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Title: Proteomic analysis of Vascular Endothelial Growth Factor (VEGF) signalling : studies of the mechanism of VEGF-induced Heat Shock Protein 27 phosphorylation and its role in endothelial cell signalling and function
Author: Britton, G.
ISNI:       0000 0004 2727 6795
Awarding Body: University College London (University of London)
Current Institution: University College London (University of London)
Date of Award: 2010
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Vascular Endothelial Growth Factor (VEGF) is essential for angiogenesis and endothelial function. Proteomic analysis of Human Umbilical Vein Endothelial Cells (HUVEC) identified Heat Shock Protein 27 (Hsp27) as a major VEGF-regulated protein. Hsp27 is implicated in actin organization, cell survival and migration, and is a potential mediator of these VEGF functions in the endothelium. Studies of pharmacological inhibitors indicated that VEGF-stimulated Hsp27 serine 82 (S82) phosphorylation was resistant to p38 mitogen-activated protein kinase inhibition and mediated by Protein Kinase C (PKC). VEGF activated Protein Kinase D (PKD), and this effect was inhibited by small interfering (si)RNAs targeting selected PKC isoforms. PKD2 siRNA inhibited VEGF-induced Hsp27 S82 phosphorylation, and PKD2 immunoprecipitated from VEGF-treated cells selectively phosphorylated Hsp27 at S82. Hsp27 siRNAs markedly inhibited VEGF-induced cell migration, increased apoptosis and reduced tubulogenesis. Furthermore, inhibition of PKC but not p38 kinase inhibited VEGF-stimulated cell migration. Overexpression of S82A and S82D Hsp27 mutants using adenoviral vectors (Ad) had no significant effect on migration. However, VEGF reduced Hsp27 oligomeric size, and Ad-overexpressed S82D Hsp27 also formed smaller oligomers than wild-type Hsp27. These findings identify a VEGF/PKC/PKD/Hsp27 S82 pathway, indicate a role for PKD and HSP27 in VEGF-induced endothelial migration, and also suggest a specific role for Hsp27 S82 phosphorylation in regulation of Hsp27 oligomerisation. Further proteomic analysis of HUVECs identified Stomatin-Like Protein 2 (SLP2) as a major component of anti-phosphotyrosine immunoprecipitates. The function of SLP2 is little understood. VEGF did not alter the amount of anti-phosphotyrosine-associated SLP2, and further investigations suggested that SLP2 may not be directly tyrosine phosphorylated. SLP2 was localized to mitochondria and co-immunoprecipitated with Prohibitin, a protein implicated in mitochondrial function. However, siRNA-mediated SLP2 knockdown did not affect mitochondrial membrane potential, apoptosis or migration of endothelial cells, and the function of this protein remains unknown.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available