Abstract 4882: Inhibitor Kappa B Kinase Regulates Endothelial Nitric Oxide Synthase Activity and Nitric Oxide Generation
The involvement of heat shock protein 90 (Hsp-90) in the regulation of eNOS activity is known. We recently demonstrated the increased interaction of Hsp-90 with IKK β in vascular endothelial cells under conditions of high glucose. However, whether this binding of IKKβ to Hsp-90 will interfere with the binding of Hsp-90 to eNOS is not clear and the region on Hsp-90 to which IKKβ binds is unknown. In this study, we examined whether the interaction of Hsp-90 with eNOS is compromised by competitive binding of IKKβ. Our attempts to demonstrate competition between eNOS and IKKβ for Hsp-90 using purified proteins confirmed that elevated levels of IKKβ can dissociate eNOS-Hsp-90 complex. Hsp-90 and eNOS with increasing concentrations of purified Gst-IKKβ were incubated for 2h at 4°C. Immunoprecipitation of this complex with anti-eNOS antibody followed by Immunoblotting using anti-Hsp-90 antibody demonstrated reduced amounts of Hsp-90 associated to eNOS as concentration of IKKβ increases (Fig 1A⇓). The supernatants were re-precipitated with anti-GST antibody and the immune complex was analyzed for the presence of Hsp-90. Immunoblot showed the presence of Hsp-90 with higher concentration of IKKβ (1B). Further, we investigated whether IKKβ can inhibit eNOS enzyme activity by dissociating Hsp-90-eNOS complex. Nitric oxide formation was measured at 23°C using the hemoglobin capture assay. Addition of IKKβ significantly blocked eNOS activity (Fig 2⇓). Also, In vitro kinase assay performed using bovine aortic endothelial cell lysates and purified proteins demonstrated possible phosphorylation of Hsp-90 by IKKβ kinase. These results show the significance of IKKβ in regulating eNOS activity.