Active Kinase Proteome Screening Reveals Novel Signal Complexity in Cardiomyopathy

Recent advances in the characterization of the phosphoproteome have been limited to measuring phosphorylation statuses, which imply but do not measure protein kinase activity directly. As such, the ability to screen, compare, and define multiple protein enzymatic activities across divergent samples remains a daunting challenge in proteomics. Here, we describe a gel-based kinase assay coupled to MS identification as an approach to map global kinase activity and assign pathway architecture to specified biologic contexts. We demonstrate the utility of this method as a platform for the comparison of proteomes based on differences in both kinase activities and for use in the de novo substrate identification for individual kinases. This approach allowed us to map the signal perturbations in the post-natal heart that were associated with activation of a myopathic cascade as mediated by the mitogen-activated protein kinase MKK6 and established the novel observation that MKK6 promotes the development of cardiomyopathy through multiple substrate interactions. Recent advances in the characterization of the phosphoproteome have been limited to measuring phosphorylation statuses, which imply but do not measure protein kinase activity directly. As such, the ability to screen, compare, and define multiple protein enzymatic activities across divergent samples remains a daunting challenge in proteomics. Here, we describe a gel-based kinase assay coupled to MS identification as an approach to map global kinase activity and assign pathway architecture to specified biologic contexts. We demonstrate the utility of this method as a platform for the comparison of proteomes based on differences in both kinase activities and for use in the de novo substrate identification for individual kinases. This approach allowed us to map the signal perturbations in the post-natal heart that were associated with activation of a myopathic cascade as mediated by the mitogen-activated protein kinase MKK6 and established the novel observation that MKK6 promotes the development of cardiomyopathy through multiple substrate interactions. Serial modification of proteins is a common mechanism for eukaryotic cellular adaptation. Within this context, protein phosphorylation is believed to be the most common form of modification. Many excellent methods describing the purification and identification of phospho-proteins are now available and have significantly contributed to our understanding of cellular regulation through signal transduction (1Knight Z.A. Schilling B. Row R.H. Kenski D.M. Gibson B.W. Shokat K.M. Phosphospecific proteolysis for mapping sites of protein phosphorylation.Nat. Biotechnol. 2003; 21: 1047-1054Google Scholar, 2Affolter M. Watts J.D. Krebs D.L. Aebersold R. Evaluation of two-dimensional phosphopeptide maps by electrospray ionization mass spectrometry of recovered peptides.Anal. Biochem. 1994; 223: 74-81Google Scholar, 3Zhou H. Watts J.D. Aebersold R. A systematic approach to the analysis of protein phosphorylation.Nat. Biotechnol. 2001; 19: 375-378Google Scholar, 4Ficarro S.B. McCleland M.L. Stukenberg P.T. Burke D.J. Ross M.M. Shabanowitz J. Hunt D.F. White F.M. Phosphoproteome analysis by mass spectrometry and its application to Saccharomyces cerevisiae.Nat. Biotechnol. 2002; 20: 301-305Google Scholar). Nevertheless, phosphorylation status alone does not unambiguously characterize the kinetic steps of signaling cascades, namely the activity of protein kinases. Indeed, there are an estimated 519 kinase-related gene products in the human genome (representing ∼3% of the coded proteome) with a unique substrate range for each kinase (5Manning G. Whyte D.B. Martinez R. Hunter T. Sudarsanam S. The protein kinase complement of the human genome.Science. 2002; 298: 1912-1934Google Scholar). As such, successfully mapping these steps in any biologic model remains a daunting challenge for proteomics. In addition to the limitation imposed by current screening methodologies, the accurate reconstruction of signaling networks may also be hindered by the prevailing hypothesis that describes signaling cascades as insulated and linear events. For example, the annotation of mitogen-activated protein kinase (MAPK) 1The abbreviations used are: MAPK, mitogen-activated protein kinase; MKKK, MAPK kinase kinase; MKK, MAPK kinase; KSE, kinase sweep; KSS, kinase substrate; MBP, myelin basic protein; 2D, two-dimensional; PAK, p21 activated kinase; AMPK, 5′-AMP activated kinase; RAK, Rho-associated kinase; IP, immunoprecipitation; CRMP, collapsin response mediator protein; PKN, protein kinase N. pathways describes a three-tier