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• W2018669255 abstract "To evaluate yeast as a high-throughput cell-based system for screening chemicals that may lead to drug development, 10,302 full-length human cDNAs (~50% of the total cDNAs) were introduced into yeast. Approximately 5.6% (583 clones) of the cDNAs repressed the growth of yeast. Notably, ~25% of the repressive cDNAs encoded uncharacterized proteins. Small chemicals can be readily surveyed by monitoring their restorative effects on the growth of yeast. The authors focused on protein kinases because protein kinases are involved in various diseases. Among 263 protein kinase cDNAs (~50% of the total) expressed in yeast, 60 cDNAs (~23%), including c-Yes, a member of the Src tyrosine kinase family, inhibited the growth of yeast. Known inhibitors for protein kinases were examined for whether they reversed the c-Yes-induced inhibition of the yeast growth. Among 85 inhibitors tested, 6 compounds (PP2, PP1, SU6656, purvalanol, radicicol, and geldanamycin) reversed the inhibition, indicating a high specificity sufficient for validating this screening system. Human c-Yes was found to interact with Hsc82, one of the yeast chaperones. Radicicol and geldanamycin probably exerted their actions through interactions with Hsc82. These results indicate that when human proteins requiring molecular chaperones for their activities are subjected to the yeast screening system, 2 groups of chemicals may be found. The actions of one group are exerted through direct interactions with the human proteins, whereas those of the other group are mediated through interactions with chaperones. To evaluate yeast as a high-throughput cell-based system for screening chemicals that may lead to drug development, 10,302 full-length human cDNAs (~50% of the total cDNAs) were introduced into yeast. Approximately 5.6% (583 clones) of the cDNAs repressed the growth of yeast. Notably, ~25% of the repressive cDNAs encoded uncharacterized proteins. Small chemicals can be readily surveyed by monitoring their restorative effects on the growth of yeast. The authors focused on protein kinases because protein kinases are involved in various diseases. Among 263 protein kinase cDNAs (~50% of the total) expressed in yeast, 60 cDNAs (~23%), including c-Yes, a member of the Src tyrosine kinase family, inhibited the growth of yeast. Known inhibitors for protein kinases were examined for whether they reversed the c-Yes-induced inhibition of the yeast growth. Among 85 inhibitors tested, 6 compounds (PP2, PP1, SU6656, purvalanol, radicicol, and geldanamycin) reversed the inhibition, indicating a high specificity sufficient for validating this screening system. Human c-Yes was found to interact with Hsc82, one of the yeast chaperones. Radicicol and geldanamycin probably exerted their actions through interactions with Hsc82. These results indicate that when human proteins requiring molecular chaperones for their activities are subjected to the yeast screening system, 2 groups of chemicals may be found. The actions of one group are exerted through direct interactions with the human proteins, whereas those of the other group are mediated through interactions with chaperones. As a model organism for eukaryotes, yeast has enormous advantages such as simple growth requirements, rapid growth, gene manipulation with great ease, and plenty of experimental tools for investigating the biological functions of target proteins. Because of the high degree of conservation between yeast and mammalian cells in fundamental biological processes such as the cell cycle and major metabolic pathways, yeast has greatly facilitated our understanding of the biological functions of mammalian proteins. In addition, these properties of yeast have drawn much attention from scientists intending to establish high-throughput screening (HTS) systems for small compounds that may lead to the development of drugs for diseases, including cancer.1Simon JA Bedalov A Yeast as a model system for anticancer drug discovery.Nat Rev Cancer. 2004; 4: 481-492Crossref PubMed Scopus (88) Google Scholar, 2Gunde T Barberis A Yeast growth selection system for detecting activity and inhibition of dimerization-dependent receptor tyrosine kinase.Biotechniques. 