Stromal cell-derived factor 2 is critical for Hsp90-dependent eNOS activation.
Endothelial nitric oxide synthase (eNOS) catalyzes the conversion of l-arginine and molecular oxygen into l-citrulline and nitric oxide (NO), a gaseous second messenger that influences cardiovascular physiology and disease. Several mechanisms regulate eNOS activity and function, including phosphorylation at Ser and Thr residues and protein-protein interactions. Combining a tandem affinity purification approach and mass spectrometry, we identified stromal cell-derived factor 2 (SDF2) as a component of the eNOS macromolecular complex in endothelial cells. SDF2 knockdown impaired agonist-stimulated NO synthesis and decreased the phosphorylation of eNOS at Ser(1177), a key event required for maximal activation of eNOS. Conversely, SDF2 overexpression dose-dependently increased NO synthesis through a mechanism involving Akt and calcium (induced with ionomycin), which increased the phosphorylation of Ser(1177) in eNOS. NO synthesis by iNOS (inducible NOS) and nNOS (neuronal NOS) was also enhanced upon SDF2 overexpression. We found that SDF2 was a client protein of the chaperone protein Hsp90, interacting preferentially with the M domain of Hsp90, which is the same domain that binds to eNOS. In endothelial cells exposed to vascular endothelial growth factor (VEGF), SDF2 was required for the binding of Hsp90 and calmodulin to eNOS, resulting in eNOS phosphorylation and activation. Thus, our data describe a function for SDF2 as a component of the Hsp90-eNOS complex that is critical for signal transduction in endothelial cells.
Vascular Biology and Therapeutics Program, Department of Pharmacology, Yale University School of Medicine, 10 Amistad Street, New Haven, CT 06520, USA.
Mauro
M
Siragusa
Department of Genetics and Complex Diseases, Harvard School of Public Health, 677 Huntington Avenue, Boston, MA 02115, USA.
Florian
F
Fröhlich
Vascular Biology and Therapeutics Program, Department of Pharmacology, Yale University School of Medicine, 10 Amistad Street, New Haven, CT 06520, USA.
Eon Joo
EJ
Park
Vascular Biology and Therapeutics Program, Department of Pharmacology, Yale University School of Medicine, 10 Amistad Street, New Haven, CT 06520, USA.
Michael
M
Schleicher
Department of Genetics and Complex Diseases, Harvard School of Public Health, 677 Huntington Avenue, Boston, MA 02115, USA.
Tobias C
TC
Walther
Vascular Biology and Therapeutics Program, Department of Pharmacology, Yale University School of Medicine, 10 Amistad Street, New Haven, CT 06520, USA. william.sessa@yale.edu.
William C
WC
Sessa
D000818:Animals; D019556:COS Cells; D002460:Cell Line; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D002853:Chromatography, Liquid; D042783:Endothelial Cells / Q000187:drug effects / Q000378:metabolism*; D004789:Enzyme Activation; D057809:HEK293 Cells; D018841:HSP90 Heat-Shock Proteins / Q000378:metabolism*; D006801:Humans; D015151:Immunoblotting; D009569:Nitric Oxide / Q000378:metabolism; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D034622:RNA Interference; D012694:Serine / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D053719:Tandem Mass Spectrometry; D042461:Vascular Endothelial Growth Factor A / Q000494:pharmacology
D018841:HSP90 Heat-Shock Proteins; D011506:Proteins; C102467:SDF2 protein, human; D042461:Vascular Endothelial Growth Factor A; D009569:Nitric Oxide; D012694:Serine; C496314:NOS3 protein, human; D052250:Nitric Oxide Synthase Type III