Aurora kinase inhibitors reveal mechanisms of HURP in nucleation of centrosomal and kinetochore microtubules
Jiun-Ming Wua, Chiung-Tong Chena, Mohane Selvaraj Coumara,c, Wen-Hsin Linb, Zi-Jie Chenb, John T.-A Hsua, Yi-Hui Penga, Hui-Yi Shiaoa, Wen-Hsing Lina, Chang-Ying Chua, Jian-Sung Wua, Chih-Tsung Lina, Ching-Ping Chena, Ching-Cheng Hsueha, Kai-Yen Changa, Li-Pin Kaob, Chi-Ying F. Huangd, Yu-Sheng Chaoa, Su-Ying Wua*, Hsing-Pang Hsieha* and Ya-Hui Chib,e*
Institute of Biotechnology and Pharmaceutical Researcha, Institute of Cellular and System Medicineb, National Health Research Institutes, Zhunan 35053, Taiwan; Centre for Bioinformaticsc, School of Life Sciences, Pondicherry University, Kalapet, Puducherry 605014, India; Institute of Biopharmaceutical Sciencesd, National Yang Ming University, Taipei 11221, Taiwan. Graduate Institute of Basic Medical Sciencee, China Medical University, Taichung 40402, Taiwan
*Corresponding authors Ya-Hui Chi
35 Keyan Road, Zhunan Township, Miaoli County, Taiwan, 35053 Ph: +886-37-246166ext37522; Fax: +886-37-587408
Email: [email protected] Hsing-Pang Hsieh
35 Keyan Road, Zhunan Township, Miaoli County, Taiwan, 35053 Ph: +886-37-246166ext35708; Fax: +886-37-586456
Email: hphsieh @nhri.org.tw Su-Ying Wu
35 Keyan Road, Zhunan Township, Miaoli County, Taiwan, 35053 Ph: +886-37-246166ext35713; Fax: +886-37-586456
ABSTRACT
The overexpression of Aurora kinases in multiple tumors makes them appealing targets for the development of anticancer therapies. This study discovered two new small molecules with a furanopyrimidine core, IBPR001 and IBPR002, targeting Aurora kinases and inducing DFG conformation change at the ATP site of Aurora A. Our results demonstrate the high potency of the IBPR compounds in reducing tumorigenesis in a colorectal cancer xenograft model in athymic nude mice. Human hepatoma up-regulated protein (HURP) is a substrate of Aurora kinase A, which plays a crucial role in the stabilization of kinetochore fibers. This study employed the novel IBPR compounds as well as MLN8237, a proven Aurora A inhibitor, as chemical probes to investigate the molecular role of HURP in mitotic spindle formation. These compounds effectively eliminated HURP phosphorylation in vivo, thereby revealing the co-existence and continuous cycling of HURP between unphosphorylated (HURP-U) and phosphorylated (HURP-P) forms that are respectively associated with microtubules emanating from centrosomes and kinetochores. Furthermore, these compounds demonstrate a spatial hierarchical preference of HURP in the attachment of microtubules extending from the mother to the daughter centrosome. The novel finding of inequality in the centrosomal microtubules revealed by these small molecules provides a versatile tool to discover new
Significance
In mitosis, microtubules continuously extend and shrink before the bilateral attachment is established. However, it is not fully elucidated regarding what molecules regulate this activity for spindle formation. Using two in-house developed small molecules that target the Aurora kinases, we show that HURP is highly dynamic, trafficking between centrosome and kinetochore driven by the Aurora A-dependent phosphorylation and PP1/PP2A-associated dephosphorylation. These compounds also demonstrate a spatial hierarchical preference of HURP in the attachment of microtubules extending from the mother to the daughter centrosome. These findings provide evidence to understand the biology of mitosis and the development of novel anti-cancer compounds.
\body
INTRODUCTION
The overexpression of Aurora kinases is closely associated with tumorigenesis (1, 2). Small molecules that inhibit the kinase activity of Aurora have attracted considerable attention for their applicability in cancer treatment; and a number of Aurora kinase inhibitors have been assessed in clinical trials (1, 3-6). Aurora kinases are serine/threonine kinases which regulate mitotic progression, centrosome maturation, and spindle assembly. Therefore, small molecules capable of inhibiting Aurora kinases can also be used as chemical probes to determine the interplay of Aurora kinases and their substrates in spindle formation.
To ensure fidelity of segregation, duplicated chromatids need to be properly attached by mitotic spindles at the kinetochores (7).At onset of mitosis, microtubules that emanate from the duplicated centrosomes gradually extend to reach the kinetochores. The formation of robust spindles relies on the cooperation between two assembly pathways: the kinetochore capture by microtubule spindles originating from centrosomes, and the RanGTP-mediated microtubule nucleation and organization in the vicinity of chromosomes (8-13). Human hepatoma up-regulated protein (HURP) is an Aurora A substrate up-regulated in hepatomas (14, 15). HURP stabilizes kinetochore fibers
(K-fibers) and promotes nucleation and crosslinking of microtubules in vitro and in vivo (16-19). In Xenopus egg extract, anti-HURP antibodies disrupt the formation of chromosome-and centrosome-induced spindles (16), suggesting the involvement of HURP in both mechanisms. HURP has also been characterized as a direct cargo of importin , involved in RanGTP-regulated spindle (Ran-spindle) assembly in the vicinity of chromosomes (17-19). Because the kinase activity of Aurora A is essential to the formation of Ran-spindles (16), HURP has been proposed to be phosphorylated at the spindle poles by Aurora A, thereby allowing its translocation to RanGTP-dependent K-fibers (17).