2005; 39: 541-549Crossref Scopus (19) Google Scholar, 3Ivey FD Wang L Demirbas D Allain C Hoffman CS Development of a fission yeast-based high-throughput screen to identify chemical regulators of cAMP phosphodiesterases.J Biomol Screen. 2008; 13: 62-71Crossref PubMed Scopus (33) Google Scholar To use yeast as a cell-based screening system, exploration of the yeast phenotypic changes induced by the expression of human proteins and surveys of compounds that can reverse these changes are obligatory requirements. Among the possible phenotypic changes, interference with yeast growth would be the most convenient for HTS systems because a huge number of compounds from diversified libraries and natural sources such as culture broths of microorganisms can be screened by simply detecting the restoration of yeast growth. The feasibility of such a yeast cell-based screening system has been tested using cDNAs from mainly human and some viral origins.4Tugendreich S Perkins E Couto J Barthmaier P Sun D Tang S et al.A streamlined processes to phenotypically profile heterologus cDNAs in parallel using yeast cell-based assays.Genome Res. 2001; 11: 1899-1912Crossref PubMed Scopus (25) Google Scholar Among 38 cDNAs tested, 12 cDNAs (30%), including those of the mitotic spindle checkpoint kinase BUB1, p38MAPK, aurora-related kinase 1, poly(ADP-ribose) polymerase 1 (PARP1), and PI3 kinase, showed significant interference with the yeast growth. PARP1 is a DNA binding protein that detects DNA strand breaks and also catalyzes adenosine diphosphate (ADP) ribosylation of various proteins. Novel inhibitors of this enzyme have been identified using a yeast strain expressing human PARP1.5Perkins E Sun D Nguyen A Tulac S Francesco M Tavana H et al.Novel inhibitors of poly(ADP-ribose) polymerase/PARP1 and PARP2 identified using a cell-based screen in yeast.Cancer Res. 2001; 61: 4175-4183PubMed Google Scholar Its activity has been implicated in diseases such as cancer, stroke, and neurotrauma, and potent inhibitors of this enzyme are now being evaluated clinically.6Komjáti K Besson VC Szabó C Poly (ADP-ribose) polymerase inhibitors as potential therapeutic agents in stroke and neurotrauma.Curr Drug Targets CNS Neurol Disord. 2005; 4: 179-194Crossref PubMed Scopus (48) Google Scholar, 7Horvath EM Szabó C Poly(ADP-ribose) polymerase as a drug target for cardiovascular disease and cancer: an update.Drug News Perspect. 2007; 20: 171-181Crossref PubMed Scopus (43) Google Scholar, 8Peralta-Leal A Rodoriguez MI Oliver FJ Poly(ADP-ribose)polymerase-1 (PARP-1) in carcinogenesis: potential role of PARP inhibitors in cancer treatment.Clin Trans Oncol. 2008; 10: 318-323Crossref PubMed Scopus (53) Google Scholar Interestingly, PARP activity is not present in yeast, indicating that yeast-based phenotypic screens can even be successful for human proteins whose homologues are not present in yeast. The completion of the sequencing of the human genome and the progress of the human cDNA projects have made it possible for most, if not all, human cDNAs to be expressed in yeast. Given that a large number of proteins probably remain to be assigned as causes of diseases, more comprehensive screening with a yeast cell-based strategy may cover a far broader range of lead compounds. Furthermore, even if they are not druggable, it is also possible that analyses of the biological alterations induced by novel compounds in human cells might reveal unexpected protein functions. In view of these points, we have designed a research project, designated the “humanized yeast project,” in which comprehensive screening of human cDNAs that inhibit yeast growth is planned. In the present study, we expressed 10,302 human cDNAs (~50% of the total cDNAs) in a budding yeast (Saccharomyces cerevisiae) and found that approximately 5.6% of the cDNAs, including those for protein kinases, interfered with the yeast growth. Protein kinases are the largest enzyme superfamily involved in cell signal transduction. The human genome contains more than 500 genes encoding protein kinases,9Manning G Whyte DB Martinez R Hunter T Sudarsanam S The protein kinase complement of the human genome.Science. 2002; 298: 1912-1934Crossref PubMed Scopus (6214) Google Scholar and at least 30% of human proteins are phosphorylated by protein kinases,10Cohen P The role of protein phosphorylation in human health and disease.Eur J Biochem. 