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

为了鉴定负责ABRE结合因子TRAB1的aba依赖性磷酸化的蛋白激酶，我们对水稻SnRK2蛋白激酶家族进行了全面分析。该家族的一些激酶，如蚕豆AAPK和拟南芥OST1，已被报道参与ABA对气孔开度的调节。因此，我们预计TRAB1激酶将包含在该激酶家族中。水稻基因组编码这个家族的10个成员，我们将其命名为SAPK1到10。通过在水稻原生质体中短暂表达表位标记的SAPKs，分析了SAPK8、9和10的活性调控，发现SAPK8、9和10被ABA快速激活。然后，我们发现，在培养的细胞原生质体中，这些ABA激活的SnRK2蛋白激酶的短暂过表达导致ABRE调控启动子的激活，这表明这些激酶参与了ABA信号传导的基因调控途径。更多的证据表明，这些ABA激活的SnRK2蛋白激酶在ABA的作用下直接磷酸化TRAB1。SAPK10活化和TRAB1磷酸化的动力学分析表明，后者紧随前者。TRAB1不仅在ABA的作用下被磷酸化，而且在高渗胁迫下也被磷酸化，这被解释为高渗活化的SAPKs磷酸化TRAB1的结果。通过共免疫沉淀实验证实了TRAB1和SAPK10在体内的物理相互作用。最后，TRAB1在体外被ABA激活的SnRK2蛋白激酶在Ser102位点磷酸化，该位点在体内响应ABA而磷酸化，对激活功能至关重要。aba激活的SnRK2 PKs也被高渗应激激活。我们之前已经证明这种激活是由PK分子的磷酸化介导的。在本研究中，我们发现ABA的激活也是通过磷酸化介导的。然而，我们发现ABA和高渗活化的机制是不同的。这一发现是基于以下观察：PKs的协同激活是通过ABA和高渗刺激在信号强度下实现的，其中对每种刺激的反应都是饱和的，并且SAPK10的c端调控域的一个小的内部缺失对高渗胁迫有反应，而对ABA没有反应。另一个重要的进展是我们通过酵母双杂交筛选确定了一个SAPK相互作用蛋白，该蛋白可以作为信号因子的支架，可能用于有效的信号传导和信号分类。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