Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.652159
Title: Discovery and characterisation of novel protein interactions with death associated protein kinase
Author: Harrison, B. J.
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2009
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Abstract:
Combinatorial peptide libraries displayed on M13 filamentous bacteriophage were used to identify peptide consensus binding sites for the kinase domain of DAPK. Peptides that bound to the DAPK core kinase domain were then isolated and sequenced leading to the discovery of binding peptides with striking homology to the SK1-4 family of transcription factors, the Promyelocytic Leukemia protein (PML) and the microtubule associating protein MAP1B. Cell growth and viability assays demonstrated that MAP1B co-operates with DAPK to reduce cell proliferation. This co-operative cell growth inhibition was independent of the p53 pathway and apoptotic (Type 1) cell death, but induced autophagic (Type II) cell death. MAP1B cooperation with DAPK was marked by a striking increase in the number of cells with membrane blebbing morphology, an effect previously shown to involve DAPK interaction with the actin cytoskeleton leading to actin-myosin contraction. This was in contrast to the known role of MAP1B that is primarily thought of as a tubulin associating protein that modifies microtubule dynamics. Therefore the role of the cytoskeleton in DAPK co-operation with MAP1B was studied in detail using immunoflurorescent cytoskeleton staining and microtubule purification assays. During DAPK transfection induced membrane blebbing, a pool of DAPK and MAP1B co-localise and co-purify with tubulin where as a separate pool is co-located to cortical actin. This DAPK and MAP1B cooperation-induced membrane blebbing involves a novel interaction with both microtubules and microfilaments. These studies highlight the utility of peptide combinatorial libraries to identify novel binding interfaces and highlight a positive role for MAP1B in DAPK dependent cytoskeletal rearrangement and the autophagic cell death program.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.652159  DOI: Not available
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