2001; 268: 5001-5010Crossref PubMed Scopus (495) Google Scholar suggesting that protein kinases play crucial roles in human physiology and pathophysiology. Consequently, protein kinases represent therapeutic targets for a range of diseases. Indeed, many successful developments of protein kinase inhibitors and HTS technologies for drug discovery against the human kinome have been reviewed recently.11Eglen RM Reisine T The current status of drug discovery against the human kinome.Assay Drug Dev Technol. 2009; 7: 22-43Crossref PubMed Scopus (69) Google Scholar This article reports the feasibility of a yeast cell-based method for surveying inhibitors of human protein kinases. This strategy was validated using human c-Yes, a tyrosine kinase, and known inhibitors of c-Yes. Budding yeast contains more than 100 known serine/threonine kinases, but no typical tyrosine kinases have been found to date.12Hunter T Plowman GD The protein kinases of budding yeast: six score and more.Trends Biochem Sci. 1997; 22: 18-22Abstract Full Text PDF PubMed Scopus (403) Google Scholar To screen for human cDNAs that repressed yeast growth, an S. cerevisiae (budding yeast) wild-type strain, W303-1A (MATa leu2-3,112 trp1-1 can1-100 ura3-1 ade2-1 his3-11,15), or BY4742 (MATα ura3Δ0 leu2Δ0 his3Δ1 lys2Δ0) was used. For the experiments involving protein kinase inhibitors, a triple-mutant yeast strain deficient in ERG4, PDR1, and PDR3, which are involved in membrane permeability and the efflux of xenobiotics, was generated from the W303-1B strain (MATα leu2-3,112 trp1-1 can1-100 ura3-1 ade2-1 his3-11,15) (Mukai et al., manuscript in preparation). To generate a triple-mutant yeast strain expressing C-terminally HA-tagged Hsp82 (Hsp82-HA) or Hsc82-HA, PCR-based HA tagging was performed according to a previously described method.13Longtine MS McKenzie III, A Demarini DJ Shah NG Wach A Brachat A et al.Additional modules for versatile and economical PCR-based gene deletion and modification in Saccharomyces cerevisiae.Yeast. 1998; 14: 953-961Crossref PubMed Scopus (4160) Google Scholar In these cells, endogenous HSP82 and HSC82 were replaced with HSP82-HA and HSC82-HA, respectively. Three types of vector plasmids, comprising a multicopy YEp-type plasmid (pYES-DEST52; Invitrogen, Carlsbad, CA), a single-copy YCp-type plasmid, and an integration YIp-type plasmid, were used for expressing human proteins in yeast. The human cDNAs were transferred from the entry clones14Goshima N Kawamura Y Fukumoto A Miura A Honma R Satoh R et al.Human protein factory for converting the transcriptome into an in vitro–expressed proteome.Nat Methods. 2008; 5: 1011-1017Crossref PubMed Scopus (211) Google Scholar to pYES-DEST52 using the Gateway LR reaction and a recombinase (Invitrogen) according to the manufacturer’s protocol. To construct the YCp-type plasmid, pYES-DEST52 was digested with Spe I, Sap I, and Stu I, and the resulting 2.7-kb fragment containing the GAL1 promoter and the Gateway cassette was isolated. This fragment was blunt-ended with a Blunting High Kit (Toyobo, Osaka, Japan) and inserted into the Sma1 site of the expression vector pRS316 (provided by the National Bio-Resource Project [NBRP] of the MEXT, Japan).15Sikorski RS Hieter P A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae.Genetics. 1989; 122: 19-27Crossref PubMed Google Scholar To construct the Yip-type plasmid, pYES-DEST52 was digested with Nhe I and SnaB I to delete the 2µ region, blunt-ended with the Blunting High Kit, and self-ligated. In all of the expression plasmids, expression of human cDNAs was under the control of the GAL1 promoter. Transformation of yeast with the plasmids was carried out according to a previously described method.16Gietz RD Woods RA Transformation of yeast by lithium acetate/single-stranded carrier DNA/polyethylene glycol method.Methods Enzymol. 