Phosphorylation of HsMis13 by Aurora B Kinase Is Essential for Assembly of Functional Kinetochore

Abstract

Chromosome movements in mitosis are orchestrated by dynamic interactions between spindle microtubules and the kinetochore, a multiprotein complex assembled onto centromeric DNA of the chromosome. Here we show that phosphorylation of human HsMis13 by Aurora B kinase is required for functional kinetochore assembly in HeLa cells. Aurora B interacts with HsMis13 in vitro and in vivo. HsMis13 is a cognate substrate of Aurora B, and the phosphorylation sites were mapped to Ser-100 and Ser-109. Suppression of Aurora B kinase by either small interfering RNA or chemical inhibitors abrogates the localization of HsMis13 but not HsMis12 to the kinetochore. In addition, non-phosphorylatable but not wild type and phospho-mimicking HsMis13 failed to localize to the kinetochore, demonstrating the requirement of phosphorylation by Aurora B for the assembly of HsMis13 to kinetochore. In fact, localization of HsMis13 to the kinetochore is spatiotemporally regulated by Aurora B kinase, which is essential for recruiting outer kinetochore components such as Ndc80 components and CENP-E for functional kinetochore assembly. Importantly, phospho-mimicking mutant HsMis13 restores the assembly of CENP-E to the kinetochore, and tension developed across the sister kinetochores in Aurora B-inhibited cells. Thus, we reason that HsMis13 phosphorylation by Aurora B is required for organizing a stable bi-oriented microtubule kinetochore attachment that is essential for faithful chromosome segregation in mitosis.The kinetochore is a super-molecular complex assembled at each centromere in eukaryotes. It provides a chromosomal attachment point for the mitotic spindle, linking the chromosome to the microtubules and functions in initiating, controlling, and monitoring the movements of chromosomes during mitosis. The kinetochore of animal cells contains two functional domains; that is, the inner kinetochore, which is tightly and persistently associated with centromeric DNA sequences throughout the cell cycle and the outer kinetochore which is composed of many dynamic protein complexes that interact with microtubules only during mitosis. The stable propagation of eukaryotic cells requires each chromosome to be accurately duplicated and faithfully segregated. During mitosis, attaching, positioning, and bi-orientating kinetochores with the spindle microtubules play critical roles in chromosome segregation and genomic stability (see Refs. 1 and 2).Mitosis is orchestrated by signaling cascades that coordinate mitotic processes and ensure accurate chromosome segregation. The key switch for the onset of mitosis is the archetypal cyclin-dependent kinase Cdk1. In addition to the master mitotic kinase Cdk1, three other protein serine/threonine kinase families are also involved, including the Polo kinases, Aurora kinases, and the NEK 3 (NIMA-related kinases) (e.g. Refs. 3 and 4). Recent studies have demonstrated the involvement of NEK kinase in stabilization of the kinetochore-microtubule attachment (e.g. Ref. 5) and the critical role of Aurora B kinase in kineto...

Mitotic Regulator SKAP Forms a Link between Kinetochore Core Complex KMN and Dynamic Spindle Microtubules

“…3B), Mis13 was then studied as the main interacting partner for SKAP. This is also consistent with our previous conclusion because Mis12 and Mis13 are thought to be the major components responsible for linking Mis12 complex with other kinetochore components (18).…”

“…Interestingly, the number of collapsed spindles (category 4) in Mis13-suppressed cells was virtually unaltered with extended cold treatment. A similar scenario was observed in CENP-E-depleted cells and SKAP-suppressed cells, suggesting that the Ndc80 complex bears direct microtubule binding and potent stabilization activity, consistent with the literature (4,18,19).…”

“…Antibodies against Mis13 were generated as described previously (18). Anti-tubulin antibody (DM1A) was purchased from Sigma.…”

Phosphorylation of Microtubule-binding Protein Hec1 by Mitotic Kinase Aurora B Specifies Spindle Checkpoint Kinase Mps1 Signaling at the Kinetochore

“…Previous studies have established that the localization of the Ndc80 complex is exterior to the inner kinetochore proteins such as the Mis12 complex (44,45), and the Ndc80 complex localizes to the interior of CENP-E (35,46). Thus, the Ndc80 complex is postulated to link microtubule-binding proteins and chromatin-bound centromere core proteins via forming a multi-subunit complex containing KNL1 and Mis12 subcomplexes.…”

“…Our recent finding of CENP-E-Nuf2 interaction provides a novel link between spindle microtubules and the kinetochore core complex via mitotic kinesin CENP-E (35). Our recent study showed that phosphoregulation of Mis13 by Aurora B governs accurate kinetochore-microtubule attachment by recruiting outer kinetochore proteins and correcting aberrant kinetochore-microtubule attachment errors (45). In addition to error correction, Aurora B is implicated in SAC signaling (47).…”