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

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... ified 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. Molecular & Cellular Proteomics 4: 673-682, 2005. 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 (1-4). 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 ϳ3% of the coded proteome) with a unique substrate range for each kinase (5). 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) 1 pathways describes a three-tier modular structure involving sequential activation from module to module (6). 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 (7) . 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 (8). 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 (9). Conversely, other models were predicated on the use of MKK6 as a selective activation source for p38 kinases (10, 11). 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 From the ‡Molecular Medicine Program,