2002; 350: 87-96Crossref PubMed Scopus (2068) Google Scholar Precultures of yeast before galactose-induced expression of c-Yes were carried out in media containing 2% glucose or 2% raffinose. In experiments involving time-dependent induction of c-Yes, the cells were precultured in the raffinose media because the glucose-induced repression of the GAL1 promoter continued for a while after the removal of glucose, whereas raffinose did not have this effect. Therefore, when the precultures were carried out in the presence of raffinose, the GAL1 promoter was immediately activated after the addition of galactose. Induction of c-Yes expression was initiated in media containing 2% galactose after washing out glucose or raffinose in the precultures. To assess the effects of human proteins on the growth of yeast, wild-type yeast cells harboring pYES-DEST52 containing a human cDNA were grown in the noninducing (glucose) liquid medium at 30 °C for ~12 h until the cultures reached saturation. After 2 washes with sterile water, the cells were resuspended in the inducing (galactose) medium and serially diluted by 5-fold. The resulting cell suspensions were spotted onto plates containing the noninducing or inducing medium and incubated at 30 °C for 2 days. As a control, yeast cells harboring the empty vector were treated in the same way. The effects of known protein kinase inhibitors on the c-Yes-induced repression of yeast growth were examined using the triple-mutant yeast strain that expressed c-Yes through the YCp-type plasmid. The protein kinase inhibitors were all dissolved in DMSO. The inhibitor solutions were serially diluted by 2-fold with DMSO, and their final concentrations ranged from 100 µM to 1.52 nM (18 different concentrations). The final concentration of DMSO was adjusted to 1%. The restorative effects of the protein kinase inhibitors on the c-Yes-induced growth retardation of yeast were assayed by measuring the cell densities after culture of the cells in the presence of the inhibitors or 1% DMSO alone. Yeast cells harboring expression plasmids for c-Yes were grown in the noninducing (glucose) liquid medium at 30 °C for ~12 h until the cultures reached saturation. The cells were washed twice with sterile water and suspended in the inducing (galactose) liquid medium. The suspensions were diluted with the medium to adjust their optical densities at 600 nm to 0.01. The resulting cell suspensions were dispensed into 96-well plates at 99 µL/well, and 1 µL of an inhibitor was added. The plates were incubated with 30 °C, and the optical densities were determined after specified intervals (24, 48, and 72 h). The ratios of the optical densities between the cultures in the presence of inhibitors and the control cultures (DMSO alone) were calculated. Harvested cells were washed twice with distilled water and lysed using glass beads in a lysis buffer consisting of 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 5 mM EDTA, 1.0% Triton X-100, 1 mM dithiothreitol [DTT], 1 mM phenylmethylsulfonyl fluoride [PMSF], and a protease inhibitor cocktail (Nacalai Tesque, Kyoto, Japan). After centrifugation of the lysates at 13,200 rpm for 30 min, the supernatants were analyzed by immunoblotting with the following primary antibodies: anti-c-Yes (1:5000; BD Biosciences Pharmingen, San Diego, CA), anti-HA (1:2000; MBL, Nagoya, Japan), anti-α-tubulin (generously provided by Dr. Andrea Baines, Sir William Dunn School of Pathology, University of Oxford), and anti-phosphotyrosine (Millipore, Billerica, MA). As the secondary antibodies, horseradish peroxidase (HRP)–conjugated antimouse IgG antibody and HRP-conjugated antirabbit IgG antibody (1:5000; Invitrogen) were used. The triple-mutant yeast strains expressing the HA-tagged yeast chaperones (Hsp82-HA or Hsc82-HA) were engineered to produce c-Yes-V5 fusion proteins under the control of the GAL1 promoter. The cells harboring the c-Yes-V5 expression plasmid were precultured in the noninducing medium containing raffinose. The culture medium was then replaced with the inducing medium containing galactose, and the cells were cultured for 2 h. The supernatants of cell lysates were obtained as described above and treated with anti-V5 antibody-conjugated agarose beads (Sigma). The immunoprecipitates were subjected to immunoblotting to analyze whether c-Yes coimmunoprecipitated with Hsp82 and/or Hsc82. c-Yes in the precipitates was detected using an anti-c-Yes antibody, whereas Hsp82 and Hsc82 were detected using an anti-HA antibody. The triple-mutant yeast strain harboring the expression plasmid for c-Yes was cultured in the presence of galactose for 2 h. Radicicol (0.78 µM) or geldanamycin (6.3 µM) was added at zero time. After 2 h of culture, total RNA was isolated using an RNeasy