Identification of the Substrates and Interaction Proteins of Aurora Kinases from a Protein-Protein Interaction Model
2003; Elsevier BV; Volume: 3; Issue: 1 Linguagem: Inglês
10.1074/mcp.m300072-mcp200
ISSN1535-9484
AutoresAn-Chi Tien, Ming-Hong Lin, Li Su, Yi‐Ren Hong, Tai-Shan Cheng, Yuan-Chii G. Lee, Wey‐Jinq Lin, Ivan H. Still, Chi‐Ying F. Huang,
Tópico(s)Cellular transport and secretion
ResumoThe increasing use of high-throughput and large-scale bioinformatics-based studies has generated a massive amount of data stored in a number of different databases. The major need now is to explore this disparate data to find biologically relevant interactions and pathways. Thus, in the post-genomic era, there is clearly a need for the development of algorithms that can accurately predict novel protein-protein interaction networks in silico. The evolutionarily conserved Aurora family kinases have been chosen as a model for the development of a method to identify novel biological networks by a comparison of human and various model organisms. Our search methodology was designed to predict and prioritize molecular targets for Aurora family kinases, so that only the most promising are subjected to empirical testing. Four potential Aurora substrates and/or interacting proteins, TACC3, survivin, Hec1, and hsNuf2, were identified and empirically validated. Together, these results justify the timely implementation of in silico biology in routine wet-lab studies and have also allowed the application of a new approach to the elucidation of protein function in the post-genomic era. The increasing use of high-throughput and large-scale bioinformatics-based studies has generated a massive amount of data stored in a number of different databases. The major need now is to explore this disparate data to find biologically relevant interactions and pathways. Thus, in the post-genomic era, there is clearly a need for the development of algorithms that can accurately predict novel protein-protein interaction networks in silico. The evolutionarily conserved Aurora family kinases have been chosen as a model for the development of a method to identify novel biological networks by a comparison of human and various model organisms. Our search methodology was designed to predict and prioritize molecular targets for Aurora family kinases, so that only the most promising are subjected to empirical testing. Four potential Aurora substrates and/or interacting proteins, TACC3, survivin, Hec1, and hsNuf2, were identified and empirically validated. Together, these results justify the timely implementation of in silico biology in routine wet-lab studies and have also allowed the application of a new approach to the elucidation of protein function in the post-genomic era. One possible path toward understanding the biological function of a target gene is through the discovery of how it interfaces with known protein-protein interaction networks. We are only now beginning to appreciate the nature and complexity of these networks, and construction of such a network using the traditional biochemical approaches still remains a significant challenge. Recently, the application of high-throughput technologies, such as large-scale yeast two-hybrid analysis, has generated an enormous amount of data (1Ito T. Chiba T. Ozawa R. Yoshida M. Hattori M. Sakaki Y. A comprehensive two-hybrid analysis to explore the yeast protein interactome..Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 4569-4574Google Scholar, 2Newman J.R. Wolf E. Kim P.S. A computationally directed screen identifying interacting coiled coils from Saccharomyces cerevisiae..Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 13203-13208Google Scholar, 3Uetz P. Giot L. Cagney G. Mansfield T.A. Judson R.S. Knight J.R. Lockshon D. Narayan V. Srinivasan M. Pochart P. Qureshi-Emili A. Li Y. Godwin B. Conover D. Kalbfleisch T. Vijayadamodar G. Yang M. Johnston M. Fields S. Rothberg J.M. A comprehensive analysis of protein-protein interactions in Saccharomyces cerevisiae..Nature. 2000; 403: 623-627Google Scholar, 4Fromont-Racine M. Mayes A.E. Brunet-Simon A. Rain J.C. Colley A. Dix I. Decourty L. Joly N. Ricard F. Beggs J.D. Legrain P. Genome-wide protein interaction screens reveal functional networks involving Sm-like proteins..Yeast. 