Peptides modified by myristoylation activate eNOS in endothelial cells through Akt phosphorylation.
1. Myristoylated pseudosubstrate of PKCzeta (mPS) - a synthetic myristoylated peptide with a sequence (13 amino acids) mimicking the endogenous PKCzeta pseudosubstrate region -- is considered a selective cell-permeable inhibitor of PKCzeta. We present strong evidence that in endothelial cells the action of mPS is not limited to inhibition of PKC activity and that myristoylation of certain peptides can activate eNOS (endothelial nitric oxide synthase) through Akt phosphorylation. 2. mPS at micromolar concentrations (1-10 microM) induced profound phosphorylation of eNOS, Akt, ERK 1/2, and p38 MAPK in cultured pulmonary artery endothelial cells (PAEC). The same changes were observed after treatment of PAEC with a myristoylated scrambled version of mPS (mScr), whereas a cell-permeable version of PKCzeta pseudosubstrate fused to the HIV-TAT membrane-translocating peptide did not induce analogous changes, suggesting that myristoylation confers new properties on the peptides consisting of activation of different signaling pathways in endothelial cells. 3. In addition to mPS and mScr, a number of other myristoylated peptides induced phosphorylation of eNOS suggesting that myristoylation of peptides can activate eNOS by mechanisms unrelated to inhibition of PKC. All active myristoylated peptides contained basic amino acids motif and were longer than six amino acids. 4. Activation of eNOS by myristoylated peptides was dependent on the PI3K/Akt pathway and the rise of intracellular calcium and was associated with an elevation of cGMP levels in PAEC and with relaxation of precontracted isolated pulmonary artery segments. 5. Myristoylated peptides can be considered a new class of activators of NO production in endothelial cells and that using mPS as a specific inhibitor of PKC should be done with caution, especially in endothelial cells.
Krotova
Karina
K
Department of Medicine, University of Florida, VA Medical Center, Gainesville, FL 32610, USA.
Hu
Hanbo
H
Xia
Shen-Ling
SL
Belayev
Leonid
L
Patel
Jawaharlal M
JM
Block
Edward R
ER
Zharikov
Sergey
S
D041721:3T3-L1 Cells; D000818:Animals; D002118:Calcium / Q000502:physiology; D002478:Cells, Cultured; D006152:Cyclic GMP / Q000096:biosynthesis; D042783:Endothelial Cells / Q000187:drug effects*; D005229:Fatty Acids, Monounsaturated / Q000737:chemistry*; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry; D051379:Mice; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism*; D009928:Organ Specificity / Q000187:drug effects; D010446:Peptide Fragments / Q000737:chemistry* / Q000494:pharmacology*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism / Q000502:physiology; D010766:Phosphorylation / Q000187:drug effects; D010919:Placebos / Q000494:pharmacology; D011493:Protein Kinase C / Q000737:chemistry / Q000494:pharmacology; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism* / Q000502:physiology; D011651:Pulmonary Artery / Q000166:cytology / Q000187:drug effects; D013552:Swine; D014664:Vasodilation / Q000187:drug effects; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism; D000099293:Protein Kinase C-lambda
D002118:Calcium; D006152:Cyclic GMP; D005229:Fatty Acids, Monounsaturated; D007527:Isoenzymes; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D052250:Nitric Oxide Synthase Type III; D010446:Peptide Fragments; D019869:Phosphatidylinositol 3-Kinases; D010919:Placebos; D011493:Protein Kinase C; D051057:Proto-Oncogene Proteins c-akt; D048051:p38 Mitogen-Activated Protein Kinases; D000099293:Protein Kinase C-lambda; C488488:protein kinase C zeta II, mouse; C054211:9-tetradecenoic acid