Mini Kit (Qiagen, Piscataway, NJ) according to the manufacturer’s protocol. RT-PCR was conducted with ReverTra Ace-α (Toyobo) using 0.8 µg of each total RNA and specific primers that amplified the c-Yes cDNA. As an internal control, the mRNA of α-tubulin was detected. The primers used were 5′-TATGTAGCGCCTGCAGATTCCATTC-3′ and 5′-CCATATCAACCAGCTGTGGAAGCTTCA-3′ for c-Yes and 5′-CAAGAGGCCATTACACCGTTGGTAGA-3′ and 5′-CACCCCTGTATAACAGACAAGTAGCCA-3′ for α-tubulin. To screen the human proteins that repressed the growth of yeast, the full-length human cDNAs of 10,302 (~50% of the total cDNAs) were introduced into yeast cells using a multicopy-type yeast expression vector in which the expression of the human cDNAs was galactose inducible. Figure 1 shows a representative example of the inhibition of yeast growth. The galactose-induced expression of human c-Yes caused repression of the yeast growth, whereas cultures in the noninducing condition (glucose medium) showed no effect on the growth. c-Yes is a plasma membrane-associated tyrosine kinase that belongs to the Src family as described below. Among the cDNAs, 583 clones (~5.6% of the tested clones) showed inhibitory effects on yeast growth. We did not examine the expressions of mRNAs or proteins encoded by cDNAs that showed no effect on the yeast growth. Table 1 lists representatives of the human proteins that inhibited the yeast growth. These proteins included proteins that play crucial roles in a wide variety of cell behaviors, such as cell proliferation, cytoskeleton, DNA replication, protein kinases, and others, which may be promising targets for screening chemicals. Interestingly, ~25% of the human proteins that showed inhibitory effects on the yeast growth had unknown cellular functions. Studies of the molecular mechanisms by which the yeast growth is repressed by these uncharacterized proteins and the identification of small compounds that can reverse the repression of yeast growth will greatly contribute to clarifying the physiological functions of these proteins.Table 1Representatives of Human Proteins That Inhibited Yeast GrowthFunctionsProteinsProliferationc-Ha-ras1 p21, RAS p21 protein activator 1Cell cycleCyclin A1, cyclin B2, cyclin E1CytoskeletonTubulin beta 2A, tubulin beta 6Cell divisionKatanin p60 subunit A-like 1Cell adhesionCatenin beta 1, TCF4, occludin, selectin P, junction mediating and regulatory proteinCell motilityActin alpha 2, actin gamma 1, actin gamma 2, RhoA, RhoB, RhoC, actin-binding Rho-activating proteinVesicular transportRAB18, chromatin-modifying protein 5Chromatin structureHistone cluster 1 H3a, histone cluster 1 H3iDNA replicationF-box protein helicase 18DNA repairPARP1, PARP6TranscriptionEts-2, MafK, MafGRNA modificationTransformer 2 alpha, transformer 2 beta, RNA binding protein with multiple splicing, IGF2BP1, IGF2BP3Protein degradationProteasome subunit alpha type 3, praja ring finger 1Tumor suppressionp53, IRF1Protein kinases60 kinases (see Table 2)Not annotated101 proteins (not shown) Open table in a new tab We focused on protein kinases because they are strongly related to various diseases. Among the 263 (50% of the total) protein kinases tested, 60 protein kinases (~23%) caused repression of the yeast growth (Table 2). Table 3 shows the percentages of the repressive enzymes in different kinase subfamilies. The tyrosine kinase subfamily showed a high score of 35.5%. Hsp90, a molecular chaperone in vertebrates, binds to its client proteins during or immediately after their synthesis and thereby guarantees the proper folding of the client proteins. Because the numerous signaling proteins whose mutations and aberrant expression cause cancers are clients, Hsp90 is an emerging therapeutic target for treatment of cancer.17Messaoudi S Peyrat JF Brion JD Alami M Recent advances in Hsp90 inhibitors as antitumor agents.Anticancer Agents Med Chem. 2008; 8: 761-782Crossref PubMed Scopus (101) Google Scholar Src family members, including c-Yes, require Hsp90 for maintaining their functions. Hereafter, c-Yes was used for the validation of the yeast-based screening system for small molecules because if yeast homologues of Hsp90 interact with c-Yes, we would be able to identify the molecules that inhibit the function of Hsp90 as well as those that inhibit c-Yes directly.Table 2Human Protein