Use this URL to cite or link to this record in EThOS: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.790630
Title: Structural and biochemical characterisation of PKCι assemblies and substrate targeting mechanisms
Author: Ivanova, M.
ISNI:       0000 0004 8498 7446
Awarding Body: UCL (University College London)
Current Institution: University College London (University of London)
Date of Award: 2017
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Abstract:
The PKCι (protein kinase C isoform ι) plays crucial roles in the formation and maintenance of cell polarity through the formation of the Par complex (PKCι, Par6, Cdc42, Par3), a master regulator of epithelial apical-basal polarity. The Par complex is an apical polarity marker and is directly opposed by the baso-lateral markers Lgl, Dlg and Scribble by mutual antagonism. The molecular basis for the mutual antagonism between the complexes is unclear but may involve direct inhibition of PKCι activity. Conversely, several proteins able to inhibit PKCι are also functionally validated substrates, for example Lgl, Par3 and Kibra. The focus of this thesis has been to explore the molecular details of PKCι regulation and RIPR-dependent substrate recruitment. Different forms of PKCι have been characterised using biophysical, biochemical and structural biology methods. An active mature PKCι kinase domain structure was defined at high resolution. Comparison with a Par3-inhibited conformer reveals how inhibition perturbs the active conformer. To probe the molecular interaction between PKCι and RIPR-dependent substrates, Llgl1 and Periphillin1, peptides from these proteins were characterised and crystallised with the PKCι kinase domain. Amino-terminal sequences flanking their PKC consensus motif make contacts exclusively with the PKCι C-lobe. Surprisingly, both Llgl1 and Periphillin1 peptides structures lacked a bound nucleotide within of PKCι. In contrast, an ATP-bound PKCι lacked the Periphillin substrate peptide. These data suggest that RIPR-dependent substrate peptides may influence nucleotide loading of PKCι. Finally, the thesis explored a second mechanism for Par6-PKCι recruitment to the apical domain through Crb-Pals1 interaction. The basis for Crb intracellular domain interaction with Pals1 and the Par complex was defined indicating contacts from the Pals-PDZ-SH3 unit are required to engage Crb. The higher Pals1 affinity for Crb than the Par complex, suggests a hierarchy of substrate interactions to recruit Par6-PKCι through the Crb complex.
Supervisor: McDonald, N. Q. Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.790630  DOI: Not available
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