Junbin Qian

PP1/Repo-Man Dephosphorylates Mitotic Histone H3 at T3 and Regulates Chromosomal Aurora B Targeting

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.

Aurora B Defines Its Own Chromosomal Targeting by Opposing the Recruitment of the Phosphatase Scaffold Repo-Man

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.

Spindle Checkpoint Silencing: PP1 Tips the Balance

The spindle checkpoint is a mitotic surveillance mechanism that delays anaphase until all sister chromatids are correctly attached to microtubules from opposite poles. Recent studies reveal that protein kinase Aurora B is a key regulator of spindle checkpoint activation whereas protein phosphatase PP1 antagonizes Aurora B and induces checkpoint silencing. Chromosome biorientation stretches the kinetochores and spatially separates centromeric Aurora B from its kinetochore substrates, comprising several PP1-interacting proteins (PIPs). The ensuing dephosphorylation of these PIPs creates docking sites for the bulk recruitment of PP1 to the kinetochores. We propose that this tension-induced targeting of PP1 triggers checkpoint silencing by the dephosphorylation of kinetochore and checkpoint components, including Aurora B substrates. In addition, PP1 also directly inactivates a kinetochore-associated pool of Aurora B and silences checkpoint signaling by opposing the centromeric targeting of Aurora B.

Development of a peptide that selectively activates protein phosphatase-1 in living cells.

The first cell-penetrating peptide that 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.

Cdk1 orders mitotic events through coordination of a chromosome-associated phosphatase switch

RepoMan is a scaffold for signalling by mitotic phosphatases at the chromosomes. During (pro)metaphase, RepoMan-associated protein phosphatases PP1 and PP2A-B56 regulate the chromosome targeting of Aurora-B kinase and RepoMan, respectively. Here we show that this task division is critically dependent on the phosphorylation of RepoMan by protein kinase Cyclin-dependent kinase 1 (Cdk1), which reduces the binding of PP1 but facilitates the recruitment of PP2A-B56. The inactivation of Cdk1 in early anaphase reverses this phosphatase switch, resulting in the accumulation of PP1-RepoMan to a level that is sufficient to catalyse its own chromosome targeting in a PP2A-independent and irreversible manner. Bulk-targeted PP1-RepoMan also inactivates Aurora B and initiates nuclear-envelope reassembly through dephosphorylation-mediated recruitment of Importin β. Bypassing the Cdk1 regulation of PP1-RepoMan causes the premature dephosphorylation of its mitotic-exit substrates in prometaphase. Hence, the regulation of RepoMan-associated phosphatases by Cdk1 is essential for the timely dephosphorylation of their mitotic substrates.

4D-networking by mitotic phosphatases

Faithful progression through mitosis is critically dependent on the timely phosphorylation and dephosphorylation of a host of proteins. The involved protein kinases and phosphatases are embedded in interconnected feedback and feedforward circuits that ensure swift and robust phase transitions. Here we review recent evidence showing that protein phosphatases are modulators of the mitotic entry but also organize the mitotic exit through an orderly dephosphorylation of their substrates. In addition, phosphatases spatiotemporally restrict the phosphorylation of key regulatory proteins and oppose kinases to control highly dynamic mitotic processes, including chromosome congression and checkpoint signaling. In accordance with their important role as nodes in phosphorylation networks, mitotic protein phosphatases are tightly regulated in four dimensions.

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.

An Attachment-Independent Biochemical Timer of the Spindle Assembly Checkpoint

The spindle assembly checkpoint (SAC) generates a diffusible protein complex that prevents anaphase until all chromosomes are properly attached to spindle microtubules. A key step in SAC initiation is the recruitment of MAD1 to kinetochores, which is generally thought to be governed by the microtubule-kinetochore (MT-KT) attachment status. However, we demonstrate that the recruitment of MAD1 via BUB1, a conserved kinetochore receptor, is not affected by MT-KT interactions in human cells. Instead, BUB1:MAD1 interaction depends on BUB1 phosphorylation, which is controlled by a biochemical timer that integrates counteracting kinase and phosphatase effects on BUB1 into a pulse-generating incoherent feedforward loop. We propose that this attachment-independent timer serves to rapidly activate the SAC at mitotic entry, before the attachment-sensing MAD1 receptors have become fully operational. The BUB1-centered timer is largely impervious to conventional anti-mitotic drugs, and it is, therefore, a promising therapeutic target to induce cell death through permanent SAC activation.