To test whether the genetic interaction reflects a physical interaction between Bora and Aurora-A, binding assays were performed in Drosophila tissue culture cells. Drosophila S2 cells were transfected with Aurora-A and Bora-GFP, and protein lysates were subjected to immunoprecipitation by anti-GFP. Since Aurora-A is specifically detected in the immunoprecipitate, it is concluded that Bora can bind to Aurora-A in vivo. To test whether this is due to a direct interaction, in vitro binding experiments were performed. In vitro translated Aurora-A binds to a GST-Bora fusion-protein but not to GST alone. While the nonconserved C terminus of Bora is dispensible for Aurora-A binding, the interaction is abrogated by deleting the conserved region (BoraΔ2) or a region N-terminal to the conserved part (BoraΔ1). Interestingly, the interaction is also observed between in vitro translated human Aurora-A and MBP-HsBora. Human Aurora-A can even bind to Drosophila MBP-Bora in vitro. The interaction with Aurora-A seems to be essential for Bora function since the N-terminal 404 amino acids of Bora (almost identical to BoraΔ3) can rescue the bora and aurA37 mutant phenotypes, while the C terminus (amino acids 404–539) does not. Thus, Bora and its homologs act as binding partners of Aurora-A (Hutterer, 2006).
Several Aurora-A regulators—like TPX2 also act as substrates for the kinase. To test whether Bora can be phosphorylated by Aurora-A, in vitro kinase assays were performed. Drosophila Aurora-A expressed and purified from E. coli can phosphorylate bacterially expressed myelin basic protein tagged Bora (MBP-Bora) but not MBP alone. Interestingly, the kinase activity of Aurora-A toward Bora is as potent as toward myelin basic protein, which is often used as a model substrate. Similarly, human Aurora-A can phosphorylate the human Bora homolog. To test which region of Bora is phosphorylated, Bora deletions were used in the kinase assay. Deletion of 125 amino acids from the N terminus of Bora (BoraΔ2) eliminates phosphorylation by Aurora-A, while deletion of the C terminus from amino acid 209 onward (BoraΔ5) does not affect it. Interestingly, Bora is still phosphorylated when the N-terminal 67 amino acids are deleted (BoraΔ1), suggesting that direct binding to Aurora-A is not necessary for Bora to act as a substrate. These experiments suggest that the N terminus of Bora is phosphorylated by Aurora-A (Hutterer, 2006).
To test whether Bora can influence the kinase activity of Aurora-A, recombinant human Bora was used in an in vitro kinase assay with myelin basic protein as a substrate. Addition of Bora increases Aurora-A activity in a dose-dependent manner, and a 2.5-fold maximum increase in kinase activity was observed. Aurora-A is regulated by phosphorylation in the activation loop of the kinase. Since Aurora-A can autophosphorylate, any kinase preparation may be partially active, and this might explain the modest degree of activation by recombinant Bora. Consistent with this, when Aurora-A is inactivated by pretreatment with protein phosphatase 1 (PP1), addition of Bora induces an over 7-fold increase in kinase activity. Analogous experiments with the Drosophila homologs reveal that Drosophila Bora similarly activates the Drosophila kinase, showing that it acts as a kinase activator as well. Taken together, these results demonstrate that Bora is an activator of Aurora-A (Hutterer, 2006).
Mutation of the autophosphorylation site of Aurora-A to alanine renders the kinase inactive, and an interesting question is whether the stimulation of Aurora-A by Bora bypasses the need for autophosphorylation. It was found that addition of Bora does not restore activity to the mutant kinase, suggesting that activation by Bora requires autophosphorylation of Aurora-A (Hutterer, 2006).
To determine the subcellular localization of Bora in SOP cells, live imaging was performed of a Bora-GFP fusion protein, which can rescue both bora and aurA37 mutant phenotypes. Histone-RFP is used to label chromosomes and indicates the cell-cycle stage. Constructs were specifically expressed by neuralized-Gal4 in SOP cells and dividing cells were imaged in whole living pupae. In interphase, Bora is a nuclear protein. When chromosomes condense, however, Bora is released from the nucleus. It is completely excluded from the nucleus by late prophase and is uniformly distributed in the cytoplasm after nuclear envelope breakdown. In telophase, Bora enters both daughter cells where it relocates into the nucleus. Bora does not have an obvious nuclear localization signal. However, it was found that the first 125 amino acids of the protein are sufficient for nuclear retention, suggesting that they contain the sequence that mediates nuclear import. Live imaging of GFP-Aurora-A together with Histone-RFP allows correlation of the localization of Aurora-A with Bora. In interphase, the two proteins are in distinct compartments. Nuclear release of Bora coincides with centrosome separation and strong recruitment of Aurora-A to the maturing centrosomes. Since both centrosome separation and maturation defects are observed in aurora-A mutants, these results suggest that release of Bora coincides with Aurora-A activation (Hutterer, 2006).
