Cdh1 is a HECT of an activator.

Cdh1 is a well-established activator of APC/C, a RING-type ubiquitin ligase. In this issue of Molecular Cell, Wan et al. (2011) report an APC/C-independent role of Cdh1 during development as an activator for Smurf1, a HECT-type ubiquitin ligase.

Control of APC/C-dependent ubiquitin chain elongation by reversible phosphorylation.

Most metazoan E3 ligases contain a signature RING domain that promotes the transfer of ubiquitin from the active site of E2 conjugating enzymes to lysine residues in substrates. Although these RING-E3s depend on E2 enzymes for catalysis, how they turn on their E2s at the right time and place remains poorly understood. Here we report a phosphorylation-dependent mechanism that ensures timely activation of the E2 Ube2S by its RING-E3, the anaphase-promoting complex (APC/C); while phosphorylation of a specific serine residue in the APC/C coactivator Cdc20 prevents delivery of Ube2S to the APC/C, removal of this mark by PP2A(B56) allows Ube2S to bind the APC/C and catalyze ubiquitin chain elongation. PP2A(B56) also stabilizes kinetochore-microtubule attachments to shut off the spindle checkpoint, suggesting that cells regulate the E2-E3 interplay to coordinate ubiquitination with critical events during cell division.

Tracking spindle checkpoint signals from kinetochores to APC/C.

Accurate chromosome segregation during mitosis is critical for maintaining genomic stability. The kinetochore--a large protein assembly on centromeric chromatin--functions as the docking site for spindle microtubules and a signaling hub for the spindle checkpoint. At metaphase, spindle microtubules from opposing spindle poles capture each pair of sister kinetochores, exert pulling forces, and create tension across sister kinetochores. The spindle checkpoint detects improper kinetochore-microtubule attachments and translates these defects into biochemical activities that inhibit the anaphase-promoting complex or cyclosome (APC/C) throughout the cell to delay anaphase onset. A deficient spindle checkpoint leads to premature sister-chromatid separation and aneuploidy. Here, we review recent progress on the generation, propagation, transmission, and silencing of the spindle checkpoint signals from kinetochores to APC/C.

The Cdc20-binding Phe box of the spindle checkpoint protein BubR1 maintains the mitotic checkpoint complex during mitosis.

The spindle checkpoint ensures accurate chromosome segregation by monitoring kinetochore-microtubule attachment. Unattached or tensionless kinetochores activate the checkpoint and enhance the production of the mitotic checkpoint complex (MCC) consisting of BubR1, Bub3, Mad2, and Cdc20. MCC is a critical checkpoint inhibitor of the anaphase-promoting complex/cyclosome, a ubiquitin ligase required for anaphase onset. The N-terminal region of BubR1 binds to both Cdc20 and Mad2, thus nucleating MCC formation. The middle region of human BubR1 (BubR1M) also interacts with Cdc20, but the nature and function of this interaction are not understood. Here we identify two critical motifs within BubR1M that contribute to Cdc20 binding and anaphase-promoting complex/cyclosome inhibition: a destruction box (D box) and a phenylalanine-containing motif termed the Phe box. A BubR1 mutant lacking these motifs is defective in MCC maintenance in mitotic human cells but is capable of supporting spindle-checkpoint function. Thus, the BubR1M-Cdc20 interaction indirectly contributes to MCC homeostasis. Its apparent dispensability in the spindle checkpoint might be due to functional duality or redundant, competing mechanisms.

A sequential multi-target Mps1 phosphorylation cascade promotes spindle checkpoint signaling.

The master spindle checkpoint kinase Mps1 senses kinetochore-microtubule attachment and promotes checkpoint signaling to ensure accurate chromosome segregation. The kinetochore scaffold Knl1, when phosphorylated by Mps1, recruits checkpoint complexes Bub1-Bub3 and BubR1-Bub3 to unattached kinetochores. Active checkpoint signaling ultimately enhances the assembly of the mitotic checkpoint complex (MCC) consisting of BubR1-Bub3, Mad2, and Cdc20, which inhibits the anaphase-promoting complex or cyclosome bound to Cdc20 (APC/CCdc20) to delay anaphase onset. Using in vitro reconstitution, we show that Mps1 promotes APC/C inhibition by MCC components through phosphorylating Bub1 and Mad1. Phosphorylated Bub1 binds to Mad1-Mad2. Phosphorylated Mad1 directly interacts with Cdc20. Mutations of Mps1 phosphorylation sites in Bub1 or Mad1 abrogate the spindle checkpoint in human cells. Therefore, Mps1 promotes checkpoint activation through sequentially phosphorylating Knl1, Bub1, and Mad1. This sequential multi-target phosphorylation cascade makes the checkpoint highly responsive to Mps1 and to kinetochore-microtubule attachment.

The Bub1-Plk1 kinase complex promotes spindle checkpoint signalling through Cdc20 phosphorylation.