2000; 17: 95-110Google Scholar). This has led researchers to often face the dilemma of how to effectively utilize the vast information gathered through these large-scale studies. Investigators relying solely on a traditional wet-lab approach for making decisions or setting research priorities are likely to find themselves outpaced by peers who combine in silico biology with empirical methods. Thus, there is clearly a need to develop a systematic and stepwise approach that can predict or prioritize potential targets in silico to aid in a greater understanding of how complex biological systems work. Given the advantages provided by an in silico approach, it seems reasonable to propose that it will become an essential tool for initially evaluating novel hypotheses and will offer an improved rationale for target prioritization, which will in theory result in only the most promising targets needing to be subjected to empirical testing. The goal of this study, therefore, was to create a virtual protein-protein interaction model using the concepts that protein-protein interactions require precise spatial proximity (compartmentalization) and temporal synchronicity (cell-cycle stage).Given the availability of information for different model organisms, the evolutionarily conserved family of Aurora family kinases was selected as a model to identify novel biological networks from yeast to humans. Aurora, a family of mitotic serine/threonine kinases, has been conserved throughout evolution, as reflected by the presence of their homologues in a variety of model organisms, including Saccharomyces cerevisiae, Caenorhabditis elegans, Drosophila, Xenopus, mouse, and human. The Aurora family consists of one member in S. cerevisiae (Ipl1); two in C. elegans (AIR-1 and AIR-2); two in Drosophila (Aurora-A and IAL), and three members in humans (Aurora-A, Aurora-B, and Aurora-C; reviewed in Refs. 5Giet R. Prigent C. Aurora/Ipl1p-related kinases, a new oncogenic family of mitotic serine-threonine kinases..J. Cell Sci. 1999; 112: 3591-3601Google Scholar, 6Bischoff J.R. Plowman G.D. The Aurora/Ipl1p kinase family: Regulators of chromosome segregation and cytokinesis..Trends Cell Biol. 1999; 9: 454-459Google Scholar, 7Nigg E.A. Mitotic kinases as regulators of cell division and its checkpoints..Nat. Rev. Mol. Cell. Biol. 2001; 2: 21-32Google Scholar). In yeast, Ipl1 is normally localized to the spindle pole body (8Kim J.H. Kang J.S. Chan C.S. Sli15 associates with the ipl1 protein kinase to promote proper chromosome segregation in Saccharomyces cerevisiae..J. Cell Biol. 1999; 145: 1381-1394Google Scholar), and, during mitosis, it is mainly associated with the kinetochore and the mitotic spindles (9He X. Rines D.R. Espelin C.W. Sorger P.K. Molecular analysis of kinetochore-microtubule attachment in budding yeast..Cell. 2001; 106: 195-206Google Scholar). In C. elegans, AIR-1 is localized at the centrosomes (10Schumacher J.M. Ashcroft N. Donovan P.J. Golden A. A highly conserved centrosomal kinase, AIR-1, is required for accurate cell cycle progression and segregation of developmental factors in Caenorhabditis elegans embryos..Development. 1998; 125: 4391-4402Google Scholar), while AIR-2 is localized at the kinetochore and midbody (11Schumacher J.M. Golden A. Donovan P.J. AIR-2: An Aurora/Ipl1-related protein kinase associated with chromosomes and midbody microtubules is required for polar body extrusion and cytokinesis in Caenorhabditis elegans embryos..J. Cell Biol. 1998; 143: 1635-1646Google Scholar). In Drosophila, only the centrosome-associated Aurora-A has been thoroughly studied (12Glover D.M. Leibowitz M.H. McLean D.A. Parry H. Mutations in aurora prevent centrosome separation leading to the formation of monopolar spindles..Cell. 1995; 81: 95-105Google Scholar). In humans, Aurora-A is localized to the centrosome in prophase, subsequently spreading to the mitotic spindles/centrosomes, where it remains until the end of mitosis (13Kimura M. Kotani S. Hattori T. Sumi N. Yoshioka T. Todokoro K. Okano Y. Cell cycle-dependent expression and spindle pole localization of a novel human protein kinase, Aik, related to Aurora of Drosophila and yeast Ipl1..J. Biol. Chem. 1997; 272: 13766-13771Google Scholar). In contrast, Aurora-B is found at the kinetochore, mainly at the midzone, during anaphase. Aurora-C is localized to