Structure-Guided Exploration of SDS22 Interactions with Protein Phosphatase PP1 and the Splicing Factor BCLAF1.

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.

Split-BioID: a proximity biotinylation assay for dimerization-dependent protein interactions.

The biotin identification (BioID) protocol uses a mutant of the biotin ligase BirA (BirA*) fused to a protein-of-interest to biotinylate proximate proteins in intact cells. Here, we show that two inactive halves of BirA* separately fused to a catalytic and regulatory subunit of protein phosphatase PP1 reconstitute a functional BirA* enzyme upon heterodimerization of the phosphatase subunits. We also demonstrate that this BirA* fragment complementation approach, termed split-BioID, can be used to screen for substrates and other protein interactors of PP1 holoenzymes. Split-BioID is a novel and versatile tool for the identification of (transient) interactors of protein dimers.

The selective inhibition of protein phosphatase-1 results in mitotic catastrophe and impaired tumor growth.

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 mitotic-arrest 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.

Metals in the active site of native protein phosphatase-1.

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.

The PP1 binding code: a molecular-lego strategy that governs specificity.

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.

The molecular basis for substrate specificity of the nuclear NIPP1:PP1 holoenzyme.

Regulation of protein phosphatase 1 (PP1) is controlled by a diverse array of regulatory proteins. However, how these proteins direct PP1 specificity is not well understood. More than one-third of the nuclear pool of PP1 forms a holoenzyme with the nuclear inhibitor of PP1, NIPP1, to regulate chromatin remodeling, among other essential biological functions. Here, we show that the PP1-binding domain of NIPP1 is an intrinsically disordered protein, which binds PP1 in an unexpected manner. NIPP1 forms an α helix that engages PP1 at a unique interaction site, using polar rather than hydrophobic contacts. Importantly, the structure also reveals a shared PP1 interaction site outside of the RVxF motif, the ΦΦ motif. Finally, we show that NIPP1:PP1 substrate selectivity is determined by altered electrostatics and enhanced substrate localization. Together, our results provide the molecular basis by which NIPP1 directs PP1 substrate specificity in the nucleus.