The spindle checkpoint senses unattached kinetochores and inhibits the Cdc20-bound anaphase-promoting complex or cyclosome (APC/C), to delay anaphase, thereby preventing aneuploidy. A critical checkpoint inhibitor of APC/C(Cdc20) is the mitotic checkpoint complex (MCC). It is unclear whether MCC suffices to inhibit all cellular APC/C. Here we show that human checkpoint kinase Bub1 not only directly phosphorylates Cdc20, but also scaffolds Plk1-mediated phosphorylation of Cdc20. Phosphorylation of Cdc20 by Bub1-Plk1 inhibits APC/C(Cdc20) in vitro and is required for checkpoint signalling in human cells. Bub1-Plk1-dependent Cdc20 phosphorylation is regulated by upstream checkpoint signals and is dispensable for MCC assembly. A phospho-mimicking Cdc20 mutant restores nocodazole-induced mitotic arrest in cells depleted of Mad2 or BubR1. Thus, Bub1-Plk1-mediated phosphorylation of Cdc20 constitutes an APC/C-inhibitory mechanism that is parallel, but not redundant, to MCC formation. Both mechanisms are required to sustain mitotic arrest in response to spindle defects.

Substrate-specific activation of the mitotic kinase Bub1 through intramolecular autophosphorylation and kinetochore targeting.

During mitosis of human cells, the kinase Bub1 orchestrates chromosome segregation through phosphorylating histone H2A and the anaphase-promoting complex/cyclosome activator Cdc20. Bub1-mediated H2A-T120 phosphorylation (H2A-pT120) at kinetochores promotes centromeric sister-chromatid cohesion, whereas Cdc20 phosphorylation by Bub1 contributes to spindle checkpoint signaling. Here, we show that phosphorylation at the P+1 substrate-binding loop of human Bub1 enhances its activity toward H2A but has no effect on its activity toward Cdc20. We determine the crystal structure of phosphorylated Bub1. A comparison between structures of phosphorylated and unphosphorylated Bub1 reveals phosphorylation-triggered reorganization of the P+1 loop. This activating phosphorylation of Bub1 is constitutive during the cell cycle. Enrichment of H2A-pT120 at mitotic kinetochores requires kinetochore targeting of Bub1. The P+1 loop phosphorylation of Bub1 appears to occur through intramolecular autophosphorylation. Our study provides structural and functional insights into substrate-specific regulation of a key mitotic kinase and expands the repertoire of kinase activation mechanisms.

Phospho-H2A and cohesin specify distinct tension-regulated Sgo1 pools at kinetochores and inner centromeres.

Accurate chromosome segregation requires coordination between the dissolution of sister-chromatid cohesion and the establishment of proper kinetochore-microtubule attachment. During mitosis, sister-chromatid cohesion at centromeres enables the biorientation of and tension across sister kinetochores. The complex between shugoshin and protein phosphatase 2A (Sgo1-PP2A) localizes to centromeres in mitosis, binds to cohesin in a reaction requiring Cdk-dependent phosphorylation of Sgo1, dephosphorylates cohesin-bound sororin, and protects a centromeric pool of cohesin from mitotic kinases and the cohesin inhibitor Wapl. Cleavage of centromeric cohesin by separase allows sister chromatids connected to microtubules from opposing poles to be evenly partitioned into daughter cells. The centromeric localization of Sgo1 requires histone H2A phosphorylation at T120 (H2A-pT120) by the kinase Bub1. The exact role of H2A-pT120 in Sgo1 regulation is, however, unclear. Here, we show that cohesin and H2A-pT120 specify two distinct pools of Sgo1-P2A at inner centromeres and kinetochores, respectively, in human cells. Bub1 inactivation delocalizes cohesin-Sgo1 to chromosome arms. Kinetochore tension triggers Sgo1 dephosphorylation and redistributes Sgo1 from inner centromeres to kinetochores. Incomplete Sgo1 redistribution causes chromosome nondisjunction. Our study suggests that Bub1-mediated H2A phosphorylation penetrates kinetochores and that this histone mark contributes to a tension-sensitive Sgo1-based molecular switch for chromosome segregation.

Defining pathways of spindle checkpoint silencing: functional redundancy between Cdc20 ubiquitination and p31(comet).

The spindle checkpoint senses unattached or improperly attached kinetochores during mitosis, inhibits the anaphase-promoting complex or cyclosome (APC/C), and delays anaphase onset to prevent aneuploidy. The mitotic checkpoint complex (MCC) consisting of BubR1, Bub3, Mad2, and Cdc20 is a critical APC/C-inhibitory checkpoint complex in human cells. At the metaphase-anaphase transition, the spindle checkpoint turns off, and MCC disassembles to allow anaphase onset. The molecular mechanisms of checkpoint inactivation are poorly understood. A major unresolved issue is the role of Cdc20 autoubiquitination in this process. Although Cdc20 autoubiquitination can promote Mad2 dissociation from Cdc20, a nonubiquitinatable Cdc20 mutant still dissociates from Mad2 during checkpoint inactivation. Here, we show that depletion of p31(comet) delays Mad2 dissociation from Cdc20 mutants that cannot undergo autoubiquitination. Thus both p31(comet) and ubiquitination of Cdc20 are critical mechanisms of checkpoint inactivation. They act redundantly to promote Mad2 dissociation from Cdc20.