XB-ART-56297J Cell Biol January 1, 2019; 218 (10): 3237-3257.
Enrichment of Aurora B kinase at the inner kinetochore controls outer kinetochore assembly.
Outer kinetochore assembly enables chromosome attachment to microtubules and spindle assembly checkpoint (SAC) signaling in mitosis. Aurora B kinase controls kinetochore assembly by phosphorylating the Mis12 complex (Mis12C) subunit Dsn1. Current models propose Dsn1 phosphorylation relieves autoinhibition, allowing Mis12C binding to inner kinetochore component CENP-C. Using Xenopus laevis egg extracts and biochemical reconstitution, we found that autoinhibition of the Mis12C by Dsn1 impedes its phosphorylation by Aurora B. Our data indicate that the INCENP central region increases Dsn1 phosphorylation by enriching Aurora B at inner kinetochores, close to CENP-C. Furthermore, centromere-bound CENP-C does not exchange in mitosis, and CENP-C binding to the Mis12C dramatically increases Dsn1 phosphorylation by Aurora B. We propose that the coincidence of Aurora B and CENP-C at inner kinetochores ensures the fidelity of kinetochore assembly. We also found that the central region is required for the SAC beyond its role in kinetochore assembly, suggesting that kinetochore enrichment of Aurora B promotes the phosphorylation of other kinetochore substrates.
PubMed ID: 31527147
PMC ID: PMC6781445
Article link: J Cell Biol
Genes referenced: birc5 bub1b cdca8 dsn1 incenp mad2l1 mis12 ndc80 picalml
Article Images: [+] show captions
|Figure 4. The central region promotes Dsn1 phosphorylation by Aurora B. (A) In vitro kinase assay for Dsn1 phosphorylation. Purified Xenopus recombinant (r) Mis12C was incubated with preactivated recombinant GST-INbox–His6-Aurora B kinase or recombinant GST-INC328-871–His6-Aurora B for the indicated times, and phosphorylation was assessed by Western blot (WB) using a phospho-specific antibody to Ser77 of Dsn1. (B) Quantification of kinase assay for Dsn1 phosphorylation shown in A. A.U., arbitrary units. (C) In vitro kinase assay for histone H3 phosphorylation (H3S10ph). Purified Xenopus recombinant H3–H4 was incubated with preactivated recombinant GST-INbox–His6-Aurora B kinase or recombinant GST-INC328-871–His6-Aurora B for the indicated times, and phosphorylation was assessed by Western blot using a phospho-specific antibody for H3S10ph. (D) Quantification of kinase assay for H3S10 phosphorylation shown in C. (E) Western blot for phosphorylation of Dsn1 in samples of immunoprecipitated (IP) recombinant Mis12Dsn1-LAP complex from WT and ΔCPC metaphase extracts, in indicated CPC conditions, in the presence of nocodazole, okadaic acid, and hesperadin as specified. Normalized phosphorylation levels are indicated below. (F) Western blot for phosphorylation of Dsn1 in samples of immunoprecipitated Mis12Dsn1-LAP complex expressed from mRNAs in ΔCPC metaphase extracts, in indicated CPC conditions. Recombinant GST-INbox–His6-Aurora B or recombinant GST-INC328–871–His6-Aurora B were activated with anti-INCENP beads in the presence of nocodazole and I-2. (G) Quantification of phosphorylation of Dsn1 from samples shown in C, normalized to GFP.|
|Figure 7. Autoinhibition of the Mis12C impedes the phosphorylation of Dsn1 by Aurora B. (A) In vitro kinase assay for Dsn1 phosphorylation. Purified recombinant (r) Mis12C was incubated with preactivated recombinant GST-INbox–His-Aurora B in the presence or absence of recombinant CENP-C2–55–LAP for the indicated times. Western blot shows Dsn1ph, Aurora B T248ph, and Mis12 at indicated time points of assay. (B) Quantification of Dsn1 phosphorylation data from in vitro kinase assay shown in A. A.U., arbitrary units. (C) Quantification of Dsn1 phosphorylation over time used to calculate initial rates. Purified recombinant Mis12C was incubated with preactivated recombinant GST-INbox–His6-Aurora B and indicated concentrations of recombinant CENP-C2–55–LAP. (D) Quantification of initial rates of Dsn1 phosphorylation calculated from data shown in C. Purified recombinant Mis12C was incubated with preactivated recombinant GST-INbox–His6-Aurora B and indicated concentrations of recombinant CENP-C2–55–LAP. Graph represents data from two independent experiments. Error bars represent SEM.|
|Figure 9. Kinetochore-localized Aurora B is required for kinetochore assembly and function. (A) Mis12C autoinhibition prevents phosphorylation by Aurora B. Away from kinetochores, the autoinhibited form of the Mis12C (orange spheres) predominates and prevents Aurora B phosphorylation of Dsn1 through burial of Dsn1S77. (B) Model for kinetochore assembly through the cooperative actions of CENP-C, the INCENP central region, and Aurora B. At the inner kinetochore, the Mis12C transiently binds to prebound CENP-C (and likely CENP-T) molecules, which relieves Mis12C autoinhibition and exposes the Dsn1 tail. In early mitosis, Aurora B is enriched at inner kinetochores by the central region of INCENP. This high local concentration of Aurora B rapidly phosphorylates the exposed Dsn1 tail to prevent dissociation of the Mis12C. Thus, the coincidence of CENP-C and the CPC at inner kinetochores stabilizes the Mis12C–CENP-C interaction to drive outer kinetochore assembly. Our data indicate that enrichment of Aurora B at the inner kinetochore is also involved in SAC signaling through the phosphorylation substrates other than Dsn1.|
References [+] :
Abe, HP1-Assisted Aurora B Kinase Activity Prevents Chromosome Segregation Errors. 2016, Pubmed