a proteomic approach for comprehensively screening substrates of protein kinases such as rho-kinase全面的蛋白质组学方法筛选rho-kinase等基质的蛋白激酶.pdfVIP

A Proteomic Approach for Comprehensively Screening Substrates of Protein Kinases Such as Rho-Kinase 1 1 1 1 1,2 1 Mutsuki Amano , Yuta Tsumura , Kentaro Taki , Hidenori Harada , Kazutaka Mori , Tomoki Nishioka , 1,2 1 1 3 1,4 Katsuhiro Kato , Takeshi Suzuki , Yosuke Nishioka , Akihiro Iwamatsu , Kozo Kaibuchi * 1 Department of Cell Pharmacology, Graduate School of Medicine, Nagoya University, Nagoya, Japan, 2 Department of Cardiology, Graduate School of Medicine, Nagoya University, Nagoya, Japan, 3 Protein Research Network, Inc., Yokohama, Japan, 4 Japan Science and Technology Agency, CREST, Kawaguchi, Japan Abstract Background: Protein 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 Findings: We 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,