modular structure involving sequential activation from module to module (6Garrington T.P. Johnson G.L. Organization and regulation of mitogen-activated protein kinase signaling pathways.Curr. Opin. Cell Biol. 1999; 11: 211-218Google Scholar). The p38 MAPK pathway is a typical representation of this kinase family. This pathway contains a phosphorylation sequence initiated by a MAPK kinase kinase (MKKK), which activates a MAPK kinase (MKK), which then in turn activates a MAPK (p38), with the MAPK then serving as the effector enzyme to stimulate or repress the activity of corresponding protein substrates by targeted phosphorylation (7Johnson G.L. Lapadat R. Mitogen-activated protein kinase pathways mediated by ERK, JNK, and p38 protein kinases.Science. 2002; 298: 1911-1912Google Scholar). Considerable experimental evidence supports the basic premise of the linear MKK/p38 MAPK pathway, yet ascribing all phenotypic outcomes exclusively to the activity of the effector kinase is most likely an oversimplification. In this regard, the relationship between activation of p38 signal cascades and the development of cardiac pathology (hypertrophy and dilatation) provides an interesting venue in which to model signaling events and their biologic response. To date, numerous studies have implicated p38 activation as a requisite step in the development of post-natal cardiac dilatation and or hypertrophy (8Han J. Molkentin J.D. Regulation of MEF2 by p38 MAPK and its implication in cardiomyocyte biology.Trends Cardiovasc. Med. 2000; 10: 19-22Google Scholar). In a portion of these studies, a clear relationship was established for p38 and its responsive transcription factor substrates, the substrates being directly validated as p38 targets and as hypertrophic agonists (9Sugden P.H. Signalling pathways in cardiac myocyte hypertrophy.Ann. Med. 2001; 33: 611-622Google Scholar). Conversely, other models were predicated on the use of MKK6 as a selective activation source for p38 kinases (10Braz J.C. Bueno O.F. Liang Q. Wilkins B.J. Dai Y.-S. Parsons S. Braunwart J. Glascock B.J. Klevitsky R. Kimball T.F. Hewett T.E. Molkentin J.D. Targeted inhibition of p38 MAPK promotes hypertrophic cardiomyopathy through upregulation of calcineurin-NFAT signaling.J. Clin. Invest. 2003; 111: 1475-1486Google Scholar, 11Liao P. Georgakopoulos D. Kovacs A. Zheng M. Lerner D. Pu H. Saffitz J. Chien K. Xiao R.-P. Kass D.A. Wang Y. The in vivo role of p38 MAP kinases in cardiac remodeling and restrictive cardiomyopathy.Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 12283-12288Google Scholar). As predicted, these later models displayed phenotypic alterations of the heart, yet the changes were solely attributed to MKK6 activation of p38. Here, we report the application of a novel proteomic screening assay to interrogate the complexity of MAPK-induced cardiac adaptation. This assay combined the utility of solid-phase kinase assays with the resolving power of nanoflow-LC-MS/MS. We have termed this assay kinase sweep (KSE)/MS. Using this assay with gel-embedded substrates, we identified a broad range of kinase activities in lysates derived from the hearts of transgenic mice expressing activated MKK6. Second, we used a modification in the assay which we termed kinase substrate (KSS)/MS, to identify a range of probable substrates for MKK6, suggesting that this kinase may influence hypertrophic adaptation through a complex signaling architecture. Together, these results provide a proof of concept that KS/MS (KSE/MS and KSS/MS) is applicable for large scale identification and analyses of both protein kinase activities and their effective substrate range. The full-length murine MKK6 gene carrying mutations at S207E and T211E (MKK6EE) was subcloned into a pBluescript backbone containing a murine α-myosin heavy chain promoter (a gift from Jeffrey Robbins, Children’s Hospital, Cincinnati, OH) and a 600-bp fragment of the human growth hormone poly(A) tail. The transgenic mice were derived as described previously (12Guy L.G. Kothary R. DeRepentigny Y. Delvoye N. Ellis J. Wall L. The β-globin locus control region enhances transcription of but does not confer position-independent expression onto the lacZ gene in transgenic mice.EMBO J. 1996; 15: 3713-3721Google Scholar). Hearts from MKK6EE and wild-type mice were excised and snap-frozen in liquid nitrogen. Total protein was extracted in modified RIPA buffer (50 mm Tris-HCl, pH 7.4, 150 mm NaCl, 1 mm EDTA, 1% Nonidet P-40, 1% glycerol) containing 1.0 μg/ml aprotinin, 0.5 μg/ml leupeptin, 0.7 μg/ml pepstatin, and 50 μg/ml PMSF and supplemented with 20 mm NaF and 5 mm sodium orthovanadate. Tissues were Dounce homogenized on ice and rotated at 4° C for 30 min. Samples were centrifuged at 10,000 × g, and soluble fractions were stored at −80° C. Total protein was measured using the bicinchoninic acid protein assay (Pierce, Rockford, IL). Samples were prepared for IEF using standard protocols as previously described (13Fernando P. Kelly J.F. Balazsi K. Slack R.S. Megeney L.A. Caspase 3 activity is required for skeletal muscle differentiation.Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 11025-11030Google Scholar). Three hundred to 400 μg of total protein was separated on immobilized pH 4–7 gradient strips (Bio-Rad, Hercules, CA). For identification of protein kinases, 0.1 mg/ml myelin basic protein (MBP) was mixed with a 10–12% polyacrylamide solution (30%T:2.6%C). In some instances, 0.1 mg/ml histone H1 or both MBP and histone H1 (0.05 mg/ml of each) were used. Gel-based kinase assays were performed as described previously for one-dimensional SDS-PAGE (14Kameshita I. Fujisawa H. A sensitive method for detection of calmodulin- dependent protein kinase II activity in sodium dodecyl sulfate-polyacrylamide gel.Anal. Biochem. 1989; 183: 139-143Google Scholar) with several modifications. All steps were performed at room temperature unless otherwise indicated. Following electrophoresis, gels were washed in three changes of a propanol-Tris buffer (50 mm Tris-HCl, pH 8.0, 20% 2-propanol). Gels were then soaked in three changes of a DTT-Tris buffer (50 mm Tris-HCL, pH 8.0, 5.0 mm DTT). A denaturing solution (50 mm Tris, pH 8.0, 20 mm DTT, 6.0 m guanidine-HCl) facilitated protein unfolding within the gel. Gel-bound proteins were refolded in a protein folding buffer (50 mm Tris-HCl, pH 8.0, 5.0 mm DTT, 0.04% Tween-40, 150 mm NaCl, and 5.0 mm MgCl2). The solution was changed four times and then once prior to an overnight incubation at 4 °C. Subsequent changes of protein folding buffer were carried out the following day (8–10 times). Prior to the kinase reaction, gels were soaked in two changes of pre-kinase reaction buffer (20 mm MOPS, pH 7.4, 25 mm β-glycerophosphate, 5 mm EGTA, 1 mm DTT, 1 mm sodium orthovanadate, and 50 mm MgCl2) for 30 min each. Gels were then incubated in a kinase buffer (20 mm MOPS, pH 7.4, 25 mm β-glycerophosphate, 5 mm EGTA, 1 mm DTT, 1 mm sodium orthovanadate including 5 μCi/μl [γ-32P]ATP) and 500 μm cold ATP for 1.5–2 h. Incubation was performed with occasional agitation. In order to minimize nonspecific background and precipitate incorporated radiolabel, gels were washed with several changes of a TCA wash solution (5% TCA, 1% sodium pyrophosphate). Finally, the gels were air-dried (Bio-Rad), and kinases were detected by autoradiography. Autophosphorylated proteins were excluded from analyses by running replicate gels that did not contain embedded substrate. The radiolabeled spots appearing on these gels were compared with spots on gels that embedded substrate. protein spots from both gels did not A to polyacrylamide substrate was used for the In this substrates for the kinase of are to the Following the electrophoresis, gels were as described for the incubation in a pre-kinase buffer as the was soaked for in a kinase buffer containing a activated kinase The was then washed and as from gel-based kinase assays were with their corresponding spots were excised and in 50 mm were and for using standard protocols R. T. in A of Scholar). All were using a mass coupled with a Samples were onto a × CA). were to a × for using a gradient of min and 5 min. The to the was The mass was to in the In order to the of kinases with the MBP were excluded from analyses using an in MBP The were the using the kinase analyses were performed using protein lysates from wild-type and MKK6EE mice as described previously (13Fernando P. Kelly J.F. Balazsi K. Slack R.S. Megeney L.A. Caspase 3 activity is required for skeletal muscle differentiation.Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 11025-11030Google Scholar). and analyses were performed as described previously (13Fernando P. Kelly J.F. Balazsi K. Slack R.S. Megeney L.A. Caspase 3 activity is required for skeletal muscle differentiation.Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 11025-11030Google Scholar). MKK6 and and and Cell were used as by the To to the of the signaling we to the post-natal heart as a To this we transgenic mice an activated MKK6 cardiac expression was by using the α-myosin heavy chain promoter A. J. analysis of cardiac gene 1994; Scholar, Wang L. Balazsi K. DeRepentigny Y. Kothary R. Megeney L.A. MEF2 is cardiac hypertrophy and is required for post-natal growth of the Biol. 1999; Scholar) MKK6EE mice at these analysis of MKK6EE mice evidence of a cardiac with and a in in comparison to wild-type A and In the from MKK6EE hearts were from wild-type we that of activated MKK6EE in the of kinase activity with or in of protein expression Indeed, MKK6 activation been to the substrate of MKK6 between the targets of and an that from the of MKK6 to in comparison to G. C. M. activation of p38 mitogen-activated protein kinase on signal Biol. 2000; Scholar). results that the signaling pathways in MKK6 activation were complex As such, was that the standard models for MAPK signaling were and the use of screening were required to identify the kinases and As a step in screening the we performed standard two-dimensional of cardiac protein lysates derived from wild-type and MKK6EE transgenic mice by analysis of these using a of to a in This screening approach differences between the yet the of this approach was to The in from the detection to be dependent on the of proteins and did not provide global or of not In the from this other proteomic did not provide a measure of protein on these we to a proteomic in which global kinase activity be coupled to kinase We previously that kinase activities of muscle protein lysates be following IEF and SDS-PAGE (13Fernando P. Kelly J.F. Balazsi K. Slack R.S. Megeney L.A. Caspase 3 activity is required for skeletal muscle differentiation.Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 11025-11030Google Scholar). we that alterations to this protein be to kinase activities and identification through the use of lysates were to IEF and SDS-PAGE with a of a kinase substrate to the in this The was with the use of a buffer to protein and of enzymatic gels were then to a modified kinase assay and then to identify kinase identification of the kinase then be using We have termed this assay As a proof of we MKK6 and p38 proteins to the with gels containing We proteins in these gels with mass and within for MKK6 and which kinase activity A and protein spots were then from the and identified by as MKK6 and p38 A and the MS analysis was using an MBP to any signal that may have from the of the KS/MS was to KS/MS A is The of in was and prepared for MS The corresponding MS for the is on the was to KS/MS analyses as in A. The from an of the in is on the For both A and the were using the Following the of these kinases in the we then the utility of this method for multiple kinase activities from protein Total protein lysates were extracted from wild-type and MKK6EE and kinase activity maps were identification of these proteins that the wild-type heart to a kinase activity with a cellular kinase activity for as protein kinase C and and p21 activated kinase and a kinase activity associated with the cellular A and The kinase activity that was from MKK6EE protein lysates was divergent from the using wild-type lysates For example, in addition to of MKK6 and MKK6EE lysates also enzymatic activity for three kinases of 5′-AMP activated kinase Rho-associated kinase and the kinase protein kinase To the from the screen, we use of standard kinase kinases were from wild-type and MKK6EE cardiac protein lysates and incubated with MBP in a in kinase The results of the kinase assay were with the results in the assay for each protein for and which the utility of in multiple kinase activities from and kinase activity maps The an enzymatic activity map that was with the for cardiac Nevertheless, of these kinases do not contain the requisite that are typical of MAPK substrates D. A. in MAPK signaling to transcription Biol. 2002; Scholar). is probable then that a portion of these kinases were to the hypertrophy and not MKK6 directly. to signaling alterations from we our kinase assay to identify probable kinase substrates for MKK6 this KS/MS protein lysates as the kinase MBP gels were then incubated with a activated kinase in an incubation buffer by MS identification of the protein we the with MKK6 and to identify corresponding substrates in cardiac protein The in phosphorylation of and identification for multiple substrates not an established for this effector kinase cardiac hypertrophy Wang L. Balazsi K. DeRepentigny Y. Kothary R. Megeney L.A. MEF2 is cardiac hypertrophy and is required for post-natal growth of the Biol. 1999; Scholar). identified and kinases as targeted substrates A and these also identified other probable targets for MKK6 in cardiac protein including and control proteins A and we also identified and proteins to be by kinases C and status for these MKK6 targets were using a of for and analysis in wild-type and MKK6EE heart The phosphorylation of cellular proteins an mechanism for eukaryotic cellular adaptation. The pathways that these have a in signaling screening now available for the identification of have established their utility in as as the of cellular the current that signaling events on methods that linear and the of signal transduction cascades remains a and In this we describe an proteomic screening assay used to interrogate the complexity of MAPK Using this we demonstrate that MAPK in cardiac in an analysis of protein kinase activity using gel-based methods been previously (14Kameshita I. Fujisawa H. A sensitive method for detection of calmodulin- dependent protein kinase II activity in sodium dodecyl sulfate-polyacrylamide gel.Anal. Biochem. 