C‐JUN N‐terminal kinase (JNKα1) binds eNOS and phosphorylates at S116, differentiating it from p38 and ERK phosphorylation

Abstract

We have previously shown that the mitogen activated protein kinases (MAPK), p38 and ERK bind endothelial nitric oxide synthase (eNOS) with submicromolar affinity via 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 MAPK. In the present study, eNOS binding by c‐Jun N‐terminal kinase (JNKα1) was examined using optical biosensing. Similar to p38 and ERK, JNK evinced binding to eNOS (31 nM KD) but not nNOS as measured by fit to a one‐state global model. Rate constants were determined (kon = 4125 M−1s−1 and koff = 1.3 × 10−4 s−1). In vitro kinase assays and immunoblotting identified phosphorylation by JNK at S116, contrasting with ERK, which phosphorylated S602, and p38, which phosphorylated both sites. Phosphorylation by JNK at S116 did not alter the conformational manifold, where phosphorylation at S602 by ERK and p38 does. In HMEC1 cells we observe that phosphorylation at these MAPK sites happens concurrently with activating sites (S1177), creating antagonistically phosphorylated eNOS. Further, we examine how phosphorylation of eNOS alters the cellular interactions of the MAP kinases. These results underscore the importance of MAPK interactions with eNOS showing that each MAPK creates a unique phosphorylation pattern. These findings strengthen the emerging paradigm of eNOS as a junction of multiple signaling pathways.Support or Funding InformationNIH 1 R15 GM110634‐01A1 | R25GM111565This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.