As HURP expression is cell cycle-dependent and limited to prophase through anaphase, investigating how HURP is temporally regulated by phosphorylation would require rapid inhibition of the kinase activity of Aurora A, which is not achievable using RNAi or other genetic methods (15, 19). Here we utilize the in-house developed Aurora kinase inhibitors to dissect the Aurora-HURP pathway in the formation of spindles. This study reports the discovery and characterization of two new Aurora inhibitors IBPR001 and IBPR002, that efficiently eliminate HURP phosphorylation in mitosis. The efficacy of the two IBPR compounds to HURP dephosphorylation is better than MLN8237 and VX-680. The rapid in vivo elimination of HURP phosphorylation provides evidence to support the notion of a dynamic equilibrium between the two forms of HURP regulated
by Aurora A-mediated phosphorylation, each playing a role in the differential assembly of centrosomal and kinetochore microtubules. These results also implicate that the symmetric distribution of HURP to centrosomal microtubules requires kinase activity of Aurora A.
RESULTS
Synthesis and Characterization of IBPR Compounds Targeting Aurora Kinases We have reported a lead compound with a furanopyrimidine core capable of inhibiting Aurora kinase activity (20, 21).Using this structure as a scaffold, we synthesized (Fig. S1) more than 200 analogues and identified two compounds, IBPR001 and IBPR002 (Fig. 1A), that demonstrate potent Aurora inhibitory activity (Fig. 1B).
To determine the specificity of these IBPR compounds, we performed in vitro activity profiling for 57 kinases associated with cancer (in a contract to Ricerca Biosciences). Of the kinases tested, IBPR002 demonstrated the strongest inhibitory activity against Aurora A. The inhibition of IBPR002 at 1.0 M is listed in Supplementary Table S1. All but 11 of the profiled kinases showed <50% inhibition at 1.0 M. The effectiveness of IBPR002 as an inhibitor of mitosis-associated kinases PLK1 (-14% inhibition at 1.0 M) and NEK2 (61% inhibition at 1.0 M, IC50=0.532 M) was less pronounced, compared with the inhibition of Aurora A (101% inhibition at 1.0 M, IC50=41 nM, Fig. 1B and Supplementary Table S1).
Crystal structures of Aurora A in complex with IBPR compounds and VX-680 VX-680 is a first-generation small molecule that inhibits the catalytic activity of Aurora kinases through competitive interactions with the ATP-binding site (3). Structures of the
Aurora A kinase domain (amino acids 123-401) in complex with IBPR001 (PDB code:
4JBO) and VX-680 (PDB code: 4JBQ) were solved to provide insight into the interaction
of the compounds with the protein (summary of X-ray data and structure refinement is listed in Supplementary Table S2). Although both compounds were well suited to the ATP binding site and formed conserved hydrogen bonds with Glu211 and Ala213 in the hinge region (22),the diphenylurea moiety of IBPR001 extends into the Aurora A back pocket, which was unoccupied in the Aurora A/VX-680 complex (Fig. 1C). Moreover, the Aurora A kinase domain adopts the DFG (Asp-Phe-Gly)-in conformation to accommodate the phenylurea group of IBPR001 which forms two hydrogen bonds with the conserved Glu181 of Aurora A. On the other hand, Phe275 of the DFG motif (23) at the activation loop (A-loop) points toward the cyclopropyl group of VX-680.
The complex structure of the Aurora A kinase domain with IBPR002 was also solved (PDB code: 4JBP) to a resolution of 2.45Å (Supplementary Table S2). The structure of IBPR002 was well superimposed with IBPR001, except for the additional piperidinol group extending toward the solvent exposed area.
IBPR001 and IBPR002 reduce tumorigenesis in mice
Aurora kinases have emerged as promising chemotherapeutic targets for cancer, due to the pivotal role they play in mitosis and their overexpression in malignant cells (5). To
evaluate whether IBPR compounds are able to reduce tumorigenesis in vivo, we applied a colorectal cancer xenograft model in athymic nude mice. Ten male mice in each group were inoculated subcutaneously with Aurora A-overexpressing HCT116 colorectal cancer cells. When the tumor size reached ≥100 mm3, mice were intravenously (i.v.) administrated IBPR002 or VX-680 via the tail veins, at a dosage of 50 mg/kg/day, 5 daily doses per week, for 2 consecutive weeks. Tumor size was observed for an additional 13 days following the final injection. IBPR002 significantly (P < 0.05) inhibited the growth of xenograft colorectal cancer cells, in a similar manner toVX-680 (Fig. 1D).
IBPR compounds eliminate HURP phosphorylation in vivo and in vitro
The expression of HURP is cell-cycle regulated [Fig. S2A and references (15, 19)]. HURP has been shown to promote the nucleation and crosslinking of microtubules in vitro and in vivo (16, 17, 24), however, whether this activity requires phosphorylation remains unclear. We examined whether VX-680 inhibits HURP phosphorylation and found that the efficacy is less than optimal (Fig. 2A). The results of Western blotting indicate that the anti-HURP phosphorylation activity of IBPR001 and IBPR002 exceed that of VX-680 (Figs. 2A,B). We also tested HURP phosphorylation in cells treated with a reported Aurora A-selective inhibitor, MLN8237. In the same manner as IBPR compounds, MLN8237 eliminated HURP phosphorylation, albeit with reduced degrees
of effectiveness. In contrast, AZD1152 (a reported Aurora B inhibitor) failed to reduce HURP phosphorylation (Figs. 2A and 2B). The inhibition of HURP phosphorylation was further verified by an in vitro kinase assay (Fig. 2C), suggesting that IBPR001 and MLN8237 play a direct role in the inhibition of Aurora A-mediated HURP phosphorylation. In cells the depletion of Aurora A (using RNA silencing), but not other
mitotic kinases such as Aurora B, CDK1 or NEK2, eliminated the expression of HURP-P
(Fig. S2B,C). These results indicate that IBPR compounds inhibit the Aurora A-mediated HURP phosphorylation in vivo.
