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• W2080484776 abstract "Bisindolylmaleimide compounds such as GF109203X are potent inhibitors of protein kinase C (PKC) activity. Although bisindolylmaleimides are not entirely selective for PKC and are known to inhibit a few other protein kinases, these reagents have been extensively used to study the functional roles of PKC family enzymes in cellular signal transduction for more than a decade. Here, we establish a proteomics approach to gain further insights into the cellular effects of this compound class. Functional immobilization of suitable bisindolylmaleimide analogues in combination with the specific purification of cellular binding proteins by affinity chromatography led to the identification of several known and previously unknown enzyme targets. Subsequent in vitro binding and activity assays confirmed the protein kinases Ste20-related kinase and cyclin-dependent kinase 2 (CDK2) and the non-protein kinases adenosine kinase and quinone reductase type 2 as novel targets of bisindolylmaleimide inhibitors. As observed specifically for CDK2, minor chemical variation of the ligand by immobilizing the closely related bisindolylmaleimides III, VIII, and X dramatically affected target binding. These observed changes in affinity correlated with both the measured IC50 values for in vitro CDK2 inhibition and results from molecular docking into the CDK2 crystal structure. Moreover, the conditions for affinity purification could be adapted in a way that immobilized bisindolylmaleimide III selectively interacted with either PKCα or ribosomal S6 protein kinase 1 only after activation of these kinases. Thus, we have established an efficient technique for the rapid identification of cellular bisindolylmaleimide targets and further demonstrate the comparative selectivity profiling of closely related kinase inhibitors within a cellular proteome. Bisindolylmaleimide compounds such as GF109203X are potent inhibitors of protein kinase C (PKC) activity. Although bisindolylmaleimides are not entirely selective for PKC and are known to inhibit a few other protein kinases, these reagents have been extensively used to study the functional roles of PKC family enzymes in cellular signal transduction for more than a decade. Here, we establish a proteomics approach to gain further insights into the cellular effects of this compound class. Functional immobilization of suitable bisindolylmaleimide analogues in combination with the specific purification of cellular binding proteins by affinity chromatography led to the identification of several known and previously unknown enzyme targets. Subsequent in vitro binding and activity assays confirmed the protein kinases Ste20-related kinase and cyclin-dependent kinase 2 (CDK2) and the non-protein kinases adenosine kinase and quinone reductase type 2 as novel targets of bisindolylmaleimide inhibitors. As observed specifically for CDK2, minor chemical variation of the ligand by immobilizing the closely related bisindolylmaleimides III, VIII, and X dramatically affected target binding. These observed changes in affinity correlated with both the measured IC50 values for in vitro CDK2 inhibition and results from molecular docking into the CDK2 crystal structure. Moreover, the conditions for affinity purification could be adapted in a way that immobilized bisindolylmaleimide III selectively interacted with either PKCα or ribosomal S6 protein kinase 1 only after activation of these kinases. Thus, we have established an efficient technique for the rapid identification of cellular bisindolylmaleimide targets and further demonstrate the comparative selectivity profiling of closely related kinase inhibitors within a cellular proteome. The protein kinase C (PKC) 1The abbreviations used are: PKC, protein kinase C; 16-BAC, 16-benzyldimethyl-n-hexadecylammonium chloride; AK, adenosine kinase; BisIII, bisindolylmaleimide