CDK1-CCNB1 creates a spindle checkpoint-permissive state by enabling MPS1 kinetochore localization.

Spindle checkpoint signaling is initiated by recruitment of the kinase MPS1 to unattached kinetochores during mitosis. We show that CDK1-CCNB1 and a counteracting phosphatase PP2A-B55 regulate the engagement of human MPS1 with unattached kinetochores by controlling the phosphorylation status of S281 in the kinetochore-binding domain. This regulation is essential for checkpoint signaling, since MPS1S281A is not recruited to unattached kinetochores and fails to support the recruitment of other checkpoint proteins. Directly tethering MPS1S281A to the kinetochore protein Mis12 bypasses this regulation and hence the requirement for S281 phosphorylation in checkpoint signaling. At the metaphase-anaphase transition, MPS1 S281 dephosphorylation is delayed because PP2A-B55 is negatively regulated by CDK1-CCNB1 and only becomes fully active once CCNB1 concentration falls below a characteristic threshold. This mechanism prolongs the checkpoint-responsive period when MPS1 can localize to kinetochores and enables a response to late-stage spindle defects. By acting together, CDK1-CCNB1 and PP2A-B55 thus create a spindle checkpoint-permissive state and ensure the fidelity of mitosis.

A PP2A-B55 recognition signal controls substrate dephosphorylation kinetics during mitotic exit.

PP2A-B55 is one of the major phosphatases regulating cell division. Despite its importance for temporal control during mitotic exit, how B55 substrates are recognized and differentially dephosphorylated is unclear. Using phosphoproteomics combined with kinetic modeling to extract B55-dependent rate constants, we have systematically identified B55 substrates and assigned their temporal order in mitotic exit. These substrates share a bipartite polybasic recognition determinant (BPR) flanking a Cdk1 phosphorylation site. Experiments and modeling show that dephosphorylation rate is encoded into B55 substrates, including its inhibitor ENSA, by cooperative action of basic residues within the BPR. A complementary acidic surface on B55 decodes this signal, supporting a cooperative electrostatic mechanism for substrate selection. A further level of specificity is encoded into B55 substrates because B55 displays selectivity for phosphothreonine. These simple biochemical properties, combined with feedback control of B55 activity by the phosphoserine-containing substrate/inhibitor ENSA, can help explain the temporal sequence of events during exit from mitosis.

KIF4A and PP2A-B56 form a spatially restricted feedback loop opposing Aurora B at the anaphase central spindle.

The mitotic kinase Aurora B is concentrated at the anaphase central spindle by the kinesin MKlp2 during mitotic exit and cytokinesis. This pool of Aurora B phosphorylates substrates including the kinesin KIF4A to regulate central spindle length. In this paper, we identify a counteracting system in which PP2A-B56γ and -ε, but not PP2A-B56α, -ß, and -δ, are maintained at the central spindle by KIF4A. Biochemical assays show that PP2A-B56γ can dephosphorylate the T799 Aurora B site on KIF4A and thereby counteract the Aurora B- and microtubule-stimulated ATPase activity of KIF4A. In agreement with these observations, combined silencing of PP2A-B56γ and -ε resulted in increased phosphorylation of KIF4A T799 and decreased central spindle growth in anaphase B. Furthermore, reduced turnover of regulatory phosphorylation on another Aurora B substrate MKlp1 was observed, suggesting that PP2A-B56γ and -ε play a general role opposing Aurora B at the central spindle. KIF4A and PP2A-B56γ and -ε therefore create a spatially restricted negative feedback loop counteracting Aurora B in anaphase.

The BEG (PP2A-B55/ENSA/Greatwall) pathway ensures cytokinesis follows chromosome separation.

Cytokinesis follows separase activation and chromosome segregation. This order is ensured in budding yeast by the mitotic exit network (MEN), where Cdc14p dephosphorylates key conserved Cdk1-substrates exemplified by the anaphase spindle-elongation protein Ase1p. However, in metazoans, MEN and Cdc14 function is not conserved. Instead, the PP2A-B55α/ENSA/Greatwall (BEG) pathway controls the human Ase1p ortholog PRC1. In this pathway, PP2A-B55 inhibition is coupled to Cdk1-cyclin B activity, whereas separase inhibition is maintained by cyclin B concentration. This creates two cyclin B thresholds during mitotic exit. Simulation and experiments using PRC1 as a model substrate show that the first threshold permits separase activation and chromosome segregation, and the second permits PP2A-B55 activation and initiation of cytokinesis. Removal of the ENSA/Greatwall (EG) timer module eliminates this second threshold, as well as associated delay in PRC1 dephosphorylation and initiation of cytokinesis, by uncoupling PP2A-B55 from Cdk1-cyclin B activity. Therefore, temporal order during mitotic exit is promoted by the metazoan BEG pathway.

Functional specialisation of yeast Rho1 GTP exchange factors.

Rho GTPases are regulated in complex spatiotemporal patterns that might be dependent, in part at least, on the multiplicity of their GTP exchange factors (GEFs). Here, we examine the extent of and basis for functional specialisation of the Rom2 and Tus1 GEFs that activate the yeast Rho1 GTPase, the orthologue of mammalian RhoA. First, we find that these GEFs selectively activate different Rho1-effector branches. Second, the synthetic genetic networks around ROM2 and TUS1 confirm very different global in vivo roles for these GEFs. Third, the GEFs are not functionally interchangeable: Tus1 cannot replace the essential role of Rom2, even when overexpressed. Fourth, we find that Rom2 and Tus1 localise differently: Rom2 to the growing bud surface and to the bud neck at cytokinesis; Tus1 only to the bud neck, but in a distinct pattern. Finally, we find that these GEFs are dependent on different protein co-factors: Rom2 function and localisation is largely dependent on Ack1, a SEL1-domain-containing protein; Tus1 function and localisation is largely dependent on the Tus1-interacting protein Ypl066w (which we name Rgl1). We have revealed a surprising level of diversity among the Rho1 GEFs that contributes another level of complexity to the spatiotemporal control of Rho1.