Targeting aurora2 kinase in oncogenesis: a structural bioinformatics approach to target validation and rational drug design.
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The aurora kinases are a novel oncogenic family of mitotic serine/threonine kinases (S/T kinases) that are overexpressed in a number of solid tumors, including pancreas and colorectal cancer. A PSI-BLAST search [National Center for Biotechnology Information (NCBI)] with the sequence of the S/T kinase domain of human aurora1 kinase [also known as AUR1, ARK2, AIk2, AIM-1, and STK12] and human aurora2 kinase (also known as AUR2, ARK1, AIK, BTAK, and STK15) showed a high sequence similarity to the three-dimensional structures of bovine cAMP-dependent kinase [Brookhaven Protein Data Bank code 1CDK], murine cAMP-dependent kinase (1APM), and Caenorhabditis elegans twitchin kinase (1KOA). When the aurora1 or aurora2 sequence was input into the tertiary structure prediction programs THREADER and 3D-PSSM (three-dimensional position-sensitive scoring matrix), the top structural matches were 1CDK, 1APM, and 1KOA, confirming that these domains are structurally conserved. The structural models of aurora1 and aurora2 were built using 1CDK as the template structure. Molecular dynamics and docking simulations, targeting the ATP binding site of aurora2 with adenylyl imidodiphosphate (AMP-PNP), staurosporine, and six small molecular S/T kinase inhibitors, identified active-site residues that interact with these inhibitors differentially. The docked structures of the aurora2-AMP-PNP and aurora2-staurosporine complexes indicated that the adenine ring of AMP-PNP and the indolocarbazole moiety of staurosporine have similar positions and orientations and provided the basis for the docking of the other S/T kinase inhibitors. Inhibitors with isoquinoline and quinazoline moieties were recognized by aurora2 in which H-89 and 6,7-dimethoxyquinazoline compounds exhibited high binding energies compared with that of staurosporine. The calculated binding energies for the docked small-molecule inhibitors were qualitatively consistent with the IC(50) values generated using an in vitro kinase assay. The aurora2 structural model provides a rational basis for site-directed mutagenesis of the active site; design of novel H-89, staurosporine, and quinazoline analogues; and the screening of the available chemical database for the identification of other novel, small-molecular entities. (+info)
Functional significance of the specific sites phosphorylated in desmin at cleavage furrow: Aurora-B may phosphorylate and regulate type III intermediate filaments during cytokinesis coordinatedly with Rho-kinase.
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Aurora-B is a protein kinase required for chromosome segregation and the progression of cytokinesis during the cell cycle. We report here that Aurora-B phosphorylates GFAP and desmin in vitro, and this phosphorylation leads to a reduction in filament forming ability. The sites phosphorylated by Aurora-B; Thr-7/Ser-13/Ser-38 of GFAP, and Thr-16 of desmin are common with those related to Rho-associated kinase (Rho-kinase), which has been reported to phosphorylate GFAP and desmin at cleavage furrow during cytokinesis. We identified Ser-59 of desmin to be a specific site phosphorylated by Aurora-B in vitro. Use of an antibody that specifically recognized desmin phosphorylated at Ser-59 led to the finding that the site is also phosphorylated specifically at the cleavage furrow during cytokinesis in Saos-2 cells. Desmin mutants, in which in vitro phosphorylation sites by Aurora-B and/or Rho-kinase are changed to Ala or Gly, cause dramatic defects in filament separation between daughter cells in cytokinesis. The results presented here suggest the possibility that Aurora-B may regulate cleavage furrow-specific phosphorylation and segregation of type III IFs coordinatedly with Rho-kinase during cytokinesis. (+info)
Phosphorylation by aurora B converts MgcRacGAP to a RhoGAP during cytokinesis.
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Cell division is finely controlled by various molecules including small G proteins and kinases/phosphatases. Among these, Aurora B, RhoA, and the GAP MgcRacGAP have been implicated in cytokinesis, but their underlying mechanisms of action have remained unclear. Here, we show that MgcRacGAP colocalizes with Aurora B and RhoA, but not Rac1/Cdc42, at the midbody. We also report that Aurora B phosphorylates MgcRacGAP on serine residues and that this modification induces latent GAP activity toward RhoA in vitro. Expression of a kinase-defective mutant of Aurora B disrupts cytokinesis and inhibits phosphorylation of MgcRacGAP at Ser387, but not its localization to the midbody. Overexpression of a phosphorylation-deficient MgcRacGAP-S387A mutant, but not phosphorylation-mimic MgcRacGAP-S387D mutant, arrests cytokinesis at a late stage and induces polyploidy. Together, these findings indicate that during cytokinesis, MgcRacGAP, previously known as a GAP for Rac/Cdc42, is functionally converted to a RhoGAP through phosphorylation by Aurora B. (+info)
The small molecule Hesperadin reveals a role for Aurora B in correcting kinetochore-microtubule attachment and in maintaining the spindle assembly checkpoint.
