Vascular endothelial growth factor induces heat shock protein (HSP) 27 serine 82 phosphorylation and endothelial tubulogenesis via protein kinase D and independent of p38 kinase.
Proteomic analysis identified HSP27 phosphorylation as a major change in protein phosphorylation stimulated by Vascular Endothelial Growth Factor (VEGF) in Human Umbilical Vein Endothelial Cells (HUVEC). VEGF-induced HSP27 phosphorylation at serines 15, 78 and 82, but whereas HSP27 phosphorylation induced by H2O2 and TNFalpha was completely blocked by the p38 kinase inhibitor, SB203580, VEGF-stimulated serine 82 phosphorylation was resistant to SB203580 and small interfering(si)RNA-mediated knockdown of p38 kinase and MAPKAPK2. The PKC inhibitor, GF109203X, partially reduced VEGF-induced HSP27 serine 82 phosphorylation, and SB203580 plus GF109203X abolished phosphorylation. VEGF activated Protein Kinase D (PKD) via PKC, and siRNAs targeted to PKD1 and PKD2 inhibited VEGF-induced HSP27 serine 82 phosphorylation. Furthermore recombinant PKD selectively phosphorylated HSP27 at serine 82 in vitro, and PKD2 activated by VEGF in HUVECs also phosphorylated HSP27 selectively at this site. Knockdown of HSP27 and PKDs markedly inhibited VEGF-induced HUVEC migration and tubulogenesis, whereas inhibition of the p38 kinase pathway using either SB203580 or siRNAs against p38alpha or MAPKAPK2, had no significant effect on the chemotactic response to VEGF. These findings identify a novel pathway for VEGF-induced HSP27 serine 82 phosphorylation via PKC-mediated PKD activation and direct phosphorylation of HSP27 by PKD, and show that PKDs and HSP27 play major roles in the angiogenic response to VEGF.
Evans
Ian M
IM
Centre for Cardiovascular Biology and Medicine, Department of Medicine, The Rayne Institute, University College London, 5 University Street, London WC1E 6JJ, United Kingdom.
Britton
Gary
G
Zachary
Ian C
IC
D002465:Cell Movement / Q000187:drug effects; D003094:Collagen / Q000378:metabolism; D042783:Endothelial Cells / Q000166:cytology / Q000187:drug effects / Q000201:enzymology; D004727:Endothelium / Q000098:blood supply* / Q000187:drug effects / Q000201:enzymology*; D006360:Heat-Shock Proteins / Q000378:metabolism*; D006801:Humans; D018919:Neovascularization, Physiologic / Q000187:drug effects*; D010766:Phosphorylation / Q000187:drug effects; D010768:Phosphoserine / Q000378:metabolism*; D011493:Protein Kinase C / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000378:metabolism; D042461:Vascular Endothelial Growth Factor A / Q000494:pharmacology*; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism
D006360:Heat-Shock Proteins; D034741:RNA, Small Interfering; D042461:Vascular Endothelial Growth Factor A; D010768:Phosphoserine; D003094:Collagen; C089212:protein kinase D; D011493:Protein Kinase C; D048051:p38 Mitogen-Activated Protein Kinases