Cargando…

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

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 interact...

Descripción completa

Detalles Bibliográficos
Autores principales: 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.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: John Wiley and Sons Inc. 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9063426/
https://www.ncbi.nlm.nih.gov/pubmed/35182051
http://dx.doi.org/10.1002/2211-5463.13384
_version_ 1784699162128809984
author 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.
author_facet 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.
author_sort Solone, Xzaviar K. V.
collection PubMed
description 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 (JNK1(α1)), p38α, and ERK2 were characterized using optical biosensing with full‐length NOS3 and NOS3 specific peptides and phosphopeptides. Like p38α and ERK2, JNK1(α1) exhibited high‐affinity binding to full‐length NOS3 (K (D) 15 nm). Rate constants exhibited fast‐on, slow‐off binding (k (on) = 4106 m (−1)s(−1); k (off) = 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 JNK1(α1,) 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. JNK1(α1) 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).
format Online
Article
Text
id pubmed-9063426
institution National Center for Biotechnology Information
language English
publishDate 2022
publisher John Wiley and Sons Inc.
record_format MEDLINE/PubMed
spelling pubmed-90634262022-05-04 MAP kinases differentially bind and phosphorylate NOS3 via two unique NOS3 sites 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. FEBS Open Bio Research Articles 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 (JNK1(α1)), p38α, and ERK2 were characterized using optical biosensing with full‐length NOS3 and NOS3 specific peptides and phosphopeptides. Like p38α and ERK2, JNK1(α1) exhibited high‐affinity binding to full‐length NOS3 (K (D) 15 nm). Rate constants exhibited fast‐on, slow‐off binding (k (on) = 4106 m (−1)s(−1); k (off) = 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 JNK1(α1,) 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. JNK1(α1) 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). John Wiley and Sons Inc. 2022-03-15 /pmc/articles/PMC9063426/ /pubmed/35182051 http://dx.doi.org/10.1002/2211-5463.13384 Text en © 2022 The Authors. FEBS Open Bio published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies https://creativecommons.org/licenses/by/4.0/This is an open access article under the terms of the http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.
spellingShingle Research Articles
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.
MAP kinases differentially bind and phosphorylate NOS3 via two unique NOS3 sites
title MAP kinases differentially bind and phosphorylate NOS3 via two unique NOS3 sites
title_full MAP kinases differentially bind and phosphorylate NOS3 via two unique NOS3 sites
title_fullStr MAP kinases differentially bind and phosphorylate NOS3 via two unique NOS3 sites
title_full_unstemmed MAP kinases differentially bind and phosphorylate NOS3 via two unique NOS3 sites
title_short MAP kinases differentially bind and phosphorylate NOS3 via two unique NOS3 sites
title_sort map kinases differentially bind and phosphorylate nos3 via two unique nos3 sites
topic Research Articles
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9063426/
https://www.ncbi.nlm.nih.gov/pubmed/35182051
http://dx.doi.org/10.1002/2211-5463.13384
work_keys_str_mv AT solonexzaviarkv mapkinasesdifferentiallybindandphosphorylatenos3viatwouniquenos3sites
AT caldaraamberl mapkinasesdifferentiallybindandphosphorylatenos3viatwouniquenos3sites
AT wellsbrady mapkinasesdifferentiallybindandphosphorylatenos3viatwouniquenos3sites
AT qiaohao mapkinasesdifferentiallybindandphosphorylatenos3viatwouniquenos3sites
AT wadelydiar mapkinasesdifferentiallybindandphosphorylatenos3viatwouniquenos3sites
AT salernojohnc mapkinasesdifferentiallybindandphosphorylatenos3viatwouniquenos3sites
AT helmskatya mapkinasesdifferentiallybindandphosphorylatenos3viatwouniquenos3sites
AT smithkatherineer mapkinasesdifferentiallybindandphosphorylatenos3viatwouniquenos3sites
AT mcmurryjonathanl mapkinasesdifferentiallybindandphosphorylatenos3viatwouniquenos3sites
AT chrestensencarola mapkinasesdifferentiallybindandphosphorylatenos3viatwouniquenos3sites