Aurora B phosphorylates centromeric MCAK and regulates its localization and microtubule depolymerization activity

BACKGROUND: Sister kinetochores must bind microtubules in a bipolar fashion to equally segregate chromosomes during mitosis. The molecular mechanisms underlying this process remain unclear. Aurora B likely promotes chromosome biorientation by regulating kinetochore-microtubule attachments. MCAK (mitotic centromere-associated kinesin) is a Kin I kinesin that can depolymerize microtubules. These two proteins both localize to mitotic centromeres and have overlapping mitotic functions, including regulation of microtubule dynamics, proper chromosome congression, and correction of improper kinetochore-microtubule attachments. RESULTS: We show that Aurora B phosphorylates and regulates MCAK both in vitro and in vivo. Specifically, we mapped six Aurora B phosphorylation sites on MCAK in both the centromere-targeting domain and the neck region. Aurora B activity was required to localize MCAK to centromeres, but not to spindle poles. Aurora B phosphorylation of serine 196 in the neck region of MCAK inhibited its microtubule depolymerization activity. We found that this key site was phosphorylated at centromeres and anaphase spindle midzones in vivo. However, within the inner centromere there were pockets of both phosphorylated and unphosphorylated MCAK protein, suggesting that phosphate turnover is crucial in the regulation of MCAK activity. Addition of alpha-p-S196 antibodies to Xenopus egg extracts or injection of alpha-p-S196 antibodies into cells caused defects in chromosome positioning and/or segregation. CONCLUSIONS: We have established a direct link between the microtubule depolymerase MCAK and Aurora B kinase. Our data suggest that Aurora B both positively and negatively regulates MCAK during mitosis. We propose that Aurora B biorients chromosomes by directing MCAK to depolymerize incorrectly oriented kinetochore microtubules.     

A perikinetochoric ring defined by MCAK and Aurora-B as a novel centromere domain

Mitotic Centromere-Associated Kinesin (MCAK) is a member of the kinesin-13 subfamily of kinesin-related proteins. In mitosis, this microtubule-depolymerising kinesin seems to be implicated in chromosome segregation and in the correction of improper kinetochore-microtubule interactions, and its activity is regulated by the Aurora-B kinase. However, there are no published data on its behaviour and function during mammalian meiosis. We have analysed by immunofluorescence in squashed mouse spermatocytes, the distribution and possible function of MCAK, together with Aurora-B, during both meiotic divisions. Our results demonstrate that MCAK and Aurora-B colocalise at the inner domain of metaphase I centromeres. Thus, MCAK shows a “cone”-like three-dimensional distribution beneath and surrounding the closely associated sister kinetochores. During the second meiotic division, MCAK and Aurora-B also colocalise at the inner centromere domain as a band that joins sister kinetochores, but only during prometaphase II in unattached chromosomes. During chromosome congression to the metaphase II plate, MCAK relocalises and appears as a ring below each sister kinetochore. Aurora-B also relocalises to appear as a ring surrounding and beneath kinetochores but during late metaphase II. Our results demonstrate that the redistribution of MCAK at prometaphase II/metaphase II centromeres depends on tension across the centromere and/or on the interaction of microtubules with kinetochores. We propose that the perikinetochoric rings of MCAK and Aurora-B define a novel transient centromere domain at least in mouse chromosomes during meiosis. We discuss the possible functions of MCAK at the inner centromere domain and at the perikinetochoric ring during both meiotic divisions.     

Aurora B regulates MCAK at the mitotic centromere

Chromosome orientation and alignment within the mitotic spindle requires the Aurora B protein kinase and the mitotic centromere-associated kinesin (MCAK). Here, we report the regulation of MCAK by Aurora B. Aurora B inhibited MCAK's microtubule depolymerizing activity in vitro, and phospho-mimic (S/E) mutants of MCAK inhibited depolymerization in vivo. Expression of either MCAK (S/E) or MCAK (S/A) mutants increased the frequency of syntelic microtubule-kinetochore attachments and mono-oriented chromosomes. MCAK phosphorylation also regulates MCAK localization: the MCAK (S/E) mutant frequently localized to the inner centromere while the (S/A) mutant concentrated at kinetochores. We also detected two different binding sites for MCAK using FRAP analysis of the different MCAK mutants. Moreover, disruption of Aurora B function by expression of a kinase-dead mutant or RNAi prevented centromeric targeting of MCAK. These results link Aurora B activity to MCAK function, with Aurora B regulating MCAK's activity and its localization at the centromere and kinetochore.     

