A proteomic approach for comprehensively screening substrates of protein kinases such as rho-kinase

Mutsuki Amano, Yuta Tsumura, Kentaro Taki, Hidenori Harada, Kazutaka Mori, Tomoki Nishioka, Katsuhiro Kato, Takeshi Suzuki, Yosuke Nishioka, Akihiro Iwamatsu, Kozo Kaibuchi

Research output: Contribution to journalArticlepeer-review

30 Citations (Scopus)


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.

Original languageEnglish
Article numbere8704
JournalPloS one
Issue number1
Publication statusPublished - 14-01-2010
Externally publishedYes

All Science Journal Classification (ASJC) codes

  • Biochemistry, Genetics and Molecular Biology(all)
  • Agricultural and Biological Sciences(all)
  • General


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