Identification of Clock-Controlled CPC-3 Phosphorylation Sites Necessary for Rhythmic P-eIF2a Activity in Neurospora crassa
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Date
2021-04-24
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Abstract
The circadian clock is an intrinsic time-keeping mechanism that is conserved between many species, ranging from cyanobacteria to mammals. Due to circadian clock conservation between organisms, understanding how the clock functions in a model organism can give us insights into the human circadian clock and how it impacts our health. It is known that the circadian clock regulates multiple biological pathways by controlling rhythmic gene expression at the transcriptional level. However, recent evidence in fungi and mammals suggested that translation is also regulated by the clock. In the clock model organism Neurospora crassa, the translation initiation factor eIF2α is rhythmically phosphorylated by the kinase CPC-3. The phosphorylation of eIF2α (P-eIF2α) peaks during the subjective daytime, and rhythmic P-eIF2α accumulation requires clock control of CPC-3 activity. My project involves investigating a potential mechanism of N. crassa CPC-3 activation that may be regulated by the clock. Specifically, I am examining three predicted CPC-3 autophosphorylation sites that may control its activity and that have been shown to have a significant time-of-day difference in constant dark conditions (T874, T879, and S238). I created two point mutations for each site, one that blocks phosphorylation at the site (converted to alanine) and one that mimics constant phosphorylation at the site (converted to glutamic acid). Mutations mimicking constant phosphorylation at T874 and S238 did not significantly alter the rhythms of P-eIF2α. However, removing phosphorylation and mimicking constant phosphorylation at T879 completely inhibited the phosphorylation of eIF2α. Furthermore, there is no P- eIF2α present in these mutants when the cell is under amino acid starvation conditions. Based on these data, we concluded that phosphorylation of T879 abolishes CPC-3 activity. Further studies should aim to determine if the phosphorylation at this specific site is rhythmic, and if the phosphorylation is specifically under control of the clock.
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Circadian, fungus, Neurospora, translation