The ubiquitous protein Ser/Thr phosphatase-1 (PP1) interacts with dozens of regulatory proteins that are structurally unrelated. However, most of them share a short, degenerate "RVxF"-type docking motif. Using a broad in silico screening based on a stringent definition of the RVxF motif, in combination with a multistep biochemical validation procedure, we have identified 78 novel mammalian PP1 interactors. A global analysis of the validated RVxF-based PP1 interactome not only provided insights into the conserved features of the RVxF motif but also led to the discovery of additional common PP1 binding elements, described as the "SILK" and "MyPhoNE" motifs. In addition to the doubling of the known mammalian PP1 interactome, our data contribute to the design of PP1 interaction networks. Notably, an interaction network linking PP1 interactors discloses a pleiotropic role of PP1 in cell polarity.

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 transient mitotic histone H3 phosphorylation by various protein kinases regulates chromosome condensation and segregation, but the counteracting phosphatases have been poorly characterized [1-8]. We show here that PP1γ is the major histone H3 phosphatase acting on the mitotically phosphorylated (ph) residues H3T3ph, H3S10ph, H3T11ph, and H3S28ph. In addition, we identify Repo-Man, a chromosome-bound interactor of PP1γ [9], as a selective regulator of H3T3ph and H3T11ph dephosphorylation. Repo-Man promotes H3T11ph dephosphorylation by an indirect mechanism but directly and specifically targets H3T3ph for dephosphorylation by associated PP1γ. The PP1γ/Repo-Man complex opposes the protein kinase Haspin-mediated spreading of H3T3ph to the chromosome arms until metaphase and catalyzes the net dephosphorylation of H3T3ph at the end of mitosis. Consistent with these findings, Repo-Man modulates in a PP1-dependent manner the H3T3ph-regulated chromosomal targeting of Aurora kinase B and its substrate MCAK. Our study defines a novel mechanism by which PP1 counteracts Aurora B.

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Aurora B is the catalytic subunit of the chromosomal passenger complex (CPC), which coordinates mitotic processes through phosphorylation of key regulatory proteins. In prometaphase, the CPC is enriched at the centromeres to regulate the spindle checkpoint and kinetochore-microtubule interactions. Centromeric CPC binds to histone H3 that is phosphorylated at T3 (H3T3ph) by Aurora B-stimulated Haspin. PP1/Repo-Man acts antagonistically to Haspin and dephosphorylates H3T3ph at the chromosome arms but is somehow prevented from causing a net dephosphorylation of centromeric H3T3ph during prometaphase. Here, we show that Aurora B phosphorylates Repo-Man at S893, preventing its recruitment by histones. We also identify PP2A as a mitotic interactor of Repo-Man that dephosphorylates S893 and thereby promotes the targeting of Repo-Man to chromosomes and the dephosphorylation of H3T3ph by PP1. Thus, Repo-Man-associated PP1 and PP2A collaborate to oppose the chromosomal targeting of Aurora B. We propose that the reciprocal feedback regulation of Haspin and Repo-Man by Aurora B generates a robust bistable response that culminates in the centromeric targeting of the CPC during prometaphase.

The first cell-penetrating peptidethat activates protein phosphatase-1 (PP1) by disrupting a subset of PP1 complexes in living cells has been developed. Activated PP1 rapidly dephosphorylates its substrates, counteracting kinase activity inside cells. Activation of PP1 can thus be a novel approach to study PP1 function and to counteract Ser/Thr kinase activity under pathologically increased kinase signaling.

Human FBP21 (formin-binding protein 21) contains a matrin-type zinc finger and two tandem WW domains. It is a component of the spliceosomes and interacts with several established splicing factors. Here we demonstrate for the first time that FBP21 is an activator of pre-mRNA splicing in vivo and that its splicing activation function and interaction with the splicing factor SIPP1 (splicing factor that interacts with PQBP1 and PP1) are both mediated by the two tandem WW domains of group III. We determined the solution structure of the tandem WW domains of FBP21 and found that the WW domains recognize peptide ligands containing either group II (PPLP) or group III (PPR) motifs. The binding interfaces involve both the XP and XP2 grooves of the two WW domains. Significantly, the tandem WW domains of FBP21 are connected by a highly flexible region, enabling their simultaneous interaction with two proline-rich motifs of SIPP1. The strong interaction between SIPP1 and FBP21 can be explained by the conjugation of two low affinity interactions with the tandem WW domains. Our study provides a structural basis for understanding the molecular mechanism underlying the functional implication of FBP21 and the biological specificity of tandem WW domains.Gene expression in eukaryotic cells involves several steps, including transcription, mRNA processing, and export. Pre-mRNA splicing takes place in the spliceosome, a highly dynamic ribonucleoprotein particle that consists of five small nuclear RNAs and at least 150 proteins. Small nuclear ribonucleoproteins (snRNPs) 3 and numerous protein factors are essential for the formation of the active spliceosome (1, 2). In budding yeast, the splicing factor Prp40 participates in crossintron bridging by interacting with the branch point-binding protein (BBP) and the U5 snRNP component Prp8. Prp40 contacts the 5Ј splice site and interacts with BBP, bringing the 5Ј splice site and the branch point in spatial proximity. These interactions are believed to be conserved in mammals (3-5). FBP21 (formin-binding protein 21), the mammalian Prp40-like protein, colocalizes with splicing factors in nuclear storage sites for pre-mRNA splicing factors. In addition, FBP21 is a component of the mammalian spliceosomal A/B complex and is associated with U2 snRNPs (6). FBP21 interacts directly with the splicing factors U1 snRNP protein U1C, the core snRNP proteins SmB and SmBЈ, and the branch point-binding protein SF1/mBBP, suggesting that it may also play a role in crossintron bridging of U1 and U2 snRNPs in the spliceosomes. FBP21 contains a matrin-type zinc finger and two group III WW domains ( Fig. 1) that are structurally related to those of the established splicing factors U1C and Prp40, respectively (6, 7). The binding of FBP21 to splicing factors is mediated by its tandem WW domains, which represent interaction modules for proline-rich ligands (4, 8, 9). Although the above data strongly suggest that FBP21 has a role in pre-mRNA splicing, there are no in vivo data to support this contention. The splicing...

Protein Ser/Thr phosphatase-1 (PP1) associates with a host of proteins to form substrate-specific holoenzymes. Sds22 and Inhibitor-3 (I3) are two independently described ancient interactors of PP1. We show here by various approaches that Sds22 and I3 form a heterotrimeric complex with PP1, both in cell lysates and after purification. The stability of the complex depended on functional PP1 interaction sites in Sds22 and I3, indicating that PP1 is sandwiched between Sds22 and I3. Intriguingly, I3 could not be replaced in this complex by another PP1 interactor with the same PP1 binding motif. In vitro, Sds22 and I3 were potent inhibitors of PP1, but with only some substrates. The inhibition by Sds22 could be reproduced with synthetic Sds22 fragments comprising leucine-rich repeats (LRR) 2 and 5. Sds22 and LRR5 also slowly converted PP1 into a conformation that was inactive with all tested substrates. Cell lysates that were prepared under conditions that prevented the Sds22-induced inactivation of PP1 contained a catalytically inactive complex of Sds22, PP1, and I3, indicating that this complex exists in vivo. Therefore, our studies show that a pool of PP1 is complexly controlled by both Sds22 and I3.