Regulation of PP1 holoenzyme dynamics by p97-mediated structural remodelling

蛋白磷酸酶-1 （PP1）通过广泛的底物去磷酸化来调节无数的细胞过程，这依赖于PP1全酶与替代亚基的组装。我们之前的研究表明，新合成的PP1首先被SDS22和抑制剂-3 （I3）捕获在一个非活性复合体中，该复合体需要被六聚体aaa - atp酶p97分解，以允许PP1与各种激活亚基结合。与p97介导的降解过程相反，p97对PP1的分解可以独立于泛素化发生。我们新的生化和结构数据现在表明，一类sep结构域适配器以多价方式介导SDS22-PP1-I3 （SPI）复合物加载到p97上。通过SDS22亚基的直接接触，SPI复合物被锁定在p97的n结构域上。I3中的一个内部识别位点随后作为环路插入p97的中心通道。I3穿过通道将I3从PP1上剥离并拆卸整个复合物。在阐明p97如何在SPI分解中起作用之后，我们现在需要了解SDS22和I3如何协同调节PP1，以及它们的结合和解离如何控制PP1全酶对细胞条件（如应激）的适应。具体来说，我们的目标是概括和解读在综合应激反应中从非活性SPI复合物到特定PP1全酶的转变。为此，我们将应用复杂的生化重构，精制Förster共振能量转移（FRET）分析以及结合细胞验证的晶体学。令人惊讶的是，我们的新数据还发现，最丰富的sep结构域适配器p47并不直接针对PP1。使用交联方法以及底物捕获和邻近生物素化策略，我们的目标是探索泛素对p47介导的PP1或其他迄今未知的p97-p47细胞底物的可能调节作用，同时考虑到泛素结合和p97调节活性是p47所独有的。预期的结果将揭示一个多功能和重要的蛋白磷酸酶是如何调节控制关键的细胞过程和应激反应的。

Protein phosphatase-1 (PP1) regulates a myriad of cellular processes by dephosphorylating a broad spectrum of substrates, which relies on assembly of PP1 holoenzymes with alternative subunits. We previously showed that newly synthesized PP1 is first trapped in an inactive complex by SDS22 and inhibitor-3 (I3) that needs to be disassembled by the hexameric AAA-ATPase p97 to allow association of PP1 with various activating subunits. In contrast to p97-mediated degradative processes, PP1 disassembly by p97 can occur independently of ubiquitination. Our new biochemical and structural data now demonstrate that a class of SEP-domain adapters mediates loading of the SDS22-PP1-I3 (SPI) complex onto p97 in a multivalent manner. The SPI complex is then locked onto the N-domain of p97 with direct contacts of the SDS22 subunit. An internal recognition site in I3 is subsequently inserted as a loop into the central channel of p97. Threading of I3 through the channel strips I3 off PP1 and disassembles the whole complex. After clarifying how p97 functions in SPI disassembly, we now need to understand how SDS22 and I3 cooperate in regulating PP1 and how their binding and dissociation may govern PP1 holoenzyme adaptation to cellular conditions such as stress. Specifically, we will aim to recapitulate and decipher the transition from the inactive SPI complex to a specific PP1 holoenzyme during the integrated stress response. To do so, we will apply sophisticated biochemical reconstitution, refined Förster resonance energy transfer (FRET) assays as well as crystallography combined with validation in cells. Surprisingly, our new data also uncovered that the most abundant SEP-domain adapter, p47, does not directly target PP1. Using crosslinking approaches as well as substrate trapping and proximity biotinylation strategies, we aim to explore a possible regulatory role of ubiquitin for p47-mediated targeting of either PP1 or of other, so far unknown, cellular substrates of p97-p47 taking into account ubiquitin binding and p97-modulating activities that are unique to p47. The expected results will reveal how a versatile and important protein phosphatase is regulated to control key cellular processes and stress responses.