Direct interaction of the cell division cycle 37 homolog inhibits endothelial nitric oxide synthase activity.
Endothelial NO synthase (eNOS) via the production of NO in the endothelium plays a key role in cardiovascular biology and is tightly regulated by co- and posttranslational mechanisms, phosphorylation, and protein-protein interactions. The cell division cycle 37 homolog (Cdc37) is a key heat shock protein 90 (Hsp90) cochaperone for protein kinase clients, and Akt/Hsp90 interaction is dependent on Cdc37. Because both Hsp90 and Akt are key eNOS regulatory proteins, we hypothesized that Cdc37 interacts with eNOS as part of the regulatory complex. In the present study, we demonstrate by coimmunoprecipitation and affinity purification in bovine aortic endothelial cells (BAECs) that Cdc37 is complexed with eNOS, Hsp90, and Akt. In addition, cell fractionation data indicate that Cdc37 is found in caveolae with eNOS. Further analysis by in vitro binding assays reveals a direct interaction between purified Cdc37 and eNOS. Incubation of purified Cdc37 with purified wild-type eNOS decreases eNOS activity in vitro. Overexpression of wild-type Cdc37 in BAECs inhibits eNOS activity and NO release, whereas overexpression of S13A-Cdc37 mutant in BAECs increases eNOS activity and NO release. Taken together, these data suggest that Cdc37 has a direct regulatory interaction with eNOS and may play an important role in mediating the eNOS protein complex formation as well as subsequent eNOS phosphorylation and activation.
Harris
M Brennan
MB
Department of Kinesiology, College of William & Mary, Williamsburg, VA 23187-8795, USA. mbharr@wm.edu
Bartoli
Manuela
M
Sood
Sarika G
SG
Matts
Robert L
RL
Venema
Richard C
RC
D000818:Animals; D001011:Aorta; D002417:Cattle; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000201:enzymology; D004789:Enzyme Activation; D004791:Enzyme Inhibitors; D018841:HSP90 Heat-Shock Proteins / Q000235:genetics / Q000502:physiology*; D052250:Nitric Oxide Synthase Type III / Q000037:antagonists & inhibitors / Q000378:metabolism*; D010766:Phosphorylation; D011993:Recombinant Fusion Proteins / Q000494:pharmacology; D042461:Vascular Endothelial Growth Factor A / Q000494:pharmacology
D004791:Enzyme Inhibitors; D018841:HSP90 Heat-Shock Proteins; D011993:Recombinant Fusion Proteins; D042461:Vascular Endothelial Growth Factor A; D052250:Nitric Oxide Synthase Type III