the centrosomes from anaphase to cytokinesis (14Kimura M. Matsuda Y. Yoshioka T. Okano Y. Cell cycle-dependent expression and centrosome localization of a third human aurora/Ipl1-related protein kinase, AIK3..J. Biol. Chem. 1999; 274: 7334-7340Google Scholar). Taken together, this information indicates that all Aurora variants from different species are localized to the mitotic apparatus.Several potential substrates and interacting proteins of the Aurora variants have been identified for the different model organisms. In yeast, it has been demonstrated that Ndc10 (15Biggins S. Severin F.F. Bhalla N. Sassoon I. Hyman A.A. Murray A.W. The conserved protein kinase Ipl1 regulates microtubule binding to kinetochores in budding yeast..Genes Dev. 1999; 13: 532-544Google Scholar), Sli15, Dam1 (kinetochore proteins) (16Cheeseman I.M. Drubin D.G. Barnes G. Simple centromere, complex kinetochore: Linking spindle microtubules and centromeric DNA in budding yeast..J. Cell Biol. 2002; 157: 199-203Google Scholar), Cin8 (a kinesin protein) (9He X. Rines D.R. Espelin C.W. Sorger P.K. Molecular analysis of kinetochore-microtubule attachment in budding yeast..Cell. 2001; 106: 195-206Google Scholar), and Histone H3 (17Hsu J.Y. Sun Z.W. Li X. Reuben M. Tatchell K. Bishop D.K. Grushcow J.M. Brame C.J. Caldwell J.A. Hunt D.F. Lin R. Smith M.M. Allis C.D. Mitotic phosphorylation of histone H3 is governed by Ipl1/aurora kinase and Glc7/PP1 phosphatase in budding yeast and nematodes..Cell. 2000; 102: 279-291Google Scholar) serve as substrates of Ipl1, thus implying a regulatory role for Ipl1 in kinetochore-microtubule attachment. Sli15 physically interacts with Ipl1 via an INCENP box in its C-terminal region (18Uren A.G. Wong L. Pakusch M. Fowler K.J. Burrows F.J. Vaux D.L. Choo K.H. Survivin and the inner centromere protein INCENP show similar cell-cycle localization and gene knockout phenotype..Curr. Biol. 2000; 10: 1319-1328Google Scholar, 19Tanaka T.U. Rachidi N. Janke C. Pereira G. Galova M. Schiebel E. Stark M.J. Nasmyth K. Evidence that the Ipl1-Sli15 (Aurora kinase-INCENP) complex promotes chromosome bi-orientation by altering kinetochore-spindle pole connections..Cell. 2002; 108: 317-329Google Scholar). It has been suggested that this complex possibly regulates bi-orientation of chromosomes during mitosis. In C. elegans, AIR-1 is required for centrosome maturation and the proper localization of centrosomal proteins, such as CeGrip and ZYG-9 (20Hannak E. Kirkham M. Hyman A.A. Oegema K. Aurora-A kinase is required for centrosome maturation in Caenorhabditis elegans..J. Cell Biol. 2001; 155: 1109-1116Google Scholar). In Drosophila, Aurora-A is able to phosphorylate and interact with the centrosomal protein D-TACC (21Giet R. McLean D. Descamps S. Lee M.J. Raff J.W. Prigent C. Glover D.M. Drosophila Aurora A kinase is required to localize D-TACC to centrosomes and to regulate astral microtubules..J. Cell Biol. 2002; 156: 437-451Google Scholar). In the frog, the Aurora-A homologue Eg2 (22Littlepage L.E. Wu H. Andresson T. Deanehan J.K. Amundadottir L.T. Ruderman J.V. Identification of phosphorylated residues that affect the activity of the mitotic kinase Aurora-A..Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 15440-15445Google Scholar) can phosphorylate a motor protein, Eg5 (23Giet R. Uzbekov R. Cubizolles F. Le Guellec K. Prigent C. The Xenopus laevis aurora-related protein kinase pEg2 associates with and phosphorylates the kinesin-related protein XlEg5..J. Biol. Chem. 1999; 274: 15005-15013Google Scholar). Survivin, which has been implicated in both the control of cell division and the inhibition of apoptosis, associates biochemically with Aurora-A (24Gigoux V. L'Hoste S. Raynaud F. Camonis J. Garbay C. Identification of Aurora kinases as RasGAP Src homology 3 domain-binding proteins..J. Biol. Chem. 2002; 277: 23742-23746Google Scholar) and Aurora-B (24Gigoux V. L'Hoste S. Raynaud F. Camonis J. Garbay C. Identification of Aurora kinases as RasGAP Src homology 3 domain-binding proteins..J. Biol. Chem. 2002; 277: 23742-23746Google Scholar, 25Wheatley S.P. Carvalho A. Vagnarelli P. Earnshaw W.C. INCENP is required for proper targeting of Survivin to the centromeres and the anaphase spindle during mitosis..Curr. Biol. 2001; 11: 886-890Google Scholar). Thus, based upon these known