title: A proteomic approach for comprehensively screening substrates of protein kinases such as Rho-kinase.
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.
  
  
  RESULTS
  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
  This method would enable identification of novel specific substrates for kinases such as Rho-kinase with high sensitivity.
authors.affiliation: Department of Cell Pharmacology, Graduate School of Medicine, Nagoya University, Nagoya, Japan.
authors.forename: Mutsuki
authors.initials: M
authors.lastname: Amano
authors.forename: Yuta
authors.initials: Y
authors.lastname: Tsumura
authors.forename: Kentaro
authors.initials: K
authors.lastname: Taki
authors.forename: Hidenori
authors.initials: H
authors.lastname: Harada
authors.forename: Kazutaka
authors.initials: K
authors.lastname: Mori
authors.forename: Tomoki
authors.initials: T
authors.lastname: Nishioka
authors.forename: Katsuhiro
authors.initials: K
authors.lastname: Kato
authors.forename: Takeshi
authors.initials: T
authors.lastname: Suzuki
authors.forename: Yosuke
authors.initials: Y
authors.lastname: Nishioka
authors.forename: Akihiro
authors.initials: A
authors.lastname: Iwamatsu
authors.forename: Kozo
authors.initials: K
authors.lastname: Kaibuchi
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D020134:Catalytic Domain; D002846:Chromatography, Affinity; D000089662:Doublecortin Protein; D006367:HeLa Cells; D006801:Humans; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010766:Phosphorylation; D040901:Proteomics; D051381:Rats; D013379:Substrate Specificity; D054460:rho-Associated Kinases / Q000737:chemistry / Q000378:metabolism*
chemical_list: C000714848:Dcx protein, rat; D000089662:Doublecortin Protein; D054460:rho-Associated Kinases