Structural and biochemical characterisation of the Rho effector kinase PKN2

The Rho effector kinase Protein Kinase N2 (PKN2) is a serine/threonine kinase with crucial functions as a key regulator of cytoskeletal organisation and cell migration in addition to roles in disease progression. Understanding of PKN function at a molecular level is currently limited in terms of their overall structural organisation, how activating partners co-operate to promote PKN activation and finally what features are recognised by PKNs to target their substrates. The aims of this thesis were to address these questions using electron microscopy, biochemical and biophysical methods. The thesis has four components. First, biochemical characterisation revealed that recombinant PKN2 activation-loop phosphorylation requires both RhoA and PDK1. In vitro characterisation of a novel, single case patient-derived PKN2 mutation revealed an overall reduced enzymatic activity compared to WT PKN2 and potential differences in putative binding partners identified by co-IP/MS. Second, the architecture and dimeric nature of RhoA-PDK1-activated PKN2 are explored by negative-stain electron microscopy revealing a low-resolution map of an asymmetric PKN2 dimer stabilised through chemical cross-linking. The map permits tentative docking of existing crystal structures and homology models of PKN2 domains. Efforts to obtain a higher resolution cryo-EM structure were hampered by sample conformational flexibility and instability. Third, a local motif flanking the phospho-acceptor site was uncovered using PamGene microarray technology. Work focused on characterising PKN2 phosphorylation of CLIP170, a microtubule associated protein and putative PKN2 substrate. In vitro phosphorylation was monitored over time permitting mapping and ordering of phospho-sites within the serine-rich regulatory region of CLIP170. Efforts to develop an assay to validate phosphorylation of CLIP170 by endogenous PKN2 from mouse brain lysate revealed the need for better PKN2 small molecule inhibitors. The final chapter describes the development of PKN2 inhibitor compounds. Several compounds improved on existing benchmarked compounds and will be taken forward for selectivity screening and validation in cell-based assays