III; BisVIII, bisindolylmaleimide VIII; BisX, bisindolylmaleimide X; CaMKII, calmodulin-dependent kinase II; CDK2, cyclin-dependent kinase 2; DAG, diacylglycerol; EGF, epidermal growth factor; GSK3, glycogen synthase kinase 3; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoline; NQO2, quinone-reductase type 2; PS, phosphatidylserine; RICK, Rip-like interacting caspase-like apoptosis-regulatory protein (CLARP) kinase; ROCK, Rho-dependent protein kinase; Rsk, ribosomal S6 protein kinase; SLK, Ste20-related kinase. family of serine/threonine kinases comprises at least 11 isozymes, which can be divided into three subgroups based on biochemical criteria such as differential co-factor requirements. Classical PKCs (α, β1, β2, and γ) depend on both diacylglycerol (DAG) and Ca2+ for kinase activation, novel PKCs (δ, ε, η, θ, and μ) require only DAG, and the activity of the atypical PKCs (ζ and λ) is regulated by neither of the two co-factors (1Newton A.C. Protein kinase C: Structural and spatial regulation by phosphorylation, cofactors, and macromolecular interactions..Chem. Rev. 2001; 101: 2353-2364Google Scholar). Individual PKC isozymes have been implicated in various aspects of cellular physiology involving biological processes as diverse as cell proliferation and differentiation, apoptosis, ion channel regulation, glycogenolysis, and protein secretion (2Dempsey E.C. Newton A.C. Mochly-Rosen D. Fields A.P. Reyland M.E. Insel P.A. Messing R.O. Protein kinase C isozymes and the regulation of diverse cell responses..Am. J. Physiol. Lung Cell Mol. Physiol. 2000; 279: L429-L438Google Scholar, 3Goekjian P.G. Jirousek M.R. Protein kinase C in the treatment of disease: signal transduction pathways, inhibitors, and agents in development..Curr. Med. Chem. 1999; 6: 877-903Google Scholar). Moreover, different PKCs have been implicated as potential targets for therapeutic intervention in various diseases including several types of cancer and heart failure (4Barry O.P. Kazanietz M.G. Protein kinase C isozymes, novel phorbol ester receptors and cancer chemotherapy..Curr. Pharm. Des. 2001; 7: 1725-1744Google Scholar, 5Vlahos C.J. McDowell S.A. Clerk A. Kinases as therapeutic targets for heart failure..Nat. Rev. Drug Discov. 2003; 2: 99-113Google Scholar). In the majority of previous studies, analysis of PKC function in cultured cells has been performed with pharmacological tools that either stimulate or interfere with its cellular activity. Phorbol esters such as phorbol myristate acetate bind with high affinity to the C1-domain of classical and novel PKC isozymes and thereby induce kinase activity through the same membrane translocation mechanisms as the endogenous C1-interacting ligand DAG. Small molecule inhibitors of PKC either interfere with C1-domain-mediated kinase activation (e.g. Calphostin C) or directly block PKC kinase activity through ATP-competitive interaction with the nucleotide-binding pocket. Inhibitors belonging to the bisindolylmaleimide class of compounds fall into the second category. GF109203X, the most popular of these bisindolylmaleimide inhibitors of PKC activity, has been used in numerous studies contributing to more than 1000 published articles about cellular PKC function (6Toullec D. Pianetti P. Coste H. Bellevergue P. Grand-Perret T. Ajakane M. Baudet V. Boissin P. Boursier E. Loriolle F. Duhamel L. Charon D. Kirilovsky J. The bisindolylmaleimide GF 109203X is a potent and selective inhibitor of protein kinase C..J. Biol. Chem. 1991; 266: 15771-15781Google Scholar). Due to this widespread application of PKC inhibitors such as GF109203X, their selectivity is a highly relevant issue for many experimental studies and has previously been examined by parallel activity measurements of different recombinant protein kinases in the presence of bisindolylmaleimide compounds (7Davies S.P. Reddy H. Caivano M. Cohen P. Specificity and mechanism of action of some commonly