interactions, the Aurora family members are capable of executing a wide range of biological functions in cells, and the in silico quest for potential substrates and Aurora's protein interaction networks may represent the first concerted step toward understanding the molecular basis of the regulation of Aurora family kinases.The first stage of this study involved assessment of computational tools for the access of public bioinformatics resources and the translation of raw large-scale high-throughput-based information to create the possible protein-protein interaction network for the Aurora family kinases. This involved a stepwise combination of extensive literature searches, database analyses of various yeast protein-protein interactions, subcellular localization, homology searches, and analysis of expression databases, resulting in the identification of four potential substrates and/or interacting proteins for the human Aurora variants. Second, these prioritized targets were subjected to traditional empirical wet-lab approaches to validate the predicted biochemical interactions, resulting not only in the confirmation of the findings reported earlier (21Giet R. McLean D. Descamps S. Lee M.J. Raff J.W. Prigent C. Glover D.M. Drosophila Aurora A kinase is required to localize D-TACC to centrosomes and to regulate astral microtubules..J. Cell Biol. 2002; 156: 437-451Google Scholar, 24Gigoux V. L'Hoste S. Raynaud F. Camonis J. Garbay C. Identification of Aurora kinases as RasGAP Src homology 3 domain-binding proteins..J. Biol. Chem. 2002; 277: 23742-23746Google Scholar), but also the elucidation of novel, previously unrecorded biochemical interactions.In summary, building such a protein-protein interaction search platform not only makes accessible a new approach to discovery of Aurora function, but also highlights the potential extrapolations of similar analysis for other as yet poorly characterized proteins in the post-genomic era.EXPERIMENTAL PROCEDURESConstruction of the Protein-Protein Interaction Network—Identification of the protein-protein interactions used the keyword search function provided at dip.doe-mbi.ucla.edu, with the interactions further classified into two categories defined by a requirement for small-scale experiments (orange line) or high-throughput analogs (blue dashed line) as illustrated in Fig. 2. Proteins without assigned names or without characterized functions as listed at www.stanford.edu/Saccharomyces were excluded using this search. Protein annotation is also provided at the above web site and also in Gene Ontology annotation (www.geneontology.org), permitting selection of proteins that fit the inclusion criteria (keywords: spindle pole and kinetochore). The following web site, www.proteome.com/databases/YPD/, provides an excellent search engine; however, this web site is not publicly accessible.Finding Homologues for the Different Model Organisms—The proteins in the map (see Fig. 2A) were further analyzed using the National Center for Biotechnology Information BLAST software (version 2.0) and the BLAST search provided at www.wormbase.org or flybase.bio.indiana.edu. The selection of some homologues was based on a search of the literature. Protein sequences were all translated from their sequences in the GenBank database. Homologues were aligned using the CLUSTAL W program.Cell Culture, Transfection, Co-immunoprecipitation, and Kinase Assay—Human 293T cells from the American Type Culture Collection (Manassas, VA) were maintained at 37 °C in a 5% CO2 incubator, and grown in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated fetal bovine serum and 100 μg/ml penicillin/streptomycin (Life Technologies, Grand Island, NY). Ectopic expression of Flag-tagged Aurora-A, -B, and -C, hemagglutinin (HA) 1The abbreviations used are: HA, hemagglutinin; GFP, green fluorescence protein; GST, glutathione S-transferase. -tagged Hec1 and hsNuf2, green fluorescence protein (GFP)-tagged TACC1, TACC2, and TACC3 in 293T cells was performed with LipofectAMINETM, according to the manufacturer's instructions. Forty-eight hours after transfection, cells were lysed with RIPA buffer (150 mm NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 1% Nonidet P-40, 50 mm Tris, pH 8.0). Equal amounts of total lysates (500 μg) were immunoprecipitated with 2 μg of anti-Flag (Sigma, St. Louis, MO), anti-HA (3F10; Roche Molecular Biochemicals, Indianapolis, IN) or anti-GFP (Roche Molecular Biochemicals) monoclonal antibody at 4 °C for 2–4 h. Protein A agarose beads (Upstate Biotechnology, Lake Placid, NY) were added and incubated for another 2 h at 4 °C. Beads were washed three times with buffer containing 10 mm HEPES, pH 7.0, 2 mm MgCl2, 50 mm NaCl, 5 mm EGTA, 0.1% Triton X-100, and 60 mm 2-glycerophosphate and twice with TBS buffer (38.5 mm Tris, pH 7.4, and 150 mm NaCl), respectively. Immune complexes were either subjected to SDS-PAGE or resuspended in kinase reaction buffer (25 mm Tris, pH 7.4, 10 mm MgCl2, 10% glycerol, 1 mm dithiothreitol, 1 mm Na3VO4, 1 mm NaF, 20 μm ATP, and 10 μCi [γ-32P]ATP) with 1 μg of purified recombinant glutathione S-transferase (GST)-Aurora-A, GST-Aurora-B, or His-Aurora-C at 30 °C for 30 min. Alternatively, 1 μg of GST-Hec1 and GST-hsNuf2 was incubated with 1 μg of each recombinant Aurora family kinase to perform the kinase reaction at 30 °C for 10 min. The kinase reaction mixtures or cell lysates were resuspended in SDS sample buffer and separated using 12.5 or 8% SDS-polyacrylamide gels. Proteins were transferred to polyvinylidene difluoride membranes and detected using autoradiography or probed with 1:1000 dilution of anti-GFP, anti-Flag, or anti-HA antibody. The complexed IgGs were detected by incubation with secondary antibodies conjugated to horseradish peroxidase, and developed using the ECL system (Amersham Pharmacia Biotech, Piscataway, NJ).Yeast Two-Hybrid Analysis—Standard techniques were used for the yeast two-hybrid system (26Chien C.T. Bartel P.L. Sternglanz R. Fields S. The two-hybrid system: A method to identify and clone genes for proteins that interact with a protein of interest..Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 9578-9582Google Scholar, 27Fields S. Song O. A novel genetic system to detect protein-protein interactions..Nature. 1989; 340: 245-246Google Scholar, 28Zhu L. Yeast GAL4 two-hybrid system. A genetic system to identify proteins that interact with a target protein..Methods Mol. Biol. 1997; 63: 173-196Google Scholar). Briefly, each Aurora family gene and predicted candidate genes were cloned in frame with the GAL4 DNA binding domain (GAL4 BD) in the pGBT-9 vector or fused to the GAL4 activation domain (GAL4 AD) in the plasmid pACT-2 (MARCHMAKER Two-Hybrid System; Clontech, Palo Alto, CA). The yeast strain Y190 was cotransformed with GAL4 BD and GAL4 AD. Positive clones were able to grow on Trp, Leu, and His dropout media supplemented with 3-aminotriazole (and an inhibitor of HIS3) and turn blue during the β-galactosidase filter assay. To determine β-galactosidase activity, we have adopted the procedure previously reported (29Harshman K.D. Moye-Rowley W.S. Parker C.S. Transcriptional activation by the SV40 AP-1 recognition element in yeast is mediated by a factor similar to AP-1 that is distinct from GCN4..Cell. 1988; 53: 321-330Google Scholar). Briefly, overnight-cultured yeast cells, adjusted to the same optical density, were collected and resuspended in 100 ml Tris, pH 7.4, and 0.05% Triton X-100. The resuspended cells were repeatedly frozen and thawed for five times at −80 °C followed by addition of Z-buffer (150 mm phosphate buffer, pH 7, 10 mm KCl, and 1 mm MgSO4) with O-nitrophenyl-β-d-galactopyranoside as the substrate (4 mg/ml final concentration). The reactions were carried out at 30 °C and stopped by addition of 250 mm sodium carbonate. The enzyme activity was determined by measuring the absorbance at a wavelength of 420 nm.RESULTSConstructing a Protein-Protein Interaction Network Centered on the Aurora Yeast Homologue Ipl1—It is becoming increasingly apparent that protein interaction networks are extremely complex. Therefore, there is an increasing need to quickly elucidate where new, uncharacterized proteins interface with these networks. As a step toward developing such networks, we investigated the applicability of a bioinformatics system centered on the interactions of the Aurora kinase family. To facilitate a predictive understanding of the interactive biology of the human Aurora family kinases with respect to the various model organisms, the information generated from large-scale yeast protein-protein interaction databases and microarray studies