Inhibition of Endothelial Nitric Oxide Synthase Activity by Proline-Rich Tyrosine Kinase 2 in Response to Fluid Shear Stress and Insulin
Abstract
In native and primary cultures of endothelial cells, fluid shear stress elicits the tyrosine phosphorylation of the endothelial NO synthase (eNOS), however, the consequences of this modification on enzyme activity are unclear. We found that fluid shear stress induces the association of eNOS with the proline-rich tyrosine kinase 2 (PYK2) in endothelial cells and that the eNOS immunoprecipitated from eNOS- and PYK2-overexpressing HEK293 cells was tyrosine-phosphorylated on Tyr657. In mouse carotid arteries, the overexpression of wild-type PYK2, but not a dominant-negative PYK2, decreased eNOS activity (≈50%), whereas in murine lung endothelial cells, the downregulation of PYK2 (small interfering RNA) increased ionomycin-induced NO production. Mutation of Tyr657 to the phosphomimetic residues aspartate (D) or glutamate (E) abolished enzyme activity, whereas a nonphosphorylatable mutant (phenylalanine [F]) showed activity comparable to the wild-type enzyme. Moreover, normal flow-induced vasodilatation was apparent in carotid arteries from eNOS −/− mice overexpressing either the wild-type eNOS or the Y657F mutant, whereas no flow-induced vasodilatation was apparent in arteries expressing the Y657E eNOS mutant. Insulin also activated PYK2 and stimulated eNOS in endothelial cells expressing the Y657F mutant but not wild-type eNOS. These data indicate that PYK2 mediates the tyrosine phosphorylation of eNOS on Tyr657 in response to fluid shear stress and insulin stimulation and that this modification attenuates the activity of the enzyme. The PYK2-dependent inhibition of NO production may serve to keep eNOS activity low and limit the detrimental consequences of maintained high NO output, ie, the generation of peroxynitrite.
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Mutation of Tyr657 causes complete loss of NO synthesis and l-citrulline formation, suggesting a blockade of intramolecular electron transfer.
“The mutation of Tyr657 (292) causes complete loss of NO synthesis and l-citrulline formation, suggesting a blockade of the intramolecular electron transfer.”
From Sources of Vascular Nitric Oxide and Reactive Oxygen Species and Their Regulation (2019) · cites this paper as [292]
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- Authors
- Fisslthaler, B., Loot, A. E., Mohamed, A., Busse, R., & Fleming, I.
- Affiliations
- Goethe University Frankfurt (DE)
- Venue
- Circulation Research
- Published
- 2008-06-20
- Volume
- 102
- Issue
- 12
- Pages
- 1520-1528
- ISSN
- 0009-7330 (ISSN-L)
- 1524-4571
- OpenAlex
- W2057919250
- Cited by
- 99
- References
- 27
- Citations by year
- 2026: 3 · 2025: 3 · 2024: 4 · 2023: 9 · 2022: 3 · 2021: 9 · 2020: 5 · 2019: 7 · 2018: 6 · 2017: 9 · 2016: 10 · 2015: 1 · 2014: 5 · 2013: 5 · 2012: 2
- FWCI
- 4.06
- Type
- journal article
- Language
- en
- Open access
- closed
- Locations
- Circulation Research (publishedVersion)
- PubMed (publishedVersion)
- Funders
- Deutsche Forschungsgemeinschaft (SFB 553)
- Topics
- Health Sciences › Medicine › Physiology › Nitric Oxide and Endothelin Effects
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein Kinase Regulation and GTPase Signaling
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Angiogenesis and VEGF in Cancer
- Keywords
- endothelial nitric oxide synthase, Pyk2, fluid shear stress, insulin, tyrosine phosphorylation, eNOS inhibition, nitric oxide production, peroxynitrite, endothelial cells, carotid artery, pulmonary endothelial cells
- MeSH terms
- Animals, Aorta, Cells, Cultured, Enzyme Activation, Epithelial Cells, Insulin, Lung, Nitric Oxide, Recombinant Fusion Proteins, Rheology, Stress, Mechanical, Ionomycin, Point Mutation, Mutation, Missense, Sus scrofa, RNA Interference, RNA, Small Interfering, Endothelial Cells, Mice, Focal Adhesion Kinase 2, Nitric Oxide Synthase Type II, Nitric Oxide Synthase Type III
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