Use this URL to cite or link to this record in EThOS: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.764259
Title: Regulation of eIF2B by phosphorylation
Author: Kousar, Rehana
Awarding Body: University of Manchester
Current Institution: University of Manchester
Date of Award: 2013
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Abstract:
The ability to sense and respond to environmental cues is crucial for the survival of all organisms. This response is often manifested by exerting control at different levels of gene expression, i.e. transcription, translation and post translation levels. Global control of protein synthesis is frequently exercised at the initial step of translation initiation and is generally achieved by changes in the phosphorylation state of initiation factors or the regulators that interact with them. The formation of ternary complex (TC) is considered first step of translation initiation and depends on the recycling of inactive eIF2-GDP to active eIF2-GTP form. This nucleotide exchange reaction is catalyzed by the eukaryotic initiation factor-2B (eIF2B). eIF2B is composed of a regulatory sub-complex of alphaβdelta subunits and a catalytic sub-complex of the γε subunits. The guanine nucleotide exchange activity of eIF2B is regulated by phosphorylation of eIF2alpha and additionally in mammalian cells, by direct phosphorylation of eIF2B at multiple sites in ε subunit, where most of the catalytic activity of eIF2B resides. Recent unpublished studies in the Pavitt laboratory identified novel phosphorylation sites by Mass Spectrometry in γ and ε subunits of eIF2B catalytic sub-complex. In order to study the functional significance of these phospho-sites for translation initiation, Site Directed Mutagenesis (SDM) was performed to generate Ser to Ala mutants. All mutations are viable and have no significant growth defect on rich or minimal media; however the significance of these sites in yeast growth became apparent by growing yeast in different stress conditions (e.g. Rapamycin, Torin1, amino acid starvation and 1-butanol). Effects on the phosphorylation pattern at these sites were monitored by using custom generated phospho-specific antibodies. All phosphorylation events appear independent of the eIF2alpha kinase (Gcn2p in yeast). The phosphorylation of ε-S528 depends on the presence of ε-S525. This study finds that addition of rapamycin, Torin1, amino acid starvation and butanol, which each inhibits global translation initiation, alters the phosphorylation pattern at ε-S435, ε-S525 and ε-S528 sites. Linking growth to phosphorylation, it appears that phosphorylation at ε-S435 and ε-S525 is directly proportional to growth. Phosphorylation of ε-S435 is necessary for effect of eIF2alpha-Ser51 phosphorylation on protein synthesis while phosphorylation of ε-S528 seems to be a target of various mechanisms. This study also suggests that eIF2Bε may be a key player of the cell cycle progression and phosphorylation changes can serve as marker for the regulation of eIF2B activity. The kinases responsible for phosphorylation at these sites are not yet known in yeast. Further investigation is required to find the functional significance of alterations in phosphorylation pattern to definitively establish eIF2Bε phosphorylation as a mechanism for regulating eIF2B activity in yeast. Models are presented to account for the results obtained that show how phosphorylation of eIF2Bε at these sites may contribute to the control of protein synthesis.
Supervisor: Pavitt, Graham ; Crosthwaite, Susan Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.764259  DOI: Not available
Keywords: eIF2B ; Translation initiation ; Phosphorylation ; Regulation ; Cell cycle
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