used protein kinase inhibitors..Biochem. J. 2000; 351: 95-105Google Scholar). Importantly, as well documented in the comprehensive selectivity study by Cohen and colleagues, various bisindolylmaleimide derivatives blocked the activities of the ribosomal S6 protein kinase 2 (Rsk2) and its close relative mitogen and stress-activated protein kinase 1 as potently as PKCα in vitro. Moreover, some other kinases including glycogen synthase kinase 3 (GSK3) and p70 ribosomal S6 protein kinase (S6K) were also inhibited, albeit to a lesser extent. In addition to these alternative kinase targets, GF109203X was reported as potent inhibitor of voltage-dependent sodium channels and the 5-hydroxytryptamine3 receptor (8Coultrap S.J. Sun H. Tenner Jr., T.E. Machu T.K. Competitive antagonism of the mouse 5-hydroxytryptamine3 receptor by bisindolylmaleimide I, a “selective” protein kinase C inhibitor..J. Pharmacol. Exp. Ther. 1999; 290: 76-82Google Scholar, 9Lingameneni R. Vysotskaya T.N. Duch D.S. Hemmings Jr., H.C. Inhibition of voltage-dependent sodium channels by Ro 31–8220, a “specific” protein kinase C inhibitor..FEBS Lett. 2000; 473: 265-268Google Scholar). But, as these previous studies only covered a tiny fraction of the potential bisindolylmaleimide targets expressed in the proteome of a mammalian cell or organism, tools for a more comprehensive and less biased analysis of PKC inhibitor selectivity are needed. To study kinase inhibitor selectivity on a proteome-wide scale, affinity purification methods combined with mass spectrometry have the potential to reveal the relevant cellular drug targets in cases where suitable compound derivatives can be obtained for immobilization on chromatography beads. In several earlier studies, this straightforward “chemical proteomics” approach has been employed and could deliver some additional information about the proteins targeted by small molecule inhibitors (10Knockaert M. Gray N. Damiens E. Chang Y.T. Grellier P. Grant K. Fergusson D. Mottram J. Soete M. Dubremetz J.F. Le Roch K. Doerig C. Schultz P. Meijer L. Intracellular targets of cyclin-dependent kinase inhibitors: Identification by affinity chromatography using immobilised inhibitors..Chem. Biol. 2000; 7: 411-422Google Scholar, 11Lolli G. Thaler F. Valsasina B. Roletto F. Knapp S. Uggeri M. Bachi A. Matafora V. Storici P. Stewart A. Kalisz H.M. Isacchi A. Inhibitor affinity chromatography: profiling the specific reactivity of the proteome with immobilized molecules..Proteomics. 2003; 3: 1287-1298Google Scholar, 12Ding S. Wu T.Y. Brinker A. Peters E.C. Hur W. Gray N.S. Schultz P.G. Synthetic small molecules that control stem cell fate..Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 7632-7637Google Scholar, 13Brown D. Superti-Furga G. Rediscovering the sweet spot in drug discovery..Drug Discov. Today. 2003; 8: 1067-1077Google Scholar). We have recently established an efficient proteomics method to identify the cellular targets of the p38 kinase inhibitor SB 203580 (14Godl K. Wissing J. Kurtenbach A. Habenberger P. Blencke S. Gutbrod H. Salassidis K. Stein-Gerlach M. Missio A. Cotten M. Daub H. An efficient proteomics method to identify the cellular targets of protein kinase inhibitors..Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 15434-15439Google Scholar). In this study, affinity chromatography was performed under highly optimized biochemical conditions and led to the identification of a set of previously unknown SB 203580 targets, demonstrating that this widely used reagent is far less selective for p38 than assumed (14Godl K. Wissing J. Kurtenbach A. Habenberger P. Blencke S. Gutbrod H. Salassidis K. Stein-Gerlach M. Missio A. Cotten M. Daub H. An efficient proteomics method to identify the cellular targets of protein kinase inhibitors..Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 15434-15439Google Scholar). Here, we have adapted this proteomics approach to identify the cellular targets of bisindolylmaleimides. In addition to protein kinases known to be affected by these compounds, we found various other specifically interacting enzymes and could verify them by in vitro binding and activity assays as novel targets of bisindolylmaleimide inhibitors. Interestingly, cyclin-dependent kinase 2 (CDK2) interaction with bisindolylmaleimides was dramatically affected through subtle chemical variation of the immobilized inhibitors. This differential binding could be correlated with the derivatives’ in vitro potency toward CDK2 kinase activity and their respective affinities according to molecular docking analysis. Cell culture media and LipofectAMINE were purchased from Invitrogen (San Diego, CA). Radiochemicals, epoxy-activated Sepharose 6B and protein G Sepharose were obtained from Amersham Biosciences (Uppsala, Sweden). 2-[1-(3-Aminopropyl)-1H-indol-3-yl]-3-(1H-indol-3-yl) maleimide (BisIII), 2-[1-(3-aminopropyl)-indol-3-yl]-3-(1-methyl-indol-3-yl) maleimide (BisVIII), 2-[1-(3-aminopropyl)-6,7,8,9-tetrahydropyridol [1,2-a] indol-3-yl]-3-(1-methyl-indol-3-yl) maleimide (BisX), and histone H1 were obtained from Calbiochem (La Jolla, CA). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolin (MTT) was obtained from Sigma (St. Louis, MO). NADH was purchased from Roche (Basel, Switzerland). Menadion was obtained from Calbiochem. All other reagents were obtained from Sigma. Antibodies used in this study were: rabbit polyclonal anti-Rsk1, mouse monoclonal anti-GSK3α/β, rabbit polyclonal anti-CDK2 (all from Santa Cruz Biotechnology, Santa Cruz, CA), rabbit polyclonal anti-p38 antibody (Cell Signaling Technology, Beverly, MA), rabbit polyclonal anti-RICK antibody (Affinity BioReagents, Golden, CO), mouse monoclonal anti-PKCα (Upstate Biotechnology, Lack Placid, NY), mouse monoclonal anti-FLAG clone M2 (Sigma), and mouse monoclonal anti-c-myc clone 9E10 (Upstate Biotechnology). Human CDK2/cyclinA was purchased from Upstate Biotechnology. The DNA sequences coding for full-length Ste20-related kinase (SLK) and full-length adenosine kinase (AK) were PCR-amplified from human spleen and liver cDNA libraries. The SLK cDNA was fused to a N-terminal FLAG-tag, whereas the AK cDNA was fused to a N-terminal myc-tag and cloned into a pRK5 expression vector (15Daub H. Blencke S. Habenberger P. Kurtenbach A. Dennenmoser J. Wissing J. Ullrich A. Cotten M. Identification of SRPK1 and SRPK2 as the major cellular protein kinases phosphorylating hepatitis B virus core protein..J. Virol. 2002; 76: 8124-8137Google Scholar, 16Spychala J. Datta N.S. Takabayashi K. Datta M. Fox I.H. Gribbin T. Mitchell B.S. Cloning of human adenosine kinase cDNA: Sequence similarity to microbial ribokinases and fructokinases..Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1232-1237Google Scholar, 17Yamada E. Tsujikawa K. Itoh S. Kameda Y. Kohama Y. Yamamoto H. Molecular cloning and characterization of a novel human STE20-like kinase, hSLK..Biochim. Biophys. Acta. 2000; 1495: 250-262Google Scholar). All three bisindolylmaleimide compounds were dissolved in 100% dimethylsulfoxide. Stocks were spectrophotometrically normalized (Spectramax Plus 384; Molecular Devices, Sunnyvale, CA) using their absorption maxima at 372 nm and 460 nm and stored under argon at −20 °C in the dark. For immobilization, drained epoxy-activated Sepharose 6B was resuspended in 2 volumes of 20 mm BisIII, BisVIII, or BisX dissolved in 50% dimethylformamide/0.1 M Na2CO3 pH 11. After adding of 10 mm NaOH, coupling was performed overnight at 30 °C in the dark. After three washes with 50% dimethylformamide/0.1 M Na2CO3, remaining reactive groups were blocked with 1 m ethanolamine, pH 11. Subsequent washing steps were performed according to the manufacturer's instructions. To generate the control matrix (Ctrl), epoxy-activated Sepharose 6B was directly reacted with 1 m ethanolamine pH 11 and equally treated as described above. The matrices were stored at 4 °C in the dark. COS-7, HeLa, or HuH-7 cells were cultured in Dulbecco′s modified Eagle′s medium supplemented with 10% fetal bovine serum. COS-7 cells were transiently transfected as previously described (15Daub H. Blencke S. Habenberger P. Kurtenbach A. Dennenmoser J. Wissing J. Ullrich A. Cotten M. Identification of SRPK1 and SRPK2 as the major cellular protein kinases phosphorylating hepatitis B virus core protein..J. Virol. 