Study on signal transduction mechanisms involved in abscisic acid-responsive transcription

为了鉴定负责ABRE结合因子TRAB 1的ABA依赖性磷酸化的蛋白激酶，我们对水稻SnRK 2蛋白激酶家族进行了全面分析。该家族中的一些激酶，如蚕豆AAPK和拟南芥OST 1，已被报道参与阿坝对气孔开度的调节。因此，我们预期TRAB 1激酶将被包括在该激酶家族中。水稻基因组编码该家族的10个成员，我们将其命名为SAPK 1至10。通过在水稻培养细胞原生质体中瞬时表达表位标记的SAPKs，分析了它们的活性调节，发现SAPK 8、9和10被阿坝快速激活。然后，我们表明，这些ABA激活的SnRK 2蛋白激酶在培养的细胞原生质体中的瞬时过表达导致ABRE调节启动子的激活，这表明这些激酶参与阿坝信号的基因调控途径。我们进一步展示了几条证据 ...更多信息 这些ABA激活的SnRK 2蛋白激酶直接磷酸化TRAB 1以响应阿坝。SAPK 10激活和TRAB 1磷酸化的动力学分析表明，后者紧随前者。TRAB 1不仅在阿坝的作用下被磷酸化，而且在高渗胁迫下也被磷酸化，这被解释为TRAB 1被高渗激活的SAPKs磷酸化的结果。TRAB 1和SAPK 10在体内的物理相互作用通过免疫共沉淀实验证明。最后，TRAB 1在体外被ABA激活的SnRK 2蛋白激酶的Ser 102磷酸化，Ser 102在体内响应阿坝而被磷酸化并且是激活功能的关键。我们以前已经证明，这种激活是由PK分子的磷酸化介导的。在这项研究中，我们发现阿坝激活也是由磷酸化介导的。但是，我们发现阿坝和高渗激活的机制是不同的。这一发现是基于这样的观察，即通过阿坝和高渗刺激在对每个刺激的反应饱和的信号强度下实现PK的协同激活，SAPK 10的C-末端调控域的一个小的内部缺失对高渗胁迫有反应，但对阿坝没有反应。杂交筛选SAPK相互作用蛋白，其将充当信号传导因子的支架，其可用于有效的信号传导和信号分选。少

In order to identify protein kinases responsible for the ABA-dependent phosphorylation of an ABRE binding factor, TRAB1, we performed a comprehensive analysis of the rice SnRK2 protein kinase family. Some kinases, such as fava bean AAPK and Arabidopsis OST1, of this family had been reported to be involved in the ABA regulation of stomata apertures. Thus we expected that TRAB1 kinase would be included in this kinase family. Rice genome encode 10 members of this family, which we named SAPK1 through 10. By expressing epitope-tagged SAPKs transiently in rice cultured cell protoplasts, we analyzed for their activity regulation and found that SAPK8, 9 and 10 were rapidly activated by ABA. We then showed that transient overexpression in cultured cell protoplasts of these ABA-activated SnRK2 protein kinases led to the activation of an ABRE regulated promoter, suggesting that these kinases are involved in the gene regulation pathway of ABA signaling. We further showed several lines of evidence … More that these ABA-activated SnRK2 protein kinases directly phosphorylate TRAB1 in response to ABA. Kinetic analysis of SAPK10 activation and TRAB1 phosphorylation indicated that the latter immediately followed the former. TRAB1 was found to be phosphorylated not only in response to ABA but also in response to hyperosmotic stress, which was interpreted as the consequence of phosphorylation of TRAB1 by hyperosmotically activated SAPKs. Physical interaction between TRAB1 and SAPK10 in vivo was demonstrated by a co-immunoprecipitation experiment. Finally, TRAB1 was phosphorylated in vitro by the ABA-activated SnRK2 protein kinases at Ser102, which is phosphorylated in vivo in response to ABA and critical for the activation function.ABA-activated SnRK2 PKs are also activated by hyperosmotic stress. We previously have demonstrated that this activation is mediated by the phosphorylation of the PK molecule. In this research, we showed that the ABA activation was also mediated by phosphorylation. However, we found that the mechanisms are different between ABA and hyperosmotic activation. This finding was based on the observations that the synergistic activation of the PKs was achieved by the ABA and hyperosmotic stimulation at the signal strengths where the response to each stimulation is saturated, and that a small internal deletion at the C-terminal regulatory domain of SAPK10 responded to hyperosmotic stress but not to ABA.Another important progress was that we identified by the yeast two-hybrid screening a SAPK interacting protein that would function as a scaffold for signaling factors, which may serve for effective signaling and signal sorting. Less