Regulation of PP1 activity by binding and p97-mediated dissociation of SDS22 and Inhibitor-3
Protein-phosphatase-1 (PP1) catalyzes a major fraction of all dephosphorylation reactions in eukaryotic cells, regulating numerous cellular pathways. To achieve substrate specificity, PP1 interacts with a myriad of interaction partners, which have inhibiting, substrate targeting and substrate specifying properties. During PP1 biogenesis, two of these interaction partners, SDS22 and Inhibitor-3 (I3), bind to PP1. The three proteins form a ternary complex (SPI complex), in which PP1 is inactive. The SPI complex was shown to be recognized by the AAA-ATPase p97, together with its adapter protein p37. Upon recognition, I3 is translocated through the p97 pore, resulting in I3 unfolding and dissociation from PP1. SDS22 also dissociates from PP1 during this process, though the mechanism of dissociation is unknown. Further, the process of SPI disassembly by p97 is uniquely ubiquitin-independent, in contrast to other known p97-dependent pathways. The role of SPI and its individual components in cells is controversial. Both SDS22 and I3 have demonstrated involvement in PP1 inhibition and stability, yet they are also shown to be crucial for PP1 functions during mitosis. This study aimed to elucidate molecular details of the SPI disassembly reaction and explore how SPI formation and p97-mediated SPI disassembly contribute to PP1 regulation beyond PP1 biogenesis.
High resolution FRET-based assays were established to determined kinetic parameters of the SPI disassembly reaction catalyzed by p97. To overcome FRET detection limits, the parameters were determined through the association of the PP1 interacting protein NIPP1 to PP1 after SPI disassembly. Kinetic parameters at 37 °C were KM = 342 *÷ 1.37 nM and kcat = 2.70 *÷ 1.25 min-1, demonstrating that the reaction achieved half its maximal velocity at a concentration of 342 nM SPI, with approximately 2.7 SPI molecules per p97 molecule being catalyzed per minute at 37 °C. The values reveal that SPI as a substrate of p97 is efficiently disassembled, displaying even more effective targeting than poly-ubiquitinated substrates, based on published data.
FRET assays monitoring the dissociation and association of SDS22 and I3 to PP1 revealed that both proteins dissociate simultaneously, while NIPP1 binds with a delay. The delayed binding of NIPP1 reveals that SDS22 is not displaced by a third PP1 interacting protein. Instead, experiments performed with a complex of SDS22 and PP1, lacking I3, demonstrated that I3 was essential for SDS22 dissociation and also aided in the association of SDS22 to PP1. These results indicate direct involvement of I3 in the SDS22-PP1 interaction, possibly with a role in PP1 metal ion exchange. Further, SDS22 was also necessary for efficient I3 disassembly, suggesting a role in SPI targeting to p97 and p37.
This study establishes that SPI disassembly releases active PP1. This was shown through dephosphorylation assays with purified components. Both, a synthetic (OMFP) and an endogenous PP1 substrate (eIF2α) were effectively dephosphorylated following SPI disassembly, and dependent on p97 activity. Activity could only be observed under conditions where re-association of SDS22 and I3 was prevented. To show SPI reassembly directly, p97 was inhibited with CB-5083 at the end of the SPI disassembly reaction. Under p97 inhibition, constant SPI disassembly ceases, which resulted in re-binding of SDS22 and I3 to PP1, as shown by FRET. Demonstrating SPI reassembly indicates a post-biogenesis relevance for the formation of the SPI complex.