2002; 76: 8124-8137Google Scholar). Epidermal growth factor (EGF) stimulation of starved HeLa cells was carried out as described (18Blencke S. Ullrich A. Daub H. Mutation of threonine 766 in the epidermal growth factor receptor reveals a hotspot for resistance formation against selective tyrosine kinase inhibitors..J. Biol. Chem. 2003; 278: 15435-15440Google Scholar). COS-7 and HeLa cells were usually lysed in Triton X-100 lysis buffer (TL-buffer) containing 50 mm HEPES, pH 7.5, 150 mm NaCl, 0.5% Triton X-100, 1 mm EDTA, 1 mm EGTA, 3 mm CaCl2 plus additives (10 mm sodium fluoride, 1 mm orthovanadate, 10 μg/ml aprotinin, 10 μg/ml leupeptin, 1 mm phenylmethylsulfonyl fluoride, 0.2 mm dithiothreitol) and co-factors (100 μg/ml phosphatidylserine (PS), 20 μg/ml DAG)). For analytical in vitro association experiments, lysates were precleared by centrifugation and equilibrated to 1 m NaCl. Then 300 μl of the high salt lysate were incubated together with 20 μl of drained bisindolylmaleimide matrix (Bis matrix) for 3 h at 4 °C. Afterward, the Bis matrices were washed twice with 500 μl of TL-buffer plus 1 m NaCl and once with 500 μl of TL-buffer; both steps were performed without additives, and the co-factor concentrations were reduced to one-tenth. In the experiments designed to detect specific SLK and AK binding to BisIII beads, CaCl2 and the co-factors were not included in the lysis and wash buffers. When the stimulation-dependent binding of Rsk1 to BisIII beads was analyzed, CaCl2 and the co-factors were omitted from the lysis and wash buffers and the NaCl concentration was kept at 150 mm. For activation-dependent detection of PKCα binding from HuH-7 cells, the lysis buffer was 20 mm HEPES, pH 7.5, 150 mm NaCl, 0.25% Triton X-100, 0.1 mm EDTA, 0.2 mm EGTA plus additives. Where indicated, 0.5 mm CaCl2 plus co-factors were added. Co-factor concentrations were also reduced to one-tenth in the respective washing buffer, which was lysis buffer without additives. Bound proteins were eluted by boiling of the affinity beads in 1.5x SDS sample buffer. After SDS-PAGE, proteins were transferred to nitrocellulose membrane and immunoblotted with the corresponding antibodies. The preparative binding experiments using 2.5 × 109 cells, 16-benzyldimethyl-n-hexadecylammonium chloride (16-BAC)/SDS-PAGE separation and mass spectrometric analysis were carried out essentially as described, with the modification that 3 mm CaCl2, 100 μg/ml PS, and 20 μg/ml DAG were included and glycerol was omitted from the lysis buffer (14Godl K. Wissing J. Kurtenbach A. Habenberger P. Blencke S. Gutbrod H. Salassidis K. Stein-Gerlach M. Missio A. Cotten M. Daub H. An efficient proteomics method to identify the cellular targets of protein kinase inhibitors..Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 15434-15439Google Scholar). Moreover, the BisIII column was washed with buffer containing 3 mm CaCl2, 20 μg/ml PS, and 4 μg/ml DAG after sample loading. Enzyme activity and inhibition were determined spectrophotometrically (Spectramax Plus 384; Molecular Devices) by measuring the reduction of MTT at 610 nm and 30 °C. In this assay, we used NADH as an electron donor for the menadion reduction and MTT for the continuous re-oxidation of menadiol. The reactions (200 μl) were performed in 96-well plates, containing 50 mm KxHxPO4, pH 7.5, 1 μl of quinone-reductase type 2 (NQO2), 40 μm menadion, 200 μm MTT, and increasing concentrations of NADH (0–1000 