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.

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.