IBPR001 and MLN8237 disrupt nucleation and bundling of K-fibers
We employed IBPR compounds and MLN8237 as chemical probes to gain insight into the association of the Aurora A-HURP pathway in spindle formation. Nocodazole inhibits mitotic progression by disrupting the assembly of microtubules. Under treatment with 300-400 nM nocodazole (25), microtubules form kinetochore-associated bundles, but fail to nucleate at centrosomes, suggesting there exist different nucleation pathways for centrosomal and kinetochore microtubules (Fig. 3A). We found that HURP is >95% phosphorylated under this condition (Fig. 3B, lanes 2 and 8) and exclusively associated with kinetochore (labeled with CREST, Fig. 3A, left panels) but not centrosomal (labeled
with Pericnetrin, Fig. 3A, right panels) microtubules (Fig. 3A). The addition of IBPR001/ or MLN8237 to nocodazole-treated cells efficiently converted HURP-P to HURP-U within one hour of treatment (Fig. 3B, lanes 3-6 and 9-12), accompanied by de-polymerization of the kinetochore microtubule bundles (Fig. 3A,C). It appears that the reduction of HURP intensity was not a result of degradation (26) because the total expression level of HURP had not altered with the treatment of MG132, a proteosome inhibitor (Fig. 3B). Contrast to IBPR001 and MLN8237, assembly of K-fibers was not interfered by an Aurora B-selective inhibitor AZD1152 (Fig. 3A,C). The co-localization of HURP-P with kinetochore microtubule bundles (Fig. 3A) and the disassembly of kinetochore microtubules in conjunction with HURP-P dephosphorylation (Figures 3A,C) suggest that HURP-P, rather than HRUP-U, was required for the nucleation of the kinetochore microtubules.
HURP cycles between centrosomes and kinetochores through phosphorylation Establishing robust mitotic spindles requires the cooperation of both K-fibers and centrosomal microtubules (9, 27). In control cells, HURP co-localized with mitotic spindles in early prophase, and gradually moved to the plus end of the spindles in the vicinity of mitotic chromosomes along with mitotic progression (Fig. 4A, left panels)
(28). A lack of adequate equipment with which to detect HURP-P in cells prevented us from determining the phosphorylation status of HURP at the plus and minus ends of mitotic spindles. Nevertheless, the use of IBPR compounds and MLN8237 to block Aurora kinase activity clearly demonstrates that HURP-P can be converted to HURP-U (Fig. 3B). In the compound treated cells, HURP resided primarily at the minus end of the microtubules, close to the centrosomes (Fig. 4A, right panels and Fig. S2D,E). It should be noted that nocodazole was not added to these cells in order to maintain tubulin polymerization. Among the cells treated with IBPR001, nearly 20% were monoastral (Fig. 4A, phenotype-1), which is similar to the phenotype observed in Aurora A-depleted cells (29, 30). The remainder of the cells treated with IBPR compounds exhibited microtubules emanating from separated poles, with HURP localized to one (Fig. 4A, phenotype-2) or both (Fig. 4A, phenotype-3). Similar phenotypes were observed in cells treated with MLN8237 (Fig. S2D). The spatial relationship between HURP and centrosomes was also demonstrated by immunofluorescence staining of -tubulin (Fig. S2E). Unlike normally progressing cells which form bi-orientated spindles in metaphase (Fig. S2E, left panels), IBPR001 and IBPR002 disrupted bipolarity (Fig. S2E, right panels). HURP may surround the un-separated centrosomes (Fig. S2E, phenotype-1), associate with centrosomal microtubules projecting toward chromosomes (Fig. S2E,
phenotype-2), or wrap around one (Fig. S2E, phenotype-3) or two (Fig. S2E, phenotype-4) of the separated centrosomes.
HURP is associated with the minus end of centrosomal microtubules when treated with IBPR compounds and MLN8237 (Fig. 4A,S2D,S2E). However, in the presence of nocodazole, treatment with IBPR compounds caused HURP to disperse into the cytoplasm, instead of accumulation around the centrosomes (Fig. 3). This raises the question of whether tubulin polymerization at centrosomes is essential to this process. By removing nocodazole from cells pre-treated with IBPR001, centrosomal microtubules were re-established in conjunction with the accumulation of HURP at the minus ends, similar to the phenotypes observed in Fig. 4A (Fig. 4C). This differed from the control cells, in which HURP was located at both the plus and minus ends of microtubules (Fig. 4C). We also observed the expression of HURP-U in control cells upon nocodazole removal (Fig. 4D, lanes 1-4, marked with *). In contrast, treatment with IBPR001 led to the preservation of HURP-U (Fig. 4D, lanes 6-9). These results verified the notion that HURP-U is indeed associated with centrosomal microtubules. Moreover, they also suggest that polymerization of centrosomal microtubules precedes HURP-U association.
These above data imply the existence of an underlying mechanism associated with HURP dephosphorylation. The protein phosphatase family targets multiple mitotic
structures such as chromosomes, centrosomes, and spindles in assisting mitotic progression (31). For example, Aurora B and protein phosphatase 1 (PP1) act antagonistically for the phosphorylation of histone H3 serine 10 in chromosome condensation (31, 32). To determine whether protein phosphatase is responsible for HURP dephosphorylation, we compared HURP Western profiles in nocodazole-arrested cells treated with IBPR001 alone or co-treated with Calyculin A, an inhibitor of PP1 and protein phosphatase 2A (PP2A) (33). The IBPR001-induced HURP dephosphorylation was eliminated by Calyculin A (Fig. 4E), suggesting that the dephosphorylation of HURP is associated with PP1/PP2A activity. Consistently, co-treatment of Calyclin A and IBPR001 enables HURP to associate with the nucleated microtubule bundles similar to control (Fig. 4F).
Collectively, these results suggest that the two forms of HURP (HURP-P and HURP-U) cycle between centrosomes and kinetochores through Aurora A-dependent phosphorylation and protein phosphatase-regulated dephosphorylation in the establishment of mitotic spindles.
IBPR001 and MLN8237 result in an asymmetric association of HURP to centrosomal microtubules
During quantification of the HURP morphology resulting from treatment of the IBPR compounds, we observed the association of HURP with one of the spindle poles that resides closer to the chromosomes in approximately 10% of cells (Fig. S2E, phenotype-3). This observation raises the question of which centrosome [mother or daughter, the mother centrosome refer to the centrosome which contains the centriole of the eldest mother as well as that of the newborn daughter. Conversely, the daughter centrosome contains the centriole of the second oldest mother and that of the newborn daughter (34, 35)] that HURP may preferentially associate with. Mitotic spindles are perceived as a symmetric structure connecting to kinetochore of the duplicated chromatids with equal tension, prior to separation (11, 27). Conversely, asymmetric cell divisions were observed in neural and male germ stem cells in which the mother centrosome oriented toward the stem cell niche while the daughter centrosome migrated through chromosomes to the opposite side of the mother centrosome (36, 37).
