Biochemistry and Pharmacology - Theses
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ItemMechanisms regulating ribosome biogenesis by AKT( 2015)Ribosomes are essential for cell growth and proliferation, and their biogenesis is a highly energy consuming process that requires exquisite regulation. Aberrant ribosome biogenesis underlies diseases of ribosomes, the so-called “ribosomopathies”. Deregulated ribosome biogenesis is now considered a characteristic of transformed cells, presenting a specific target for cancer treatment. One global regulator of ribosome biogenesis is the transcription factor c-MYC, which selectively transcribes an RNA Polymerase I (Pol I) specific regulon required for synthesis of the ribosomal RNA (rRNA), a key rate-limiting step in ribosome biogenesis. Recently it has been demonstrated that the kinase AKT also mediates Pol I-driven ribosomal DNA (rDNA) transcription and ribosome biogenesis to a similar extent as c-MYC. Importantly, maximal activation of rDNA transcription, ribosome biogenesis, and cell growth is achieved through the co-operative activities of both c- MYC and AKT. While previous work has outlined c-MYC’s major regulatory role as a transcription factor, the mechanism(s) by which AKT alone or in co-operation with c-MYC regulates rDNA transcription by Pol I remains unclear. This thesis aims to identify and elaborate upon potential mechanisms of AKT-driven ribosome biogenesis regulation. I hypothesise that AKT phosphorylates and subsequently activates Pol I complex components and associated factors, some of which may be c-MYC transcriptional targets. To test this hypothesis, the Pol I complex and associated proteins were immunoprecipitated and high sensitivity mass spectrometry was used to characterise the members of the complex and to identify phosphorylated proteins, providing insight into potential regulators of Pol I function. I found that the endogenous nucleolar protein treacle associates within the Pol I complex, providing insight into this protein previously implicated in promoting rDNA transcription by Pol I through an unknown mechanism, and which may be transcriptionally regulated by c- MYC. Further mass spectrometric analysis of phosphorylated proteins associated with the Pol I complex consistently identified p-peptides corresponding to treacle, including its putative AKT phosphorylation site (S1350 (human)), also identified in a bioinformatics analysis using Scansite. Furthermore, immunoprecipitated treacle could be phosphorylated by purified AKT in vitro demonstrating that treacle is a direct AKT substrate. In order to establish phosphorylation of treacle as a possible mechanism by which AKT regulates rDNA transcription by Pol I, I examined the effects of creating a phosphoinhibitory mutation in treacle at the AKT consensus phosphorylation site (S1191 (mouse)), replacing the serine with an alanine (S1191A). I found that while wild type treacle localised to the nucleolus (the site of rDNA transcription by Pol I), the phosphoinhibitory S1191A treacle mutant was dispersed throughout the cytoplasm. This suggests that AKT phosphorylation of treacle at S1191/S1350 is required for treacle’s nucleolar localisation and potentially its ability to promote rDNA transcription by Pol I there. Subsequent phosphoproteomic analysis further identified the inverse relationship between this phosphorylation site and phosphorylation of treacle at another site (S853) and further mass spectrometric analysis of immunoprecipitated samples also reveal treacle’s additional affiliation with components of the pre-rRNA methylation complex. This study has therefore uncovered aspects of key new regulatory mechanisms by which AKT can drive rDNA transcription and ribosome biogenesis through treacle, thereby revealing novel potential therapeutic targets for addressing dysregulated ribosome biogenesis.