was accessed to recreate protein-protein interaction networks centered on Ipl1, the yeast homologue of the Aurora kinases. This proposed interaction network model was based upon the notion that some of the interacting proteins and/or substrates for the human Aurora family kinases may also be evolutionarily conserved, interacting with each other in the same temporal and spatial configurations. Construction of this model involved five bioinformatics steps, and the predicted interactions then verified empirically.Step 1. Identification of Ipl1 Interacting Proteins from Published Small-scale Experimental Studies—The first step in the generation of the interaction model involved an extensive search of the literature to acquire the currently published experimental data for interaction networks centered on Ipl1. The keyword used in the literature searches was Ipl1. However, a functional network is not only limited to physical protein-protein interactions but also includes genetic and biochemical interactions. Thus, we combined all available genetic, biochemical, and physical interaction data centered on Ipl1. This data is summarized in Fig. 1 , which shows that Ipl1 physically (the orange line) and genetically (8Kim J.H. Kang J.S. Chan C.S. Sli15 associates with the ipl1 protein kinase to promote proper chromosome segregation in Saccharomyces cerevisiae..J. Cell Biol. 1999; 145: 1381-1394Google Scholar) interacts with Sli15 (19Tanaka T.U. Rachidi N. Janke C. Pereira G. Galova M. Schiebel E. Stark M.J. Nasmyth K. Evidence that the Ipl1-Sli15 (Aurora kinase-INCENP) complex promotes chromosome bi-orientation by altering kinetochore-spindle pole connections..Cell. 2002; 108: 317-329Google Scholar) and Dam1 (30Cheeseman I.M. Enquist-Newman M. Muller-Reichert T. Drubin D.G. Barnes G. Mitotic spindle integrity and kinetochore function linked by the Duo1p/Dam1p complex..J. Cell Biol. 2001; 152: 197-212Google Scholar) and phosphorylates Sli15 (30Cheeseman I.M. Enquist-Newman M. Muller-Reichert T. Drubin D.G. Barnes G. Mitotic spindle integrity and kinetochore function linked by the Duo1p/Dam1p complex..J. Cell Biol. 2001; 152: 197-212Google Scholar), Ndc10 (15Biggins S. Severin F.F. Bhalla N. Sassoon I. Hyman A.A. Murray A.W. The conserved protein kinase Ipl1 regulates microtubule binding to kinetochores in budding yeast..Genes Dev. 1999; 13: 532-544Google Scholar), Dam1 (30Cheeseman I.M. Enquist-Newman M. Muller-Reichert T. Drubin D.G. Barnes G. Mitotic spindle integrity and kinetochore function linked by the Duo1p/Dam1p complex..J. Cell Biol. 2001; 152: 197-212Google Scholar), and Cin8 (9He X. Rines D.R. Espelin C.W. Sorger P.K. Molecular analysis of kinetochore-microtubule attachment in budding yeast..Cell. 2001; 106: 195-206Google Scholar) (the pink line). Moreover, processes that are reversibly controlled by protein phosphorylation require not only a protein kinase, but also a protein phosphatase. Therefore, the biochemical interactions of the phosphatase (Glc7) and Ndc10 (31Sassoon I. Severin F.F. Andrews P.D. Taba M.R. Kaplan K.B. Ashford A.J. Stark M.J. Sorger P.K. Hyman A.A. Regulation of Saccharomyces cerevisiae kinetochores by the type 1 phosphatase Glc7p..Genes Dev. 1999; 13: 545-555Google Scholar) (black line) were also included in our model.Fig. 1The biochemical and physical interaction networks for the yeast protein Ipl1, a homologue of the human Aurora serine/threonine family kinases. This figure is a summary of the results of the literature search for Ipl1 substrates and cooperators, which are the basis of the protein-protein interaction networks. Ipl1 phosphorylates Sli15, Cin8, Ndc10, and Dam1 (pink lines with balls labeled P, which stands for phosphorylation). Glc7 serves as a phosphatase counteracting Ipl1 function and dephosphorylates Ndc10 (black line). Physical interactions between two proteins are illustrated by orange lines.View Large Image Figure ViewerDownload (PPT)Step 2. Establishment of the Protein-Protein Interaction Network by Analysis of Public Accessible Databases—Ipl1 interacting molecules, as summarized in Fig. 1, were used as templates for reconstructing the interaction networks by accessing the different yeast protein-protein interaction databases (data collections at dip.doe-mbi.ucla.edu and portal.curagen.com). These include small-scale (orange line) and high-throughput (blue dashed line) yeast two-hybrid experiments. To date, there are four different comprehensive large-scale yeast protein-protein interaction databases, which, despite discrepancies among the different reports (32Deane C.M. Salwinski L. Xenarios I. Eisenberg D. Protein interactions: Two methods for assessment of the reliability of high throughput observations..Mol. Cell. Proteomics. 