Outer dense fiber 2 (Odf2) was identified as a major component of the sperm tail cytoskeleton, which is a component of the centrosomal scaffold preferentially associated with the appendages of the mother centriole in somatic cells (38). Human Cenexin1 is an Odf2-related protein preferentially associated with the centrosome which contain the mother centriole (39) (Fig. 5A). To verify whether a spatial hierarchy exists in the
HURP-centrosome association, we monitored the localization of HURP and Cenexin1 in IBPR001/or MLN8237 treated cells. As a result, the centrosome that was stained positive for Cenexin1 was always (30 out of 30 in IBPR001/or MLN8237 treated asymmetric cells) associated with HURP (Fig. 5B).
The formation and function of the complex containing HURP, EG5, and TPX2 depends on Aurora A for the conversion of aster-like to spindle-like structures (16). We co-immunostained IBPR001-treated cells with HURP, EG5, and TPX2 to determine whether the asymmetric distribution of HURP is unique, or rather, a phenomenon common to other Aurora A-regulated proteins. Unlike EG5 or TPX2, HURP appears to locate unequally to centrosomal esters in prophase cells; however, distribution became symmetric when the cell cycle entered prometaphase (Fig. 5C). Similar phenotypes were observed in cells treated with IBPR001. Thus, the phenotype of asymmetry was not generated by the Aurora kinase inhibitors. Rather, these compounds assisted in revealing the asymmetric nature of HURP (as expressed by its association with microtubules emanating from the mother centrosome, Fig. 5B) through the inhibition of Aurora A kinase activity and subsequent HURP dephosphorylation.
DISCUSSION
Small molecules that inhibit Aurora kinase activity have been extensively developed for their potential use in inhibiting tumor growth (3, 5, 6). Most attention has been focused on the correlation between the kinase activity of Aurora A and mitotic progression (3, 4, 6). However, how these compounds influence the molecular properties of the substrate remains unclear. Our use of small molecules demonstrates that Aurora A-mediated HURP phosphorylation is required to initiate and stabilize microtubules emanating from the kinetochore, but is not required for those originating at the centrosome. We also identified a previously uncharacterized function of HURP, a preferential association with the mother centrosome. To the best of our knowledge, these findings represent the first direct experimental evidence that correlate HURP phosphorylation and microtubule nucleation between centrosomes and kinetochores in mammalian cells. These findings are summarized in Schemes 6A-E.
Rapid inhibition of kinase activities is necessary to identify the functional role of protein phosphorylation in spindle formation, which generally reaches completion within one hour. VX-680 and MLN8237 inhibit Aurora A kinase activity more effectively than IBPR compounds in vitro [Fig. 2C and references (3, 40)]; however, our results suggest that IBPR compounds are more effective inhibitors of HURP phosphorylation in vivo
(Fig. 2A,B). The discrepancies between in vitro and in vivo inhibition can be attributed to compound pharmacokinetics and/or co-factors binding to Aurora A in vivo. The crystal structures reveal that the Aurora A kinase alters conformation of the activation loop in accommodating IBPR001 from VX-680. The activation loop in protein kinases is important for the activity regulation and substrate binding (41). The difference in the conformation of the activation loop between Aurora A/IBPR001 and Aurora A/VX-680 may confer the sensitivity of Aurora A to HURP phosphorylation. Thus, the two IBPR compounds discovered in this study could help to reveal the molecular mechanism underlying spindle formation and perhaps lead to more effective clinical treatments of cancer in the future.
During spindle formation, microtubules emanating from the duplicated centrosomes continuously extend and shrink before the bilateral attachment is established. Our results suggest the phosphorylation status of HURP at two extreme ends (i.e. centrosome and kinetochore) of the mitotic spindles are different and exhibit with distinct function in tubulin nucleation (Figs. 3-4). These results raised additional questions regarding how HURP migrates between these two loci during the dynamic process of spindle formation. If the process of HURP phosphorylation is unidirectional, with nocodazole treatment, HURP should remain as phosphorylated and always associate with kinetochore
microtubules (Fig. 3A). In this setting, the inhibition of Aurora A activity (by IBPR compounds or MLN8237) should not influence HURP phosphorylation, but this was not the case (Fig. 3B), suggesting that the Aurora A-mediated HURP phosphorylation can be reversed. By releasing cells from nocodazole arrest, we showed that HURP dephosphorylation resumed (Fig. 4D, lanes 1-4) and HURP re-associates with the centrosomal microtubules (Fig. 4C, control). These results imply, first, the existence of an underlying mechanism that is associated with HURP dephosphorylation, which was demonstrated to involve the PP1/PP2A activity (Fig. 4E-F). Second, HURP is highly dynamic, trafficking between centrosomes and kinetochores driven by the mechanisms of Aurora A-dependent phosphorylation and PP1/PP2A-associated dephosphorylation (Fig. 6F). Third, although tubulin flux is not required for HURP phosphorylation or dephosphorylation, tubulin polymerization may facilitate HURP dephosphorylation in order to generate a balanced HURP phosphorylation status and consequently the establishment of robust bipolar spindles between centrosomes and kinetochores (Fig.
4C-D, 6F). In addition to provide mechanistic explanation for HURP in spindle
establishment, the assays developed in this study enable enrichment of HURP in its phosphorylated form at the kinetochore and in its unphosphorylated form at the centrosomes. This methodology may help to reveal new biomolecules that participate in
the nucleation and bundling of kinetochore or centrosomal microtubules.