1989; 183: 139-143Google Scholar, kinase assay as a method to identify kinase 2002; Scholar). In these instances, proteins were to one-dimensional based on mass through a polyacrylamide the to and kinase protein kinases were the of this assay provides several protein kinases may the Using an gel-based we incorporated a polyacrylamide coupled with mass to unambiguously identify protein kinases, termed these allowed us to between activated MKK6 murine model of cardiomyopathy several interesting and results following the application of from the activation of signaling p38 MAPK, we identified kinases that cellular and In is that as an step in in the context, activation of been implicated in events the development of cardiac hypertrophy R. N. J. protein kinase activity in hearts with 2001; Scholar). activity been to be a of hypertrophy S. Y. M. T. M. Y. N. inhibition of kinase the of hypertrophy to cardiac in heart Cell 2003; Scholar, M. H. T. J. Y. K. T. S. A. inhibition of hypertrophy in in on 2003; and been to activation of activity to hypertrophy in The PKN, gene transcription in through a response J. 2000; Scholar). In addition to the both and are to be of the of the method is the of substrate that is within the gel. MBP is a substrate for a of protein kinases R. M. G. of multiple in vivo phosphorylation sites in myelin basic Biol. Scholar, M. S. Krebs of multiple protein kinases the in protein phosphorylation that the in Biol. a of kinases have substrate In this the method may be with a For example, we both MBP and histone H1 in a of kinase activities were using the two substrates we were to a kinase substrate range A novel of the KS/MS approach is the ability to identify substrates of a protein termed IEF samples in a and a protein kinase in the kinase incubation we were to identify several substrates of MKK6. of these substrates are to be targeted by kinases yet this is the to imply that these proteins as MKK6 substrates C and of these proteins are to be in and have been to For example, is a protein that with and for into 2001; Scholar) and been to cardiac hypertrophy and and of and for 2001; Scholar). are that and a of proteins termed were identified as through of the L. To or not to in Scholar). been to have on cardiac by the of by cardiomyocyte T. H. A. N. J. K. M. M. Fujisawa H. H. of in cardiac through of its with and growth factor Scholar, T. H. A. N. M. K. M. H. of in cardiac development by Cell Biol. Scholar). Finally, is to that collapsin response mediator was also identified as an MKK6 substrate. proteins to and activity is to be by at in A. H. for in dependent J. Scholar). As such, is to that MKK6 may the of cardiomyocyte hypertrophy by targeted phosphorylation of these and is the phosphorylation and inhibition of these proteins that the of the hypertrophy of cardiac activity and regulation with pathways signaling hypertrophy and 2000; Scholar). The prevailing hypothesis for MAPK-induced cardiac hypertrophy describes a linear relationship in which the effector kinase as the of the hypertrophic signal by cardiac transcription factor activity O.F. Molkentin J.D. of kinases in cardiac hypertrophy and 2002; Scholar, J.D. signaling the cardiac hypertrophic response in with the Scholar). This model that the and that cardiomyocyte hypertrophy is the of effector kinase results challenge this model and that MAPK of cardiac hypertrophy from both effector dependent and activation of The application of KS/MS on the of proteins by and then by the of an using gel-based is a these methods and advances in proteomic have been with methods of by R. M. 2003; Scholar, H. D. for analysis in the 2002; Scholar, The application of mass spectrometry to Biotechnol. 2003; 21: Scholar). have the of through analyses including the detection of proteins and proteins within We are to the of the KS/MS by coupled to To date, proteomic in signaling have been limited to annotation of the protocols are based on the of from a protein by mass identification of the the of these outcomes to the phosphoproteome maps have that the KS/MS this signal protein to protein As such, application of the KS/MS provide a platform for signaling in complex biologic We for with

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