Src kinase activates endothelial nitric-oxide synthase by phosphorylating Tyr-83

Area of Science:

• Biochemistry
• Cell Biology
• Molecular Biology

Background:

• Endothelial nitric-oxide synthase (eNOS) activity is modulated by serine/threonine phosphorylation.
• The role of eNOS tyrosine phosphorylation in regulating its function remains less understood.
• Oxidant stress is a significant factor influencing endothelial cell function and eNOS activity.

Purpose of the Study:

• To investigate the mechanisms of eNOS tyrosine phosphorylation in response to oxidant stress.
• To identify the specific tyrosine residues involved in eNOS phosphorylation.
• To determine the functional consequences of eNOS tyrosine phosphorylation on nitric oxide (NO) production.

Main Methods:

• Treatment of bovine aortic endothelial cells (BAECs) with hydrogen peroxide and pervanadate (PV) to induce oxidant stress.
• Inhibition studies using the Src kinase inhibitor PP2.
• Co-immunoprecipitation assays to assess eNOS and c-Src interaction.
• Site-directed mutagenesis and domain mapping in COS-7 cells to identify phosphorylation sites.
• Measurement of nitric oxide (NO) release.

Main Results:

• Oxidant stress (H2O2 and PV) significantly increased eNOS tyrosine phosphorylation in BAECs.
• The Src kinase inhibitor PP2 blocked eNOS tyrosine phosphorylation.
• eNOS and c-Src were found to co-immunoprecipitate, indicating a physical interaction.
• Tyr-83 was identified as the major site of eNOS tyrosine phosphorylation.
• Phosphorylation of Tyr-83 was associated with a 3-fold increase in basal NO release.
• Mutation of Tyr-83 to phenylalanine (Y83F) attenuated stimulated NO production.

Conclusions:

• Src-mediated tyrosine phosphorylation of eNOS at Tyr-83 is a key mechanism regulating eNOS activity.
• This phosphorylation event enhances nitric oxide production in endothelial cells.
• Understanding eNOS tyrosine phosphorylation provides insights into endothelial cell signaling pathways under oxidant stress.