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

et al. (2010) A Proteomic Approach for Comprehensively Screening Substrates of Protein Kinases Such as Rho-Kinase. PLoS ONE 5(1): e8704. doi:10.1371/journal.pone.0008704 A Proteomic Approach for Comprehensively Screening Substrates of Protein Kinases Such as Rho-Kinase Mutsuki Amano 0 Yuta Tsumura 0 Kentaro Taki 0 Hidenori Harada 0 Kazutaka Mori 0 Tomoki Nishioka 0 Katsuhiro Kato 0 Takeshi Suzuki 0 Yosuke Nishioka 0 Akihiro Iwamatsu 0 Kozo Kaibuchi 0 Gian Maria Fimia, INMI, Italy 0 1 Department of Cell Pharmacology, Graduate School of Medicine, Nagoya University , Nagoya , Japan , 2 Department of Cardiology, Graduate School of Medicine, Nagoya University , Nagoya , Japan , 3 Protein Research Network, Inc., Yokohama, Japan, 4 Japan Science and Technology Agency , CREST, Kawaguchi , Japan 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 Rhokinase with high sensitivity. - Funding: This research was supported in part by Grant-in-Aid for Scientific Research on Priority Areas (20058012, 17024024) and Grant-in-Aid for Scientific Research (S) (20227006) and (C) (20590308) from the Ministry of Education, Culture, Sports, Science and Technology of Japan (MEXT). This research was also supported in part by Japan Science and Technology Agency, CREST (26-J-Jc08). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. 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 vitro kinase assays have been used to identify potential substrates for specific kinases for many years. As an extension of this method, genome-wide screening of substrates for 87 yeast protein kinases has been performed using protein microarrays containing 4,400 yeast proteins [1]. However, this method requires a large number of recombinant proteins, and the native conformation of substrates may be lost on the plates. One of the recent phosphoproteomic strategies is the semi-quantitative liquid chromatography tandem mass spectrometry (LC-MS/MS) approach combined with phosphopeptide enrichment, in which proteins or peptides from cells treated with agonists and protein kinase inhibitors are labeled with stable isotope or isobaric reagent iTRAQ ([2,3] for reviews). Two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) has also been used to identify potential substrates for ERK from the cells treated with a MEK inhibitor [4]. Both methods require specific antagonists, agonists and/or RNA interference to identify the responsible kinases. Thus, screening of direct substrates for specific kinases is still laborious and difficult. Protein kinases share common catalytic domain structures composed of a small N-terminal lobe and a large C-terminal lobe. The cleft between these lobes is the active center that binds to both ATP and the substrate. In spite of highly analogous structures, protein kinases exhibit striking substrate specificity partly due to their surface charge and hydrophobicity [5]. In addition to the active center, several kinases, such as MAPK, GSK3 and PDK1, have been reported to associate with substrates through extra docking sites, which may confer substrate specificity and facilitate phosphorylation efficiency [6]. Nevertheless, the interaction between protein kinases and substrates is transient and not very stable, such that utilizing the interaction to identify substrates has been thought to be difficult, with a few exceptions. However, recent improvement in the sensitivity of mass spectrometry is expected to make it possible to detect substrate proteins weakly associated with the catalytic domain of protein kinases. Here, we developed a method combining affinity column chromatography, using the active catalytic fragment of protein kinase as a bait, and shotgun LC-MS/MS to efficiently screen the kinase substrates. We employed Rho-kinase/ROCK/ROK, a Ser/Thr protein kinase belonging to the AGC family of kinases, as a model protein kinase. Rho-kinase is an effector of small GTPase Rho and is implicated in various cellular functions, including cell migration, cell adhesion, smooth muscle contraction, cytokinesis and neurite retraction [7,8]. Here, we describe our discovery of more than a hundred proteins that specifically interacted with Rho-kinase, some of which functioned as Rho-kinase substrates. Affinity column chromatography of Rho-kinase To screen potential substrates of Rho-kinase, we examined whether the active catalytic fragment of Rho-kinase (Rho-kinasecat) interacts with its substrates by affinity column chromatography. Rat brain cytosol or peripheral membrane (P2) fractions concentrated by ammonium sulfate precipitation were loaded onto a glutathione-sepharose affinity column on which GST, GSTRho-kinase-cat, or GST-Rho-kinase-cat-KD, a kinase-deficient mutant of Rho-kinase, was immobilized (Figure 1A, B). GSTPKN-cat, another Rho effector belonging to the PKC subfamily in the AGC family of kinases, was also subjected to affinity column chromatography. The proteins bound to the affinity columns were then eluted by addition of 50 mM and 1 M NaCl, and then 10 mM glutathione. Numerous proteins were d (...truncated)