Regulation of PP1 holoenzyme dynamics by p97-mediated structural remodelling

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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.