Although centrosome-derived microtubules are initially radial, they begin growing with directional bias such that the density of microtubules between centrosomes and the mitotic chromosomes is greater than between centrosomes and the cell cortex (42). These observations suggest a lack of equality in the extension of microtubules to the cortex or to chromosomes. It remains unclear what elements, including microtubule-associated proteins or motor proteins, cause this directional activity (42). HURP is one element that participates in this asymmetry through its association with microtubules that grow toward the mitotic chromosomes rather than the cortex [Fig. 4A and reference (17)]. Using IBPR001/ or MLN8237 to block Aurora A activity, we identify a previously undescribed phenotype in which HURP preferentiality resides in the microtubules initiated from the mother centrosome (Fig. 5B-C). Similar to HURP, TPX2 is a Ran-regulated spindle assembly factor, which is enriched near the spindle poles and required for K-fiber formation (16, 17, 28). However, TPX2 does not present asymmetric distribution (Fig. 5C). This phenotype suggests that HURP plays a unique role in generating an additional dimension of asymmetry associated with mitotic centrosomal microtubules.
Asymmetry has been observed in many forms of cell division, in which spindles organize asters with various dynamics, associate with various molecules or subcellular
domains, and perform various functions (43). For example, in budding yeast, one spindle pole nucleates more stable microtubules than the other (43). In the zygote of C. elegans, the anterior aster of the asymmetrically-positioned spindle is large and has many microtubules, whereas the posterior aster appears flattened and smaller with fewer astral microtubules (44, 45). In Drosophila neuroblasts, the astral microtubules on the basal spindle pole are induced to depolymerize, while those of the apical aster are stabilized, resulting in a larger apical and smaller basal aster that together constitute an asymmetric spindle (44, 46). Intriguingly, Aurora A is required for the asymmetric localization of aPKC (atypical protein kinase C) to prevent it from localizing to the basal cortex in
Drosophila neuroblasts (47, 48). In aura loss-of-function mutants, supernumerary
self-renewal neuroblasts are produced, whereas neuronal differentiation is reduced (47). As with Aurora A, Hurp allows efficient sorting of MTOC (microtubule organizing center) into distinct poles, efficient congression of chromosomes, and the establishment of bipolarity in mouse oocytes, by promoting microtubule stability in the central domain of the spindle (49). In summary, our results provide evidence to establish the involvement of both Aurora A kinase activity and HURP in microtubule nucleation and the symmetry of mitotic spindles in cultured mammalian cells. Beyond Aurora A and HURP, whether other cell-division molecules contribute to this process warrants future investigation (50).
Materials and Methods
Cell culture, plasmids and transfection. HeLa and 293T cells were maintained in high glucose Dulbecco's Modified Eagle Medium (DMEM, Invitrogen) supplemented with 10% fetal bovine serum (FBS, Biological Industries), 2 mM L-glutamine and antibiotics. Human colorectal cancer HCT116 cell line was obtained from American Type Culture Collection (ATCC). Cells were maintained in McCoy’s 5A Medium (Gibco) containing 10% FBS (Gibco).
Antibodies and reagents. The antibodies were obtained from the following resources. Abcam: mouse anti--tubulin (ab11316), mouse anti-TPX2 (ab32795); Sigma-Aldrich: mouse anti-α-tubulin (T5168), mouse anti-Actin (A1978); Santa Cruz Biotechnology: goat anti-HURP (sc-68540); Covance: rabbit anti-Pericentrin (PRB-432C); Cell Signaling: mouse anti-Cyclin E1 (4129); BD Transduction Laboratories: mouse anti-Aurora A (610939); Invitrogen: rabbit anti-anti-Aurora B (36-5200), rabbit anti-PTTG
(34-1500); Bethyl Laboratories: rabbit anti-HURP (A300-853A); MBL International: mouse
anti-BUBR1 (K0169-3); ProteinTech Group: rabbit anti-ODF2/Cenexin1, rabbit
anti-NEK2 (629402); Cortex Biochem: CREST antiserum. Epitomics: rabbit anti-EG5
from Sigma-Aldrich; MG132 was from Calbiochem (474790). MLN8237 and AZD1152 were purchased from Selleckchem.
RNAi. Experimentally verified FlexiTube double-stranded siRNAs of AURKA (SI02223305), AURKB (SI02622032), HURP (SI02654169), CDK1 (SI00299719) and
NEK2 (SI00605640 and SI00605647) were from QIAGEN. Small interfering RNAs were
introduced into HeLa using the Lipofectamine RNAiMax transfection reagent (Invitrogen) following the manufacturer’s protocol.
Immunofluorescence and confocal microscopy. Cells were fixed in 4% paraformaldehyde in PBS for 30 minutes and permeablized with 0.1% TritonX-100 for 5 minutes at room temperature. For -tubulin, Pericentrin and Cenexin1 staining, cells were fixed in methanol for 10 minutes at -20oC. Cells were incubated with 1% BSA in PBS for 30 minutes to block nonspecific binding. Primary antibodies were added at dilutions of 1:100 to 1:1000 and incubated for 1.5 hours at room temperature. After three washes with PBS, cells were probed with corresponding fluorescent (488, 594 or Alexa-647)-conjugated secondary antibodies (Invitrogen). Cell nuclei were counterstained with Hoechst33342 (Invitrogen). Cells were mounted onto glass slides with ProLong Gold antifade reagent (Invitrogen), and were visualized using a Leica TCS SP5 confocal
microscope. Images were processed by the Imaris 7.2.1 (Bitplane) software.
Western Blotting. Total cell lysates were extracted with ice-cold RIPA buffer [50 mM HEPES, pH 7.3, 150 mM NaCl, 2 mM EDTA, 20 mM -gylcerophosphate, 0.1 mM Na3VO4, 1mM NaF, 0.5 mM DTT and protease inhibitor cocktail (Roche)] containing 1% NP-40 plus mild sonication. Phosphatase inhibitors were not supplied for shrimp alkaline phosphatase (SAP, Fermentas) treatment. Lysates were analyzed by SDS-PAGE, transferred to polyvinylidene fluoride (PVDF, GE Healthcare) membrane and blotted with antibodies. Alkaline phosphatase-conjugated secondary antibodies (Sigma-Aldrich) were added, and the blots were developed by chemiluminescence following the manufacturer’s protocol (Perkin Elmer).