Re-association of SDS22 and I3 caused the displacement of other regulatory PP1 subunits. Even in the absence of p97 and p37, SDS22 and I3 displaced NIPP1 from PP1 in a cooperative manner. While NIPP1 could be displaced by SDS22 and I3, SPI disassembly and NIPP1 binding to PP1 was strictly dependent on p97 activity. This was highlighted by the observation that SPI was stable in the presence of high concentrations of NIPP1 over long time periods. These results indicate that PP1 availability for holophosphatase formation and activity is governed by alternating binding of SDS22 and I3, and p97-mediated SPI disassembly. The purpose of PP1 regulation by p97 and SPI could be to prevent PP1 from existing free in the cell, as the PP1 catalytic domain is highly indiscriminate towards substrates, which would cause signaling problems in the cell. Another hypothesis is that the ability of SDS22 and I3 to take apart PP1 holoenzymes contributes to exchange of PP1 holoenzyme subunits. Together with p97-mediated SPI disassembly, this would maintain of a pool of free PP1, available for holoenzyme formation, as well as regulate dynamic PP1 subunit exchange. This study contributed insights into the convergence of two major, evolutionarily conserved cellular regulators, PP1 and p97. Future studies may build upon the concepts presented here to elucidate precisely how these mechanisms contribute to cellular health and disease.
High resolution FRET-based assays were established to determined kinetic parameters of the SPI disassembly reaction catalyzed by p97. To overcome FRET detection limits, the parameters were determined through the association of the PP1 interacting protein NIPP1 to PP1 after SPI disassembly. Kinetic parameters at 37 °C were KM = 342 *÷ 1.37 nM and kcat = 2.70 *÷ 1.25 min-1, demonstrating that the reaction achieved half its maximal velocity at a concentration of 342 nM SPI, with approximately 2.7 SPI molecules per p97 molecule being catalyzed per minute at 37 °C. The values reveal that SPI as a substrate of p97 is efficiently disassembled, displaying even more effective targeting than poly-ubiquitinated substrates, based on published data.
FRET assays monitoring the dissociation and association of SDS22 and I3 to PP1 revealed that both proteins dissociate simultaneously, while NIPP1 binds with a delay. The delayed binding of NIPP1 reveals that SDS22 is not displaced by a third PP1 interacting protein. Instead, experiments performed with a complex of SDS22 and PP1, lacking I3, demonstrated that I3 was essential for SDS22 dissociation and also aided in the association of SDS22 to PP1. These results indicate direct involvement of I3 in the SDS22-PP1 interaction, possibly with a role in PP1 metal ion exchange. Further, SDS22 was also necessary for efficient I3 disassembly, suggesting a role in SPI targeting to p97 and p37.
This study establishes that SPI disassembly releases active PP1. This was shown through dephosphorylation assays with purified components. Both, a synthetic (OMFP) and an endogenous PP1 substrate (eIF2α) were effectively dephosphorylated following SPI disassembly, and dependent on p97 activity. Activity could only be observed under conditions where re-association of SDS22 and I3 was prevented. To show SPI reassembly directly, p97 was inhibited with CB-5083 at the end of the SPI disassembly reaction. Under p97 inhibition, constant SPI disassembly ceases, which resulted in re-binding of SDS22 and I3 to PP1, as shown by FRET. Demonstrating SPI reassembly indicates a post-biogenesis relevance for the formation of the SPI complex.
Re-association of SDS22 and I3 caused the displacement of other regulatory PP1 subunits. Even in the absence of p97 and p37, SDS22 and I3 displaced NIPP1 from PP1 in a cooperative manner. While NIPP1 could be displaced by SDS22 and I3, SPI disassembly and NIPP1 binding to PP1 was strictly dependent on p97 activity. This was highlighted by the observation that SPI was stable in the presence of high concentrations of NIPP1 over long time periods. These results indicate that PP1 availability for holophosphatase formation and activity is governed by alternating binding of SDS22 and I3, and p97-mediated SPI disassembly. The purpose of PP1 regulation by p97 and SPI could be to prevent PP1 from existing free in the cell, as the PP1 catalytic domain is highly indiscriminate towards substrates, which would cause signaling problems in the cell. Another hypothesis is that the ability of SDS22 and I3 to take apart PP1 holoenzymes contributes to exchange of PP1 holoenzyme subunits. Together with p97-mediated SPI disassembly, this would maintain of a pool of free PP1, available for holoenzyme formation, as well as regulate dynamic PP1 subunit exchange. This study contributed insights into the convergence of two major, evolutionarily conserved cellular regulators, PP1 and p97. Future studies may build upon the concepts presented here to elucidate precisely how these mechanisms contribute to cellular health and disease.