Dynamics of protein phosphorylation on Ser/Thr/Tyr in Bacillus subtilis
Dynamics of protein phosphorylation on Ser/Thr/Tyr in Bacillus subtilis
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DOI：
10.1002/pmic.200700232
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发表时间：
2007-10-01
期刊：
影响因子：
3.4
通讯作者：
Hecker, Michael
中科院分区：
文献类型：
--
作者：
Eymann, Christine;Becher, Dorte;Hecker, Michael
采用Pro-Q Diamond染色和[P-33]标记相结合的二维凝胶法分析枯草芽孢杆菌的Ser/Thr/Tyr磷酸化蛋白组。在呈指数增长的枯草芽孢杆菌细胞中，通过Pro-Q Diamond染色和/或[(33)p]标记可以鉴定27个蛋白，另外一个蛋白被[(33)p]单独标记，总共有28个可能磷酸化的蛋白。这些蛋白主要参与基本碳代谢的酶促反应和替代sigma因子sigma的调控(B)。我们还发现了磷酸化蛋白质组的显著变化，包括一些蛋白质的磷酸化和去磷酸化率增加，以及在应激或饥饿反应中检测到四种新磷酸化的蛋白质。用质谱法测定了9种蛋白丝氨酸或苏氨酸残基的磷酸化位点，包括已知的Crh、PtsH和RsbV的磷酸化位点。此外，我们能够鉴定AroA， Pyk和YbbT的新的磷酸化位点。有趣的是，在指数增长过程中发现了RsbRA、B、C和D的磷酸化，这是一种多组分蛋白复合物中涉及环境应激信号的四种蛋白。对于RsbRA、B和D，用质谱（MS）验证了它们c端一个保守苏氨酸残基的磷酸化（分别为T171、T186、T181）。
The Ser/Thr/Tyr phosphoproteome of Bacillus subtilis was analyzed by a 2-D gel-based approach combining Pro-Q Diamond staining and [P-33]-labeling. In exponentially growing B. subtilis cells 27 proteins could be identified after staining with Pro-Q Diamond and/or [(33)p]-labeling and one additional protein was labeled solely by [(33)p] resulting in a total of 28 potentially phosphorylated proteins. These proteins are mainly involved in enzymatic reactions of basic carbon metabolism and the regulation of the alternative sigma factor sigma(B). We also found significant changes of the phosphoproteome including increased phosphorylation and dephosphorylation rates of some proteins as well as the detection of four newly phosphorylated proteins in response to stress or starvation. For nine proteins, phosphorylation sites at serine or threonine residues were determined by MS. These include the known phosphorylation sites of Crh, PtsH, and RsbV. Additionally, we were able to identify novel phosphorylation sites of AroA, Pyk, and YbbT. Interestingly, the phosphorylation of RsbRA, B, C, and D, four proteins of a multicomponent protein complex involved in environmental stress signaling, was found during exponential growth. For RsbRA, B, and D, phosphorylation of one of the conserved threonine residues in their C-termini were verified by MS (T171, T186, T181, respectively).