Characterization of ribosomal S6 protein phosphorylation and possible control of ribosome biogenesis in arabidopsis cell culture

 
 
 
 

Characterization of ribosomal S6 protein phosphorylation and possible control of ribosome biogenesis in arabidopsis cell culture

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Title: Characterization of ribosomal S6 protein phosphorylation and possible control of ribosome biogenesis in arabidopsis cell culture
Author: Kim, Sunghan
Description: Regulation of translation through control of ribosome biogenesis is the primary means of adjusting the growth and proliferation potential of an organism in response to nutrition and abiotic stresses, which is conserved among all eukaryotes. The phosphorylation status of rpS6, one of the small ribosomal subunit proteins, has been identified as a key parameter of this response in which TOR (target of rapamycin) kinase has been implicated as the master regulator. In order to have a better insight to the translational regulation mediated through ribosomal S6 phosphorylation in plants, the phosphorylation status of rpS6 and its upstream regulator kinase, S6K1, was studied under different physiological conditions, starvation and osmotic stress in particular. A novel kinase assay system which allows pulling down a specific kinase from cells as bound to the specific GST-fusion protein substrate has been developed to facilitate this goal. The results of the kinase assays suggest that the phytohormone auxin and a nutrient sensing signal are acting in parallel to control the pathway regulating rpS6 phosphorylation and translational activity in Arabidopsis cell culture. The same principle of this novel kinase assay method was also applied in a larger scale to identify the kinase as well as any other regulatory elements interacting with the substrate by mass spectrometry. Several Arabidopsis proteins with chromatin related functions were identified as interacting partners of rpS6. Of particular interest among them is a nucleolus targeted histone deacetylase, HD2B, as recent evidences in yeast suggest that TOR controls ribosome biogenesis through modulation of histone deacetlyase activity. Thus, the finding made by this study may provide a clue to explain the molecular mechanism of ribosome biogenesis in which rpS6 plays a crucial role in connecting the functions of TOR and histone deacetylase. Regulation of translation through control of ribosome biogenesis is the primary means of adjusting the growth and proliferation potential of an organism in response to nutrition and abiotic stresses, which is conserved among all eukaryotes. The phosphorylation status of rpS6, one of the small ribosomal subunit proteins, has been identified as a key parameter of this response in which TOR (target of rapamycin) kinase has been implicated as the master regulator. In order to have a better insight to the translational regulation mediated through ribosomal S6 phosphorylation in plants, the phosphorylation status of rpS6 and its upstream regulator kinase, S6K1, was studied under different physiological conditions, starvation and osmotic stress in particular. A novel kinase assay system which allows pulling down a specific kinase from cells as bound to the specific GST-fusion protein substrate has been developed to facilitate this goal. The results of the kinase assays suggest that the phytohormone auxin and a nutrient sensing signal are acting in parallel to control the pathway regulating rpS6 phosphorylation and translational activity in Arabidopsis cell culture. The same principle of this novel kinase assay method was also applied in a larger scale to identify the kinase as well as any other regulatory elements interacting with the substrate by mass spectrometry. Several Arabidopsis proteins with chromatin related functions were identified as interacting partners of rpS6. Of particular interest among them is a nucleolus targeted histone deacetylase, HD2B, as recent evidences in yeast suggest that TOR controls ribosome biogenesis through modulation of histone deacetlyase activity. Thus, the finding made by this study may provide a clue to explain the molecular mechanism of ribosome biogenesis in which rpS6 plays a crucial role in connecting the functions of TOR and histone deacetylase.
Permanent Link: http://rave.ohiolink.edu/etdc/view?acc_num=osu1072819298
http://hdl.handle.net/2374.OX/5056
Date: 2004

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