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Title: Affinity isolation and characterisation of PtdIns(3,4,5)P3 binding proteins from brain tissue
Author: Cook, Ian Haston
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2007
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The aim of this project was to identify proteins capable of specifically binding PtdIns(3,4,5)P3 in the brain and then to establish the cellular significance of their ability to respond to P13K signalling. Potential phosphoinositide binding proteins from mouse brain cytosolic fractions, were captured using matrices carrying the tethered homologs of PtdIns(3,4,5)P3. Individual PtdIns(3,4,5)P3 bead binding assays enabled mass spectrometric identification of over 100 proteins. They included several proteins with established phosphoinositide binding domains and many proteins with no reported phosphoinositide binding properties. Ten proteins or domains were selected for further analysis using recombinant protein based in vitro lipid binding assays. Four were proven to bind a range of phosphoinositides. These were the isolated PH domain from PLC-12 which preferentially bound PtdIns(4,5)P2 over PtdIns(3,4,5)P3 and PtdIns(4,5)P2 equally, the ENTH domain of Epsin2 which preferentially bound PtdIns(3,4)P2 over PtdIns(3,4,5)P3 over PtdIns(4,5)P2 and the Cullin homology domain from Cullin 4b which preferentially bound PtdIns(3,4,5)P3 over PtdIns(4,5)P2. As Cullin 4b had a high specificity for PtdIns(3,4,5)P3, a novel observation, the Cullin family was studied in greater detail. Cullin proteins are core components of E3 ubiquitin ligases. All of the Cullins, both full length and isolated Cullin homology domains bound to PtdIns(3,4,5)P3 beads but different family members showed different phosphoinositide specificity. Precedent has shown that a large subset, but not all, PI3K-effectors translocate in response to activation of class I PI3K. Hence we sought to find evidence for a physiological role for PI3K activity in the regulation of Cullins by studying their cellular distribution. This work provides evidence that some Cullins translocate only weakly in response to PI3K activation.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available