Proteome-wide Identification of Cellular Targets Affected by Bisindolylmaleimide-type Protein Kinase C Inhibitors

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. M. H. efficient proteomics method to identify the cellular targets of protein kinase Natl. Acad. Sci. U. S. A. 2003; 100: Scholar). In this affinity chromatography was performed highly biochemical conditions and led to the identification of a of previously unknown SB 203580 targets, that this used is less selective for p38 than (14Godl K. Wissing J. Kurtenbach A. Habenberger P. Blencke S. Gutbrod H. Salassidis K. Stein-Gerlach M. Missio A. M. H. efficient proteomics method to identify the cellular targets of protein kinase Natl. Acad. Sci. U. S. A. 2003; 100: Scholar). Here, we have adapted this proteomics approach to identify the cellular targets of In addition to protein kinases known to be affected by these we various other specifically interacting enzymes and could by in vitro binding and activity assays as novel targets of bisindolylmaleimide inhibitors. cyclin-dependent kinase 2 (CDK2) interaction with bisindolylmaleimides was dramatically affected through chemical variation of the immobilized inhibitors. differential binding could be correlated with the in vitro CDK2 kinase activity and their to molecular docking Cell and were from and protein were obtained from and were obtained from was obtained from was from was obtained from other reagents were obtained from used in this study mouse from mouse mouse and mouse was from The for Ste20-related kinase and adenosine kinase were from and The was to a the was to a and into a H. Blencke S. Habenberger P. Kurtenbach A. J. Wissing J. A. M. Identification of and as the cellular protein kinases Scholar, J. N.S. K. M. T. of adenosine kinase to and Natl. Acad. Sci. U. S. A. Scholar, E. K. S. H. and of a novel 2000; Scholar). three bisindolylmaleimide compounds were in were using their at and and at in the was in 2 of BisIII, BisVIII, or in of was performed at in the three with were blocked with 1 Subsequent were performed to the To the control was directly with 1 11 and as The were at in the or cells were cultured in with cells were as previously H. Blencke S. Habenberger P. Kurtenbach A. J. Wissing J. A. M. Identification of and as the cellular protein kinases Scholar). growth of cells was as S. A. H. of in the epidermal growth receptor a for selective kinase Biol. Chem. 2003; Scholar). and cells were in 1 1 3 sodium 1 1 and co-factors in vitro were by and to 1 of the high were with of bisindolylmaleimide for 3 at the were with of 1 and with of both were performed and the co-factor were to In the to specific and binding to and the co-factors were not in the and the binding of to was and the co-factors were from the and and the was at of PKCα binding from the was co-factors were were also to in the which was proteins were by of the affinity in proteins were to membrane and with the The binding using 16-benzyldimethyl-n-hexadecylammonium and mass analysis were as with the that 3 PS, and were and was from the (14Godl K. Wissing J. Kurtenbach A. Habenberger P. Blencke S. Gutbrod H. Salassidis K. Stein-Gerlach M. Missio A. M. H. efficient proteomics method to identify the cellular targets of protein kinase Natl. Acad. Sci. U. S. A. 2003; 100: Scholar). Moreover, the was with 3 PS, and after activity and inhibition were by the of at and In this we used as an for the and for the of The were performed in 1 of quinone-reductase type 2 MTT, and of in the presence of different a we the values for the different The was by the different its from cells to a were to with protein for 3 at the were three with of and with of kinase 1 and kinase activity assays were performed in a of to of was with of kinase the and for at The kinase were by addition of 1 and of protein and for at CDK2 assays were performed in a of of and BisIII, BisVIII, and as were in kinase for at The kinase were by 2 and of and for at kinase were by of were by and using for IC50 were performed with BisIII, BisVIII, and were with into The were using into CDK2 The were by into potential function was used for were with the function to to chromatography aspects of binding to be to identify bisindolylmaleimide derivatives with suitable functional that inhibitor immobilization protein kinase The of bisindolylmaleimides is to the kinase inhibitor We a binding for bisindolylmaleimides to the known from a of reported protein kinase M.E. P. Protein kinase inhibition by in of the molecular interaction with Biol. Scholar, E. L. T. M. K. S. M. K. J. kinase 2 in with and reveal with the 2003; Scholar, H. Structural analysis of the kinase in with and family selective kinase Des. 1999; 7: Scholar). to identify the three bisindolylmaleimide derivatives III, VIII, and X VIII, and as for immobilization on chromatography beads. have a in which is most from the and thereby functional immobilization on The of these derivatives in with the closely related