PKCzeta decreases eNOS protein stability via inhibitory phosphorylation of ERK5.
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.
Aab Cardiovascular Research Institute and Department of Medicine, University of Rochester Medical Center, 601 Elmwood Ave., Rochester, NY 14642, USA.
Patrizia
P
Nigro
Jun-ichi
J
Abe
Chang-Hoon
CH
Woo
Kimio
K
Satoh
Carolyn
C
McClain
Michael R
MR
O'Dell
Hakjoo
H
Lee
Jae-Hyang
JH
Lim
Jian-dong
JD
Li
Kyung-Sun
KS
Heo
Keigi
K
Fujiwara
Bradford C
BC
Berk
D000818:Animals; D001011:Aorta / Q000378:metabolism; D001057:Apolipoproteins E / Q000235:genetics / Q000378:metabolism; D016466:CHO Cells; D002478:Cells, Cultured; D006224:Cricetinae; D003412:Cricetulus; D042783:Endothelial Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D005260:Female; D006801:Humans; D015151:Immunoblotting; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D018613:Microscopy, Confocal; D048054:Mitogen-Activated Protein Kinase 7 / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D052250:Nitric Oxide Synthase Type III / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D011485:Protein Binding; D011493:Protein Kinase C / Q000235:genetics / Q000378:metabolism*; D055550:Protein Stability; D034622:RNA Interference; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D000099221:Protein Kinase C zeta
D001057:Apolipoproteins E; D048054:Mitogen-Activated Protein Kinase 7; D052250:Nitric Oxide Synthase Type III; D011493:Protein Kinase C; D014409:Tumor Necrosis Factor-alpha; D000099221:Protein Kinase C zeta