Tripin/hSgo2 recruits MCAK to the inner centromere to correct defective kinetochore attachments

hSgo2 (previously annotated as Tripin) was recently reported to be a new inner centromere protein that is essential for centromere cohesion (Kitajima et al., 2006). In this study, we show that hSgo2 exhibits a dynamic distribution pattern, and that its localization depends on the BUB1 and Aurora B kinases. hSgo2 is concentrated at the inner centromere of unattached kinetochores, but extends toward the kinetochores that are under tension. This localization pattern is reminiscent of MCAK, which is a microtubule depolymerase that is believed to be a key component of the error correction mechanism at kinetochores. Indeed, we found that hSgo2 is essential for MCAK to localize to the centromere. Delocalization of MCAK accounts for why cells depleted of hSgo2 exhibit kinetochore attachment defects that go uncorrected, despite a transient delay in the onset of anaphase. Consequently, these cells exhibit a high frequency of lagging chromosomes when they enter anaphase. We confirmed that hSgo2 is associated with PP2A, and we propose that it contributes to the spatial regulation of MCAK activity within inner centromere and kinetochore.     

Mitotic centromere-associated kinesin (MCAK) mediates paclitaxel resistance

Paclitaxel has powerful anticancer activity, but some tumors are inherently resistant to the drug, whereas others are initially sensitive but acquire resistance during treatment. To deal with this problem, it will be necessary to understand the mechanisms of drug action and resistance. Recent studies indicate that paclitaxel blocks cell division by inhibiting the detachment of microtubules from centrosomes. Here, we demonstrate that mitotic centromere-associated kinesin (MCAK), a kinesin-related protein that destabilizes microtubules, plays an important role in microtubule detachment. Depletion of MCAK altered mitotic spindle morphology, increased the frequency of lagging chromosomes, and inhibited the proliferation of WT CHO cells, confirming that it is an essential protein for cell division. In contrast, MCAK depletion rescued the proliferation of mutant paclitaxel-dependent cell lines that are unable to divide because of defective spindle function resulting from altered α-tubulin or class III β-tubulin overexpression. In concert with the correction of mitotic defects, loss of MCAK reversed an aberrantly high frequency of microtubule detachment in the mutant cells and increased their sensitivity to paclitaxel. The results indicate that MCAK affects cell sensitivity to mitotic inhibitors by modulating the frequency of microtubule detachment, and they demonstrate that changes in a microtubule-interacting protein can reverse the effects of mutant tubulin expression.     

Mutations in the ATP-binding domain affect the subcellular distribution of mitotic centromere-associated kinesin (MCAK)

Mitotic centromere-associated kinesin (MCAK) is important for anaphase chromosome segregation. MCAK is diffusely localized to both the cytoplasm and the nucleus during interphase. At prophase MCAK is recruited to mitotic centromeres. It is associated with centromeres throughout mitosis and then returns to exhibiting a diffuse nuclear and cytoplasmic localization during interphase. MCAK has several predicted nuclear localization sequences. The subcellular distribution of expressed deletion constructs of GFP-MCAK suggest that the nucleocytoplasmic ratio of MCAK protein is dependent on a balance between several predicted nuclear localization sequences (NLS) and a putative nuclear exclusion sequence (NES) in the amino-terminal region of MCAK. Amino acid substitutions in the ATP-binding domain of the MCAK motor affect nuclear localization, which, in turn, influences the degree of centromere binding.     

