Kostelecky, B.D.; (2009) An investigation of PKC isoform functional specificity. Doctoral thesis , UCL (University College London).

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Abstract

Protein kinase C (PKC) isozymes are vital signalling proteins in many intracellular processes including cell survival, proliferation and migration. As such, changes in their expression levels have been linked to many types of cancer. The various PKC family members provoke differential responses in cancer highlighting the need for study of individual isoforms. This investigation of PKC has aimed to determine how kinase domain structure, regulatory region interactions and binding partners confer functional specificity to individual PKC isoforms. X-ray crystallographic, biochemical and biophysical studies have been employed to explore the architecture of these PKC interactions. A panel of recombinant PKC kinase domains has been cloned, expressed and purified to characterise their maturation, activities and structures. Kinetic constants have been determined for several PKC kinase domains in various phosphorylation states. Additionally, inhibition by novel low molecular weight inhibitors provided by collaborators at Cancer Research Technologies (CRT) has been probed. The PKCζ kinase domain has been crystallised with one of the CRT inhibitors and the structure determined at 2.8Å resolution. A panel of PKC isoform regulatory regions has also been expressed and purified. The results presented here show it is possible to reconstitute an intact PKC holoenzyme complex after expression of the domains as individual polypeptides. The protocols and materials developed during this thesis project will be further used in the laboratory with the aim of crystallising a PKC holoenzyme complex. This thesis also presents the crystal structure of PKCε-binding partner 14-3-3 bound to an asymmetric PKCε di-phosphorylated peptide determined at 2.2Å resolution. The PKCε di-phosphorylated peptide in the crystal structure was derived from the PKCε V3 variable region containing one consensus 14-3-3-phospho-binding motif and one divergent 14-3-3-binding motif. A thermodynamic analysis of the interaction between 14-3-3 and the PKCεV3 di-phosphopeptide reveals an increased affinity more than two orders of magnitude greater than the singly phosphorylated species. Together, the results of this study provide a multifaceted examination of PKC functional specificity by isoform-specific low molecular weight inhibitors, regulatory domains and binding partner interactions and provide a solid platform for exploring further aspects of PKC regulation.

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