State Key Laboratory of Agricultural Microbiology, College of Animal Sciences and Technology, Huazhong Agricultural University, Wuhan, Hubei 430070, China; The Cooperative Innovation Center for Sustainable Pig Production, Wuhan, Hubei 430070, China; Hubei Provincial Engineering Laboratory for Pig Precision Feeding and Feed Safety Technology, Wuhan, Hubei 430070, China.
State Key Laboratory of Agricultural Microbiology, College of Animal Sciences and Technology, Huazhong Agricultural University, Wuhan, Hubei 430070, China; The Cooperative Innovation Center for Sustainable Pig Production, Wuhan, Hubei 430070, China; Hubei Provincial Engineering Laboratory for Pig Precision Feeding and Feed Safety Technology, Wuhan, Hubei 430070, China.
Cell Signal. 2021 Jun;82:109956. doi: 10.1016/j.cellsig.2021.109956. Epub 2021 Feb 16.
ATG4B facilitates autophagy by promoting autophagosome maturation through the reversible lipidation and delipidation of LC3. Recent reports have shown that phosphorylation of ATG4B regulates its activity and LC3 processing, leading to modulate autophagy activity. However, the mechanism about how ATG4B phosphorylation is involved in amino acid deprivation-induced autophagy is unclear. Here, we combined the tandem affinity purification with mass spectrometry (MS) and identified the ATG4B-interacting proteins including its well-known partner gamma-aminobutyric acid receptor-associated protein (GABARAP, a homolog of LC3) and phosphofructokinase 1 platelet isoform (PFKP). Further immunoprecipitation assays showed that amino acid deprivation strengthened the interaction between ATG4B and PFKP. By genetic depletion of PFKP using CRISPR/Cas9, we uncovered that PFKP loss reduced the degradation of LC3-II and p62 due to a partial block in autophagic flux. Furthermore, MS analysis of Flag-tagged ATG4B immunoprecipitates identified phosphorylation of ATG4B serine 34 residue (S34) and PFKP serine 386 residue (S386) under amino acid deprivation condition. In vitro kinase assay validated that PFKP functioning as a protein kinase phosphorylated ATG4B at S34. This phosphorylation could enhance ATG4B activity and p62 degradation. In addition, PFKP S386 phosphorylation was important to ATG4B S34 phosphorylation and autophagy in HEK293T cells. In brief, our findings describe that PFKP, a rate-limiting enzyme in the glycolytic pathway, functions as a protein kinase for ATG4B to regulate ATG4B activity and autophagy under amino acid deprivation condition.
ATG4B 通过促进 LC3 的可逆脂质化和去脂质化来促进自噬体成熟，从而促进自噬。最近的报道表明，ATG4B 的磷酸化调节其活性和 LC3 加工，从而调节自噬活性。然而，ATG4B 磷酸化如何参与氨基酸剥夺诱导的自噬的机制尚不清楚。在这里，我们将串联亲和纯化与质谱（MS）相结合，鉴定了 ATG4B 的相互作用蛋白，包括其众所周知的伙伴γ-氨基丁酸受体相关蛋白（GABARAP，LC3 的同源物）和磷酸果糖激酶 1 血小板同工型（PFKP）。进一步的免疫沉淀实验表明，氨基酸剥夺增强了 ATG4B 和 PFKP 之间的相互作用。通过 CRISPR/Cas9 基因敲除 PFKP，我们发现 PFKP 缺失由于自噬通量的部分阻断而减少了 LC3-II 和 p62 的降解。此外，对 Flag 标记的 ATG4B 免疫沉淀物进行 MS 分析，鉴定出在氨基酸剥夺条件下 ATG4B 丝氨酸 34 残基（S34）和 PFKP 丝氨酸 386 残基（S386）的磷酸化。体外激酶实验验证了 PFKP 作为一种蛋白激酶，在 S34 位磷酸化 ATG4B。这种磷酸化可以增强 ATG4B 的活性和 p62 的降解。此外，PFKP S386 磷酸化对 ATG4B S34 磷酸化和 HEK293T 细胞中的自噬很重要。总之，我们的研究结果表明，PFKP，糖酵解途径中的限速酶，作为一种蛋白激酶，在氨基酸剥夺条件下调节 ATG4B 的活性和自噬。
