Src-mediated Phosphorylation of Hsp90 in Response to Vascular Endothelial Growth Factor (VEGF) Is Required for VEGF Receptor-2 Signaling to Endothelial NO Synthase

Abstract

Nitric oxide (NO) release from endothelial cells, via endothelial NO synthase (eNOS) activation, is central to the proangiogenic actions of vascular endothelial growth factor (VEGF). VEGF signaling to eNOS is principally mediated byan Akt-dependent phosphorylation of eNOS and by increased association of eNOS to the molecular chaperone, heatshock protein 90 kDa (Hsp90). Herein, we report that VEGFR-2 activation induces tyrosine phosphorylation of VEGF receptor 2 (VEGFR-2)-associated Hsp90␤. Tyrosine phosphorylation of Hsp90␤ in response to VEGF is dependent on internalization of the VEGFR-2 and on Src kinase activation. Furthermore, we demonstrate that c-Src directly phosphorylates Hsp90 on tyrosine 300 residue and that this event is essential for VEGF-stimulated eNOS association to Hsp90 and thus NO release from endothelial cells. Our work identifies Y300 phosphorylation of Hsp90 as a novel regulated posttranslational modification of the chaperone and demonstrates its importance in the proangiogenic actions of VEGF, namely by regulating NO release from endothelial cells. INTRODUCTIONVascular endothelial growth factor (VEGF) is a potent proangiogenic cytokine that activates three specific receptor tyrosine kinases (RTK): VEGF receptor (VEGFR)-1, -2, and -3. Gene knockout studies revealed that VEGF and VEGFRs are essential for the development of the vasculature in mouse embryos (Fong et al., 1995;Shalaby et al., 1995;Carmeliet et al., 1996;Dumont et al., 1998). In addition to its functional roles in vasculogenesis, VEGF plays key roles in adult physiological and pathological angiogenesis such as wound healing and tumor vascularization. VEGFR-2 mediates most of the functional and biochemical effects of VEGF in endothelial cells including proliferation, migration, survival, and nitric oxide (NO) release. These effects are produced through the activation of multiple signaling pathways, such as the phosphoinositide-3 kinase (PI-3 kinase)/ Akt, p38 mitogen-activated protein kinase (MAPK), phospholipase C (PLC)-␥, and Erk/MAPK, after the activation of VEGFR-2 (Rousseau et al., 1997;Takahashi and Shibuya, 1997;Gerber et al., 1998;Takahashi et al., 2001). VEGF-mediated activation of the Ser/Thr kinase Akt leads to phosphorylation of serine 1179 on bovine endothelial NO synthase (eNOS; Ser 1177 in the human form), which increases eNOS catalytic activity and results in release of NO from endothelial cells (Fulton et al., 1999;Dimmeler et al., 1999). In turn, NO released from endothelium contributes to the proangiogenic effects of VEGF. Indeed, VEGF-mediated vascular permeability, angiogenesis, and endothelial cell precursor mobilization are markedly impaired in eNOS-deficient mice (Fukumura et al., 2001;Aicher et al., 2003).Heat-shock protein 90 kDa (Hsp90) is a multifunctional molecular chaperone, and its role in the prevention of protein aggregation and in the refolding of denatured proteins has been studied in much detail (Whitesell and Lindquist, 2005). Hsp90 is also a regulator of the activity of signaling molecules su...

Hsp90 is expressed and represents a therapeutic target in human oesophageal cancer using the inhibitor 17-allylamino-17-demethoxygeldanamycin

“…Moreover, we detected tyrosine phosphorylation of Hsp90, which recently has been connected to Hsp90 functions in vascular endothelial growth factor receptor-2 signalling (Duval et al, 2007). However, the functional consequence of Hsp90 tyrosine phosphorylation in our model system is not understood and warrants further investigation.…”

Conformational dynamics of the molecular chaperone Hsp90

“…Phosphorylation at S225 and S254 in Hsp90 causes a decreased affinity for the aryl hydrocarbon receptor (AhR) (Ogiso et al, 2004). In another example, c-src directly phosphorylates Hsp90 on Y300 and unlike other clients this phosphorylation event is required for the binding of eNOS to Hsp90 (Duval et al, 2007). Further studies will be required to determine the effect of phosphorylation on the conformation of Hsp90.…”

Hsp90 phosphorylation, Wee1 and the cell cycle

“…27 A recent study reported that c-Src directly phosphorylates Tyr300 of human Hsp90β. 32,33 This is essential for VEGF-stimulated endothelial nitric oxide synthase (eNOS) association with Hsp90 and thus is necessary for nitric oxide release from endothelial cells. 33 Another study reported that tyrosinephosphorylated Hsp90 repressed the function of ionotropic P2X 7 receptors.…”

“…32,33 This is essential for VEGF-stimulated endothelial nitric oxide synthase (eNOS) association with Hsp90 and thus is necessary for nitric oxide release from endothelial cells. 33 Another study reported that tyrosinephosphorylated Hsp90 repressed the function of ionotropic P2X 7 receptors. These receptors serve as ligand-gated ion channels and are responsible for ATPdependent lysis of macrophages.…”