Research Article: A Proteomic Approach for Comprehensively Screening Substrates of Protein Kinases Such as Rho-Kinase

Date Published: January 14, 2010

Publisher: Public Library of Science

Author(s): Mutsuki Amano, Yuta Tsumura, Kentaro Taki, Hidenori Harada, Kazutaka Mori, Tomoki Nishioka, Katsuhiro Kato, Takeshi Suzuki, Yosuke Nishioka, Akihiro Iwamatsu, Kozo Kaibuchi, Gian Maria Fimia.

Abstract: 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.

Partial Text: Protein phosphorylation is one of the most ubiquitous and essential mechanisms mediating intracellular signal transduction in various cellular processes. About 500 protein kinases are encoded in the human genome, where these are mainly divided into two groups, Ser/Thr protein kinases and Tyr protein kinases. Kinases recognize and phosphorylate their specific substrates and modulate their functions. In the cell, numerous proteins are continuously and dynamically phosphorylated and dephosphorylated under the control of complex signaling networks. Comprehensive screening of substrates for kinases is necessary to increase understanding of the signaling networks in which protein kinases participate. However, it remains difficult to efficiently screen the physiological substrates of protein kinases.

In this study, we found that combining affinity column chromatography using the catalytic domain of Rho-kinase and shotgun LC-MS/MS analysis is powerful for identification of substrates, including known substrates and novel ones, such as DCX, APP and AP180. We also found that Rho-kinase phosphorylates DCX not only in vitro, but also in vivo. Thus, compared to previous methods, our method can easily enrich substrates for specific kinases and can be applied for screening other weak enzyme-substrate interactions. One of the advantages of this method is the ability to screen protein kinase substrates, including transmembrane proteins, in their native states. However, it lacks information concerning direct phosphorylation, phosphorylation sites, or in vivo phosphorylation. Combining this simple method with a conventional method, such as 2D-DIGE, would be more informative and powerful for screening novel substrates of specific kinases and would increase understanding of the phosphorylation network.



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