Role of DNA binding in the regulation and function of ribosomal S6 kinase 2

Ribosomal protein S6 kinase (S6K) is a member of the AGC family of Ser Thr kinases which also includes PKA, PKB (Akt), PKCs etc. Biochemical and genetic studies in cell-based and animal models have provided evidence that S6K is a principal player in the regulation of cell growth, size and energy metabolism. Two major signal transduction pathways, phosphatidylinositide 3-kinases (PI3K) and mammalian target of rapamycin (mTOR), coordinate the activity of S6Ks in response to extracellular and intracellular stimuli, such as growth factors, mitogens, metabolites and nutrients. In an activate state, S6Ks translocate to discrete cellular compartments/multienzyme complexes, where they interact with and phosphorylate diverse substrates implicated in the regulation of translation, RNA processing, cytoskeletal rearrangement, cell growth and survival. A growing body of evidence links S6K signalling to various human pathologies, including diabetes, ageing and cancer.
In mammalian cells, there are two isoforms of S6K, termed S6K1 and S6K2. Nucleocytoplasmic shuttling has been reported for both isoforms of S6Ks, but their nuclear functions are not well understood. This grant application is based on our novel findings which imply the role of S6K2 in the regulation of transcription. We provide the evidence that S6K2 possesses an AT-hook DNA-binding domain at the C-terminal autoinhibitory domain, which mediates specific interaction with DNA. We also found that the activity of S6K2 is induced by direct interaction with DNA in a dose-dependent manner. Furthermore, mutational studies of the AT-hook motif revealed its importance in the regulation of S6K2 pro-survival signalling.
To further advance these original findings, we propose to study molecular mechanisms of S6K2-DNA interaction and an alternative model of S6K2 activation upon DNA binding by employing a range of biophysical, biochemical and cellular approaches. Our efforts will be also focused on exploring a cooperative mode of interaction between S6K2 and transcription factors in targeting promoter and enhancer regions of a specific set of genes, implicated in the regulation of S6K2-mediated cellular processes. Mammalian cell models and xenograft studies in nude mice will be employed to study the role of the S6K2 AT-hook motif in the regulation of transcription, ribosome biogenesis, cell survival and growth. It is expected that the proposed study will give deeper insight into nuclear functions of S6K2 and its implication in the regulation of transcription. Unique features of the S6K2-DNA interaction are not only of academic interest, but may also provide novel insights applicable to drug development.

Ribosomal protein S6 kinase (S6K) is implicated in the regulation of cell growth, survival and glucose metabolism in response to extracellular stimuli and stresses. Deregulation of S6K signaling has been linked to various human pathologies, including obesity, diabetes and cancer. To date, nuclear functions of S6Ks are not well understood. We have recently discovered a novel feature of S6K2 regulation involving specific interaction with DNA via the AT-hook motif and the activation of its kinase activity. These original findings are fur-ther supported by mutational studies which reveal the importance of the AT-hook motif in S6K2 pro-survival signalling. We hypothesize that S6K2 has a potential to form regulatory complexes with transcription factors which may deliver the kinase at promoter regions of target genes in a sequence-specific manner. At DNA, the AT-hook motif can interact with the minor groove of AT-rich sequences, leading to a conformational change and activation of S6K2. In an activated state, S6K2 may modulate gene expression through phosphoryla-tion of transcription factors and/or protein-protein interactions. In the proposed study, we will focus to: a) investigate the molecular basis of the S6K2/DNA interaction; b) test an al-ternative model of S6K2 activation by DNA binding; c) explore a cooperation between S6K2 and transcription factors in targeting prompter/enhancer regions of a specific set of genes; d) investigate physiological relevance of the DNA binding activity of S6K2, focusing in particular on the regulation of transcription, ribosome biogenesis, cell growth and sur-vival. The proposed study may uncover a second member of a protein kinase family, in addition to DNA-PK, with a potential to be activated upon direct bindsing to DNA. It might also establish a platform for the development of small molecule compounds capable to block S6K2/DNA interaction and relevant S6K2-mediated functions.

The primary benefit of the proposed research relates to increased knowledge of the regulation and function of S6K2, which we will intend to disseminate through peer-reviewed journals, national and international conferences, and free-to-access websites. We will also make S6K reagents and technical expertise freely available and will attempt to stimulate research in this field. Considering the current gap in knowledge relating to S6K2 regulation and function and our novel findings that the activity of S6K2 can be regulated by DNA binding, we believe the proposed research has the potential to significantly contribute to a better understanding of the roles of S6K2 in health and disease, and in the long-term, development of better treatment for diseases in which S6K2 is implicated. In particular, the study of the mechanism of S6K2 activation by DNA binding may present an opportunity for developing a novel way to modulate the activity of S6K2 and its downstream effects. We have an on-going S6K drug discovery programme which is primarily focused on developing small molecule inhibitors for S6K1. Where possible, we will feed findings from the proposed study into our ongoing drug discovery project and stimulate the development of a HTP screen for the identification of small molecule compounds capable to specifically inhibit the S6K2/DNA interaction. The proposed work therefore has the potential to initiate the development of novel therapeutic drugs in the long-term, as well as fostering positive economic outcomes, considering the rapidly growing global pharmaceutical market for protein kinase inhibitors. Another potential benefit of this research relates to the experience and skills gained by the staff involved in this project, which includes undergraduate and internship students who will work on the project. The proposed research offers a diverse range of techniques including biochemical and molecular biology techniques, bacterial and baculoviral expression of recombinant proteins, lentiviral overexpression and downregulation, mammalian cell biology, in vitro kinase assays and SPR analysis. Effective time-management, co-ordination and communication skills will therefore be critical for successful completion of this highly multidisciplinary project.