horylation, ubiquitylation, SUMOylation, acetylation, and glycosylation. These PTMs can dictate protein activity, function, fate, and subcellular localization. A highthroughput, unbiased, and cost-effective method that allows for global measurement of different types of PTMs in a given cell or LBH589 biological activity tissue could have a huge impact on the study of 
protein dynamics. Our microarray based approach would specifically work to meet this need. We view this method as a parallel to the DNA microarray technology. The latter uses in vitro hybridization on a microarray to measure mRNA abundance in cell extracts, whereas ours uses in vitro phosphorylation of specific substrates on a protein microarray to profile kinase activity in cell lysates. Also similar to DNA microarrays, this new method can be further exploited by profiling phosphorylomes at multiple time points and with different cell/ tissue types, thus, providing a dynamic global picture of the rapid changes in kinase activity that occur both in cellulo and in vivo. This method is also potentially amenable to the measurement of other PTM enzyme families, such as ubiquitin and SUMO E3 ligases, acetyltransferases, and proteases. Further, the method is capable of examining different sample types, developmental stages, and physiological states. We and others have demonstrated that novel biological functions and pathways can be discovered from the use of functional protein microarrays when coupled with sophisticated bioinformatics analyses and in-depth in vivo characterization. This often occurs when a covalent PTM reaction is studied under highly simplified in vitro reaction conditions and testing a single PTM enzyme at a time. The successful examples include identification of substrates of protein kinases , acetyltransferases , and ubiquitin E3 ligases . However, these approaches risk the physiologically 7685384 relevant environment, such as the context of protein complexes, adapter proteins, inhibitors, and subcellular 16392774 compartmentalization. Also, such methods are not amenable to the characterization of global PTM activity. The use of cell/tissue lysates for profiling kinase activity not only allowed us to examine the dynamic changes in kinase activity as a whole, but also preserved the physiologically relevant microenvironments for the kinases to execute their activity on the functional protein microarrays. Furthermore, the relevant adapters and/or required scaffold proteins are also readily available in these reactions. Indeed, using HGF/c-Met activation in cultured cells and xenograft tissues as a proof-of-principle, we were able to reproducibly recover differentially phosphorylated proteins that are known to be phosphorylated upon activation of the HGF/cMet signaling pathway. The fidelity of the method was validated by the recovery of a high percentage of known downstream targets associated with HGF-induced cell growth, cell cycle progression, and cell survival, including AKT and MAPK. More importantly, the discovery and confirmation of novel components downstream of the HGF/c-Met signaling pathway demonstrates the usefulness of this method. Our findings underscore the complexity of the signaling events that mediate the phenotypes associated with HGF and tumorigenesis. The ability to monitor the phosphorylation of these proteins simultaneously should provide a valuable technology for network analyses and drug target discovery. Although the use of cell lysates to discover kinase-substrate relationships ha
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