Hsp90 Ensures the Transition from the Early Ca2+-dependent to the Late Phosphorylation-dependent Activation of the Endothelial Nitric-oxide Synthase in Vascular Endothelial Growth Factor-exposed Endothelial Cells

Abstract

Vascular endothelial growth factor (VEGF) exerts its angiogenic effects partly through the activation of endothelial nitric-oxide synthase (eNOS). Association with heat shock protein 90 (hsp90) and phosphorylation by Akt were recently shown to separately activate eNOS upon VEGF stimulation in endothelial cells. Here, we examined the interplay between these different mechanisms in VEGF-exposed endothelial cells. We documented that hsp90 binding to eNOS is, in fact, the crucial event triggering the transition from the Ca 2؉ -dependent activation of eNOS to the phosphorylation-mediated potentiation of its activity by VEGF. Accordingly, we showed that early VEGF stimulation first leads to the Ca 2؉ /calmodulin disruption of the caveolin-eNOS complex and promotes the association between eNOS and hsp90. eNOS-bound hsp90 can then recruit VEGF-activated (phosphorylated) Akt to the complex, which in turn can phosphorylate eNOS. Further experiments in transfected COS cells expressing either wild-type or S1177A mutant eNOS led us to identify the serine 1177 as the critical residue for the hsp90-dependent Akt-mediated activation of eNOS. Finally, we documented that although the VEGF-induced phosphorylation of eNOS leads to a sustained production of NO independently of a maintained increase in [Ca 2؉ ] i , this late stage of eNOS activation is strictly conditional on the initial VEGFinduced Ca 2؉ -dependent stimulation of the enzyme. These data establish the critical temporal sequence of events leading to the sustained activation of eNOS by VEGF and suggest new ways of regulating the production of NO in response to this cytokine through the ubiquitous chaperone protein, hsp90. Nitric oxide (NO)1 contributes to the cardiovascular homeostasis through its profound effects on blood pressure, vascular remodeling, platelet aggregation, and angiogenesis (1). Under normal conditions, the endothelial isoform of NO synthase (eNOS) expressed both in endothelial cells (EC) and cardiac myocytes is the major source of NO in the cardiovascular system (2). eNOS was originally identified as a particulate enzyme, associated primarily with the plasma membrane and with intracellular organelles such as the Golgi complex (3). Recent findings suggest that trafficking of eNOS from plasmalemmal to intracellular structures is part of a physiological cycle highly sensitive to the state of cell activation (4 -8). The dynamic trafficking of eNOS between cell surface and intracellular compartments is supported in part by its close interaction with the scaffold protein caveolin (4). In addition to regulating eNOS subcellular localization, this stable interaction with caveolin leads to the inhibition of the enzyme activity in basal conditions. Upon increases in intracellular calcium ([Ca 2ϩ ] i ), Ca 2ϩ /calmodulin (CaM) displaces the inhibitory binding of caveolin to eNOS and allows enzyme activation (9). Similarly, increasing vascular flow and pressure in an in situ artery perfusion model rapidly leads to activation of caveolar eNOS with apparent eNOS ...

S-nitrosylation of Hsp90 promotes the inhibition of its ATPase and endothelial nitric oxide synthase regulatory activities

“…The data in these studies suggest that Hsp90 promotes the transition of eNOS-Ca 2ϩ dependency to eNOS-Ca 2ϩ independency by synergizing with Akt, thus lending a molecular basis for a calcium-independent operative mechanism. It has also been proposed that Hsp90 acts as a scaffold protein for eNOS and Akt, facilitating eNOS phosphorylation by Akt (19,50). This hypothesis fits well with the biochemical and structural data available, which show that there are different ␣␤␣ structural motifs in the M domain of Hsp90 where the two proteins could interact without a structural overlapping ( Fig.…”

“…A direct interaction between eNOS and Hsp90 has been demonstrated (27), and Hsp90 has been implicated in many of the positive regulatory mechanisms of eNOS, both Ca 2ϩ -dependent and -independent (46)(47)(48)(49). Two different studies report cooperativity between Hsp90 and Akt in relation to VEGF and insulin (50,51). The data in these studies suggest that Hsp90 promotes the transition of eNOS-Ca 2ϩ dependency to eNOS-Ca 2ϩ independency by synergizing with Akt, thus lending a molecular basis for a calcium-independent operative mechanism.…”

NOSTRIN: A protein modulating nitric oxide release and subcellular distribution of endothelial nitric oxide synthase

“…In vascular endothelial growth factor (VEGF)-triggered endothelial cells, hsp90 mediates the interaction between eNOS and Akt by inducing the transition from the ''early'' Ca 2ϩ -dependent to the ''late'' phosphorylation-dependent activation of eNOS (13). It is conceivable that NOSTRIN may modulate eNOS (de)phosphorylation, thereby affecting subcellular distribution and, thus, activity of eNOS.…”

“…Posttranslational modifications are efficiently complemented by multiple proteinprotein interactions that help regulate eNOS activity with respect to time and space. For instance, chaperone hsp90 bound to eNOS may mediate vascular endothelial growth factor-induced eNOS phosphorylation by promoting the interaction between eNOS and Akt (12,13). At the plasma membrane, eNOS is complexed to and inhibited by the master components of caveolae, i.e., caveolin-1 in endothelial cells (9,14) and caveolin-3 in cardiac myocytes (15).…”

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

“…Hsp90 is also a regulator of the activity of signaling molecules such as c-Src, Akt, and eNOS and contributes to their activation. Hsp90 is known to associate with eNOS and Akt after VEGF stimulation, which contributes to increase eNOS catalytic activity (Garcia-Cardena et al, 1998;Brouet et al, 2001a;Fontana et al, 2002). In addition to a direct allosteric effect of Hsp90 on eNOS, it is thought that binding of Hsp90 to Akt prevents the phosphatase 2A-mediated dephosphorylation of Thr 308 of Akt, which maintains its positive functional effects on eNOS catalytic activity (Sato et al, 2000;Takahashi and Mendelsohn, 2003).…”