Crystal structure of the motor domain of the human kinetochore protein CENP-E

The human kinetochore is a highly complex macromolecular structure that connects chromosomes to spindle microtubules (MTs) in order to facilitate accurate chromosome segregation. Centromere-associated protein E (CENP-E), a member of the kinesin superfamily, is an essential component of the kinetochore, since it is required to stabilize the attachment of chromosomes to spindle MTs, to develop tension across aligned chromosomes, to stabilize spindle poles and to satisfy the mitotic checkpoint. Here we report the 2.5A resolution crystal structure of the motor domain and linker region of human CENP-E with MgADP bound in the active site. This structure displays subtle but important differences compared to the structures of human Eg5 and conventional kinesin. Our structure reveals that the CENP-E linker region is in a "docked" position identical to that in the human plus-end directed conventional kinesin. CENP-E has many advantages as a potential anti-mitotic drug target and this crystal structure of human CENP-E will provide a starting point for high throughput virtual screening of potential inhibitors.     

Sequential Assembly of Centromeric Proteins in Male Mouse Meiosis

The assembly of the mitotic centromere has been extensively studied in recent years, revealing the sequence and regulation of protein loading to this chromosome domain. However, few studies have analyzed centromere assembly during mammalian meiosis. This study specifically targets this approach on mouse spermatocytes. We have found that during prophase I, the proteins of the chromosomal passenger complex Borealin, INCENP, and Aurora-B load sequentially to the inner centromere before Shugoshin 2 and MCAK. The last proteins to be assembled are the outer kinetochore proteins BubR1 and CENP-E. All these proteins are not detected at the centromere during anaphase/telophase I and are then reloaded during interkinesis. The loading sequence of the analyzed proteins is similar during prophase I and interkinesis. These findings demonstrate that the interkinesis stage, regularly overlooked, is essential for centromere and kinetochore maturation and reorganization previous to the second meiotic division. We also demonstrate that Shugoshin 2 is necessary for the loading of MCAK at the inner centromere, but is dispensable for the loading of the outer kinetochore proteins BubR1 and CENP-E.     

MCAK facilitates chromosome movement by promoting kinetochore microtubule turnover

Mitotic centromere-associated kinesin (MCAK)/Kif2C is the most potent microtubule (MT)-destabilizing enzyme identified thus far. However, MCAK's function at the centromere has remained mechanistically elusive because of interference from cytoplasmic MCAK's global regulation of MT dynamics. In this study, we present MCAK chimeras and mutants designed to target centromere-associated MCAK for mechanistic analysis. Live imaging reveals that depletion of centromere-associated MCAK considerably decreases the directional coordination between sister kinetochores. Sister centromere directional antagonism results in decreased movement speed and increased tension. Sister centromeres appear unable to detach from kinetochore MTs efficiently in response to directional switching cues during oscillatory movement. These effects are reversed by anchoring ectopic MCAK to the centromere. We propose that MCAK increases the turnover of kinetochore MTs at all centromeres to coordinate directional switching between sister centromeres and facilitate smooth translocation. This may contribute to error correction during chromosome segregation either directly via slow MT turnover or indirectly by mechanical release of MTs during facilitated movement.     

Yeast Kar3 is a minus-end microtubule motor protein that destabilizes microtubules preferentially at the minus ends.

Mutants of the yeast Kar3 protein are defective in nuclear fusion, or karyogamy, during mating and show slow mitotic growth, indicating a requirement for the protein both during mating and in mitosis. DNA sequence analysis predicts that Kar3 is a microtubule motor protein related to kinesin, but with the motor domain at the C-terminus of the protein rather than the N-terminus as in kinesin heavy chain. We have expressed Kar3 as a fusion protein with glutathione S-transferase (GST) and determined the in vitro motility properties of the bacterially expressed protein. The GST-Kar3 fusion protein bound to a coverslip translocates microtubules in gliding assays with a velocity of 1-2 microns/min and moves towards microtubule minus ends, unlike kinesin but like kinesin-related Drosophila ncd. Taxol-stabilized microtubules bound to GST-Kar3 on a coverslip shorten as they glide, resulting in faster lagging end, than leading end, velocities. Comparison of lagging and leading end velocities with velocities of asymmetrical axoneme-microtubule complexes indicates that microtubules shorten preferentially from the lagging or minus ends. The minus end-directed translocation and microtubule bundling of GST-Kar3 is consistent with models in which the Kar3 protein crosslinks internuclear microtubules and mediates nuclear fusion by moving towards microtubule minus ends, pulling the two nuclei together. In mitotic cells, the minus end motility of Kar3 could move chromosomes polewards, either by attaching to kinetochores and moving them polewards along microtubules, or by attaching to kinetochore microtubules and pulling them polewards along other polar microtubules.(ABSTRACT TRUNCATED AT 250 WORDS)     