While Aurora-A is required for a subset of mitotic events, Cdc2 is essential for all steps of mitosis. How Cdc2 activates Aurora-A is unclear. To test whether Cdc2 regulates the release of Bora into the cytoplasm, Bora localization was examined in string mutants. String is the Drosophila homolog of the Cdc25 phosphatase, and in string mutants, Cdc2 is not activated. Antibody staining of Drosophila embryos reveals that endogenous Bora shows the same dynamic localization during the cell cycle as the functional GFP fusion protein. In string mutant embryos, however, Bora was never observed in the cytoplasm, indicating that Cdc2 activation is required for the release of Bora from the nucleus. To test whether Cdc2 might directly phosphorylate Bora, in vitro kinase assays were performed. Both Bora and HsBora are phosphorylated by recombinant Cdk1. Although the in vivo relevance of Cdk1 phosphorylation remains to be tested, these experiments show that Bora is released into the cytoplasm at the onset of mitosis in a Cdc2-dependent manner (Hutterer, 2006).
In a genetic screen for mutations affecting the development of Drosophila external sensory (ES) organs, mutations were identified in aurora-A (Berdnik, 2002). In these mutants, Numb fails to localize asymmetrically and the proteins γ-Tubulin and Centrosomin are not recruited to centrosomes during mitosis, leading to spindle abnormalities. Two other mutations from the same screen caused similar phenotypes but are not allelic to aurora-A. Both alleles affect the same gene, which has been named bora (for aurora borealis) to indicate its similarity with aurora-A. Flies that are homozygous for bora on the head and eye were generated by the ey-Flp/FRT system. These flies frequently show duplicated hairs and sockets, a phenotype indicative of defects in asymmetric cell division. To determine whether this morphological defect results from cell-fate transformations, the SOP cell progeny were analyzed by using different molecular markers. The socket cell expresses the transcription factor Suppressor of Hairless (Su(H)), whereas the sheath cell can be recognized by expression of Prospero. All four cells express the transcription factor Cut, and the hair cell can be distinguished from the neuron based on its larger size. In bora mutant ES organs, four equally sized Cut-positive cells are found, two of which express Su(H), while no Prospero-positive cell can be detected. Thus in bora mutants, inner cells are transformed into additional outer cells, which is a phenotype characteristic of a defect in Numb localization (Berdnik, 2002; Bhalerao, 2005). Indeed, whereas in wild-type SOP cells Numb localizes asymmetrically into a crescent in mitosis and segregates into one of the two daughter cells, in bora mutant SOP cells, the protein is uniformly cortical in metaphase and equally distributed into both daughter cells. Defects in asymmetric localization (although at lower frequency) are also observed for the Numb binding partner Pon (Partner of Numb), but localization of Gαi and Pins is normal. Gαi and Pins are required for Numb localization and can act as markers for the polarization of SOP cells, which already occurs in interphase. Thus, bora is required for the asymmetric localization of cell fate determinants during mitosis but is not essential for polarization of SOP cells in general (Hutterer, 2006).
To further explore the phenotypic similarity with aurora-A, centrosome maturation was analyzed in bora mutants. In wild-type SOP cells, several proteins including γ-Tubulin and Centrosomin are recruited to centrosomes during mitosis. In bora mutant SOP cells, however, Centrosomin recruitment is either weak or not detected at all. Frequently, only one or two closely spaced Centrosomin dots were detected, indicating defects in centrosome separation. Thus, bora mutants recapitulate all aspects of the aurora-A mutant phenotype in SOP cells (Hutterer, 2006).
To test whether Aurora-A is active in bora mutants, phosphospecific antibodies were used against D-TACC, a substrate of Aurora-A. In wild-type cells, phosphorylated D-TACC is found at centrosomes and on the mitotic spindle. In both aurA37 and bora mutants, however, P-D-TACC staining is significantly reduced and not enriched on any intracellular structures. These results suggest that Bora is required for the activation of Aurora-A during mitosis (Hutterer, 2006).
To determine which gene is affected in bora mutants, the mutation was narrowed down to the cytological interval 75B-C by P-element and deficiency mapping. Single-nucleotide polymorphism (SNP) mapping was used for further refinement and sequencing of candidate genes in the respective region. This revealed that both mutants carry lesions in a transcript that has been annotated as CG6897 by the Drosophila sequencing consortium. bora15 is a 14 base-pair out-of-frame deletion in the coding region, which introduces a stop codon after amino acid 162, while bora18 is a G-to-A transition that affects a splice-acceptor site. Both alleles are lethal during pupal stages when homozygous, transheterozygous, or hemizygous over Df(3L)Cat, suggesting that they are either null or strong hypomorphic alleles. Flies carrying large bora15 or bora18 mutant clones frequently show duplication of hairs and sockets. These defects can be rescued by expression of a Bora-GFP fusion-protein under the control of scabrous-Gal4, indicating that CG6897 is indeed responsible for the bora mutant phenotype (Hutterer, 2006).
