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

Xzaviar K V Solone, Amber L Caldara, Brady Wells, Hao Qiao, Lydia R Wade, John C Salerno, Katy A Helms, Katherine E R Smith, Jonathan L McMurry, Carol A Chrestensen

FEBS open bio 2022 May

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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-1 s-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). © 2022 The Authors. FEBS Open Bio published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.

Citation

Xzaviar K V Solone, Amber L Caldara, Brady Wells, Hao Qiao, Lydia R Wade, John C Salerno, Katy A Helms, Katherine E R Smith, Jonathan L McMurry, Carol A Chrestensen. MAP kinases differentially bind and phosphorylate NOS3 via two unique NOS3 sites. FEBS open bio. 2022 May;12(5):1075-1086