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

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.

内质网（endoplasmic reticulum, ER）的选择性自噬，称为内质网自噬（ER-phagy），是内质网重塑的重要调控因子，在环境变化过程中对维持细胞稳态至关重要。我们近期的研究表明，FAM134家族成员在应激诱导的内质网自噬中发挥关键作用。然而，其激活机制在很大程度上仍未明确。本研究中，我们分析了雷帕霉素机制性靶标（mechanistic target of rapamycin, mTOR）抑制条件下，FAM134的磷酸化作为驱动FAM134介导的内质网自噬的触发因素。通过无偏倚激酶抑制剂筛选，我们发现CK2在mTOR抑制后，对FAM134B和FAM134C介导的内质网自噬不可或缺。此外，本研究证实内质网自噬受体受泛素化事件调控，且E1抑制剂处理可抑制Torin1诱导的内质网自噬流。利用超分辨率显微镜技术，我们证明CK2活性是形成高密度FAM134B与FAM134C簇的必要条件。同时，FAM134B和FAM134C的致密聚集依赖于二者的磷酸化依赖性泛素化。使用CK2抑制剂SGC-CK2-1处理，或对FAM134B、FAM134C的磷酸化位点进行突变，均可阻断FAM134蛋白的泛素化、高密度簇的形成，以及Torin1诱导的内质网自噬流。据此我们提出：内质网自噬受体的CK2依赖性磷酸化，先于泛素依赖的内质网自噬流激活过程。