The phenotypic similarity suggests a close connection between Bora and Aurora-A. To test whether bora and aurora-A interact genetically, rescue experiments were performed with the hypomorphic aurora-A allele aurA37 (Berdnik, 2002). Overexpression of Bora-GFP with scabrous-Gal4 does not cause a phenotype by itself but can rescue the bristle duplications, which are observed in aurA37 mutants. Antibody staining reveals that both the defects in Numb localization and the centrosome defects are rescued by Bora-GFP. While Numb is mislocalized and centrosome maturation is impaired in all SOP cells of aurA37 mutant flies, asymmetric Numb localization is rescued to 77% in metaphase SOP cells and centrosome maturation to 35% upon overexpression of Bora-GFP. In contrast to aurA37 clones, eyFlp/FRT clones of aurora-A null mutants die early after clone induction. Overexpression of Bora-GFP cannot inhibit this cell lethal effect suggesting that Bora can increase the activity of Aurora-A but not compensate for the complete loss of kinase activity. Taken together, these results suggest that Bora is a rate-limiting regulator of Aurora-A activity (Hutterer, 2006).
Reference names in red indicate recommended papers.
Bayliss, R., Sardon, T., Vernos, I. and Conti, E. (2003). Structural basis of Aurora-A activation by TPX2 at the mitotic spindle. Mol. Cell 12(4): 851-62. 14580337
Berdnik, D. and Knoblich, J. (2002). Drosophila Aurora-A is required for centrosome maturation and actin-dependent asymmetric protein localization during mitosis. Curr. Biol. 12: 640-647. 11967150
Bhalerao, S., Berdnik, D., Torok, T. and Knoblich, J. A. (2005). Localization-dependent and -independent roles of numb contribute to cell-fate specification in Drosophila. Curr Biol. 15(17): 1583-90. 16139215
Betschinger, J. and Knoblich, J. A. (2004). Dare to be different: asymmetric cell division in Drosophila, C. elegans and vertebrates. Curr. Biol. 14: R674-R685. 15324689
Cotteret, S. and Chernoff, J. (2005). Pak GITs to Aurora-A. Dev. Cell 9: 573-574. Medline abstract: 16256730
Hirota, T., et al. (2003). Aurora-A and an interacting activator, the LIM protein Ajuba, are required for mitotic commitment in human cells. Cell 114: 585-598. 13678582
Hutterer, A., Berdnik, D., Wirtz-Peitz, F., Zigman, M., Schleiffer, A., Knoblich, J. A. (2006). Mitotic activation of the kinase Aurora-A requires its binding partner Bora. Dev. Cell 11(2): 147-57. 16890155
Kufer, T. A., et al. (2002). Human TPX2 is required for targeting Aurora-A kinase to the spindle. J. Cell Biol. 158(4): 617-23. 12177045
Kufer, T., Nigg, E. and Sillje, H. (2003), Regulation of Aurora-A kinase on the mitotic spindle. Chromosoma 112: 159-163. 14634755
Marumoto, T., et al. (2003). Aurora-A kinase maintains the fidelity of early and late mitotic events in HeLa cells. J. Biol. Chem. 278: 51786-51795. 14523000
Maton, G., et al. (2003). Cdc2-cyclin B triggers H3 kinase activation of Aurora-A in Xenopus oocytes. J. Biol. Chem. 278: 21439-21449. 12670933
Maton, G., et al. (2005). Differential regulation of Cdc2 and Aurora-A in Xenopus oocytes: a crucial role of phosphatase 2A, J. Cell Sci. 118: 2485-2494. 15923661
Pratt, S., et al. (2005). The LIM protein Ajuba influences p130Cas localization and Rac1 activity during cell migration. J. Cell Biol. 168: 813-824. 15728191
Pugacheva, E. and Golemis, E. (2005). The focal adhesion scaffolding protein HEF1 regulates activation of the Aurora-A and Nek2 kinases at the centrosome. Nat. Cell Biol. 7: 937-946. 16184168
Tio, M., et al. (2001). cdc2 links the Drosophila cell cycle and asymmetric division machineries. Nature 409: 1063-1067. 11234018
Zhao, Z., et al. (2005). The GIT-associated kinase PAK targets to the centrosome and regulates Aurora-A. Mol. Cell 237-249. 16246726
date revised: 22 February 2007
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