MAP Kinases Mediate Regulation of eNOS through Phosphorylation of Different Sites

Abstract

Mitogen activated protein kinases (MAPK) p38 and ERK, have both been reported to bind endothelial nitric oxide synthase (eNOS) with submicromolar affinity via hypothesized interactions with a pentabasic non‐canonical MAPK binding sequence in the autoinhibitory insertion of eNOS. The neuronal isoform, which lacks the pentabasic motif, did not bind either MAPK. In the present study, eNOS phosphorylation by MAPKs (P38, ERK, JNK) was examined using in vitro kinase assays and immunoblotting. JNK phosphorylation at pS116 contrasts with ERK, which phosphorylated pS602, and p38, which phosphorylated both sites. Literature reports that JNK1/2 activation does not alter eNOS activity while prior work in our lab shows that ERK phosphorylation negativity impacts NO output. We will present in vitro results comparing the direct impact of each kinase on eNOS activity using the oxyhemoglobin (Oxy‐Hb) assay. In HMEC‐1 cells and in vitro we observe that phosphorylation at these MAPK sites happens concurrently with activating sites (pS1179), creating antagonistically phosphorylated eNOS. These results underscore the importance of MAPK interactions with eNOS showing that each MAPK creates a unique phosphorylation pattern and NO production outcome. These findings strengthen the emerging paradigm of eNOS as a junction of multiple signaling pathways.Support or Funding InformationR15GM110634‐01A1 supported this work.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.