A proteomic approach for comprehensively screening substrates of protein kinases such as Rho-kinase.
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.
Amano
Mutsuki
M
Department of Cell Pharmacology, Graduate School of Medicine, Nagoya University, Nagoya, Japan.
Tsumura
Yuta
Y
Taki
Kentaro
K
Harada
Hidenori
H
Mori
Kazutaka
K
Nishioka
Tomoki
T
Kato
Katsuhiro
K
Suzuki
Takeshi
T
Nishioka
Yosuke
Y
Iwamatsu
Akihiro
A
Kaibuchi
Kozo
K
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*
C000714848:Dcx protein, rat; D000089662:Doublecortin Protein; D054460:rho-Associated Kinases