Nicotiana tabacum osmotic stress-activated kinase is regulated by phosphorylation on Ser-154 and Ser-158 in the kinase activation loop
Nicotiana tabacum osmotic stress-activated kinase is regulated by phosphorylation on Ser-154 and Ser-158 in the kinase activation loop
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DOI：
10.1074/jbc.m601977200
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发表时间：
2006-11-10
影响因子：
4.8
通讯作者：
Dobrowolska, Grazyna
中科院分区：
文献类型：
--
作者：
Burza, Anna Maria;Pekala, Izabela;Dobrowolska, Grazyna
NtOSAK（Nicotiana tabacum osmotic stress-activated protein kinase，烟草渗透压应激激活蛋白激酶）是SnRK 2亚家族的成员，在高渗胁迫下被迅速激活。我们以前的结果以及其他人提供的数据表明，磷酸化参与了SnRK 2激酶的激活。在这里，我们已经映射的监管磷酸化位点的NtOSAK的质谱与碰撞诱导的肽片段。我们发现，活性NtOSAK，从NaCl处理的烟草BY-2细胞中分离，在激酶激活环中的Ser-154和Ser-158上被磷酸化。NtOSAK三维结构的预测表明，Ser-154和Ser-158的磷酸化触发酶构象的变化，导致其活化。通过对细菌和玉米原生质体中表达的NtOSAK进行定点突变，证实了Ser-154和Ser-158磷酸化参与NtOSAK活性的调节。我们的数据显示，Ser-158的磷酸化是NtOSAK激活所必需的，而Ser-154的磷酸化最有可能促进Ser-158的磷酸化。在BY-2细胞中Ser-154和Ser-158上NtOSAK磷酸化的时间过程与NtOSAK活性相关，表明NtOSAK在体内受这些残基的可逆磷酸化调节。重要的是，Ser-154和Ser-158在所有SnRK 2亚家族成员中是保守的，这表明这些位点的磷酸化可能是SnRK 2激活的一般机制。
NtOSAK (Nicotiana tabacum osmotic stress-activated protein kinase), a member of the SnRK2 subfamily, is activated rapidly in response to hyperosmotic stress. Our previous results as well as data presented by others indicate that phosphorylation is involved in activation of SnRK2 kinases. Here, we have mapped the regulatory phosphorylation sites of NtOSAK by mass spectrometry with collision-induced peptide fragmentation. We show that active NtOSAK, isolated from NaCl-treated tobacco BY-2 cells, is phosphorylated on Ser-154 and Ser-158 in the kinase activation loop. Prediction of the NtOSAK three-dimensional structure indicates that phosphorylation of Ser-154 and Ser-158 triggers changes in enzyme conformation resulting in its activation. The involvement of Ser-154 and Ser-158 phosphorylation in regulation of NtOSAK activity was confirmed by site-directed mutagenesis of NtOSAK expressed in bacteria and in maize protoplasts. Our data reveal that phosphorylation of Ser-158 is essential for NtOSAK activation, whereas phosphorylation of Ser-154 most probably facilitates Ser-158 phosphorylation. The time course of NtOSAK phosphorylation on Ser-154 and Ser-158 in BY-2 cells subjected to osmotic stress correlates with NtOSAK activity, indicating that NtOSAK is regulated by reversible phosphorylation of these residues in vivo. Importantly, Ser-154 and Ser-158 are conserved in all SnRK2 subfamily members, suggesting that phosphorylation at these sites may be a general mechanism for SnRK2 activation.