Acknowledgements
We thank the staff at beamline BL13B1 and BL13C1 at National Synchrotron Radiation Research Centre (NSRRC), Taiwan, and the staff at beamline SP12B2 at SPring-8, Japan, for technical assistance. We thank Dr. Chang-Tze Rickey Yu (Graduate Institute of Biomedicine and Biomedical Technology, National Chi Nan University, Taiwan) for technical suggestions. We also thank Dr. Lindsay Sawyer (Institute of Structural & Molecular Biology, University of Edinburgh, UK) and Dr. Yung-Chi Cheng (Department
of Pharmacology, Yale University, USA) for critical comments on this manuscript.
Author contributions
Y.-H.C. and S.-Y.W. developed the concept, designed experiments, analyzed the data and wrote the manuscript. H.-P.H. designed and initiated chemical synthesis of the IBPR series compounds and provided critical opinions to the manuscript. Y.-H.C., J.-M.W., W. -H. L., L. -P. K. and Z.-J.C. carried out confocal microscopy and Western analyses. S.-Y.W. supervised crystal structure determination. Y.-H.P., J.-S.W. and C.-C.H. carried out the structural biology study. M.S.C., C.-Y.C., H.-Y.S. and C.-T.L. contributed to chemical synthesis of the BPR1K series compounds. C.-D.C., K.-Y.C. and C.-P.C. contributed to the mouse tumorigenesis study. J.T.-A.H. and W.-S.L. contributed to the in vitro Aurora kinase A activity assay. C.-Y.H. provided constructs of HURP. Y.-S.C. contributed to the project direction. Correspondence and requests for materials should be addressed to and Y.-H.C. ([email protected]), H.-P.H. ([email protected]) and S.-Y.W. ([email protected]).
Financial Disclosure
Work in the authors’ laboratories was supported by grants to Y.-H.C. (NHRI 00A1-CSPP11-014 and 01A1-CSPP13-014), S.-Y.W. (NHRI BP-100-PP-01 and NSC-96-2113-M-400-001-MY3) and H.-P.H. (NSC-95-2113-M-400-001-MY3 and NSC-100-2325-B-400-003).
Competing Interest
We declare there are no financial, personal, or professional interests that could be construed to have influenced this paper.
Abbreviations: HURP, human hepatoma up-regulated protein; K-fiber, kinetochore microtubule fiber; siRNA, small interfering RNA; IC50, half-maximal inhibitory concentration; SEM, standard error of mean.
References
1. Lens SM, Voest EE, Medema RH (2010) Shared and separate functions of polo-like kinases and aurora kinases in cancer. Nat Rev Cancer 10:825-841.
2. Weaver BA, Cleveland DW (2006) Does aneuploidy cause cancer? Curr Opin Cell
Biol 18:658-667.
3. Harrington EA, et al. (2004) VX-680, a potent and selective small-molecule inhibitor of the Aurora kinases, suppresses tumor growth in vivo. Nat Med 10:262-267.
4. Manfredi MG, et al. (2007) Antitumor activity of MLN8054, an orally active small-molecule inhibitor of Aurora A kinase. Proc Natl Acad Sci U S A 104:4106-4111. 5. Katayama H, Sen S (2010) Aurora kinase inhibitors as anticancer molecules.
Biochim Biophys Acta 1799:829-839.
6. Gorgun G, et al. (2010) A novel Aurora-A kinase inhibitor MLN8237 induces cytotoxicity and cell-cycle arrest in multiple myeloma. Blood 115:5202-5213. 7. Pinsky BA, Biggins S (2005) The spindle checkpoint: tension versus attachment.
Trends Cell Biol 15:486-493.
8. Tillement V, et al. (2009) Spindle assembly defects leading to the formation of a monopolar mitotic apparatus. Biol Cell 101:1-11.
9. Maiato H, Rieder CL, Khodjakov A (2004) Kinetochore-driven formation of kinetochore fibers contributes to spindle assembly during animal mitosis. J Cell
Biol 167:831-840.
10. O'Connell CB, Khodjakov AL (2007) Cooperative mechanisms of mitotic spindle formation. J Cell Sci 120:1717-1722.
11. Rieder CL (2005) Kinetochore fiber formation in animal somatic cells: dueling mechanisms come to a draw. Chromosoma 114:310-318.
12. Gadde S, Heald R (2004) Mechanisms and molecules of the mitotic spindle. Curr
Biol 14:R797-R805.
13. Waters JC, Salmon E (1997) Pathways of spindle assembly. Curr Opin Cell Biol 9:37-43.
14. Tsou AP, et al. (2003) Identification of a novel cell cycle regulated gene, HURP, overexpressed in human hepatocellular carcinoma. Oncogene 22:298-307.
15. Yu CT, et al. (2005) Phosphorylation and stabilization of HURP by Aurora-A: implication of HURP as a transforming target of Aurora-A. Mol Cell Biol 25:5789-5800.
16. Koffa MD, et al. (2006) HURP is part of a Ran-dependent complex involved in spindle formation. Curr Biol 16:743-754.
17. Sillje HH, Nagel S, Korner R, Nigg EA (2006) HURP is a Ran-importin beta-regulated protein that stabilizes kinetochore microtubules in the vicinity of chromosomes. Curr Biol 16:731-742.
18. Wilde A (2006) "HURP on" we're off to the kinetochore! J Cell Biol 173:829-831. 19. Wong J, Lerrigo R, Jang CY, Fang G (2008) Aurora A regulates the activity of
HURP by controlling the accessibility of its microtubule-binding domain. Mol Biol
Cell 19:2083-2091.
20. Coumar MS, et al. (2010) Identification, SAR studies, and X-ray co-crystallographic analysis of a novel furanopyrimidine aurora kinase A inhibitor.
ChemMedChem 5:255-267.
21. Coumar MS, et al. (2010) Fast-forwarding hit to lead: aurora and epidermal growth factor receptor kinase inhibitor lead identification. J Med Chem 53:4980-4988. 22. Cheetham GM, et al. (2002) Crystal structure of aurora-2, an oncogenic
serine/threonine kinase. J Biol Chem 277:42419-42422.
