A proteomic approach for comprehensively screening substrates of protein kinases such as Rho-kinase.
A proteomic approach for comprehensively screening substrates of protein kinases such as Rho-kinase.
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DOI：
10.1371/journal.pone.0008704
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发表时间：
2010-01-14
期刊：
影响因子：
3.7
通讯作者：
Kaibuchi K
中科院分区：
文献类型：
--
作者：
Amano M;Tsumura Y;Taki K;Harada H;Mori K;Nishioka T;Kato K;Suzuki T;Nishioka Y;Iwamatsu A;Kaibuchi K
蛋白激酶是多种细胞过程中信号转导途径的主要组成部分。激酶直接与下游底物相互作用并磷酸化，从而调节其功能。尽管为了更充分地理解各个激酶的信号传导网络而鉴定底物的重要性，但是搜索底物的有效方法仍然探索得很少。我们结合质谱和亲和柱层析的蛋白激酶的催化结构域筛选潜在的底物。以Rho激酶/ROCK/ROK的活性催化片段为模型诱饵，从大鼠脑细胞质中获得了约300个相互作用蛋白，其中包括以前报道的Rho激酶底物蛋白。一些新的相互作用的蛋白质，包括doublecortin，磷酸化的Rho激酶在体外和体内。该方法将能够以高灵敏度鉴定激酶如Rho-激酶的新型特异性底物。
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. 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. This method would enable identification of novel specific substrates for kinases such as Rho-kinase with high sensitivity.
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