GF109203X known as bisindolylmaleimide used in most studies are in of of the three derivatives were to to the BisIII, BisVIII, and affinity As is the most closely related of the used PKC inhibitor GF109203X, the was used for the purification of cellular targets relevant for pharmacological analysis The only the two compounds is that a of the of the target identification we from cells on a immobilized affinity several proteins were specifically by a combination of and in the proteins were and by protein were and by mass spectrometry of the cellular binding proteins were protein kinases known to be by bisindolylmaleimide compounds such as different PKC isozymes, and both These results various for the of the purification method (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). the Rho-dependent protein kinase 1 was in the the kinase activity of the closely related was previously to be to 1 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). is to a cellular protein with a affinity for immobilized In we and calmodulin-dependent kinase as protein kinase targets of bisindolylmaleimide inhibitors. Moreover, some of the most protein two non-protein kinase targets, which were and of proteins is in synthase in a To small of the immobilized bisindolylmaleimides the of specifically cellular cell cells was used for in vitro on an with either control or the closely related BisIII, BisVIII, and as affinity purification and several proteins were from the by and by small changes on the bisindolylmaleimide of the immobilized ligand affected the affinity selectivity for cellular targets, as from the both and in specific protein binding the and the interacted with a protein not by either or control beads. Subsequent purification of this protein using from cells as of the and which were from a and as the serine/threonine kinase In this purification approach using either BisIII, BisVIII, or also the and mass of GSK3, and NQO2, which were the most proteins by chromatography and from cells with In we the of the related bisindolylmaleimide affinity by analysis with specific for CDK2, RICK, and and p38 were as and both kinases not with of the immobilized bisindolylmaleimide compounds As further in of the other kinases interacted with control beads. PKCα and were from the and on the three different affinity to a extent. In binding was more for the and with the affinity CDK2 not bind to immobilized BisIII, a affinity for and interacted with the Thus, the and analysis of the and protein of the BisIII, BisVIII, and a and of the selectivity and of the used bisindolylmaleimide compounds their cellular targets. The is to quinone by its in to be was recently to the activation of the cancer in cells high of this enzyme S. the to and Pharm. Des. 2003; Scholar, K. A. M. R. R. of 2 to of and to Biol. Chem. Scholar). To that with the also into inhibition of activity by in we performed a using as an for K. R. Sun P. D. S. of a Scholar). In this to its The the of MTT, which can be measured at only a affinity for To with previously reported activity we the of for and a of which is in with earlier results K. R. Sun P. D. S. of a Scholar). the of activity at different and in the presence of different of or we could that the inhibition of activity is with to The of with to inhibition was as in the of Thus, we could as a cellular bisindolylmaleimide albeit of its activity at than for potent inhibition of known protein kinase targets. The non-protein kinase adenosine to and was of the cellular proteins with the is a target Inhibition of its activity the of adenosine from and adenosine is known to as a with potent and inhibition of activity is as a therapeutic for the treatment of and and several potent inhibitors of activity have been reported A. S. K. J. H. K. and of as potent adenosine kinase Med. Chem. Scholar, kinase inhibitors. enzyme and activity of Med. Chem. 2000; Scholar, III, kinase inhibitors. enzyme and activity of Med. Chem. 2000; Scholar). To binding to a of was expressed in Cell was with either control or to of proteins with As in expressed specifically interacted with and was not by the control Moreover, the activity of recombinant with an IC50 of 1 in the presence of not Thus, these establish as a cellular target of bisindolylmaleimide inhibitors. mass spectrometry analysis of proteins SLK, a of the family of serine/threonine kinases, as a potential protein kinase target of this inhibitor class several including a protein kinase and was previously as of the and of by SLK, a Ste20-related 1999; Scholar, K. P. J. 3 of the Ste20-related kinase and an kinase and an Biol. 2000; Scholar). To as a we the binding of a of to the by the affinity was by with As in specifically interacted with the binding to the control was specific binding was also observed for a of that only the protein kinase not Moreover, potently activity with an IC50 of in vitro in the presence of and as a pharmacological inhibitor of has been identification