23. Martin MP, et al. (2012) A novel mechanism by which small molecule inhibitors induce the DFG flip in Aurora A. ACS Chem Biol 7:698-706.
24. Santarella RA, Koffa MD, Tittmann P, Gross H, Hoenger A (2007) HURP wraps microtubule ends with an additional tubulin sheet that has a novel conformation of tubulin. J Mol Biol 365:1587-1595.
25. Jordan MA, Thrower D, Wilson L (1992) Effects of vinblastine, podophyllotoxin and nocodazole on mitotic spindles. Implications for the role of microtubule dynamics in mitosis. J Cell Sci 102 ( Pt 3):401-416.
26. Song L, Rape M (2010) Regulated degradation of spindle assembly factors by the anaphase-promoting complex. Mol Cell 38:369-382.
27. Tanaka TU (2010) Kinetochore-microtubule interactions: steps towards bi-orientation. EMBO J 29:4070-4082.
28. Wong J, Fang G (2006) HURP controls spindle dynamics to promote proper interkinetochore tension and efficient kinetochore capture. J Cell Biol 173:879-891. 29. Ducat D, Zheng Y (2004) Aurora kinases in spindle assembly and chromosome
segregation. Exp Cell Res 301:60-67.
30. Hannak E, Kirkham M, Hyman AA, Oegema K (2001) Aurora-A kinase is required for centrosome maturation in Caenorhabditis elegans. J Cell Biol 155:1109-1116. 31. Ceulemans H, Bollen M (2004) Functional diversity of protein phosphatase-1, a
cellular economizer and reset button. Physiol Rev 84:1-39.
33. Wakimoto T, Matsunaga S, Takai A, Fusetani N (2002) Insight into binding of calyculin A to protein phosphatase 1: isolation of hemicalyculin a and chemical transformation of calyculin A. Chem Biol 9:309-319.
34. Januschke J, Llamazares S, Reina J, Gonzalez C (2011) Drosophila neuroblasts retain the daughter centrosome. Nat Commun 2:243.
35. Yamashita YM, Mahowald AP, Perlin JR, Fuller MT (2007) Asymmetric inheritance of mother versus daughter centrosome in stem cell division. Science 315:518-521.
36. Rebollo E, et al. (2007) Functionally unequal centrosomes drive spindle orientation in asymmetrically dividing Drosophila neural stem cells. Dev Cell 12:467-474. 37. Cabernard C, Doe CQ (2007) Stem cell self-renewal: centrosomes on the move.
Curr Biol 17:R465-R467.
38. Ishikawa H, Kubo A, Tsukita S, Tsukita S (2005) Odf2-deficient mother centrioles lack distal/subdistal appendages and the ability to generate primary cilia. Nat Cell
Biol 7:517-524.
39. Soung NK, et al. (2006) Requirement of hCenexin for proper mitotic functions of polo-like kinase 1 at the centrosomes. Mol Cell Biol 26:8316-8335.
40. Sloane DA, et al. (2010) Drug-resistant aurora A mutants for cellular target validation of the small molecule kinase inhibitors MLN8054 and MLN8237. ACS
Chem Biol 5:563-576.
41. Endicott JA, Noble ME, Johnson LN (2012) The structural basis for control of eukaryotic protein kinases. Annu Rev Biochem 81:587-613.
42. Duncan T, Wakefield JG (2011) 50 ways to build a spindle: the complexity of microtubule generation during mitosis. Chromosome Res 19:321-333.
43. Barral Y, Liakopoulos D (2009) Role of spindle asymmetry in cellular dynamics.
Int Rev Cell Mol Biol 278:149-213.
44. Kaltschmidt JA, Brand AH (2002) Asymmetric cell division: microtubule dynamics and spindle asymmetry. J Cell Sci 115:2257-2264.
45. Keating HH, White JG (1998) Centrosome dynamics in early embryos of Caenorhabditis elegans. J Cell Sci 111 ( Pt 20):3027-3033.
46. Giansanti MG, Gatti M, Bonaccorsi S (2001) The role of centrosomes and astral microtubules during asymmetric division of Drosophila neuroblasts. Development 128:1137-1145.
47. Wang H, et al. (2006) Aurora-A acts as a tumor suppressor and regulates self-renewal of Drosophila neuroblasts. Genes Dev 20:3453-3463.
division: Aurora-A phosphorylates the Par complex to regulate Numb localization.
Cell 135:161-173.
49. Breuer M, et al. (2010) HURP permits MTOC sorting for robust meiotic spindle bipolarity, similar to extra centrosome clustering in cancer cells. J Cell Biol 191:1251-1260.
50. Inaba M, Yamashita YM (2012) Asymmetric stem cell division: precision for robustness. Cell Stem Cell 11:461-469.
Figure Legends
Fig. 1. Structures of IBPR compounds that induce DFG conformation change in Aurora A. (A) Chemical structures of IBPR001 (1) and IBPR002 (2). (B) IC50 of the compounds from the in vitro Aurora A and Aurora B activity assay. (C) Active sites of Aurora A in complex with IBPR001 and VX-680. The interacting residues with the inhibitors are shown in stick representation and are labeled. The DFG motif (cyan) at the activation loop (A-loop, yellow) adopts a different conformation to accommodate IBPR001 from VX-680. Hydrogen bonds between the inhibitors and Aurora A are presented in red dashed line. (D) Athymic nude mice xenograft with HCT116 cancer cells were injected intravenously with control vehicle or 50 mg/kg of VX-680 or IBPR002. Mean tumor volumes (mm3) ± SEM (n = 10/group) are shown from the initiation of treatment (~100 mm3). *, P < 0.05 compare to vehicle.
