2023
Nature

The function of ER-phagy receptors is regulated through phosphorylation-dependent ubiquitination pathways

Authors:

Rayene Berkane, Hung Ho-Xuan, Marius Glogger, Pablo Sanz-Martinez, Lorène Brunello, Tristan Glaesner, Santosh Kumar Kuncha, Katharina Holzhüter, Sara Cano-Franco, Viviana Buonomo, Paloma Cabrerizo-Poveda, Ashwin Balakrishnan, Georg Tascher, Koraljka Husnjak, Thomas Juretschke, Mohit Misra, Alexis González, Volker Dötsch, Paolo Grumati, Mike Heilemann, Alexandra Stolz

Keywords:

endoplasmic reticulum, ER, ER-phagy, FAM134, mTOR, ubiquitination, CK

Abstract:

Selective autophagy of the endoplasmic reticulum (ER), known as ER-phagy, is an important regulator of ER remodeling and essential to maintain cellular homeostasis during environmental changes. We recently showed that members of the FAM134 family play a critical role during stress-induced ER-phagy. However, the mechanisms on how they are activated remain largely unknown. In this study, we analyze phosphorylation of FAM134 as a trigger of FAM134-driven ER-phagy upon mTOR (mechanistic target of rapamycin) inhibition. An unbiased screen of kinase inhibitors reveals CK2 to be essential for FAM134B- and FAM134C-driven ER-phagy after mTOR inhibition. Furthermore, we provide evidence that ER-phagy receptors are regulated by ubiquitination events and that treatment with E1 inhibitor suppresses Torin1-induced ER-phagy flux. Using super-resolution microscopy, we show that CK2 activity is essential for the formation of high-density FAM134B and FAM134C clusters. In addition, dense clustering of FAM134B and FAM134C requires phosphorylation-dependent ubiquitination of FAM134B and FAM134C. Treatment with the CK2 inhibitor SGC-CK2-1 or mutation of FAM134B and FAM134C phosphosites prevents ubiquitination of FAM134 proteins, formation of high-density clusters, as well as Torin1-induced ER-phagy flux. Therefore, we propose that CK2-dependent phosphorylation of ER-phagy receptors precedes ubiquitin-dependent activation of ER-phagy flux.