Analysis of Novel Signal Transduction Network of Gibberellins

通过反馈机制对植物激素赤霉素（giberellins， GA）内源水平的时空调节，保持植物细胞内赤霉素的稳态，并导致植物充分的形态发生以适应其不断变化的环境。我们认为遗传因子的反馈调控包含生物合成和信号转导两部分，是遗传因子内部发育程序和外部环境信号的聚合位点，并对遗传因子反馈调控的分子机制进行了探讨。到目前为止，我们已经确定了GA反馈调节的信号转导途径，包括转录GA生物合成酶的bZIP转录因子抑制SHOOT GROWTH (RSG)，通过细胞内定位调节RSG活性的14-3-3蛋白，以及磷酸化RSG的S114并促进RSG与14-3-3蛋白相互作用的Ca2+依赖性蛋白激酶CDPK1。为了更好地理解GA的信号转导，我们重点研究了接收GA信号并将其转导给RSG.1的CDPK1。在植物中，cdpkks形成一个大家族，在拟南芥中有34个基因，在水稻中有31个基因，在不编码c激酶的植物中，cdpkks在Ca2+信号传导中起核心作用。CDPKs由4个结构域组成，即n端、激酶、自抑制区和钙调蛋白样区，被认为是通过钙调蛋白激酶和钙调蛋白融合而进化而来的。为了了解一种同型cdpkk如何在这种情况下识别其特定的生理底物，我们分析了cdpk1识别其特定底物RSG的机制。结果表明，CDPK1的n端结构域在底物识别中起作用，而该区域只有一个氨基酸残基R10的突变使其失去了底物识别的活性。此外，我们成功地证明了另一种不能先天磷酸化RSG的cdpkk同型可以通过将CDPK1的n端区域而不是其先天n端区域添加到其n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端区域作为底物识别结构域，而且还揭示了底物识别和磷酸化的结构域可以在cdpks中离散地划分。cdpk1磷酸化位点的鉴定cdpk1在过量GA信号下被磷酸化。为了阐明这种磷酸化对CDPK1催化活性的意义，我们开始鉴定CDPK1的磷酸化位点。到目前为止，有报道称许多CDPKs自身磷酸化。因此，我们尝试用重组CDPK1蛋白质谱鉴定CDPK1的自磷酸化位点，并成功鉴定了n端S6和T21为自磷酸化位点。为了证实这些结果，我们制备了CDPK1的去磷组突变体（S6A T21A），并证明S6A T21A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。重组RSG和CDPK1的拉下实验表明，CDPK1脱磷突变体对底物RSG具有低亲和力。该结果提示CDPK1的一系列磷酸化反应，即CDPK1在磷酸化底物RSG后，通过降低RSG与它们之间的亲和力，导致RSG与它们分离，从而使这些位点自磷酸化。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