PKCzeta decreases eNOS protein stability via inhibitory phosphorylation of ERK5.

摘要

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