Phosphorylation of Atg5 by the Gadd45β–MEKK4-p38 pathway inhibits autophagy

Abstract

Autophagy is a lysosomal degradation pathway important for cellular homeostasis, mammalian development, cancer and immunity. Many molecular components of autophagy have been identified, but little is known about regulatory mechanisms controlling their effector functions. Here, we show that, in contrast to other p38 MAP kinase activators, the growth arrest and DNA damage 45 beta (Gadd45b)-MAPK/ERK kinase kinase 4 (MEKK4) pathway specifically directs p38 to autophagosomes. This process results in an accumulation of autophagosomes through p38-mediated inhibition of lysosome fusion. Conversely, autophagic flux is increased in p38-deficient fibroblasts and Gadd45b-deficient cells. We further identified the underlying mechanism and demonstrate that phosphorylation of the autophagy regulator autophagy-related (Atg)5 at threonine 75 through p38 is responsible for inhibition of starvation-induced autophagy. Thus, we show for the first time that Atg5 activity is controlled by phosphorylation and, moreover, that the spatial regulation of p38 by Gadd45b/MEKK4 negatively regulates the autophagic process. Macroautophagy (hereafter referred to as autophagy) is a catabolic process, by which the cell degrades cytosolic content to supply metabolic processes with nutrients in order to maintain ATP production and macromolecular synthesis. Thus, autophagy acts as an efficient recycling mechanism in eukaryotic cells.1 Cellular stress, for example, nutrient deprivation, enhances autophagy as a survival mechanism during starvation. In addition, autophagy serves important functions in development, cancer, cell death and immunity in mammals. 1,2 Autophagy is controlled by conserved key regulators known as autophagy-related (Atg) proteins.3 At the onset of the autophagy cascade, Atg6/Beclin-1 forms a complex with the class III phosphatidylinoside kinase Vps34, which induces expansion of the precursor membrane vesicle, the phagophore, via recruitment of additional Atg proteins. During expansion, the double membrane vesicle surrounds cytosolic content, and the completed vesicle, called autophagosome, finally fuses with lysosomes to degrade the autophagosomal content.3 Maturation of autophagosomes is regulated by two ubiquitin-like conjugation systems, namely the Atg8-phosphatidylethanolamine (PE) and the Atg5-12/16L1 conjugation system. The Atg5-Atg12 conjugate interacts with Atg16L1, which tethers the complex to phagophores and autophagosomes. This complex then acts as an E3-like ubiquitin ligase for microtubule-associated protein 1 light chain 3 (LC3) lipidation. The conversion of LC3 to the PE-conjugated LC3-II form and its recruitment to the membrane serves as a wellaccepted marker for autophagy.Although Atg5-independent autophagy has been described, 4 Atg5 is crucial for autophagy under most circumstances and Atg5-deficient mouse embryonic fibroblasts (MEFs) lack LC3 conversion and autophagy. Therefore, Atg5-deficient mice die postnatal owing to their inability to cope with starvation during the neonatal period. 5 In addition, Atg5 see...

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“…Previous work has shown that ERK1/2 inhibits phosphorylation of p38 (35), which is a negative regulator of autophagy (23,36). We found that p38 phosphorylation was increased in the livers of db/db mice, and the increased p38 phosphorylation was significantly decreased by Ad-CA MEK1 in the livers of db/db mice or primary hepatocytes ( Fig.…”

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