Identification of a phosphorylation site on Ulk1 required for genotoxic stress-induced alternative autophagy

Satoru Torii,Akira Nakanishi,Shigeomi Shimizu,Shinya Honda,Hiroyasu Nakano,Kenta Moriwaki,Hirofumi Yamaguchi,Satoko Arakawa

doi:10.1038/s41467-020-15577-2

Abstract

Alternative autophagy is an autophagy-related protein 5 (Atg5)-independent type of macroautophagy. Unc51-like kinase 1 (Ulk1) is an essential initiator not only for Atg5-dependent canonical autophagy but also for alternative autophagy. However, the mechanism as to how Ulk1 differentially regulates both types of autophagy has remained unclear. In this study, we identify a phosphorylation site of Ulk1 at Ser746, which is phosphorylated during genotoxic stress-induced alternative autophagy. Phospho-Ulk1746 localizes exclusively on the Golgi and is required for alternative autophagy, but not canonical autophagy. We also identify receptor-interacting protein kinase 3 (RIPK3) as the kinase responsible for genotoxic stress-induced Ulk1746 phosphorylation, because RIPK3 interacts with and phosphorylates Ulk1 at Ser746, and loss of RIPK3 abolishes Ulk1746 phosphorylation. These findings indicate that RIPK3-dependent Ulk1746 phosphorylation on the Golgi plays a pivotal role in genotoxic stress-induced alternative autophagy.

Highlights

Alternative autophagy is an autophagy-related protein 5 (Atg5)-independent type of macroautophagy
We detected various reported phosphorylation sites of Ulk[1], such as Ser[317], which is an AMPK target that is known to be phosphorylated during alternative autophagy[17]
We focused on Ser[746] (Fig. 1a), because when various phosphodeficient Ulk[1] mutants were expressed at equivalent levels in Atg5/Ulk[1] double-knockout (Atg5/Ulk1DKO) mouse embryonic fibroblasts (MEFs) (Supplementary Fig. 1a, b), most Ulk[1] mutants, but not mutant Ulk[1] (S746A), recovered the ability to perform alternative autophagy (Supplementary Fig. 1c, d)

Summary

Introduction

Alternative autophagy is an autophagy-related protein 5 (Atg5)-independent type of macroautophagy. We identify receptorinteracting protein kinase 3 (RIPK3) as the kinase responsible for genotoxic stress-induced Ulk1746 phosphorylation, because RIPK3 interacts with and phosphorylates Ulk[1] at Ser[746], and loss of RIPK3 abolishes Ulk1746 phosphorylation These findings indicate that RIPK3dependent Ulk1746 phosphorylation on the Golgi plays a pivotal role in genotoxic stressinduced alternative autophagy. III lipid kinases producing phosphatidylinositol 3-phosphate (PI3K), Atg[9] complexes, the Atg[5] conjugation system, and the microtubule-associated protein light chain 3 (LC3) conjugation system After their generation, autophagosomes fuse with lysosomes to form autolysosomes by a mechanism dependent on syntaxin 17 (Stx17)[4]. Atg5-independent alternative macroautophagy (hereafter described as alternative autophagy), which we discovered previously[5], is one such type of autophagy This autophagy machinery is named Golgi membrane‐associated degradation (GOMED)[8]. In the terminal differentiation of erythrocytes, alternative and canonical autophagy eliminate different substrates, i.e., mitochondria and ribosomes, respectively[10]

Methods

Results

Conclusion