Ismail, H.M.S.; (2009) Regulation of S6 kinase 2 by DNA binding in downstream signalling to transcription. Doctoral thesis , UCL (University College London).

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. However, homologous regions between S6K1 and S6K2 are in the kinase and kinase-extension domains, 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. Even though both S6K2 isoforms are predominantly nuclear, the nuclear functions/substrates have not yet been discovered. S6K2, unlike S6K1, is selectively recruited into a signalling complex containing PKCepsilon and B-Raf and likely controls FGF-2-mediated translation of mRNA species involved in the regulation of cell survival. In a previous study in this 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. This thesis details my attempt to understand the functional importance of the AT-hook motif in S6K2 protein. It also aims to identify the nuclear binding partners of S6K2, which could be involved in mediating its downstream signalling in the nucleus. The binding of DNA to S6K2 is shown to potentiate its kinase activity in a DNA dose-dependent manner. Generation of truncation and point mutation of S6K2-AT-hook allowed me to study the importance of this motif in kinase activity and subcellular localisation. This work revealed that the AT-hook motif has an autoinhibitory effect on S6K2 kinase activity, and in addition, affects protein subcellular localisation. A novel and specific binding partner of S6K2, but not S6K1, has been discovered, the general transcription factor YY1. The complex formation between S6K2 and YY1 is shown to be stimulated by mitogens. S6K2 is identified to bind to chromatin and nuclear matrix cell fractions. The initial identification of S6K2 as a phosphorylating kinase for histone 3 in the histone fold domain is also demonstrated. In summary, the data presented in this thesis suggests a novel function of S6K2 in chromatin-associated complexes. It also demonstrates the identification of DNA binding as a novel mode of kinase regulation, and a specific S6K2 binding partner, YY1. The ability of S6K2 to bind YY1 and to phosphorylate Histone H3 means that the possibility exists of its involvement in transcription regulation.

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