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.