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Cytokinesis, the final step of mitosis, is mediated by an actomyosin contractile ring, the formation of which is temporally and spatially regulated following anaphase onset. Aurora-B is a member of the chromosomal passenger complex, which regulates various processes during mitosis; it is not understood, however, how Aurora-B is involved in cytokinesis. Here, we show that Aurora-B and myosin-IIB form a complex in vivo during telophase. Aurora-B phosphorylates the myosin-IIB rod domain at threonine 1847 (T1847), abrogating the ability of myosin-IIB monomers to form filaments. Furthermore, phosphorylation of myosin-IIB filaments by Aurora-B also promotes filament disassembly. We show that myosin-IIB possessing a phosphomimetic mutation at T1847 was unable to rescue cytokinesis failure caused by myosin-IIB depletion. Cells expressing a phosphoresistant mutation at T1847 had significantly longer intercellular bridges, implying that Aurora-B-mediated phosphorylation of myosin-IIB is important for abscission. We propose that myosin-IIB is a substrate of Aurora-B and reveal a new mechanism of myosin-IIB regulation by Aurora-B in the late stages of mitosis.

Background: INCENP is predicted to have a coiled coil domain. Results: The coil is actually a stable single α-helix (SAH) domain that is highly extendable and directly binds microtubules. Conclusion: This flexible dog leash may allow Aurora B to associate with dynamic targets in the outer kinetochore. Significance: The SAH domain allows CPC flexibility without requiring complex dimerization., The chromosome passenger complex (CPC) is a master regulator of mitosis. Inner centromere protein (INCENP) acts as a scaffold regulating CPC localization and activity. During early mitosis, the N-terminal region of INCENP forms a three-helix bundle with Survivin and Borealin, directing the CPC to the inner centromere where it plays essential roles in chromosome alignment and the spindle assembly checkpoint. The C-terminal IN box region of INCENP is responsible for binding and activating Aurora B kinase. The central region of INCENP has been proposed to comprise a coiled coil domain acting as a spacer between the N- and C-terminal domains that is involved in microtubule binding and regulation of the spindle checkpoint. Here we show that the central region (213 residues) of chicken INCENP is not a coiled coil but a ∼32-nm-long single α-helix (SAH) domain. The N-terminal half of this domain directly binds to microtubules in vitro. By analogy with previous studies of myosin 10, our data suggest that the INCENP SAH might stretch up to ∼80 nm under physiological forces. Thus, the INCENP SAH could act as a flexible “dog leash,” allowing Aurora B to phosphorylate dynamic substrates localized in the outer kinetochore while at the same time being stably anchored to the heterochromatin of the inner centromere. Furthermore, by achieving this flexibility via an SAH domain, the CPC avoids a need for dimerization (required for coiled coil formation), which would greatly complicate regulation of the proximity-induced trans-phosphorylation that is critical for Aurora B activation.

Aurora kinase A and B share great similarity in sequences, structures, and phosphorylation motif, yet they show different localizations and play distinct crucial roles. The factors that determine such differences are largely unknown. Here we targeted Aurora A to the localization of Aurora B and found that Aurora A phosphorylates the substrate of Aurora B and substitutes its function in spindle checkpoint. In return, the centrosome targeting of Aurora B substitutes the function of Aurora A in the mitotic entry. Expressing the chimera proteins of the Auroras with exchanged N termini in cells indicates that the divergent N termini are also important for their spatiotemporal localizations and functions. Collectively, we demonstrate that functional divergence of Aurora kinases is determined by spatial compartmentalization, and their divergent N termini also contribute to their spatial and functional differentiation.

The condensin complex plays a key role in organizing mitotic chromosomes. In vertebrates, there are two condensin complexes that have independent and cooperative roles in folding mitotic chromosomes. In this study, we dissect the role of a putative Cdk1 site on the condensin II subunit CAP-D3 in chicken DT40 cells. This conserved site has been shown to activate condensin II during prophase in human cells, and facilitate further phosphorylation by polo-like kinase I. We examined the functional significance of this phosphorylation mark by mutating the orthologous site of CAP-D3 (CAP-D3(T1403A)) in chicken DT40 cells. We show that this mutation is a gain of function mutant in chicken cells; it disrupts prophase, results in a dramatic shortening of the mitotic chromosome axis, and leads to abnormal INCENP localization. Our results imply phosphorylation of CAP-D3 acts to limit condensin II binding onto mitotic chromosomes. We present the first in vivo example that alters the ratio of condensin I:II on mitotic chromosomes. Our results demonstrate this ratio is a critical determinant in shaping mitotic chromosomes.

INCENP, Borealin, Survivin, and Aurora B kinase comprise the chromosomal passenger complex, an essential regulator of mitotic events. INCENP (inner centromere protein) binds and activates Aurora B through a feedback loop involving phosphorylation of a Thr-Ser-Ser (TSS) motif near the INCENP C terminus. Here, we have examined the role of the TSS motif in vertebrate cells using an DT40 INCENPON/OFF conditional knock-out cell line in which mutants are expressed in the absence of wild-type INCENP. Our analysis confirms that regulated phosphorylation of the two serine residues (presumably by Aurora B) is critical for full activation of the kinase and is essential for cell viability. Cells expressing INCENP mutants bearing either phospho-null (TAA) or phospho-mimetic (TEE) mutations exhibit significant levels of Aurora B kinase activity but fail to undergo normal spindle elongation or complete cytokinesis. This work confirms previous suggestions that INCENP can act as a rheostat, with different INCENP mutants promoting differing degrees of kinase activation. Our results also reveal that mitotic progression is accompanied by a requirement for progressively higher levels of Aurora B kinase activity.

Mitotic kinases regulate cell division and its checkpoints, errors of which can lead to aneuploidy or genetic instability. One of these is Aurora-B, a key kinase that is required for chromosome alignment at the metaphase plate and for cytokinesis in mammalian cells. We report here that human Aurora-B is phosphorylated at Thr-232 through interaction with the inner centromere protein (INCENP) in vivo. The phosphorylation of Thr-232 occurs by means of an autophosphorylation mechanism, which is indispensable for the Aurora-B kinase activity. The activation of Aurora-B spatio-temporally correlated with the site-specific phosphorylation of its physiological substrates, histone H3 and vimentin. Overexpression of the TA mutant of Aurora-B, in which Thr-232 was changed into alanine, frequently induced multinuclearity in cells. These results indicate that the phosphorylation of Thr-232 is an essential regulatory mechanism for Aurora-B activation.