Tyrosine kinase FYN negatively regulates NOX4 in cardiac remodeling
Shouji Matsushima, … , Hiroyuki Tsutsui, Junichi Sadoshima
Shouji Matsushima, … , Hiroyuki Tsutsui, Junichi Sadoshima
Published September 1, 2016; First published August 15, 2016
Citation Information: J Clin Invest. 2016;126(9):3403-3416. https://doi.org/10.1172/JCI85624.
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Research Article Cardiology

Tyrosine kinase FYN negatively regulates NOX4 in cardiac remodeling

Abstract

NADPH oxidases (Noxes) produce ROS that regulate cell growth and death. NOX4 expression in cardiomyocytes (CMs) plays an important role in cardiac remodeling and injury, but the posttranslational mechanisms that modulate this enzyme are poorly understood. Here, we determined that FYN, a Src family tyrosine kinase, interacts with the C-terminal domain of NOX4. FYN and NOX4 colocalized in perinuclear mitochondria, ER, and nuclear fractions in CMs, and FYN expression negatively regulated NOX4-induced O2– production and apoptosis in CMs. Mechanistically, we found that direct phosphorylation of tyrosine 566 on NOX4 was critical for this FYN-mediated negative regulation. Transverse aortic constriction activated FYN in the left ventricle (LV), and FYN-deficient mice displayed exacerbated cardiac hypertrophy and dysfunction and increased ROS production and apoptosis. Deletion of Nox4 rescued the exaggerated LV remodeling in FYN-deficient mice. Furthermore, FYN expression was markedly decreased in failing human hearts, corroborating its role as a regulator of cardiac cell death and ROS production. In conclusion, FYN is activated by oxidative stress and serves as a negative feedback regulator of NOX4 in CMs during cardiac remodeling.

Authors

Shouji Matsushima, Junya Kuroda, Peiyong Zhai, Tong Liu, Shohei Ikeda, Narayani Nagarajan, Shin-ichi Oka, Takashi Yokota, Shintaro Kinugawa, Chiao-Po Hsu, Hong Li, Hiroyuki Tsutsui, Junichi Sadoshima

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Figure 4

FYN phosphorylates Y566 in the NOX4 C-terminus.

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FYN phosphorylates Y566 in the NOX4 C-terminus. (A) Phosphorylation of N...
(A) Phosphorylation of NOX4 C-terminus by FYN was examined with in vitro kinase assays (left). Protein loaded in each group was resolved by SDS-PAGE (right). The experiment was conducted 2 times. (B) MS/MS spectrum of a doubly charged ion corresponding to phosphopeptide, 559LSNQNNSpYGTRFEYNK574 (m/z 1,007.94) from NOX4. The observed y- and b-ion series confirmed the peptide sequence and localization of phospho-Y566 with a Mascot score of 37. (C) Phosphorylation of the indicated fragments from the NOX4 C-terminus by FYN was examined by in vitro phosphorylation of 6 peptides containing tyrosine residues in NOX4-CT by FYN was examined with an in vitro kinase assay. The experiment was conducted 3 times. (D) Levels of phosphorylated NOX4 and NOX4 in cultured neonatal rat CMs transduced with indicated adenoviruses were examined by immunoblotting. The experiment was conducted 3 times. (E) Levels of phosphorylated NOX4 and NOX4 in the indicated mouse hearts were examined by immunoblotting. The experiment was conducted 3 times. (F) The sequence of the C-terminus in NOX4 (upper) and NOX2 (lower). The position of Y566 is indicated with a red rectangle.