Crystal Structure of Aurora-2, an Oncogenic Serine/Threonine Kinase

Abstract

Aurora-2 is a key member of a closely related subgroup of serine/threonine kinases that plays important roles in the completion of essential mitotic events. Aurora-2 is oncogenic and amplified in various human cancers and could be an important therapeutic target for inhibitory molecules that would disrupt the cell cycle and block proliferation. We report the first crystal structure of Aurora-2 kinase in complex with adenosine. Analysis of residues in the active site suggests differences with structurally and biologically related protein kinases. The activation loop, which contains residues specific to the Aurora family of kinases, has a unique conformation. These results provide valuable insight into the design of selective and highly potent ATP-competitive inhibitors of the Aurora kinases. Aurora-2 is a key member of a closely related subgroup of serine/threonine kinases that plays important roles in the completion of essential mitotic events. Aurora-2 is oncogenic and amplified in various human cancers and could be an important therapeutic target for inhibitory molecules that would disrupt the cell cycle and block proliferation. We report the first crystal structure of Aurora-2 kinase in complex with adenosine. Analysis of residues in the active site suggests differences with structurally and biologically related protein kinases. The activation loop, which contains residues specific to the Aurora family of kinases, has a unique conformation. These results provide valuable insight into the design of selective and highly potent ATP-competitive inhibitors of the Aurora kinases. dithiothreitol 4-morpholineethanesulfonic acid adenosine 5′-(β,γ-imino)triphosphate Cyclin-dependent kinase glycogen synthase kinase a human kinase from Rous sarcoma virus Protein kinases play important roles in promoting or retarding transitions between different stages and checkpoints of the cell cycle (1Nigg E.A. Blangy A. Lane H.A. Exp. Cell Res. 1996; 229: 174-180Crossref PubMed Scopus (73) Google Scholar). In humans, a closely related subgroup of three serine/threonine protein kinases, the Aurora kinases (2Giet R. Prigent C. J. Cell Sci. 1999; 112: 3591-3601Crossref PubMed Google Scholar, 3Bischoff J.R. Plowman G.D. Trends Cell Biol. 1999; 9: 454-459Abstract Full Text Full Text PDF PubMed Scopus (310) Google Scholar), are believed to play a key role in protein phosphorylation events that regulate the mitotic phase of the cell cycle. The human Aurora kinases display distinct subcellular locations during mitosis. Aurora-2, alternatively known as Aurora-A, is up-regulated during the M phase of the cell cycle and localizes to the spindle pole during mitosis, suggesting a possible involvement in centrosomal functions. While Aurora-2 activity is maximal during prophase, Aurora-1 (Aurora-B) appears to play an important role during chromatid separation and formation of the cleavage furrow in anaphase and telophase. The role of Aurora-3 (Aurora-C) is less clear, but it has been shown to localize to centrosomes during mitosis from anaphase to cytokinesis. The Aurora kinases are specifically degraded by proteosome activity (2Giet R. Prigent C. J. Cell Sci. 1999; 112: 3591-3601Crossref PubMed Google Scholar, 4Walter A.O. Seghezzi W. Korver W. Sheung J. Lees E. Oncogene. 