The Akt kinase signals directly to endothelial nitric oxide synthase.
Endothelial nitric oxide synthase (eNOS) is an important modulator of angiogenesis and vascular tone [1]. It is stimulated by treatment of endothelial cells in a phosphatidylinositol 3-kinase (PI 3-kinase)-dependent fashion by insulin-like growth factor-1 (IGF-1) and vascular endothelial growth factor (VEGF) [2] [3] and is activated by phosphorylation at Ser1177 in the sequence RIRTQS(1177)F (in the single-letter amino acid code) [4]. The protein kinase Akt is an important downstream target of PI 3-kinase [5] [6], regulating VEGF-stimulated endothelial cell survival [7]. Akt phosphorylates substrates within a defined motif [8], which is present in the sequence surrounding Ser1177 in eNOS. Both Akt [5] [6] and eNOS [9] are localized to, and activated at, the plasma membrane. We found that purified Akt phosphorylated cardiac eNOS at Ser1177, resulting in activation of eNOS. Phosphorylation at this site was stimulated by treatment of bovine aortic endothelial cells (BAECs) with VEGF or IGF-1, and Akt was activated in parallel. Preincubation with wortmannin, an inhibitor of Akt signalling, reduced VEGF- or IGF-1-induced Akt activity and eNOS phosphorylation. Akt was detected in immunoprecipitates of eNOS from BAECs, and eNOS in immunoprecipitates of Akt, indicating that the two enzymes associate in vivo. It is thus apparent that Akt directly activates eNOS in endothelial cells. These results strongly suggest that Akt has an important role in the regulation of normal angiogenesis and raise the possibility that the enhanced activity of this kinase that occurs in carcinomas may contribute to tumor vascularization and survival.
Michell
B J
BJ
St Vincent's Institute of Medical Research, 41 Victoria Parade, Fitzroy, Victoria, 3065, Australia.
Griffiths
J E
JE
Mitchelhill
K I
KI
Rodriguez-Crespo
I
I
Tiganis
T
T
Bozinovski
S
S
de Montellano
P R
PR
Kemp
B E
BE
Pearson
R B
RB
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D002460:Cell Line; D002478:Cells, Cultured; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D016228:Endothelial Growth Factors / Q000494:pharmacology; D004730:Endothelium, Vascular / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D007334:Insulin-Like Growth Factor I / Q000494:pharmacology; D008222:Lymphokines / Q000494:pharmacology; D008969:Molecular Sequence Data; D019001:Nitric Oxide Synthase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D052250:Nitric Oxide Synthase Type III; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D011518:Proto-Oncogene Proteins; D051057:Proto-Oncogene Proteins c-akt; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D014162:Transfection; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors
D016228:Endothelial Growth Factors; D008222:Lymphokines; D011518:Proto-Oncogene Proteins; D011993:Recombinant Fusion Proteins; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors; D007334:Insulin-Like Growth Factor I; C496314:NOS3 protein, human; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III; C494918:AKT1 protein, human; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; D017868:Cyclic AMP-Dependent Protein Kinases