PKCζ decreases eNOS protein stability via inhibitory phosphorylation of ERK5

Patrizia Nigro, Jun Ichi Abe, Chang Hoon Woo, Kimio Satoh, Carolyn McClain, Michael R. O'Dell, Hakjoo Lee, Jae Hyang Lim, Jian Dong Li, Kyung Sun Heo, Keigi Fujiwara, Bradford C. Berk

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53 Scopus citations

Abstract

PKCζ has emerged as a pathologic mediator of endothelial cell dysfunction, based on its essential role in tumor necrosis factor α (TNFα)-mediated inflammation. In contrast, extracellular signal-regulated kinase 5 (ERK5) function is required for endothelial cell homeostasis as shown by activation of Krüppel-like factor 2 (KLF2), increased endothelial nitric-oxide synthase (eNOS) expression, and inhibition of apoptosis. We hypothesized that protein kinase C ζ (PKCζ) activation by TNFα would inhibit the ERK5/KLF2/eNOS pathway. TNFα inhibited the steady laminar flow-induced eNOS expression, and this effect was reversed by the dominant-negative form of PKCζ (Ad.DN-PKCζ). In addition, ERK5 function was inhibited by either TNFα or the transfection of the catalytic domain of PKCζ. This inhibition was reversed by PKCζ small interfering RNA. PKCζ was found to bind to ERK5 under basal conditions with coimmunoprecipitation and the mammalian 2-hybrid assay. Furthermore, PKCζ phosphorylates ERK5, and mutation analysis showed that the preferred site is S486. Most importantly, we found that the predominant effect of TNFα stimulation of PKCζ was to decrease eNOS protein stability that was recapitulated by transfecting Ad.ERK5S486A mutant. Finally, aortic en face analysis of ERK5/PKCζ activity showed high PKCζ and ERK5 staining in the athero-prone region. Taken together our results show that PKCζ binds and phosphorylates ERK5, thereby decreasing eNOS protein stability and contributing to early events of atherosclerosis.

Original languageEnglish (US)
Pages (from-to)1971-1979
Number of pages9
JournalBlood
Volume116
Issue number11
DOIs
StatePublished - Sep 16 2010

ASJC Scopus subject areas

  • Biochemistry
  • Immunology
  • Hematology
  • Cell Biology

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    Nigro, P., Abe, J. I., Woo, C. H., Satoh, K., McClain, C., O'Dell, M. R., Lee, H., Lim, J. H., Li, J. D., Heo, K. S., Fujiwara, K., & Berk, B. C. (2010). PKCζ decreases eNOS protein stability via inhibitory phosphorylation of ERK5. Blood, 116(11), 1971-1979. https://doi.org/10.1182/blood-2010-02-269134