TY - JOUR
T1 - A proteomic approach for comprehensively screening substrates of protein kinases such as rho-kinase
AU - Amano, Mutsuki
AU - Tsumura, Yuta
AU - Taki, Kentaro
AU - Harada, Hidenori
AU - Mori, Kazutaka
AU - Nishioka, Tomoki
AU - Kato, Katsuhiro
AU - Suzuki, Takeshi
AU - Nishioka, Yosuke
AU - Iwamatsu, Akihiro
AU - Kaibuchi, Kozo
N1 - Copyright:
Copyright 2013 Elsevier B.V., All rights reserved.
PY - 2010/1/14
Y1 - 2010/1/14
N2 - 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, were phosphorylated by Rho-kinase both in vitro and in vivo. Conclusions/Significance: This method would enable identification of novel specific substrates for kinases such as Rho-kinase with high sensitivity.
AB - 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, were phosphorylated by Rho-kinase both in vitro and in vivo. Conclusions/Significance: This method would enable identification of novel specific substrates for kinases such as Rho-kinase with high sensitivity.
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U2 - 10.1371/journal.pone.0008704
DO - 10.1371/journal.pone.0008704
M3 - Article
C2 - 20090853
AN - SCOPUS:77952515521
VL - 5
JO - PLoS One
JF - PLoS One
SN - 1932-6203
IS - 1
M1 - e8704
ER -