Sammendrag
Previous work on the calcium/calmodulin-dependent kinase kinase 2 (CaMKK2) has focussed, in particular, on the contribution of CaMKK2 as an androgen receptor target gene to the development and progression of prostate cancer. Despite the very significant volume of research on the functional contribution of CaMKK2 to disease there has been limited progress in understanding the structure and biophysical properties of the enzyme. To advance drug development it is vital to make progress in these latter areas.
The work presented in this thesis focused on structural and functional studies of CaMKK2 in prostate cancer. Firstly, we set out to improve the expression and purification of CaMKK2 in complex with calmodulin with the intention to crystallise the complex. Since our crystallisation attempts were unsuccessful we decided to pursue other avenues, and instead successfully established a new biophysical assay based on the interaction between CaMKK2 and the CaMKK2 inhibitor, STO-609. In a parallel cellular approach we studied the CaMKK2 interactome using co-immunoprecipitation and mass spectrometry to identify novel interacting partners of the kinase. Furthermore, we went on to evaluate the contribution of CaMKK2 to autophagy using functional assays.
In summary, we report a detailed co-expression and co-purification protocol for the CaMKK2:calmodulin complex, that has led to the development of a novel in vitro binding assay for CaMKK2 by exploiting the intrinsic fluorescence of STO-609. Novel phosphorylation sites in the activation segment of the kinase were detected. Furthermore, we identified a direct interaction between CaMKK2 and Gemin4, a subunit of the multiprotein SMN complex, and discovered that CaMKK2 does not regulate autophagy in prostate cancer cells, whilst STO-609 might enhance autophagy.