GABARAPs and LC3s have opposite roles in regulating ULK1 for autophagy induction
GABARAPs and LC3s have opposite roles in regulating ULK1 for autophagy induction
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DOI：
10.1080/15548627.2019.1632620
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发表时间：
2019-06-29
期刊：
影响因子：
13.3
通讯作者：
Kim, Do-Hyung
中科院分区：
文献类型：
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作者：
Grunwald, Douglas S.;Otto, Neil Michael;Kim, Do-Hyung
ULK 1（unc-51 like autophagy activating kinase 1）是MTORC 1信号传导至巨自噬/自噬的关键介质。ULK 1通过与ATG 13、RB 1CC 1/FIP 200和ATG 101相互作用而作为蛋白质复合物发挥功能。ULK 1复合物如何被调节以触发自噬诱导仍不清楚。在这项研究中，我们确定了Atg 8家族蛋白（ATG 8）在调节ULK 1活性和自噬中的作用。使用耗尽每个ATG 8亚家族的人细胞，我们发现GABARAP亚家族积极调节ULK 1活性和吞噬细胞和自噬体形成以响应饥饿。相反，LC 3亚家族负调控ULK 1活性和吞噬细胞形成。通过用单独的ATG 8成员重建ATG 8耗尽的细胞，我们鉴定GABARAP和GABARAPL 1为阳性，LC 3B和LC 3C为ULK 1活性的负调节剂。为了解决ATG 8与ULK 1结合的作用，我们突变了内源性ULK 1的LIR，以通过基因组编辑破坏ATG 8-ULK 1相互作用。该突变显著降低了ULK 1的活性、SQSTM 1的自噬降解和响应饥饿的吞噬细胞形成。该突变还抑制了响应饥饿的自噬体的形成和周转。类似于ULK 1 LIR的突变，破坏ATG 13-ATG 8相互作用抑制ULK 1活性和自噬体形成。与此相反，RB 1CC 1没有显示出任何与ATG 8的特异性结合，并且其LIR的突变不影响ULK 1活性。总之，这项研究证明了GABARAPs和LC 3对ULK 1复合物的差异结合和相反调节，以及ULK 1和ATG 13-ATG 8相互作用在自噬诱导中的重要作用。
ULK1 (unc-51 like autophagy activating kinase 1) is the key mediator of MTORC1 signaling to macroautophagy/autophagy. ULK1 functions as a protein complex by interacting with ATG13, RB1CC1/FIP200, and ATG101. How the ULK1 complex is regulated to trigger autophagy induction remains unclear. In this study, we have determined roles of Atg8-family proteins (ATG8s) in regulating ULK1 activity and autophagy. Using human cells depleted of each subfamily of ATG8, we found that the GABARAP subfamily positively regulates ULK1 activity and phagophore and autophagosome formation in response to starvation. In contrast, the LC3 subfamily negatively regulates ULK1 activity and phagophore formation. By reconstituting ATG8-depleted cells with individual ATG8 members, we identified GABARAP and GABARAPL1 as positive and LC3B and LC3C as negative regulators of ULK1 activity. To address the role of ATG8 binding to ULK1, we mutated the LIR of endogenous ULK1 to disrupt the ATG8-ULK1 interaction by genome editing. The mutation drastically reduced the activity of ULK1, autophagic degradation of SQSTM1, and phagophore formation in response to starvation. The mutation also suppressed the formation and turnover of autophagosomes in response to starvation. Similar to the mutation of the ULK1 LIR, disruption of the ATG13-ATG8 interaction suppressed ULK1 activity and autophagosome formation. In contrast, RB1CC1 did not show any specific binding to ATG8s, and mutation of its LIR did not affect ULK1 activity. Together, this study demonstrates differential binding and opposite regulation of the ULK1 complex by GABARAPs and LC3s, and an important role of the ULK1- and ATG13-ATG8 interactions in autophagy induction.