Analysis of Novel Signal Transduction Network of Gibberellins

植物激素赤霉素（GA）的内源水平通过反馈机制进行时空调控，保持GA在植物细胞中的稳态，使植物能够维持足够的形态建成以适应不断变化的环境。我们将GA生物合成和信号转导的反馈调节看作是来自内部发育程序和外部环境的信号的汇聚点，并对GA反馈调节的分子机制进行了探讨。我们确定了GA反馈调节的信号转导途径，包括bZIP转录因子抑制芽生长（RSG）其转录GA生物合成的酶、通过细胞内定位调节RSG活性的14-3-3蛋白、以及使RSG的S114磷酸化并促进RSG与14-3-3蛋白之间的相互作用的Ca 2+依赖性蛋白激酶CDPK 1。了解fe的信号转导 ...更多信息 为了研究GA的反向调节，我们将重点放在CDPK 1上，CDPK 1接收GA信号并将其转换为RSG。CDPK 1的底物识别机制在植物中，CDPKs在拟南芥中形成了一个由34个基因组成的大家族，在水稻中形成了31个基因，在植物中不编码C-激酶的Ca 2+信号转导中起着核心作用。CDPKs由N端、激酶区、自抑制区和钙调素样区4个结构域组成，被认为是通过钙调素激酶和钙调素融合进化而来。为了了解CDPKs在这种情况下如何识别其特异性生理底物，我们分析了CDPKs 1识别其特异性底物RSG的机制。结果表明，CDPK 1的N-末端结构域在底物识别中起作用，并且在该区域中仅一个氨基酸残基R10的突变消除了其底物识别活性。此外，我们成功地证明了另一种不能先天磷酸化RSG的CDPKs同种型可以通过将CDPK 1的N-末端区域添加到其N-末端而不是其先天N-末端区域来转化为RSG-激酶。我们估计这一结果是非常有趣的，因为它不仅确定了未知功能的N-末端区域，直到那时作为底物识别结构域，但也揭示了底物识别和磷酸化的结构域可以离散地划分在CDPKs中。CDPK 1磷酸化位点的鉴定CDPK 1在过量GA的信号下磷酸化。为了阐明这种磷酸化对CDPK 1催化活性的意义，我们开始鉴定CDPK 1中的磷酸化位点。迄今为止，许多CDPKs被报道为磷酸化自身。因此，我们尝试用重组CDPK 1蛋白通过质谱鉴定CDPK 1的自磷酸化位点，并成功地鉴定了N端区域的S6和T21为自磷酸化位点。为了证实这些结果，我们制备了CDPK 1的脱磷酸模拟突变体（S6 A T21 A），并证明S6 A T21 A突变体不能再被自磷酸化。接下来，我们研究了自磷酸化和底物识别之间的关系。用重组RSG和CDPK 1进行的Pull down实验表明，CDPK 1的脱磷酸模拟突变体与底物RSG的亲和力较低。这一结果证实了CDPK 1的一系列磷酸化反应，即CDPK 1在磷酸化底物RSG后使这些位点自磷酸化，通过降低底物RSG与这些位点之间的亲和力使RSG从这些位点解离。少

The spatiotemporal regulation of the endogenous level of a phytohormone Gibberellins (GA)by the feedback mechanism keeps GA homeostasis in plant cells and leads to the adequate morphogenesis of plants to adapt their ever fluctuating environment. We consider the feedback regulation which containing both of biosynthesis and signal transduction of GA as a convergent site of signals from internal developmental program and external environment, and proceed the investigation into the molecular mechanism of GA feedback regulation.Until now, we identified the signal transduction pathway of GA feedback regulation comprised of a bZIP transcription factor REPRESSION OF SHOOT GROWTH (RSG) which transcribes an enzyme of GA biosynthesis, a 14-3-3 protein which regulates RSG activity via intracellular localization, and a Ca2+-dependent protein kinase, CDPK1 which phosphorylates the S114 of RSG and promotes the interaction between RSG and the 14-3-3 protein. To understand the signal transduction of fe … More edback regulation of GA, we focused on the CDPK1 that receives the GA signal and transduce it to RSG.1. Substrate recognition mechanism of CDPK1In plants, CDPKs form a large family consisting 34 genes in Arabidopsis and 31 genes in rice, play a central role in Ca2+ signaling in plants which do not encode C-kinases. CDPKs are consisted of 4 domains, that is, N-terminal, kinase, autoinhibitory and calmodulin-like regions and thought to be evolved through fusion between calmodulin kinase and calmodulin. To understand how one isotype of CDPKs can recognize its specific physiological substrate in this situation, we analyzed the mechanism by which CDPK1recognize its specific substrate RSG. As results, we elucidated that the N-terminal domain of CDPK1 functions in the substrate recognition and mutation of only one amino acid residue, R10, in this region eliminates its activity for substrate recognition. Furthermore, we succeeded to demonstrate that another isotype of CDPKs which cannot congenitally phosphorylate RSG could be converted to a RSG-kinase by addition of N-terminal region of CDPK1 to its N-terminus instead of its innate N-terminal region. We estimate that this results is of great interest because it not only identifies the N-terminal region of unknown function until then as the substrate recognition domain but also reveal that the domains for substrate recognition and phosphorylation can be discretely divided in CDPKs.2. Identification of phosphorylation sites in CDPK1CDPK1 was phosphorylated upon the signal of excess GA. To elucidate the significance of this phosphorylation on CDPK1 catalytic activity, we started the identification of phosphorylation sites in CDPK1. Many CDPKs were reported to phosphorylate themselves until now. Accordingly, we tried to identify autophosphorylation sites of CDPK1 with recombinant CDPK1 protein by mass spectrometry and succeeded to identify S6 and T21 in N-terminal region as autophosphorylation sites. To confirm these results, we prepared a dephosphomimic mutant of CDPK1 (S6A T21A) and demonstrated that the S6A T21A mutant could not be autophosphorylated any longer. Next, we searched the relationship between autophosphorylation and substrate recognition. Pull down assay with recombinant RSG and CDPK1 demonstrated that dephosphomimic mutant of CDPK1 had low affinity to substrate RSG. This result suggestes a series of phosphorylation reaction by CDPK1, that is, CDPK1 autophosphotylates these sites after phosphorylates substrate RSG leading dissociation of RSG from them by decreasing affinity between RSG and them. Less