Use this URL to cite or link to this record in EThOS: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.790522
Title: The PDKI-atypical PKC paradigm
Author: De Naurois, J. M.
ISNI:       0000 0004 8498 3912
Awarding Body: UCL (University College London)
Current Institution: University College London (University of London)
Date of Award: 2016
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Abstract:
AGC kinases have been the subject of extensive research in cell signalling and particularly in cancer. Using several cell-based cancer models, this thesis studied two such kinases, Phosphoinositide-dependent kinase 1(PDK1), and one of its substrates, atypical Protein kinase C iota (PKCι). Using both biochemical and Förster Resonance Energy Transfer determined by fluorescent lifetime imaging microscopy, we uncovered the existence of an in vivo PDK1-PKCι protein complex in cancer cells. Using ectopic expression of wild-type and mutant proteins, the key protein motifs required for the assembly of this protein complex were established as the PIF pocket of PDK1, and the C-terminal hydrophobic tail of PKCι. This was confirmed with a cell penetrant peptide competitor. There was no modulation of the PDK1-PKCι complex or PKCι T-loop phosphorylation (T412) by upstream growth factor stimulation, contrary to the known effects of PDK1 on AKT, but consistent with PKCι being already highly phosphorylated on T412. However, prolonged treatment of cells with an ATPpocket competitive inhibitor of PDK1 (GSK2334470) resulted in PDK1-PKCι complex disassembly as measured either by co-immunoprecipitation assays or by FRET. Disassembly was associated with a nuclear shift in the cellular localisation of PKCι. Additionally, when using an open conformation PKCι mutant (A129E), there was net dephosphorylation of T412 following PDK1 inhibition. These results are consistent with a dual role for PDK1 in both maintaining active PKCι T-loop phosphorylation, as well as retaining PKCι in the cytoplasm through direct interaction. It was found that atypical PKC inhibition and PDK1 inhibition did not display similar effects in regulating proliferation. Given that PDK1 appears to be responsible for the activation loop phosphorylation of atypical PKC this implies that PDK1 inhibition may be insufficient to prevent the ratchet-like behaviour of atypical PKC phosphorylation and hence unable to mimic the direct effect of PKCι inhibition.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.790522  DOI: Not available
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