Department of Biology, University of Fribourg, Fribourg, Switzerland.
Autophagy. 2022 Feb;18(2):455-456. doi: 10.1080/15548627.2021.2002546. Epub 2021 Nov 28.
ULK1 kinase is the gatekeeper of canonical macroautophagy (hereafter referred to as autophagy) phosphorylating an array of substrates critical for autophagosome biogenesis. To uncover if ULK1 has broader functions also regulating subsequent steps of autophagosome turnover, i.e., maturation, lysosomal fusion, and degradation, we performed a set of unbiased phosphoproteomic experiments employing mouse and human cells in combination with genetic and environmental perturbations. We characterized more than 1,000 potential ULK1 target sites of which many affect proteins known to be involved in all phases of the autophagosome life cycle. To better understand which of these 1,000 phosphosites were directly phosphorylated by ULK1, in contrast to downstream kinases being activated or phosphatases being inhibited by ULK1, we developed a proteome-scale kinase assay and characterized 187 phosphosites on 157 proteins as ULK1 target sites. Interestingly, our results highlight an intricate crosstalk between ULK1 and protein phosphatases. Focusing on STRN (striatin), a regulatory subunit of PPP2/PP2A (protein phosphatase 2), we identified a positive feedback loop linked to ULK1 and promoting autophagy.
ULK1 激酶是经典巨自噬（以下简称自噬）的守门员，它磷酸化一系列对于自噬体生物发生至关重要的底物。为了揭示 ULK1 是否具有更广泛的功能，也可以调节自噬体周转的后续步骤，即成熟、溶酶体融合和降解，我们进行了一组无偏磷酸蛋白质组学实验，结合遗传和环境干扰，使用小鼠和人类细胞。我们鉴定了超过 1000 个潜在的 ULK1 靶位点，其中许多影响已知参与自噬体生命周期所有阶段的蛋白质。为了更好地理解这 1000 个磷酸化位点中的哪些是直接被 ULK1 磷酸化的，而不是下游激酶被 ULK1 激活或磷酸酶被 ULK1 抑制，我们开发了一种蛋白质组规模的激酶测定法，并鉴定了 157 个蛋白质上的 187 个磷酸化位点作为 ULK1 的靶位点。有趣的是，我们的结果强调了 ULK1 和蛋白磷酸酶之间复杂的相互作用。我们专注于 STRN（striatin），一种 PPP2/PP2A（蛋白磷酸酶 2）的调节亚基，鉴定了一个与 ULK1 相关并促进自噬的正反馈回路。