2000; 19: 4906-4916Crossref PubMed Scopus (234) Google Scholar) upon exit from mitosis. Homologous members of the Aurora kinase family have been identified in a number of different organisms (5Schumacher J.M. Golden A. Donovan P.J. J. Cell Biol. 1998; 143: 1635-1646Crossref PubMed Scopus (268) Google Scholar, 6Kimura M. Kotani S. Hattori T. Sumi N. Yoshioka T. Todokoro K. Okano Y. J. Biol. Chem. 1997; 272: 13766-13771Abstract Full Text Full Text PDF PubMed Scopus (242) Google Scholar). The Aurora kinases are overexpressed in several types of human cancer cells such as colon, breast, and other solid tumors (7Bischoff J.R. Anderson L. Zhu Y. Mossie K., Ng, L. Souza B. Schryver B. Flanagan P. Clairvoyant F. Ginther C. Chan C.S.M. Novotny M. Slamon D.J. Plowman G.D. EMBO J. 1998; 17: 3052-3065Crossref PubMed Scopus (1115) Google Scholar, 8Goepfert T.M. Brinkley B.R. Curr. Top. Dev. Biol. 2000; 49: 331-342Crossref PubMed Google Scholar, 9Sakakura C. Hagiwara A. Yasuoka R. Fujita Y. Nakanishi M. Masuda K. Shimomura K. Nakamura Y. Inazawa J. Abe T. Yamagishi H. Br. J. Cancer. 2001; 84: 824-831Crossref PubMed Scopus (203) Google Scholar). More importantly, both the Aurora-1 and Aurora-2genes are amplified in breast and colorectal cancers, while theAurora-3 gene is located in a region that is rearranged or deleted in several cancer cells (10Bicher A. Ault K. Kimmelman A. Gershenson D. Reed E. Liang B. Gynecol. Oncol. 1997; 66: 36-40Abstract Full Text PDF PubMed Scopus (43) Google Scholar, 11Hoglund M. Gorunova L. Andren-Sandberg A. Dawiskiba S. Mitelman F. Johansson B. Genes Chromosomes Cancer. 1998; 21: 8-16Crossref PubMed Scopus (53) Google Scholar). Overexpression of Aurora-2 in rodent fibroblasts induces transformation, indicating that Aurora-2 is oncogenic (7Bischoff J.R. Anderson L. Zhu Y. Mossie K., Ng, L. Souza B. Schryver B. Flanagan P. Clairvoyant F. Ginther C. Chan C.S.M. Novotny M. Slamon D.J. Plowman G.D. EMBO J. 1998; 17: 3052-3065Crossref PubMed Scopus (1115) Google Scholar). Recently Aurora-2 mRNA expression has been linked to chromosomal instability in human breast cancers (12Miyoshi Y. Iwao K. Egawa C. Noguchi S. Int. J. Cancer. 2001; 92: 370-373Crossref PubMed Scopus (227) Google Scholar). Inhibition of Aurora kinase activity in mammalian cells leads to abnormal cell growth and polyploidy (13Terada Y. Tatsuka M. Suzuki F. Yasuda Y. Fujita S. Otsu M. EMBO J. 1998; 17: 667-676Crossref PubMed Scopus (369) Google Scholar). Thus the Aurora kinases could be good targets for therapeutic treatment of cancer. The Aurora kinases share a common sequence and structure consisting of a highly conserved C-terminal catalytic domain and a short N-terminal domain that varies in size (2Giet R. Prigent C. J. Cell Sci. 1999; 112: 3591-3601Crossref PubMed Google Scholar). The N-terminal domains of Aurora-1, Aurora-2, and Aurora-3 have limited sequence similarity, and it has been proposed that they make interactions with other proteins (14Adams R.R. Carmena M. Earnshaw W.C. Trends Cell Biol. 2001; 11: 49-54Abstract Full Text Full Text PDF PubMed Scopus (476) Google Scholar, 15Wheatley S.P. Carvalho A. Vagnarelli P. Earnshaw W.C. Curr. Biol. 2001; 11: 886-890Abstract Full Text Full Text PDF PubMed Scopus (244) Google Scholar) that facilitate relocation of the Aurora enzymes from the cytoplasm to the nucleus during mitosis. The catalytic domain of Aurora family kinases contains a short C-terminal tail sequence (residues 390–403) that may aid proteosome degradation of the enzymes at the end of mitosis (4Walter A.O. Seghezzi W. Korver W. Sheung J. Lees E. Oncogene. 2000; 19: 4906-4916Crossref PubMed Scopus (234) Google Scholar, 16Glotzer M. Murray A.W. Kirschner M.W. Nature. 1991; 349: 100-101Crossref PubMed Scopus (2006) Google Scholar). Specific degradation of the Aurora enzymes upon exit from mitosis is consistent with their very low cellular levels in G1 phase. As central mediators of cell mitosis and progression through the G2/M phase of the cell cycle, Aurora protein kinases are potential targets for inhibitory molecules that would block cell proliferation and may therefore be useful as cancer therapeutics. It has been established that highly specific ATP-competitive inhibitors can be obtained against a number of different kinases with clinical uses as agents in the treatment of cancer (17Garcia-Echeverria C. Traxler P. Evans D.B. Med. Res. Rev. 2000; 20: 28-57Crossref PubMed Scopus (144) Google Scholar). Understanding the molecular constraints of the ATP-binding site of Aurora-2 and the structural basis for interactions with adenosine is an essential step in designing inhibitors for this subfamily of kinases that are both selective and potent. We have determined the crystal structure of the C-terminal catalytic kinase domain of Aurora-2 bound to adenosine at 2.9 Å resolution. This is the first report of a crystal structure of a member of the Aurora family of protein kinases. A truncated version of Aurora-2 (residues 107–403) (the same sequence as GenBankTMaccession number AF011468) with an N-terminal hexahistidine tag and a thrombin cleavage site was overexpressed in a baculovirus expression system. Aurora-2 was purified using nickel-nitrilotriacetic acid-agarose metal affinity chromatography (Qiagen, Hilden, Germany) followed by size exclusion on a Superdex 200 column (AmershamBiosciences). The hexahistidine tag was removed by overnight incubation at 4 °C with 5 units mg−1 thrombin (Calbiochem). The thrombin was removed with benzamidine-Sepharose (Amersham Biosciences), and the protein was applied to a Mono S 10/10 column (AmershamBiosciences) equilibrated in 20 mm HEPES, pH 7.3, 10% glycerol (v/v), 2 mmDTT.1Aurora-2-(107–403) was eluted with a linear gradient from 0 to 500 mm NaCl in 80 column volumes. Unphosphorylated Aurora-2-(107–403) eluted at 148 mm NaCl. The protein was dialyzed against 25 mm Tris, pH 8.0 containing 200 mm NaCl and 2 mm DTT at 4 °C, concentrated to 15 mg ml−1, and centrifuged at 100,000 ×g prior to crystallization. Aurora-2 kinase activity was assayed using a standard coupled enzyme system (18Fox T. Coll J.T. Xie X. Ford P.J. Germann U.A. Porter M.D. Pazhanisamy S. Fleming M.A. Galullo V., Su, M.S. Wilson K.P. Protein Sci. 1998; 7: 2249-2255Crossref PubMed Scopus (132) Google Scholar). Kemptide peptide (Bachem (UK) Ltd.), sequence LRRASLG, was used a substrate for Aurora-2. Reactions were carried out at 30 °C in a solution containing 100 mm HEPES (pH 7.5), 10 mmMgCl2, 25 mm NaCl, 1 mm DTT. Rates of reaction were obtained using a Molecular Devices Spectramax plate reader (Sunnyvale, CA) over 10 min. K m and IC50 values were determined using computerized nonlinear regression (Prism 3.0, GraphPad Software, San Diego, CA). Crystals of the Aurora-2-adenosine complex were grown by hanging-drop vapor diffusion from a solution of 21% polyethylene glycol 3350 and 0.2 m ammonium sulfate buffered with 0.1 m MES, pH 6.0. Adenosine (Sigma, catalog number A9251) was added to the Aurora-2 enzyme solution, to a give final concentration of 2.5 mm, immediately before crystallization. Hexagonal button-shaped crystals took ∼36 h to grow to a size of 0.4 × 0.4 × 0.2 mm. For cryogenic data collection at 100 K, crystals were harvested and then allowed to equilibrate in a similar crystallization solution, this time containing 15% glycerol, for 1 min prior to freezing in liquid nitrogen. The crystals contain a single enzyme complex per asymmetric unit. Data were reduced using MOSFLM (19Leslie A.G.W. Joint CCP4 and ESF-EACMB Newsletter on Protein Crystallography. Daresbury Laboratory, Warrington, UK1992Google Scholar) and CCP4 (20Collaborative Computational Project, Number 4 Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 760-763Crossref PubMed Scopus (19879) Google Scholar) see Table I. The structure was determined by molecular replacement (21Brünger A.T. Acta Crystallogr. Sect. A. 1991; 47: 195-204Crossref PubMed Scopus (46) Google Scholar) using a homology model of Aurora-2 based on the structure of cAMP-dependent protein kinase (22Hanks S.K. Quinn A.M. Hunter T. Science. 