDK8 represents a promising target for therapeutic manipulation of cytokine responses.
Asunto(s)
Quinasa 8 Dependiente de Ciclina/genética , Regulación de la Expresión Génica/efectos de los fármacos , Interferón gamma/farmacología , Factor de Transcripción STAT1/genética , Animales , Quinasa 8 Dependiente de Ciclina/inmunología , Quinasa 8 Dependiente de Ciclina/metabolismo , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Fibroblastos/virología , Células Hep G2 , Humanos , Interferón gamma/inmunología , Interleucina-6/inmunología , Interleucina-6/farmacología , Ratones , Análisis de Secuencia por Matrices de Oligonucleótidos , Fosforilación , Regiones Promotoras Genéticas , ARN Polimerasa II/genética , ARN Polimerasa II/inmunología , Factor de Transcripción STAT1/inmunología , Factor de Transcripción STAT1/metabolismo , Factor de Transcripción STAT3/genética , Factor de Transcripción STAT3/inmunología , Factor de Transcripción STAT5/genética , Factor de Transcripción STAT5/inmunología , Transducción de Señal/efectos de los fármacos , Transcripción Genética/efectos de los fármacos , Vesiculovirus/fisiologíaRESUMEN
The degradation and recycling of cellular components is essential for cell growth and survival. Here we show how selective and non-selective lysosomal protein degradation pathways cooperate to ensure cell survival upon nutrient limitation. A quantitative analysis of starvation-induced proteome remodeling in yeast reveals comprehensive changes already in the first three hours. In this period, many different integral plasma membrane proteins undergo endocytosis and degradation in vacuoles via the multivesicular body (MVB) pathway. Their degradation becomes essential to maintain critical amino acids levels that uphold protein synthesis early during starvation. This promotes cellular adaptation, including the de novo synthesis of vacuolar hydrolases to boost the vacuolar catabolic activity. This order of events primes vacuoles for the efficient degradation of bulk cytoplasm via autophagy. Hence, a catabolic cascade including the coordinated action of the MVB pathway and autophagy is essential to enter quiescence to survive extended periods of nutrient limitation.