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The role of arginine methylation in regulating the function of ribosomal S6 kinase 2

Khalil, M.I.M.; (2010) The role of arginine methylation in regulating the function of ribosomal S6 kinase 2. Doctoral thesis, UCL (University College London).

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Abstract

S6 kinases are serine/threonine protein kinases which play a key role in the control of cell growth and metabolism. The S6K family has two members: S6K1 and S6K2. Both isoforms are highly homologous in the kinase and kinase-extension domains and the main divergence resides in non-catalytic regions which hint at differential functions and/or regulation. Although much effort has been invested in finding S6K1 binding partners/substrates, less attention has been given to the closely related S6K2. S6K2, with the presence of a C-terminal NLS not found in S6K1 would indicates that S6K2 would has distinct nuclear functions. S6K2, unlike S6K1, is selectively recruited into a signalling complex containing PKCε and B-Raf and likely controls FGF2-mediated translation of mRNA species involved in the regulation of cell survival. In a previous study in Ivan Gout’s laboratory, S6K2 was shown to bind directly to DNA via a short motif at its extreme C-terminus, highly homologous to the AT-hook motif found in high mobility group (HMG) DNA binding proteins. To our knowledge, this study describes for the first time the modification of a protein kinase by arginine methylation. This thesis details my attempt to understand the functional importance of arginine methylation for S6K2 which is found to be at the extreme C-terminus around the AT-hook motif. Here we have shown that S6K2 can be methylated by Protein Arginine Methyltransferases (PRMTs). We demonstrate that PRMTs methylates S6K2 primarily at Arg475 and Arg477 that are evolutionarily conserved. Furthermore, the interaction between PRMTs (PRMT1, -3, and -6) and S6K2 was demonstrated using a GST pull down, and revealed the importance of AdoMet, the methyl donor, in enhancing the interaction of PRMT3 and -6 with S6K2. The interaction was also confirmed by the co-immunoprecipitation assay indicating that endogenous S6K2 forms a physical complex with endogenous PRMT1, -3, and -6 in cells. In addition, S6K activity was found to be associated with PRMTs. Importantly, the interaction between S6K2 and PRMT6 was found to be induced by serum stimulation, while PRMT1 and PRMT6 knockdown reduces methylation of S6K2. One interesting observation in this report, the specific demethylation of S6K2 upon starvation of Hek293 cells which may imply the existence of specific demethylase(s) for S6K2. We found that methylated S6K2 (wild type) found more in the nucleus than in the cytoplasm, in other words, the mutants which lacks the methylation signal (R2M) showed decreased nuclear distribution. Our results have clearly demonstrated that S6K2 mutant (R2M), lacking the methylation signal, promote serum starvation-induced apoptosis when compared to the wild type and it seems that the methylation signal leads to protection from apoptosis that follows growth factor(s) withdrawal. The molecular mechanisms still need to be clarified. Taken together, we provide the first evidence that arginine methylation serves as a regulatory modification for S6K2-mediated prosurvival effect.

Type:Thesis (Doctoral)
Title:The role of arginine methylation in regulating the function of ribosomal S6 kinase 2
Language:English
UCL classification:UCL > School of Life and Medical Sciences > Faculty of Life Sciences > Biosciences (Division of) > Structural and Molecular Biology

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