μm) in the presence of different fixed BisIII concentrations (0, 1, 30, 60 μm). Using a Lineweaver-Burk application, we determined the apparent Km (Km,app) values for the different BisIII concentrations. The Ki was calculated by plotting the different Km,app against its corresponding BisIII concentrations. Lysates from COS-7 cells transiently expressing full-length SLK fused to a N-terminal FLAG-tag were subjected to immunoprecipitation with protein G Sepharose-bound anti-FLAG antibodies for 3 h at 4 °C. After binding, the beads were washed three times with 500 μl of TL-buffer and once with 500 μl of kinase buffer (20 mm HEPES, pH 7.5, 15 mm MgCl2, 80 mm KCl, 1 mm Na3VO4, and 0.1 mm DTT). SLK kinase activity assays were performed in a total volume of 60 μl. Immunoprecipitated SLK bound to 15 μl of drained beads was mixed with 35 μl of kinase buffer containing the indicated BisIII concentrations and incubated for 10 min at 4 °C. The kinase reactions were started by addition of 100 μm ATP, 1 μCi [γ-32P]ATP, and 20 μg of myelin basic protein and incubated for 10 min at 30 °C. CDK2 assays were performed in a total volume of 50 μl. Then 100 ng of CDK2/CyclinA and BisIII, BisVIII, and BisX concentration as indicated were pre-incubated in kinase buffer for 10 min at 4 °C. The kinase reactions were started by adding 100 μm ATP, 2 μCi [γ-32P]ATP, and 10 μg of histone H1 and incubated for 20 min at 30 °C. All kinase reactions were stopped by adding 25 μl of 3× SDS sample buffer. Samples were analyzed by SDS electrophoresis and autoradiography using phosphoimaging for quantification. IC50 calculations were performed with GraFit 5.0 (Erithacus, Horley, Surey, United Kingdom). BisIII, BisVIII, and BisX were converted with Corina (www2.chemie.uni-erlangen.de/software/corina) into three-dimensional structures. The structures were docked using LigandFit 4.8 (www.accelrys.com) into CDK2 (1AQ1). The LigandFit settings were optimized by redocking staurosporine into 1AQ1. A piecewise linear potential function was used for docking, generating 15 poses per ligand. All poses were rescored with the empirical scoring function X-score (sw16.im.med.umich.edu/software/xtool/manual/). Prior to covalent coupling to chromatography beads, structural aspects of binding had to be considered to identify bisindolylmaleimide derivatives with suitable functional moieties that would allow inhibitor immobilization without abrogating protein kinase interaction. The scaffold of bisindolylmaleimides is structurally similar to the indolocarbazole kinase inhibitor staurosporine. We therefore assumed a binding mode for bisindolylmaleimides similar to the well-characterized staurosporine orientation known from a variety of reported protein kinase co-crystal structures (19Lawrie A.M. Noble M.E. Tunnah P. Brown N.R. Johnson L.N. Endicott J.A. Protein kinase inhibition by staurosporine revealed in details of the molecular interaction with CDK2..Nat. Struct. Biol. 1997; 4: 796-801Google Scholar, 20Underwood K.W. Parris K.D. Federico E. Mosyak L. Czerwinski R.M. Shane T. Taylor M. Svenson K. Liu Y. Hsiao C.L. Wolfrom S. Maguire M. Malakian K. Telliez J.B. Lin L.L. Kriz R.W. Seehra J. Somers W.S. Stahl M.L. Catalytically active MAP KAP kinase 2 structures in complex with staurosporine and ADP reveal differences with the autoinhibited enzyme..Structure. 