1988; 241: 42-52Crossref PubMed Scopus (4278) Google Scholar). The initial molecular replacement solution was subject to multiple rounds of molecular dynamics refinement (23Brünger A.T. Adams P.D. Rice L.M. Curr. Opin. Struct. Biol. 1998; 8: 606-611Crossref PubMed Scopus (81) Google Scholar) in CNX and rebuilding with QUANTA. The bound adenosine molecule could be clearly seen in a difference-Fourier map calculated at 2.9 Å resolution.Table IX-ray data and refinement summaryData collectionUnit cell parameters a (Å)a = b = 86.2, c = 75.4X-ray sourceX31 DESY, EMBL outstationSpace groupP3221Resolution (Å)18–2.9Number of observations17,936Number of unique7,275Completeness (%)97.3R symaRsym = ∑h∑j‖〈I(h)〉 −I(h)j‖/∑h∑j〈I(h)〉, where 〈I(h)〉 is the mean intensity of symmetry equivalent reflections.0.063RefinementResolution range (Å)18–2.9Reflections used6,421R-factor,R freebR-factor = ∑‖ ‖F obs‖ − ‖F calc‖ ‖/∑‖F obs‖, where F obs andF calc are the observed and calculated structure factors, respectively. R free = R-factor for 10% or data excluded from structure refinement (38).0.24, 0.29a Rsym = ∑h∑j‖〈I(h)〉 −I(h)j‖/∑h∑j〈I(h)〉, where 〈I(h)〉 is the mean intensity of symmetry equivalent reflections.b R-factor = ∑‖ ‖F obs‖ − ‖F calc‖ ‖/∑‖F obs‖, where F obs andF calc are the observed and calculated structure factors, respectively. R free = R-factor for 10% or data excluded from structure refinement (38Brünger A.T. Nature. 1992; 355: 472-474Crossref PubMed Scopus (3896) Google Scholar). Open table in a new tab The catalytic domain of Aurora-2 has the typical bilobal kinase fold (22Hanks S.K. Quinn A.M. Hunter T. Science. 1988; 241: 42-52Crossref PubMed Scopus (4278) Google Scholar,24Bossemeyer D. Engh R.A. Kinzel V. Ponstingl H. Huber R. EMBO J. 1993; 12: 849-859Crossref PubMed Scopus (375) Google Scholar), comprised of an N-terminal β-strand domain (residues 127–215) and a C-terminal α-helical domain (residues These domains are linked by a region (residues that plays an important in the catalytic active site In the crystal a number of residues located at the and of the kinase domain (residues and and residues could be in The of Aurora family kinases contains a conserved acid residues and located in a region at the of the kinase domain Å from the catalytic active This degradation from the kinase fold and is for by protein or active site of Aurora-2. interactions with adenosine bound in the catalytic active A similar to The protein of residues in the region of Aurora-2, and the of located in the activation loop, adenosine through specific are between the or of the and Aurora-2. and are essential for Aurora-2 kinase activity but to other as seen in crystal of several other protein of the Aurora-2 active site the or by residues and that is in of Aurora-2 with other protein kinases that structure and is closely related to both the family of kinases F. I. J. Nature. 1997; PubMed Scopus Google Scholar, W. Nature. 1997; PubMed Scopus Google Scholar) and the family of kinases D. Engh R.A. Kinzel V. Ponstingl H. Huber R. EMBO J. 1993; 12: 849-859Crossref PubMed Scopus (375) Google Scholar). mean calculated for for of Aurora-2 with and cAMP-dependent protein kinase are and respectively. For residues located a of the Aurora-2 active the mean with and protein kinase D. Engh R.A. Kinzel V. Ponstingl H. Huber R. EMBO J. 1993; 12: 849-859Crossref PubMed Scopus (375) Google Scholar) are and respectively. Adenosine in a at the of the α-helical and β-strand domains as observed for other protein kinases D. Engh R.A. Kinzel V. Ponstingl H. Huber R. EMBO J. 1993; 12: 849-859Crossref PubMed Scopus (375) Google Scholar, F. I. J. Nature. 1997; PubMed Scopus Google Scholar, W. Nature. 1997; PubMed Scopus Google Scholar). Specific are between the of adenosine and the region of the kinase The active site is by the loop, which contains the kinase sequence (residues in and the activation As observed in the of kinase bound to J. Biol. 1999; PubMed Scopus Google Scholar) and the family kinase Cyclin-dependent bound to F. I. J. Nature. 