Functional analysis of phosphorylation of the mitotic centromere-associated kinesin by Aurora B kinase in human tumor cells

Mitotic centromere-associated kinesin (MCAK) is the best characterized member of the kinesin-13 family and plays important roles in microtubule dynamics during mitosis. Its activity and subcellular localization is tightly regulated by an orchestra of mitotic kinases, such as Aurora B. It is well known that serine 196 of MCAK is the major phosphorylation site of Aurora B in Xenopus leavis extracts and that this phosphorylation regulates its catalytic activity and subcellular localization. In the current study, we have addressed the conserved phosphorylation site serine 192 in human MCAK to characterize its function in more depth in human cancer cells. Our data confirm that S192 is the major phosphorylation site of Aurora B in human MCAK and that this phosphorylation has crucial roles in regulating its catalytic activity and localization at the kinetochore/centromere region in mitosis. Interfering with this phosphorylation leads to a delayed progression through prometa- and metaphase associated with mitotic defects in chromosome alignment and segregation. We show further that MCAK is involved in directional migration and invasion of tumor cells, and interestingly, interference with the S192 phosphorylation affects this capability of MCAK. These data provide the first molecular explanation for clinical observation, where an overexpression of MCAK was associated with lymphatic invasion and lymph node metastasis in gastric and colorectal cancer patients.     

PLK1 phosphorylates mitotic centromere-associated kinesin and promotes its depolymerase activity

During cell division, interaction between kinetochores and dynamic spindle microtubules governs chromosome movements. The microtubule depolymerase mitotic centromere-associated kinesin (MCAK) is a key regulator of mitotic spindle assembly and dynamics. However, the regulatory mechanisms underlying its depolymerase activity during the cell cycle remain elusive. Here, we showed that PLK1 is a novel regulator of MCAK in mammalian cells. MCAK interacts with PLK1 in vitro and in vivo. The neck and motor domain of MCAK associates with the kinase domain of PLK1. MCAK is a novel substrate of PLK1, and the phosphorylation stimulates its microtubule depolymerization activity of MCAK in vivo. Overexpression of a polo-like kinase 1 phosphomimetic mutant MCAK causes a dramatic increase in misaligned chromosomes and in multipolar spindles in mitotic cells, whereas overexpression of a nonphosphorylatable MCAK mutant results in aberrant anaphase with sister chromatid bridges, suggesting that precise regulation of the MCAK activity by PLK1 phosphorylation is critical for proper microtubule dynamics and essential for the faithful chromosome segregation. We reasoned that dynamic regulation of MCAK phosphorylation by PLK1 is required to orchestrate faithful cell division, whereas the high levels of PLK1 and MCAK activities seen in cancer cells may account for a mechanism underlying the pathogenesis of genomic instability.     

Identification of novel centromere/kinetochore-associated proteins using monoclonal antibodies generated against human mitotic chromosome scaffolds

We describe the generation of 11 monoclonal antibodies that bind to the centromere/kinetochore region of human mitotic chromosomes. These antibodies were raised against mitotic chromosome scaffolds and screened for centromere/kinetochore binding by indirect immunofluorescence against purified chromosomes. Immunoblot analyses with these antibodies revealed that all of the antigens are greater than 200 kD and are components of nuclei, chromosomes, and/or chromosome scaffolds. Comparison of the immunolocalization of the antigens with that observed for the centromere-associated protein CENP-B revealed that each of these centromere/kinetochore proteins lies more peripherally to the DNA than does CENP-B. In cells normally progressing through the cell cycle, these antigens displayed four distinct patterns of centromere/kinetochore association, corresponding to a minimum of four novel centromere/kinetochore-associated proteins.     