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The proper segregation of sister chromatids in mitosis depends on bipolar attachment of all chromosomes to the mitotic spindle. We have identified the small molecule Hesperadin as an inhibitor of chromosome alignment and segregation. Our data imply that Hesperadin causes this phenotype by inhibiting the function of the mitotic kinase Aurora B. Mammalian cells treated with Hesperadin enter anaphase in the presence of numerous monooriented chromosomes, many of which may have both sister kinetochores attached to one spindle pole (syntelic attachment). Hesperadin also causes cells arrested by taxol or monastrol to enter anaphase within <1 h, whereas cells in nocodazole stay arrested for 3-5 h. Together, our data suggest that Aurora B is required to generate unattached kinetochores on monooriented chromosomes, which in turn could promote bipolar attachment as well as maintain checkpoint signaling. (+info)
CSC-1: a subunit of the Aurora B kinase complex that binds to the survivin-like protein BIR-1 and the incenp-like protein ICP-1.
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The Aurora B kinase complex is a critical regulator of chromosome segregation and cytokinesis. In Caenorhabditis elegans, AIR-2 (Aurora B) function requires ICP-1 (Incenp) and BIR-1 (Survivin). In various systems, Aurora B binds to orthologues of these proteins. Through genetic analysis, we have identified a new subunit of the Aurora B kinase complex, CSC-1. C. elegans embryos depleted of CSC-1, AIR-2, ICP-1, or BIR-1 have identical phenotypes. CSC-1, BIR-1, and ICP-1 are interdependent for their localization, and all are required for AIR-2 localization. In vitro, CSC-1 binds directly to BIR-1. The CSC-1/BIR-1 complex, but not the individual subunits, associates with ICP-1. CSC-1 associates with ICP-1, BIR-1, and AIR-2 in vivo. ICP-1 dramatically stimulates AIR-2 kinase activity. This activity is not stimulated by CSC-1/BIR-1, suggesting that these two subunits function as targeting subunits for AIR-2 kinase. (+info)
Closing the GAP: a role for a RhoA GAP in cytokinesis.
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During cell division, cytokinesis partitions the cytoplasm to form two daughter cells. Recently in Developmental Cell, Minoshima et al. (2003) establish an important connection between three regulatory molecules that coordinate this process, showing that the mitotic kinase aurora B phosphorylates MgcRacGAP to convert it to a GAP for RhoA, a small GTPase that drives cytokinesis. (+info)
Aurora B couples chromosome alignment with anaphase by targeting BubR1, Mad2, and Cenp-E to kinetochores.
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The Aurora/Ipl1 family of protein kinases plays multiple roles in mitosis and cytokinesis. Here, we describe ZM447439, a novel selective Aurora kinase inhibitor. Cells treated with ZM447439 progress through interphase, enter mitosis normally, and assemble bipolar spindles. However, chromosome alignment, segregation, and cytokinesis all fail. Despite the presence of maloriented chromosomes, ZM447439-treated cells exit mitosis with normal kinetics, indicating that the spindle checkpoint is compromised. Indeed, ZM447439 prevents mitotic arrest after exposure to paclitaxel. RNA interference experiments suggest that these phenotypes are due to inhibition of Aurora B, not Aurora A or some other kinase. In the absence of Aurora B function, kinetochore localization of the spindle checkpoint components BubR1, Mad2, and Cenp-E is diminished. Furthermore, inhibition of Aurora B kinase activity prevents the rebinding of BubR1 to metaphase kinetochores after a reduction in centromeric tension. Aurora B kinase activity is also required for phosphorylation of BubR1 on entry into mitosis. Finally, we show that BubR1 is not only required for spindle checkpoint function, but is also required for chromosome alignment. Together, these results suggest that by targeting checkpoint proteins to kinetochores, Aurora B couples chromosome alignment with anaphase onset. (+info)
Survivin is required for stable checkpoint activation in taxol-treated HeLa cells.
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Survivin is an essential chromosomal passenger protein whose function remains unclear. Here, we have used RNA interference to specifically repress Survivin in cultured HeLa cells. Immunoblot analysis showed that Survivin was no longer detectable in cultures 60 hours after transfection with Survivin-specific siRNA. Live cell analysis showed that many Survivin-depleted cells were delayed in mitosis, and immunofluorescence analysis of fixed specimens revealed that Survivin-depleted cells accumulated in prometaphase with misaligned chromosomes. The chromosomal passenger proteins, INCENP and Aurora-B, which can interact directly with Survivin, were absent from the centromeres of Survivin-depleted cells. These data contribute to the emerging picture that Survivin operates together with INCENP and Aurora-B to perform its mitotic duties. Some Survivin-depleted cells eventually exited mitosis without completing cytokinesis. This resulted in a gradual increase in the percentage of multinucleated cells in the culture. Time-lapse imaging of synchronized cultures revealed that control and Survivin-depleted cells arrested in mitosis in the presence of nocodazole; however, the latter failed to arrest in mitosis when treated with taxol. Immunofluorescence studies revealed that Survivin-depleted cells were unable to stably maintain BubR1 at the kinetochores in the presence of either taxol or nocodazole. Our data reveal that Survivin is not required for the spindle assembly checkpoint when it is activated by the loss of microtubules. However, Survivin is required for the maintenance of the checkpoint when it is activated by taxol, which is generally thought to cause a loss of spindle tension. (+info)