The Akt1-eNOS axis illustrates the specificity of kinase-substrate relationships in vivo.
Akt1 is critical for many in vivo functions; however, the cell-specific substrates responsible remain to be defined. Here, we examine the importance of endothelial nitric oxide synthase (eNOS) as an Akt1 substrate by generating Akt1-deficient mice (Akt1(-/-) mice) carrying knock-in mutations (serine to aspartate or serine to alanine substitutions) of the critical Akt1 phosphorylation site on eNOS (serine 1176) that render the enzyme "constitutively active" or "less active." The eNOS mutations did not influence several phenotypes in Akt1(-/-) mice; however, the defective postnatal angiogenesis characteristic of Akt1(-/-) mice was rescued by crossing the Akt1(-/-) mice with mice carrying the constitutively active form of eNOS, but not by crossing with mice carrying the less active eNOS mutant. This genetic rescue resulted in the stabilization of hypoxia-inducible factor 1alpha (HIF-1alpha) and increased production of HIF-1alpha-responsive genes in vivo and in vitro. Thus, Akt1 regulates angiogenesis largely through phosphorylation of eNOS and NO-dependent signaling.
Schleicher
Michael
M
Department of Pharmacology and Vascular Biology and Therapeutics Program, Amistad Building, Yale University School of Medicine, New Haven, CT 06520, USA.
Yu
Jun
J
Murata
Takahisa
T
Derakhshan
Berhad
B
Atochin
Dimitriy
D
Qian
Li
L
Kashiwagi
Satoshi
S
Di Lorenzo
Annarita
A
Harrison
Kenneth D
KD
Huang
Paul L
PL
Sessa
William C
WC
D000704:Analysis of Variance; D000818:Animals; D015153:Blotting, Western; D003433:Crosses, Genetic; D019084:Fluorescent Antibody Technique, Indirect; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000378:metabolism; D020287:In Situ Nick-End Labeling; D051379:Mice; D018345:Mice, Knockout; D009154:Mutation / Q000235:genetics; D018919:Neovascularization, Physiologic / Q000235:genetics / Q000502:physiology*; D052250:Nitric Oxide Synthase Type III / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D051057:Proto-Oncogene Proteins c-akt / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000502:physiology*; D013379:Substrate Specificity / Q000502:physiology; D014945:Wound Healing / Q000235:genetics / Q000502:physiology
C497443:Hif1a protein, mouse; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D052250:Nitric Oxide Synthase Type III; C514862:Akt1 protein, mouse; D051057:Proto-Oncogene Proteins c-akt