MAP kinases differentially bind and phosphorylate NOS3 via two unique NOS3 sites

• Published in: FEBS open bio 2022-05, Vol.12 (5), p.1075-1086
• Main Authors: Solone, Xzaviar K. V., Caldara, Amber L., Wells, Brady, Qiao, Hao, Wade, Lydia R., Salerno, John C., Helms, Katy A., Smith, Katherine E. R., McMurry, Jonathan L., Chrestensen, Carol A.
• Format: Article
• Language: English
• Subjects: Affinity; Amino acids; Antibodies; Binding Sites; Biosensors; E coli; Endothelial cells; Endothelial Cells - metabolism; ERK2; Experiments; Extracellular signal-regulated kinase; Glycerol; Humans; Immunoblotting; JNK protein; JNK1α1; Kinases; MAP kinase; Microvasculature; Mitogen-Activated Protein Kinases - metabolism; Nitric oxide; Nitric Oxide Synthase Type III - metabolism; Nitric-oxide synthase; NOS3; NOS3 protein; optical biosensing; p38α; Peptides; Peptides - metabolism; Phosphorylation; Protein kinase A; Proteins; signaling
• ISSN: 2211-5463; 2211-5463
• DOI: 10.1002/2211-5463.13384

Nitric oxide synthase 3 (NOS3) is a major vasoprotective enzyme that catalyzes the conversion of l‐arginine to nitric oxide (NO) in response to a significant number of signaling pathways. Here, we provide evidence that NOS3 interactions with MAP kinases have physiological relevance. Binding interactions of NOS3 with c‐Jun N‐terminal kinase (JNK1α1), p38α, and ERK2 were characterized using optical biosensing with full‐length NOS3 and NOS3 specific peptides and phosphopeptides. Like p38α and ERK2, JNK1α1 exhibited high‐affinity binding to full‐length NOS3 (KD 15 nm). Rate constants exhibited fast‐on, slow‐off binding (kon = 4106 m−1s−1; koff = 6.2 × 10‐5 s−1). Further analysis using synthetic NOS3 peptides revealed two MAP kinase binding sites unique to NOS3. p38α evinced similar affinity with both NOS3 binding sites. For ERK2 and JNK1α1, the affinity at the two sites differed. However, NOS3 peptides with a phosphate at either S114 or S633 did not meaningfully interact with the kinases. Immunoblotting revealed that each kinase phosphorylated NOS3 with a unique pattern. JNK1α1 predominantly phosphorylated NOS3 at S114, ERK2 at S600, and p38α phosphorylated both residues. In vitro production of NO was unchanged by phosphorylation at these sites. In human microvascular endothelial cells, endogenous interactions of all the MAP kinases with NOS3 were captured using proximity ligation assay in resting cells. Our results underscore the importance of MAP kinase interactions, identifying two unique NOS3 interaction sites with potential for modulation by MAP kinase phosphorylation (S114) and other signaling inputs, like protein kinase A (S633). Nitric oxide synthase 3 (NOS3, eNOS) is a major vasoprotective enzyme that binds MAP kinases in cells. Representative MAP kinases (JNK1α1, p38α, and ERK2) bound NOS3 with high affinity at two hypothesized binding motifs and each kinase uniquely phosphorylated NOS3. Phosphorylation adjacent to the motifs disrupted MAP kinase binding, suggesting NOS3 phosphorylation status could modulate these interactions.