Fig. 2. IBPR001 and IBPR002 efficiently eliminate HURP phosphorylation. (A) Immunoblot of HURP from HeLa cells treated with increasing concentrations of the Aurora inhibitors, VX-680, IBPR001, IBPR002, MLN8237 or AZD1152. Cells were arrested in the M phase with nocodazole (400 nM) for 24 hours followed by 1 hour co-treatment with the compounds and MG132 (5.0 g/mL). Actin immunoblot was included as a loading control. Cyclin B1 (expressed in late G2-metaphase), PTTG (expressed in prophase-metaphase) and Cyclin E1 (expressed in G1-S phase) (Supplementary Fig. S2A)
immunoblots are shown for verification of the M phase synchronization. The Western signals of HURP in the phosphorylated form (HURP-P) and the unphosphorylated form (HURP-U) are denoted. (B) Ratios of HURP-P/total HURP were quantified from the Western blot results in (A). (C) The kinase activity of Aurora A for HURP phosphorylation was assessed in an in vitro kinase assay. Full length FLAG-tagged HURP was expressed and immunoprecipitated from 293T cells as a substrate for Aurora A. The addition of active Aurora A increased the molecular size of HURP (compare lane 1 and lane 5). The molecular size of HURP failed to increase in the reactions that contain MLN8237 (lane 3) and IBPR001 (lane 4), but not AZD1152 (lane 2).
Fig. 3. IBPR compounds and MLN8237 disrupt nucleation of kinetochore microtubules. (A) HeLa cells were treated as the upper scheme, followed by co-immunofluorescence staining with two combinations of antibodies: HURP/-tubulin/CREST (a centromere marker) or HURP/-tubulin/Pericentrin (a centrosome marker). Tubulins were nucleated as thick bundles that link to kinetochores (i.e. K-fibers). DNA was stained with
Hoechst33342. Noc, nocodazole. All images are summation of z-stacks. Bars, 5 m. (B) Immunoblot of HURP from nocodazole-arrested HeLa cells treated with 1.0 M of IBPR001 or MLN8237 and 5g/mL MG132. Total cell lysates were collected at 0, 30, 60, 90 and 120 minutes after the compound treatment. (C) Statistics of HURP that form K-fibers in (A). Bars, 5 m.
Fig. 4. The IBPR compounds restrict association of HURP with centrosomal microtubules. (A) Representative HURP morphological phenotypes in HeLa cells treated with DMSO control (left panels) or 1.0 M of IBPR001/IBPR002 (right panels) for 13 hours following the thymidine release. Cells were co-immunostained with rabbit anti-HURP and mouse anti--tubulin antibodies. DNA was stained with Hoechst33342. (B) Statistics of the representative HURP morphological phenotypes presented in (A). (C) Cells were treated as the upper scheme. HURP is associated with centrosomal microtubules (stained with -tubulin) emanating from centrosomes (stained with Pericentrin) in IBPR001-treated cells upon nocodazole removal. Images are summation of z-stacks. Bars, 5 m. (D) Cells were treated as the upper scheme in (C). Cell lysates were harvested every 30 minutes after the removal of nocodazole. Immunoblots of cyclin B1 and Actin were included to indicate the cell cycle status and serve as a loading control, respectively. Immunoblot of HURP shows that HURP was kept unphosphorylated upon nocodazole removal (lanes 7-9). On the other hand, part of HURP was converted to the unphosphorylated form upon nocodazole removal in control cells (lanes 2-4, denoted by *). (E) Immunoblot of HURP in nocodazole-arrested cells treated with 1.0 M of IBPR001, 100 nM of Calyculin A, or both for one hour. Immunoblot of Actin was included as a loading control. (F) Cellular localization of HURP (green) and -tubulin (red) in nocodazole-arrested cells treated with 1.0 M of IBPR001 or co-treated with 1.0 M of IBPR001 and 100 nM of Calyculin A. Cells were fixed after 1 hour of drug treatment. A control (DMSO) cell was shown for comparison. Images are summation of z-stacks. Bars, 5 m.
Fig. 5. The unphosphorylated HURP is preferentially associated with the mother centrosome. (A) The eldest mother centriole was stained positive for Cenexin1. Centrosomes were stained positive for -tubulin. DNA was stained with Hoechst33342. (B) HURP preferentially resides with the mother centrosome that was stained positive (or stronger) for Cenexin1 in cells treated with 1.0 M IBPR001 or MLN8237. Note that the -tubulin antibody stained both centrosomes. (C) Cells were treated with DMSO control
HURP and TPX2. Bars in immunofluorescence images: 5 m. Images are maximum projection of z-stacks.
Fig. 6. Models for nucleation of centrosomal and kinetochore microtubule fibers by Aurora A-regulated HURP phorphorylation during spindle formation. (A) As a cell enters mitosis, the nuclear envelope breaks down (light blue circle), HURP is expressed and associated with the minus end of centrosomal microtubules that project toward chromosomes. As the cell cycle proceeds to promethaphase and metaphase, HURP is gradually phosphorylated and translocated to the vicinity of chromosomes to assist nucleation and stabilization of kinetochore fibers. Finally the bipolarity is established. The phosphorylated HURP forms a rod-like structure (purple bar) that links to kinetochore. (B) Treatment of 300-400 nM nocodazole enriched the phosphorylated HURP that nucleates kinetochore microtubules. (C) Continue from (B), adding IBPR001/IBPR002/MLN8237 disrupts nucleation of kinetochore microtubule. (D) Removing nocodazole under the treatment of IBPR001/IBPR002/MLN8237 re-initiates tubulin polymerization from centrosomes but not from kinetochores. HURP goes to the minus end of centrosomal microtubules that face toward the chromosomes. (E) Inhibiting HURP phosphorylation by IBPR001/IBPR002/MLN8237 abolishes nucleation of HURP in the vicinity of chromosomes. The unphosphorylated HURP distributes restrictively to the minus end of centrosomal microtubules. Because IBPR001/IBPR002/MLN8237 do not completely block separation of the duplicated centrosomes, HURP is preferentially associated with the microtubules that emanate from the mother centrosome which resides in proximity to the chromosomes. (F) Models for HURP phosphorylation and dephosphorylation with and without nocodazole. In the presence of nocodazole, the force that drives HURP phosphorylation (by Aurora A) overrides dephosphorylation (through a
PP1/PP2A-dependent pathway). Conversely, the ratio of