Microtubule capture: a concerted effort

Kinetochores direct attachment of chromosomes to microtubules of the mitotic spindle during cell division. Three recent studies in Cell, including one in this issue, reveal important new roles for two kinetochore protein complexes-Ndc80 and INCENP-Survivin-in establishing the correct attachment of chromosomes to spindle microtubules (Cheeseman et al., 2006, DeLuca et al., 2006 and Sandall et al., 2006).     

Separation of meiotic and mitotic effects of claret non-disjunctional on chromosome segregation in Drosophila.

The claret (ca) locus in Drosophila encodes a kinesin-related motor molecule that is required for proper distribution of chromosomes in meiosis in females and in the early mitotic divisions of the embryo. Here we demonstrate that a mutant allele of claret non-disjunctional (ca(nd)), non-claret disjunctional Dominant (ncdD), causes abnormalities in meiotic chromosome segregation, but is near wild-type with respect to early mitotic chromosome segregation. DNA sequence analysis of this mutant allele reveals two missense mutations compared with the predicted wild-type protein. One mutation lies in a proposed microtubule binding region of the motor domain and affects an amino acid residue that is conserved in all kinesin-related proteins reported to date. This region of the motor domain can be used to distinguish meiotic and mitotic motor function, defining an amino acid sequence criterion for classifying motors according to function. ncdD's mutant meiotic effect, but near wild-type mitotic effect, suggests that interactions of the ca motor protein with spindle microtubules differ in meiosis and mitosis.     

MCAK activity at microtubule tips regulates spindle microtubule length to promote robust kinetochore attachment

Mitotic centromere-associated kinesin (MCAK) is a microtubule-depolymerizing kinesin-13 member that can track with polymerizing microtubule tips (hereafter referred to as tip tracking) during both interphase and mitosis. MCAK tracks with microtubule tips by binding to end-binding proteins (EBs) through the microtubule tip localization signal SKIP, which lies N terminal to MCAK's neck and motor domain. The functional significance of MCAK's tip-tracking behavior during mitosis has never been explained. In this paper, we identify and define a mitotic function specific to the microtubule tip-associated population of MCAK: negative regulation of microtubule length within the assembling bipolar spindle. This function depends on MCAK's ability to bind EBs and track with polymerizing nonkinetochore microtubule tips. Although this activity antagonizes centrosome separation during bipolarization, it ultimately benefits the dividing cell by promoting robust kinetochore attachments to the spindle microtubules.     

Kid, a novel kinesin-like DNA binding protein, is localized to chromosomes and the mitotic spindle.

Microtubule-associated motor proteins are thought to be involved in spindle formation and chromosome movements in mitosis/meiosis. We have molecularly cloned cDNAs for a gene that codes for a novel member of the kinesin family of proteins. Nucleotide sequencing reveals that the predicted gene product is a 73 kDa protein and is related to some extent to the Drosophila node gene product, which is involved in chromosomal segregation during meiosis. A sequence similar to the microtubule binding motor domain of kinesin is present in the N-terminal half of the protein, and its ability to bind to microtubules is demonstrated. Furthermore we show that its C-terminal half contains a putative nuclear localization signal similar to that of Jun and is able to bind to DNA. Accordingly, the protein was termed Kid (kinesin-like DNA binding protein). Indirect immunofluorescence studies show that Kid colocalizes with mitotic chromosomes and that it is enriched in the kinetochore at anaphase. Thus, we propose that Kid might play a role(s) in regulating the chromosomal movement along microtubules during mitosis.     

