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The serine/threonine protein phosphatase-1 (PP1) complex is a key regulator of the cell cycle. However, the redundancy of PP1 isoforms and the lack of specific inhibitors have hampered studies on the global role of PP1 in cell cycle progression in vertebrates. Here, we show that the overexpression of nuclear inhibitor of PP1 (NIPP1; also known as PPP1R8) in HeLa cells culminated in a prometaphase arrest, associated with severe spindle-formation and chromosome-congression defects. In addition, the spindle assembly checkpoint was activated and checkpoint silencing was hampered. Eventually, most cells either died by apoptosis or formed binucleated cells. The NIPP1-induced mitotic arrest could be explained by the inhibition of PP1 that was titrated away from other mitotic PP1 interactors. Consistent with this notion, the mitoticarrest phenotype could be rescued by the overexpression of PP1 or the inhibition of the Aurora B kinase, which acts antagonistically to PP1. Finally, we demonstrate that the overexpression of NIPP1 also hampered colony formation and tumor growth in xenograft assays in a PP1-dependent manner. Our data show that the selective inhibition of PP1 can be used to induce cancer cell death through mitotic catastrophe. [ABSTRACT FROM AUTHOR]

Faithful chromosome segregation during mitosis is tightly regulated by opposing activities of Aurora B kinase and protein phosphatase-1 ( PP1). PP1 function at kinetochores has been linked to SDS22, but the exact localization of SDS22 and how it affects PP1 are controversial. Here, we confirm that SDS22 is required for PP1 activity, but show that SDS22 does not normally localize to kinetochores. Instead, SDS22 is kept in solution by formation of a ternary complex with PP1 and inhibitor-3 (I3). Depletion of I3 does not affect the amount of PP1 at kinetochores but causes quantitative association of SDS22 with PP1 on KNL1 at the kinetochore. Such accumulation of SDS22 at kinetochores interferes with PP1 activity and inhibits Aurora B threonine-232 dephosphorylation, which leads to increased Aurora B activity in metaphase and persistence in anaphase accompanied with segregation defects. We propose a model in which I3 regulates an SDS22-mediated PP1 activation step in solution that precedes SDS22 dissociation and transfer of PP1 to kinetochores, and which is required for PP1 to efficiently antagonize Aurora B. [ABSTRACT FROM AUTHOR]

Ser/Thr protein phosphatase 1 (PP1) is a single-domain hub protein with nearly 200 validated interactors in vertebrates. PP1-interacting proteins (PIPs) are ubiquitously expressed but show an exceptional diversity in brain, testis and white blood cells. The binding of PIPs is mainly mediated by short motifs that dock to surface grooves of PP1. Although PIPs often contain variants of the same PP1 binding motifs, they differ in the number and combination of docking sites. This molecular-lego strategy for binding to PP1 creates holoenzymes with unique properties. The PP1 binding code can be described as specific, universal, degenerate, nonexclusive and dynamic. PIPs control associated PP1 by interference with substrate recruitment or access to the active site. In addition, some PIPs have a subcellular targeting domain that promotes dephosphorylation by increasing the local concentration of PP1. The diversity of the PP1 interactome and the properties of the PP1 binding code account for the exquisite specificity of PP1 in vivo. [ABSTRACT FROM AUTHOR]

Abstract: The ubiquitously expressed protein Ser/Thr phosphatase-1 isoforms PP1α, PP1β and PP1γ1 are dynamically targeted to distinct, but overlapping cellular compartments by associated proteins. Within the nucleus of HeLa cells, EGFP-tagged PP1γ1 and PP1β were predominantly targeted to the nucleoli, while PP1α showed a more diffuse distribution. Using PP1 chimaeras and point mutants we show here that a single N-terminal residue, i.e., Gln20 for PP1α, Arg19 for PP1β and Arg20 for PP1γ1 accounts for their distinct subnuclear distribution. Our data also suggest that the N-terminus of PP1β and PP1γ1 harbours an interaction site for one or more nucleolar interactors. [Copyright &y& Elsevier]

Protein phosphatase-1 (PP1) is a major protein Ser/Thr phosphatase in eukaryotic cells. Its activity depends on two metal ions in the catalytic site, which were identified as manganese in the bacterially expressed phosphatase. However, the identity of the metal ions in native PP1 is unknown. In this study, total reflection X-ray fluorescence (TXRF) was used to detect iron and zinc in PP1 that was purified from rabbit skeletal muscle. Metal exchange experiments confirmed that the distinct substrate specificity of recombinant and native PP1 is determined by the nature of their associated metals. We also found that the iron level associated with native PP1 is decreased by incubation with inhibitor-2, consistent with a function of inhibitor-2 as a PP1 chaperone. [ABSTRACT FROM AUTHOR]

Summary SDS22 is an ancient regulator of protein phosphatase-1 (PP1). Our crystal structure of SDS22 shows that its twelve leucine-rich repeats adopt a banana-shaped fold that is shielded from solvent by capping domains at its extremities. Subsequent modeling and biochemical studies revealed that the concave side of SDS22 likely interacts with PP1 helices α5 and α6, which are distal from the binding sites of many previously described PP1 interactors. Accordingly, we found that SDS22 acts as a "third" subunit of multiple PP1 holoenzymes. The crystal structure of SDS22 also revealed a large basic surface patch that enables binding of a phosphorylated form of splicing factor BCLAF1. Taken together, our data provide insights into the formation of PP1:SDS22 and the recruitment of additional interaction proteins, such as BCLAF1. Graphical Abstract Highlights • The LRRs of SDS22 interact with PP1 helices α5 and α6 • A basic surface patch of SDS22 binds phosphorylated BCLAF1 • Many PP1 holoenzymes contain SDS22 as a third subunit • SDS22 prevents the autodephosphorylation of PP1 The function of protein phosphatase 1 (PP1) is determined by associated regulatory proteins. Heroes et al. describe the structure of the PP1 regulator SDS22, which largely consists of leucine-rich repeats. They also present a biochemically validated model of the PP1:SDS22 complex and use it as a guide for functional studies. [ABSTRACT FROM AUTHOR]