Kinases That Inhibited Yeast GrowthApproved Gene SymbolGroupApproved Gene NameAURKAOtherAurora kinase AAURKBOtherAurora kinase BBLKTKB lymphoid tyrosine kinaseBMPR1ATKLBone morphogenetic protein receptor, type IABUB1OtherBudding uninhibited by benzimidazoles 1 homologue (yeast)CDC2CMGCCell division cycle 2, G1 to S and G2 to MCDK2CMGCCyclin-dependent kinase 2CDK7CMGCCyclin-dependent kinase 7CDKL2CMGCCyclin-dependent kinase-like 2 (CDC2-related kinase)CHEK2CAMKCHK2 checkpoint homologue (Schizosaccharomyces pombe)DDR2TKDiscoidin domain receptor tyrosine kinase 2DYRK2CMGCDual-specificity tyrosine-(Y)-phosphorylation regulated kinase 2EIF2AK2OtherEukaryotic translation initiation factor 2-alpha kinase 2EIF2AK3OtherEukaryotic translation initiation factor 2-alpha kinase 3EPHA4TKEPH receptor A4EPHA7TKEPH receptor A7FERTKfer (fps/fes related) tyrosine kinaseFESTKFeline sarcoma oncogeneFGRTKGardner-Rasheed feline sarcoma viral (v-fgr) oncogene homologueFRKTKfyn-related kinaseFYNTKFYN oncogene related to SRC, FGR, YESIRAK3TKLInterleukin-1 receptor-associated kinase 3IRAK4TKLInterleukin-1 receptor-associated kinase 4MAP3K5STEMitogen-activated protein kinase kinase kinase 5MAPK14CMGCMitogen-activated protein kinase 14MAPK9CMGCMitogen-activated protein kinase 9MLKLTKLMixed lineage kinase domain-likeNEK2OtherNIMA (never in mitosis gene a)–related kinase 2NLKCMGCNemo-like kinasePAK4STEp21 protein (Cdc42/Rac)–activated kinase 4PAK6STEp21 protein (Cdc42/Rac)–activated kinase 6PDIK1LOtherPDLIM1 interacting kinase 1 likePDK3AtypicalPyruvate dehydrogenase kinase, isozyme 3PIM3CAMKpim-3 oncogenePINK1OtherPTEN induced putative kinase 1PLK2OtherPolo-like kinase 2 (Drosophila)PLK4OtherPolo-like kinase 4 (Drosophila)PRKACAAGCProtein kinase, cAMP dependent, catalytic, alphaPRKACBAGCProtein kinase, cAMP dependent, catalytic, betaPRKCAAGCProtein kinase C, alphaPRKCBAGCProtein kinase C, betaPRKCEAGCProtein kinase C, epsilonPRKXAGCProtein kinase, X-linkedPRPF4BCMGCPRP4 pre-mRNA processing factor 4 homologue B (yeast)RAF1TKLv-raf-1 murine leukemia viral oncogene homologue 1RIOK3AtypicalRIO kinase 3 (yeast)SCYL3OtherSCY1-like 3 (Saccharomyces cerevisiae)SGK3AGCSerum/glucocorticoid-regulated kinase family, member 3SIK1CAMKSalt-inducible kinase 1SRPK2CMGCSFRS protein kinase 2STK3STESerine/threonine kinase 3 (STE20 homologue, yeast)STK36OtherSerine/threonine kinase 36, fused homologue (Drosophila)TLK1OtherTousled-like kinase 1TSSK2CAMKTestis-specific serine kinase 2TTKOtherTTK protein kinaseULK2Otherunc-51-like kinase 2 (Caenorhabditis elegans)WEE2OtherWEE1 homologue 2 (S. pombe)YES1TKv-yes-1 Yamaguchi sarcoma viral oncogene homologue 1ZAKTKLSterile alpha motif and leucine zipper containing kinase AZKZAP70TKZeta-chain (TCR) associated protein kinase 70 kDaAGC: PKA, PKG, and PKC families; CAMK: calcium/calmodulin-dependent protein kinase; CMGC: CDK, MAPK, GSK3, and CLK families; STE: homologues of yeast Sterile 7, Sterile 11, and Sterile 20 kinase; TK: tyrosine kinase; TKL: tyrosine kinase-like. Approved gene symbol and approved gene name are identified according to the Hugo Gene Nomenclature Committee (http://www.genenames.org/). Group is identified according to the kinbase (http://kinase.com/kinbase/). Open table in a new tab Table 3Protein Kinase (PK) Subfamilies That Inhibited Yeast GrowthGroupNumber of PK TestedNumber of Inhibitory PK% of Inhibitory PKAGC36719.4Atypical17211.8CAMK39410.3CK1700.0CMGC381026.3Other401640.0RGC100.0STE27414.8TK311135.5TKL27622.2Total2636022.8CK1, casein kinase 1 group; RGC, receptor guanylate cyclases. The group is given according to kinbase (http://kinase.com/kinbase/). Open table in a new tab AGC: PKA, PKG, and PKC families; CAMK: calcium/calmodulin-dependent protein kinase; CMGC: CDK, MAPK, GSK3, and CLK families; STE: homologues of yeast Sterile 7, Sterile 11, and Sterile 20 kinase; TK: tyrosine kinase; TKL: tyrosine kinase-like. Approved gene symbol and approved gene name are identified according to the Hugo Gene Nomenclature Committee (http://www.genenames.org/). Group is identified according to the kinbase (http://kinase.com/kinbase/). CK1, casein kinase 1 group; RGC, receptor guanylate cyclases. The group is given according to kinbase (http://kinase.com/kinbase/). One drawback of using yeast for large-scale drug screening is the presence of the cell membrane, which may function as a permeability barrier and contains efflux pumps for small molecules. Improvements have been attempted by generating yeast strains that lack genes that are probably responsible for these disadvantages.18Gaber RF Copple DM Kennedy BK Vidal M Bard M The yeast gene ERG6 is required for normal membrane function but is not essential for biosynthesis of the cell-cycle-sparking sterol.Mol Cell Biol. 1989; : 3447-3456Google Scholar,19Balzi E Goffeau A Yeast multidrug resistance: the PDR network.J Bioenerg Biomembr. 