Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.641012
Title: Regulation of dematin by the p38 MAPK and the adaptor protein 14-3-3
Author: Baum, Holly
ISNI:       0000 0004 5350 144X
Awarding Body: University of Warwick
Current Institution: University of Warwick
Date of Award: 2014
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Abstract:
Dematin is an actin-binding and -bundling protein that was first identified in human erythrocytes where it is important for providing mechanical stability. Two splice variants exist in vivo of 48 KDa and 52 KDa, each of which comprises a disordered C-terminal core domain and a short, highly-ordered, N-terminal headpiece (HP). Expression of dematin has now been demonstrated in a wide range of tissues and functional roles are emerging for dematin as a tumour suppressor, and regulator of wound healing, via negative regulation of RhoA. Despite these advances, the in vivo regulation of dematin remains poorly understood. This thesis therefore aimed to characterise the regulation of dematin by two known cytoskeletal coordinators; the p38 mitogen-activated protein-kinase (MAPK), and the adaptor protein 14-3-3. Dematin was confirmed as a substrate of p38, and multiple phosphorylation sites were identified in both the core and HP regions of the protein using mass spectrometry and in vitro kinase assays. Moreover pulse-chase degradation analysis identified dematin as a short-lived protein, with a calculated half-life of 5.49 +/- 0.44 hours. Further experiments confirmed that this propensity for degradation is at least in part due to the presence of a PEST motif, with the half-life of the dematin_ΔPEST construct significantly increased compared to wild-type dematin at 7.64 +/- 0.72 hours. Despite identifying p38 phosphorylation sites within and flanking the PEST motif, hyper-activation or inhibition of p38 had no significant effect on protein stability. Finally, it was shown that although p38 phosphorylated the dematin HP at Ser383, this was not sufficient to disrupt the association of the HP and core regions which is known to inhibit actin bundling. Additionally dematin was identified as a novel substrate of the 14-3-3 family of adaptor proteins. The 14-3-3β isoform was shown to bind to 52 KDa dematin at two phosphorylated motifs; RKTRS269LP and RGNS333LP. It was not possible to identify the kinases that regulate these motifs, but Akt, PKA, or AMPK were ruled out as potential candidates. Immunofluorescence staining and confocal imaging confirmed that 14-3-3 regulates dematin by altering its subcellular distribution, which is likely to be via masking of an actin-binding motif. Wild-type dematin was distributed throughout the cytoplasm in C2C12 skeletal muscle cells, whereas the dematin_S269/333A binding mutant localised strongly to the F-actin cytoskeleton. This localisation result was confirmed in quantitative sedimentation experiments, which showed a statistically significant decrease in both the actin-binding (42.8%) and -bundling (30.1%) ability of dematin upon 14-3-3 association. In this thesis I have characterised both the p38 MAPK and the adaptor protein 14-3-3 as novel regulators of dematin, adding considerably to the understanding of dematin regulation in vivo. This provides a basis for further investigation into the ability of dematin to coordinate the actin cytoskeleton, which has implications for a wide array of dematin functions due to its ubiquitous tissue expression profile.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.641012  DOI: Not available
Keywords: QP Physiology
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