2002; 1: 349-356Google Scholar), have provided ∼6000 uncharacterized interactions (1Ito T. Chiba T. Ozawa R. Yoshida M. Hattori M. Sakaki Y. A comprehensive two-hybrid analysis to explore the yeast protein interactome..Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 4569-4574Google Scholar, 2Newman J.R. Wolf E. Kim P.S. A computationally directed screen identifying interacting coiled coils from Saccharomyces cerevisiae..Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 13203-13208Google Scholar, 3Uetz P. Giot L. Cagney G. Mansfield T.A. Judson R.S. Knight J.R. Lockshon D. Narayan V. Srinivasan M. Pochart P. Qureshi-Emili A. Li Y. Godwin B. Conover D. Kalbfleisch T. Vijayadamodar G. Yang M. Johnston M. Fields S. Rothberg J.M. A comprehensive analysis of protein-protein interactions in Saccharomyces cerevisiae..Nature. 2000; 403: 623-627Google Scholar, 4Fromont-Racine M. Mayes A.E. Brunet-Simon A. Rain J.C. Colley A. Dix I. Decourty L. Joly N. Ricard F. Beggs J.D. Legrain P. Genome-wide protein interaction screens reveal functional networks involving Sm-like proteins..Yeast. 2000; 17: 95-110Google Scholar). To broadly cover potential candidates in our study, we have included three steps of radial network expansion with Ipl1 at the center (Fig. 2 A). The search results showed that there are 192 known yeast proteins in this protein-protein interaction radial network (partially illustrated in Fig. 2A).Step 3. Protein-Protein Interaction in the Proper Spatial Configuration—Because intracellular events may be compartmentalized to unique intracellular locations, to provide additional specificity for target selection we also included a spatial component to further refine the construction of the model. Because the Aurora variants are localized to the mitotic apparatus in organisms ranging from yeast (9He X. Rines D.R. Espelin C.W. Sorger P.K. Molecular analysis of kinetochore-microtubule attachment in budding yeast..Cell. 2001; 106: 195-206Google Scholar) to human (7Nigg E.A. Mitotic kinases as regulators of cell division and its checkpoints..Nat. Rev. Mol. Cell. Biol. 2001; 2: 21-32Google Scholar), association with the mitotic spindle or kinetochore became a prerequisite for the selection of potential Ipl1 interacting proteins. Therefore, we further prioritized our target selections by using two spatial keywords (spindle pole and kinetochore) and searched for the information available at genome-www.stanford.edu/Saccharomyces and www.proteome.com/databases/YPD/. Thirty-three out of the 192 proteins (∼20%) were left in the map (balls containing protein names; Fig. 2A). Of these 33 proteins, four complexes were identified: the inner kinetochore (CBF3 complex), the central kinetochore (Ndc80 complex), the outer kinetochore (Dam1 complex; for review see Ref. 16Cheeseman I.M. Drubin D.G. Barnes G. Simple centromere, complex kinetochore: Linking spindle microtubules and centromeric DNA in budding yeast..J. Cell Biol. 2002; 157: 199-203Google Scholar) (circled by green dashed line; Fig. 2A), and the spindle-pole (γ-tubulin complex; Ref. 33Knop M. Pereira G. Geissler S. Grein K. Schiebel E. The spindle pole body component Spc97p interacts with the gamma-tubulin of Saccharomyces cerevisiae and functions in microtubule organization and spindle pole body duplication..EMBO J. 1997; 16: 1550-1564Google Scholar) (circled by red dashed line; Fig. 2A). This analysis raises the possibility that Ipl1 may regulate these complexes as a whole rather than regulate one or more of the individual components.Step 4. Conversion from Yeast Interaction Networks to Human Homologues—To establish the interaction networks for the mammalian system, selected candidates were subsequently converted into higher-organism proteins based on their sequence and their functional homologies in different model organisms such as C. elegans (www.wormbase
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