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Structural and functional characterisation of PKCI

Kerai, Preeti; (1999) Structural and functional characterisation of PKCI. Doctoral thesis (Ph.D), UCL (University College London). Green open access

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Abstract

PKCI is a member of the highly conserved and ubiquitous HIT (Histidine triad) family of proteins. It was originally purified as an inhibitor of protein kinase C (PKC) and proposed to bind zinc via the HIT motif. However, recent studies have shown that PKCI is not a regulator of PKC activity. In order to structurally and functionally characterise PKCI, the gene was cloned from a human T-cell cDNA library, expressed in Escherichia coli and the recombinant protein purified. The three-dimensional structure of PKCI was determined and refined at 2.2 Å resolution. The main structural feature of PKCI is a ten stranded β-sheet structure with a typical right handed twist that extends across two PKCI monomers to form a dimer. Each monomer consists of five antiparallel β-sheets and two β-helices. Circular dichroism studies show that while human PKCI does not bind zinc, maize PKCI binds zinc via Cysl07 but not the HIT motif. Although it has not been possible to detect an association between PKCI and 14-3-3, PKCI and 14-3-3 proteins appear to synergistically inhibit PKC activity. However, PKCI alone does not inhibit nor interact with PKC. To date, no definitive description of any function for PKCI has been reported. In order to elucidate a physiological function for PKCI, affinity chromatography was used to identify PKCI interacting proteins. Munc18-1 was identified as the major PKCI-binding protein in porcine brain. Munc18-1 is known to interact with syntaxin 1A and is required for synaptic vesicle exocytosis. I have shown that munc18-l and PKCI bind with an equilibrium dissociation constant of 0.3μM. Munc18-1 complexed with PKCI no longer binds to syntaxin 1A, suggesting a role for PKCI in neurotransmitter release where it may regulate SNARE complex formation. Injection of PKCI into squid giant presynaptic terminals reduces synaptic depression indicating that PKCI increases the number of synaptic vesicles available for neurotransmitter release. Immunofluorescence studies show that PKCI is localised to the plasma membrane, consistent with a potential interaction between PKCI and muncl8 in vivo. Together, these data demonstrate a potential physiological function for PKCI in synaptic vesicle exocytosis.

Type: Thesis (Doctoral)
Qualification: Ph.D
Title: Structural and functional characterisation of PKCI
Open access status: An open access version is available from UCL Discovery
Language: English
Additional information: Thesis digitised by ProQuest.
Keywords: Biological sciences; Protein kinase C
URI: https://discovery.ucl.ac.uk/id/eprint/10121851
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