Abstract
BackgroundProtein kinases are major components of signal transduction pathways in multiple cellular processes. Kinases directly interact with and phosphorylate downstream substrates, thus modulating their functions. Despite the importance of identifying substrates in order to more fully understand the signaling network of respective kinases, efficient methods to search for substrates remain poorly explored.Methodology/Principal FindingsWe combined mass spectrometry and affinity column chromatography of the catalytic domain of protein kinases to screen potential substrates. Using the active catalytic fragment of Rho-kinase/ROCK/ROK as the model bait, we obtained about 300 interacting proteins from the rat brain cytosol fraction, which included the proteins previously reported as Rho-kinase substrates. Several novel interacting proteins, including doublecortin, were phosphorylated by Rho-kinase both in vitro and in vivo.Conclusions/SignificanceThis method would enable identification of novel specific substrates for kinases such as Rho-kinase with high sensitivity.
Highlights
Protein phosphorylation is one of the most ubiquitous and essential mechanisms mediating intracellular signal transduction in various cellular processes
About 500 protein kinases are encoded in the human genome, where these are mainly divided into two groups, Ser/Thr protein kinases and Tyr protein kinases
Affinity column chromatography of Rho-kinase To screen potential substrates of Rho-kinase, we examined whether the active catalytic fragment of Rho-kinase (Rho-kinasecat) interacts with its substrates by affinity column chromatography
Summary
Protein phosphorylation is one of the most ubiquitous and essential mechanisms mediating intracellular signal transduction in various cellular processes. Kinases recognize and phosphorylate their specific substrates and modulate their functions. Numerous proteins are continuously and dynamically phosphorylated and dephosphorylated under the control of complex signaling networks. Comprehensive screening of substrates for kinases is necessary to increase understanding of the signaling networks in which protein kinases participate. It remains difficult to efficiently screen the physiological substrates of protein kinases. Protein kinases are major components of signal transduction pathways in multiple cellular processes. Kinases directly interact with and phosphorylate downstream substrates, modulating their functions. Despite the importance of identifying substrates in order to more fully understand the signaling network of respective kinases, efficient methods to search for substrates remain poorly explored
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