1995; 27: 71-76Crossref PubMed Scopus (229) Google Scholar To increase the intracellular concentrations of the small molecules to be tested, we prepared a triple-mutant yeast strain that lacked ERG4, PDR1, and PDR3 (Mukai et al., manuscript in preparation). Erg4 is involved in the biosynthesis of ergosterol, which strengthens the permeability barrier of the yeast membrane to small molecules,18Gaber RF Copple DM Kennedy BK Vidal M Bard M The yeast gene ERG6 is required for normal membrane function but is not essential for biosynthesis of the cell-cycle-sparking sterol.Mol Cell Biol. 1989; : 3447-3456Google Scholar whereas Pdr1 and Pdr3 are transcription factors that stimulate the expression of pump proteins for the efflux of xenobiotics.19Balzi E Goffeau A Yeast multidrug resistance: the PDR network.J Bioenerg Biomembr. 1995; 27: 71-76Crossref PubMed Scopus (229) Google Scholar As described below, 6 compounds including PP2 were found to recover the c-Yes-induced repression of the yeast growth. PP2 is one of the known inhibitors of c-Yes.20Hanke JH Gardner JP Dow RL Changelian PS Brissette WH Weringer EJ et al.Discovery of a novel, potent, and Src family-selective tyrosine kinase inhibitor: study of Lck- and FynT-dependent T cell activation.J Biol Chem. 1996; 271: 695-701Abstract Full Text Full Text PDF PubMed Scopus (1784) Google Scholar Cyclin A1 repressed the growth of yeast (Table 1), and radicicol, an Hsp90-mediated tyrosine kinase inhibitor (see below), reversed this repression by an unknown mechanism. To show that the triple-mutant strain is more convenient for screening chemical compounds than the wild-type strain, we compared these 2 strains with respect to the PP2- and radicicol-dependent recovery of growth. As shown in Figure 2a, the dose-dependent curve of PP2 in the triple-mutant strain was similar to that in the wild-type strain, indicating that the permeability increase by mutations did not improve the sensitivity to PP2 (EC50app = 0.54 µM in the wild-type strain and 0.29 µM in the mutant strain), but the maximum growth recovery of the mutant yeast was seen at 4-fold lower concentration of PP2 than that of the wild-type yeast. In contrast, the radicicol-dependent recovery from the cyclin A1-induced growth repression was markedly sensitive in the triple-mutant strain as compared with that in the wild-type strain (Fig. 2b, EC50app = 2.04 µM in the wild-type strain and 0.06 µM in the mutant strain). Reductions in the growth recovery at high concentrations of PP2 and radicicol are probably brought about by their cytotoxic effects. The relatively narrow concentration ranges of both compounds effective for the growth recovery may result because the concentrations causing their cytotoxicity are close to those required for the growth recovery. Nevertheless, it was an advantage of the strain with an increased permeability that a minimum amount of chemical compounds could be assayed, and therefore we decided to use the triple-mutant yeast for further experiments. Next, to validate this screening system, we examined whether the c-Yes-induced retardation of the yeast growth was specifically reversed by known inhibitors for c-Yes. Among 85 protein kinase inhibitors tested, 6 compounds (PP2, PP1 [a PP2 analog], SU6656, ra" @default.
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• W2018669255 date "2010-04-01" @default.
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• W2018669255 title "Comprehensive Screening of Human Genes with Inhibitory Effects on Yeast Growth and Validation of a Yeast Cell-Based System for Screening Chemicals" @default.
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• W2018669255 doi "https://doi.org/10.1177/1087057110363822" @default.
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