2003; 11: 627-636Google Scholar, 21Zhu X. Kim J.L. Newcomb J.R. Rose P.E. Stover D.R. Toledo L.M. Zhao H. Morgenstern K.A. Structural analysis of the lymphocyte-specific kinase Lck in complex with non-selective and Src family selective kinase inhibitors..Structure. Fold. Des. 1999; 7: 651-661Google Scholar). This comparison enabled us to identify the three commercially available bisindolylmaleimide derivatives III, VIII, and X (BisIII, VIII, and X) as interesting candidates for covalent immobilization on chromatography beads. They have a primary alkylamino moiety in common, which is most likely accessible from the outside solvent and should thereby permit functional immobilization on activated support materials. The structures of these derivatives in comparison with the closely related GF109203X (also known as bisindolylmaleimide I) used in most signaling studies are shown in Fig. 1. Equal quantities of each of the three derivatives were then covalently coupled to epoxy-activated Sepharose to generate the respective BisIII, BisVIII, and BisX affinity matrices. As BisIII is the most closely related analogue of the widely used PKC inhibitor GF109203X, the corresponding BisIII matrix was used for the preparative purification of cellular targets relevant for pharmacological analysis employing bisindolylmaleimides. The only structural distinction between the two compounds is that BisI contains a dimethylaminepropyl group instead of the corresponding primary aminepropyl moiety of BisIII (Fig. 1). For the preparative target identification experiment, we loaded total lysate from 2.5 × 109 HeLa cells on a column containing immobilized BisIII affinity material. After several washing steps, bound proteins were specifically eluted by a combination of ATP and free BisIII in the elution buffer. Fractions containing eluted proteins were pooled and separated by 16-BAC/SDS gel electrophoresis. Coomassie-stained protein spots were cut out and analyzed by mass spectrometry (Fig. 2). Several of the identified cellular binding proteins were protein kinases known to be inhibited by bisindolylmaleimide compounds such as different PKC isozymes, Rsk2, and both GSK3 isoforms. These results provided various positive controls for the functionality of the purification method (Fig. 2) (7Davies S.P. Reddy H. Caivano M. Cohen P. Specificity and mechanism of action of some commonly used protein kinase inhibitors..Biochem. J. 2000; 351: 95-105Google Scholar). Notably, the Rho-dependent protein kinase 1 (ROCK1) was identified in the high-molecular-mass region, although the kinase activity of the closely related ROCK2 was previously shown to be rather insensitive to 1 μm BisIII in vitro (7Davies S.P. Reddy H. Caivano M. Cohen P. Specificity and mechanism of action of some commonly used protein kinase inhibitors..Biochem. J. 2000; 351: 95-105Google Scholar). Therefore, ROCK1 is likely to represent a cellular protein with a relatively weak affinity for immobilized BisIII ligand. In addition, we identified SLK and calmodulin-dependent kinase IIγ (CaMKIIγ) as new putative protein kinase targets of bisindolylmaleimide inhibitors. Moreover, some of the most prominent protein spots contained two non-protein kinase targets, which were AK and NQO2. A complete list of all identified BisIII-interacting proteins is shown in Table I.Table ISpot no.Protein nameGi no.Mr (kDa)1NQO2gi 5822324258212Not identified––3Lactate dehydrogenase Bgi 4557032366384Glycerinaldehyde-3-phosphate dehydrogenasegi 31645360545GSK3βgi 20455502467446GSK3βgi 20455502467447GSK3αgi 11995474509978Adenosine kinasegi 6840802405459Adenosine kinasegi 68408024054510GSK3βgi 204555024674411GSK3βgi 204555024674412Eukaryotic translation elongation factor 1αgi 45034715014113Enolase 1gi 45035714716914HSP90αgi 131291508466015GSK3αgi 119954745099716GSK3αgi 119954745099717β-Tubulingi 180887194967218α-Tubulingi 143893094989519Glucose-6-phosphate dehydrogenasegi 10853145912620Proline-4-hydroxylasegi 200701255711621CaMKIIγgi 4227695836522Pyruvate kinase, M1 isoenzymegi 201782965793723HSP70.1gi 4623257005224HSP73gi 57298777089825PKCδgi 147359457750526PKCαgi 45060677676427Rsk2gi 47590508373628β-tubulingi 180887194967229HSP90αgi 131391508466030Eukaryotic translation elongation factor 2gi 45034839533831Not identified––32α-Glucosidase II, α subunitgi 2136145610943833HSP70RYgi 62268699430034Not identified––35Leucine-rich proteingi 1895920214520136Valyl-tRNA synthetase 2gi 545415814047637ROCK1gi 667775915817138Carbamoyl-phosphate synthase (ammonia) precursorgi 