Analysis of Novel Signal Transduction Network of Gibberellins

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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