of as such a a for further analysis of As in in CDK2 binding were observed for the three different bisindolylmaleimide affinity To these with the of the CDK2 activity to inhibition in we performed kinase assays using as and the IC50 values for the closely related BisIII, BisVIII, and In with the results from the binding was the most potent of CDK2, with only of the inhibitor kinase inhibition in vitro BisVIII, which only CDK2 in its immobilized CDK2 with a IC50 of the of CDK2 binding to the correlated with a further of inhibitor in an IC50 of 2 for the Importantly, these kinase inhibition results the in vitro binding immobilized BisIII, BisVIII, and derivatives as an to inhibitor and for a target of Moreover, this parallel affinity purification approach be to other inhibitor and the proteome-wide of inhibitor selectivity relative to closely related derivatives of the same compound class. We used molecular analysis to the of the different inhibitor measured for the related bisindolylmaleimide the bisindolylmaleimide is to the of we to BisIII, BisVIII, and into the published of CDK2 in with M.E. P. Protein kinase inhibition by in of the molecular interaction with Biol. Scholar). As for in the to BisIII, BisVIII, and well into the nucleotide-binding of of two to the which the two of the kinase The two were in and to previously for and The of for the three bisindolylmaleimides correlated with their measured in vitro CDK2 Moreover, the for was than for BisX, with the reported inhibition of CDK2 by with a IC50 M.E. P. Protein kinase inhibition by in of the molecular interaction with Biol. Scholar). Moreover, ligand was from to to BisX, their at the more in the same Importantly, both effects the binding by the ligand into the nucleotide-binding pocket. the docking approach that the of the bisindolylmaleimide compounds the protein and confirmed that the to an not kinase binding. bisindolylmaleimides that are this to a can be used for binding and identification of cellular targets. affinity purification of cellular bisindolylmaleimide targets the presence of high binding to the these the co-factors DAG, and not binding to the as for in vitro were performed at a of PKCα specifically interacted with only DAG, and were not in the of these co-factors To the affinity can be used as a to the cellular activation of a target we cells after different of is known to cellular activity through the activation of the protein kinase S.A. J. of ribosomal S6 kinase kinase docking and Biol. 2003; Scholar). were to in vitro with to and with As in from cells to the of binding was and in a cellular were for that the binding to correlated with the regulation of cellular activity these that the affinity can also be adapted for and the specific of protein kinases either in vitro or in Bisindolylmaleimide inhibitors of protein kinases have been used in signal transduction the tools to their target selectivity have been and biased (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). In this we have established a of functional bisindolylmaleimide affinity which to identify previously unknown targets of this inhibitor class. To this we used a proteomics approach to recently affinity purification for p38 cellular proteins (14Godl K. Wissing J. Kurtenbach A. Habenberger P. Blencke S. Gutbrod H. Salassidis K. Stein-Gerlach M. Missio A. M. H. efficient proteomics method to identify the cellular targets of protein kinase Natl. Acad. Sci. U. S. A. 2003; 100: Scholar). results with the bisindolylmaleimide demonstrate that this biochemical can be adapted for to the of the p38 inhibitor SB In as established for CDK2 in differential binding to closely related inhibitor a for the relative of the inhibitor compounds a kinase Due to the to identify and the cellular proteins targeted by a of inhibitor analogues based on the same chemical such a proteomics deliver alternative drug targets and their relative to other within the same experimental this type of application has the potential to a relevant in drug J. and with protein kinase inhibitors for cancer Rev. Drug Discov. 2003; 2: Scholar, S. S. A. and Chem. Biol. 2003; 7: Scholar). The of proteomics method is and protein kinases expressed at to 1000 cell can be to as from mammalian cells are used S.P. R. of proteome analysis Natl. Acad. Sci. U. S. A. 2000; Scholar). Moreover, be to further the of this affinity approach by a of to for by with analysis of protein through chromatography to mass spectrometry III, method for the comprehensive analysis of membrane 2003; Scholar). on these the and identification of kinase inhibitor targets of a or less is a which be in the In this we have used cell from cells as a protein for the identification of Importantly, bisindolylmaleimide compounds used for immobilization are Thus, established purification can be to PKC in other cell or and thereby a comprehensive this used class of signal transduction inhibitors its effects on the molecular We Blencke and for