8701816483039Not identified––40Glutamyl-prolyl tRNA synthetasegi 1615894816296941Not identified––42SLKgi 766199413279743Myosingi 1266778822653244Not identified–– Open table in a new tab To test whether small structural variations of the immobilized bisindolylmaleimides would affect the pattern of specifically retained cellular proteins, HeLa cell lysate (from 1.5 × 106 cells per sample) was used for in vitro associations on an analytical scale with either control beads or the closely related BisIII, BisVIII, and BisX matrices as affinity purification reagents. After incubation and several washing steps, bound proteins were eluted from the beads, separated by SDS-PAGE, and visualized by silver staining (Fig. 3A). Strikingly, small structural changes on the bisindolylmaleimide scaffold of the immobilized ligand strongly affected the affinity matrices’ selectivity for cellular targets, as evident from the both qualitative and quantitative differences in specific protein binding (Fig. 3A). For example, the BisVIII matrix weakly and the BisX matrix strongly interacted with a prominent 32-kDa protein not retained by either BisIII or control beads. Subsequent batch purification of this protein using freshly prepared lysate from 4 × 107 HeLa cells as starting material yielded Coomassie-stainable quantities of the BisVIII- and BisX-interacting protein, which were then excised from a one-dimensional SDS gel and identified as the serine/threonine kinase CDK2. In addition, this “semi-preparative” batch purification approach using either BisIII, BisVIII, or BisX beads also permitted the isolation and mass spectrometric detection of Rsk2, PKCα, PKCδ, GSK3, and NQO2, which were among the most prominent BisIII-interacting proteins purified by column chromatography and subsequent 16-BAC/SDS-PAGE from 2.5 × 109 HeLa cells (compare Fig. 3A with Fig. 2). In addition, we analyzed the selectivities of the related bisindolylmaleimide affinity matrices by immunblot analysis with antibodies specific for Rsk1, PKCα, GSK3α/β, CDK2, RICK, and p38. Immunoblots against RICK and p38 were included as controls, and both kinases did not interact with any of the immobilized bisindolylmaleimide compounds (Fig. 3B). As further shown in Fig. 3B, none of the other kinases interacted nonspecifically with control beads. PKCα and GSK3α/β were depleted from the lysate and retained on the three different affinity matrices to a similar extent." @default.
• W2080484776 created "2016-06-24" @default.
• W2080484776 creator A5001032583 @default.
• W2080484776 creator A5031338205 @default.
• W2080484776 creator A5053972254 @default.
• W2080484776 creator A5086644018 @default.
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• W2080484776 date "2004-05-01" @default.
• W2080484776 modified "2023-10-16" @default.
• W2080484776 title "Proteome-wide Identification of Cellular Targets Affected by Bisindolylmaleimide-type Protein Kinase C Inhibitors" @default.
• W2080484776 cites W1528985769 @default.
• W2080484776 cites W1755902553 @default.
• W2080484776 cites W1767727568 @default.
• W2080484776 cites W1911507478 @default.
• W2080484776 cites W1969416735 @default.
• W2080484776 cites W1972747946 @default.
• W2080484776 cites W1972844855 @default.
• W2080484776 cites W1983255121 @default.
• W2080484776 cites W1993860023 @default.
• W2080484776 cites W1995367007 @default.
• W2080484776 cites W2005309570 @default.
• W2080484776 cites W2019293169 @default.
• W2080484776 cites W2027828420 @default.
• W2080484776 cites W2033751505 @default.
• W2080484776 cites W2034611095 @default.
• W2080484776 cites W2051064724 @default.
• W2080484776 cites W2073206600 @default.
• W2080484776 cites W2076718655 @default.
• W2080484776 cites W2081070994 @default.
• W2080484776 cites W2090518535 @default.
• W2080484776 cites W2101447126 @default.
• W2080484776 cites W2105461348 @default.
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