This proposal is designed to investigate three different post-translational mechanisms of circadian clock control in Arabidopsis. The first is the role of protein phosphorylation, and the effect on activity, localization and stability of key clock proteins. Robust-phase specific phosphorylation of five key proteins in the Arabidopsis clock raises the question of its significance in their activity, localization and turnover. We will focus on the first validated phosphorylation-dependent protein-protein interaction in the Arabidopsis clock to begin to understand the dynamics of TOC1 and PRR3 post-translationally. Additionally, we propose a novel approach to discovering new kinases and phosphatases that act on clock proteins. We also propose to begin modeling this portion of the circadian system to address circuitry of the clock that is not based on transcription. The second is the effect chaperonins play in the maturation of the F-box protein ZEITLUPE to its functional state, necessary to the maintenance of robust circadian amplitude and period. We have identified two components new to any circadian system which likely contribute to ZTL maturation. Through effects on ZTL, these two components indirectly regulate the circadian system. The role of these two proteins in obtaining fully active ZTL, and their potential interaction is the underlying question addressed. The third mechanism is the role of the nuclear pore in the regulation of nuclear import/export of mRNA and/or protein of clock genes. The nuclear pore acts as a gatekeeper to the nucleus and all transcription factors and other regulators of nuclear function must pass through this highly complex structure. All eukaryotic circadian systems involve nucleocytoplasmic shuttling of mRNA and protein which may contribute substantially to establishing the timing delays necessary to establishing a 24 h molecular periodicity. We have identified a nuclear pore component that slows the circadian clock when absent. Understanding the molecular basis of this delay should lead to a greater understanding of how circadian timing is tied to intracellular transport. In a related way, the TOC1/PRR5 interaction appears to facilitate TOC1 nuclear entry and may also serve to recruit a kinase to the interaction. The molecular basis of this interaction and the effect on period will lead to increased understanding of the role of regulated nuclear entry in the clock. While the early heuristic models of the clock focused on transcription/translation feedback loops, recent findings across all circadian systems have highlighted the inadequacy of this view and how post- translational mechanisms contribute substantially to sustaining circadian oscillation. The studies described below will contribute to a greater understanding of circadian clock in particular, and to oscillatory feedback systems in general.
Circadian rhythms control a wide variety of processes in all eukaryotes, including recent linkages to the cell cycle and cancer. The fundamental organization of the clock as consisting of two or more, linked autoregulatory feedback loops is shared among all model systems, but the individual molecular players often vary among the kingdoms. Here post-translational mechanism of circadian clock control in Arabidopsis is investigated, with potential relevance to other eukaryotic oscillatory systems.
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