Use this URL to cite or link to this record in EThOS: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.756352
Title: Structural and biochemical studies of substrate recognition and allostery in aPKCι and PKN2
Author: Constable, Robert
ISNI:       0000 0004 7429 3053
Awarding Body: UCL (University College London)
Current Institution: University College London (University of London)
Date of Award: 2018
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Abstract:
The AGC family of protein kinases (related to protein kinases A, G and C) are a functionally and structurally distinct class of eukaryotic kinases that phosphorylate serine/threonine residues within their substrates. AGC kinases are implicated in diverse biological processes but mechanisms generating substrate specificity remain unclear. Protein kinase substrates can be recognised through sequences proximal to the phospho-acceptor, distal docking motifs or subcellular co-localisation by associated scaffold proteins. Peptide libraries revealed that AGC kinases target a basophilic (R-X-X-S/T-ψ) consensus but the contribution of distal docking motifs towards their substrate recognition, remains poorly understood. This thesis investigates structural features implicated in substrate recognition and allostery, in two related human AGC kinases; the atypical PKC (aPKC) isoform iota (PKCι) and protein kinase novel isoform 2 (PKN2), primarily using crystallographic and biochemical methods. The docking role of the FXR (Phe-X-Arg) motif, present in many cell polarity substrates of aPKC was investigated. This study supports the docking role of the FXR motif, reveals a novel allosteric pocket in the PKCι kinase domain, whilst collaborative studies in silico and in vivo indicate a broader role of the motif in polarity substrates of aPKC. Crystallographically defined allosteric pockets within the kinase domain of PKCι, were targeted in a search for small molecule ligands, including a high throughput compound library screen. The first structure of a substrate peptide bound to the PKN2 kinase domain is reported, revealing a novel peptide binding trajectory, engaging the conserved but poorly understood RYPR (Arg-Tyr-Pro-Arg) motif. This structure and preliminary biochemical data, explain the previously observed S312 phospho-acceptor preference of PKN2 in the CLIP1 protein, point to a RYPR mediated substrate docking PKN2 kinase domain dimer and suggest an extensive CLIP1-PKN2 interface potentially regulated by S755 phosphorylation in PKN2.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.756352  DOI: Not available
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