1997; PubMed Scopus Google Scholar), a of the Aurora-2 activation as residues is residues and were clearly and have been in the final which is during activation of Aurora-2, to of the region and was in in of Aurora-2 by a of with sequence and structure of Aurora-2 and related protein kinases was used to as of the Aurora-2 that the N-terminal and C-terminal catalytic an expression residues was used for The of residues in the final crystal structure suggests that this is and in Aurora-2. of the Aurora-1 and Aurora-3 in this region that enzymes have a The of both the and truncated (residues 107–403) Aurora-2 proteins were to the is for catalytic purified which were first by with a was used to determined the K m values for as as the IC50 for adenosine. The proteins = = = suggesting that the N-terminal domain of Aurora-2 has on the catalytic of the This that the truncated enzyme used in this the structure of the ATP-binding site of Aurora-2. This is different from other kinases where protein domains or to the catalytic kinase domain the of in the catalytic active site or the of enzyme V. M. J. M. Struct. Biol. 2001; 8: PubMed Scopus Google Scholar, D. 1999; PubMed Scopus Google Scholar). It has been that the N-terminal domains of Aurora-1 and Aurora-2 may interactions with other which of kinases to the nucleus during mitosis S. M. V. M. Curr. Biol. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). We have shown that the C-terminal domain of Aurora-2 is to as an catalytic and this a distinct role for the N-terminal domain during the cell cycle. The ATP-binding of Aurora-2 is and has several that have been observed in other protein kinases. The and of adenosine to the region of the Aurora-2 active site through with the of residues and of and respectively. This of the region is structurally conserved in other kinases, and interactions with adenosine are very similar to seen between adenosine N. S. S. N. 1997; PubMed Scopus Google Scholar) or F. I. J. Nature. 1997; PubMed Scopus Google Scholar, W. Nature. 1997; PubMed Scopus Google Scholar, J. Biol. 1999; PubMed Scopus Google Scholar) and other protein kinases. The a of protein kinase inhibitors that this active site S. S. Adams 1998; Full Text Full Text PDF PubMed Scopus Google Scholar), is by residues and the the and and in Aurora-2. The of adenosine is between located in the of the active site and a by residues and the and of kinase F. I. J. Nature. 1997; PubMed Scopus Google Scholar, W. Nature. 1997; PubMed Scopus Google Scholar), Cyclin-dependent kinase 2 P.D. Struct. Biol. 1996; PubMed Scopus Google Scholar, Cell Biol. 1999; PubMed Scopus Google Scholar), glycogen synthase kinase E. Coll J.T. L. J. Struct. Biol. 2001; 8: PubMed Scopus Google Scholar), and Aurora-2 conserved ATP-binding of the active site of kinases with this crystal structure play an important role in designing selective A of the Aurora-2 active site is in with similar serine/threonine and kinases, Aurora-2 several unique that can be in the design of highly selective it an target for In the of the of are that could be for the design of new inhibitors that to Aurora-2 with affinity and over closely related kinases. The of adenosine a by the and the This contains several residues that are conserved in and to in Aurora-2 2 and have and residues at this respectively. in the size and to this and in a unique and a less at the to this the the of differences in and to Aurora-2 with and the of Aurora-2 and contain a single in which the size and of the at this This is to in Aurora-2, the at which the the C-terminal kinase and this a ATP-binding in the kinase The of this active site with the of that Aurora-2 adenosine interactions with the and of the It was possible to at 2.9 Å resolution. This to specific of the