Depletion of centromeric MCAK leads to chromosome congression and segregation defects due to improper kinetochore attachments

The complex behavior of chromosomes during mitosis is accomplished by precise binding and highly regulated polymerization dynamics of kinetochore microtubules. Previous studies have implicated Kin Is, unique kinesins that depolymerize microtubules, in regulating chromosome positioning. We have characterized the immunofluorescence localization of centromere-bound MCAK and found that MCAK localized to inner kinetochores during prophase but was predominantly centromeric by metaphase. Interestingly, MCAK accumulated at leading kinetochores during congression but not during segregation. We tested the consequences of MCAK disruption by injecting a centromere dominant-negative protein into prophase cells. Depletion of centromeric MCAK led to reduced centromere stretch, delayed chromosome congression, alignment defects, and severe missegregation of chromosomes. Rates of chromosome movement were unchanged, suggesting that the primary role of MCAK is not to move chromosomes. Furthermore, we found that disruption of MCAK leads to multiple kinetochore-microtubule attachment defects, including merotelic, syntelic, and combined merotelic-syntelic attachments. These findings reveal an essential role for Kin Is in prevention and/or correction of improper kinetochore-microtubule attachments.     

Kinesin-related proteins in the mammalian testes: candidate motors for meiosis and morphogenesis.

The kinesin superfamily of molecular motors comprises proteins that participate in a wide variety of motile events within the cell. Members of this family share a highly homologous head domain responsible for force generation attached to a divergent tail domain thought to couple the motor domain to its target cargo. Many kinesin-related proteins (KRPs) participate in spindle morphogenesis and chromosome movement in cell division. Genetic analysis of mitotic KRPs in yeast and Drosophila, as well as biochemical experiments in other species, have suggested models for the function of KRPs in cell division, including both mitosis and meiosis. Although many mitotic KRPs have been identified, the relationship between mitotic motors and meiotic function is not clearly understood. We have used sequence similarity between mitotic KRPs to identify candidates for meiotic and/or mitotic motors in a vertebrate. We have identified a group of kinesin-related proteins from rat testes (termed here testes KRP1 through KRP6) that includes new members of the bimC and KIF2 subfamilies as well as proteins that may define new kinesin subfamilies. Five of the six testes KRPs identified are expressed primarily in testes. Three of these are expressed in a region of the seminiferous epithelia (SE) rich in meiotically active cells. Further characterization of one of these KRPs, KRP2, showed it to be a promising candidate for a motor in meiosis: it is localized to a meiotically active region of the SE and is homologous to motor proteins associated with the mitotic apparatus. Testes-specific genes provide the necessary probes to investigate whether the motor proteins that function in mammalian meiosis overlap with those of mitosis and whether motor proteins exist with functions unique to meiosis. Our search for meiotic motors in a vertebrate testes has successfully identified proteins with properties consistent with those of meiotic motors in addition to uncovering proteins that may function in other unique motile events of the SE.     

Human centromere chromatin protein hMis12, essential for equal segregation,is independent of CENP-A loading pathway

Kinetochores are the chromosomal sites for spindle interaction and play a vital role for chromosome segregation. The composition of kinetochore proteins and their cellular roles are, however, poorly understood in higher eukaryotes. We identified a novel kinetochore protein family conserved from yeast to human that is essential for equal chromosome segregation. The human homologue hMis12 of yeast spMis12/scMtw1 retains conserved sequence features and locates at the kinetochore region indistinguishable from CENP-A, a centromeric histone variant. RNA interference (RNAi) analysis of HeLa cells shows that the reduced hMis12 results in misaligned metaphase chromosomes, lagging anaphase chromosomes, and interphase micronuclei without mitotic delay, while CENP-A is located at kinetochores. Further, the metaphase spindle length is abnormally extended. Spindle checkpoint protein hMad2 temporally localizes at kinetochores at early mitotic stages after RNAi. The RNAi deficiency of CENP-A leads to a similar mitotic phenotype, but the kinetochore signals of other kinetochore proteins, hMis6 and CENP-C, are greatly diminished. RNAi for hMis6, like that of a kinetochore kinesin CENP-E, induces mitotic arrest. Kinetochore localization of hMis12 is unaffected by CENP-A RNAi, demonstrating an independent pathway of CENP-A in human kinetochores.