and in adenosine or observed for several other kinases. The residues and in cAMP-dependent protein kinase D. Engh R.A. Kinzel V. Ponstingl H. Huber R. EMBO J. 1993; 12: 849-859Crossref PubMed Scopus (375) Google Scholar, N. S. S. N. 1997; PubMed Scopus Google Scholar) and make a specific with the of or adenosine. In Aurora-1 and would be to make a with the of adenosine. In Aurora-2, to the of the C-terminal kinase by in a with residues and the Aurora the catalytic domain and the kinase activation are highly conserved as observed in other protein kinase J. F. D. Full Text Full Text PDF PubMed Scopus Google Scholar). of the Aurora family kinases in the activation region and with of other related kinases have identified a conserved in Aurora-2, as a potential phosphorylation site (2Giet R. Prigent C. J. Cell Sci. 1999; 112: 3591-3601Crossref PubMed Google Scholar). is in this crystal of in Aurora-2 to which a a kinase (7Bischoff J.R. Anderson L. Zhu Y. Mossie K., Ng, L. Souza B. Schryver B. Flanagan P. Clairvoyant F. Ginther C. Chan C.S.M. Novotny M. Slamon D.J. Plowman G.D. EMBO J. 1998; 17: 3052-3065Crossref PubMed Scopus (1115) Google Scholar), indicating that this is the target for an is located Å from the ATP-binding site of Aurora-2. of cAMP-dependent protein kinase with or adenosine and a peptide substrate are believed to the of protein kinases D. Engh R.A. Kinzel V. Ponstingl H. Huber R. EMBO J. 1993; 12: 849-859Crossref PubMed Scopus (375) Google Scholar, N. S. S. N. 1997; PubMed Scopus Google Scholar). located at the N-terminal end of the activation in Aurora-2, are clearly in this crystal structure and a unique conformation. In the cAMP-dependent kinase crystal and other kinase crystal the activation an of an or kinase was used for crystallization. In this Aurora-2 crystal residues and a into the active site to a The of to the of adenosine. homology with other kinases, the between the catalytic residues and in Aurora-2, which is seen in other kinase is observed 2 the N-terminal and C-terminal of the catalytic domain in Aurora-2 to to a of this enzyme that is This is in the of Aurora-2 (residues see that is in this crystal structure and a similar and to the in the active structure of cAMP-dependent the conserved in in Aurora-2, is for and make a with as observed for cAMP-dependent The of is by interactions with in the and is conserved in other similar interactions with the activation have been in Aurora-2 to in cAMP-dependent kinase and in These residues a different with to the is conserved in three human Aurora kinases and known (2Giet R. Prigent C. J. Cell Sci. 1999; 112: 3591-3601Crossref PubMed Google Scholar). A of the data J. W. D.J. Res. 1997; PubMed Scopus Google Scholar) other kinases with a located at this It is the of this upon phosphorylation of The catalytic domains of Aurora-1 and Aurora-3 share sequence homology with Aurora-2, and the ATP-binding site the three human Aurora kinases are In the of protein residues the bound adenosine which is in Aurora-2 and acid in both Aurora-1 and is In Aurora-2, the of a with the of This important role of in the structure of the C-terminal kinase suggests that it is a good target to for Aurora-1 and on this crystal structure and sequence of Aurora-1 and it is that the design of inhibitors that are selective for Aurora-2 be very It is from this crystal structure that the ATP-binding site are several that could be to for Aurora-2 over the closely related active site of and In by inhibitors that of the Aurora-2 active site it may be possible to the site at the of the activation and to The unique and interactions observed in this crystal structure facilitate the design of Aurora kinase inhibitors for the treatment of cancer. We for in designing Aurora-2 for data Fleming for of Aurora-2 enzyme by limited for in protein and for the Aurora-2 enzyme