Epidermal and hepatocyte growth factors, but not keratinocyte growth factor, modulate protein kinase Calpha translocation to the plasma membrane through 15(S)-hydroxyeicosatetraenoic acid synthesis.
Activation of protein kinase C (PKC) involves its recruitment to the membrane, where it interacts with its activator(s). We expressed PKCalpha fused to green fluorescent protein and examined its real time translocation to the plasma membrane in living human corneal epithelial cells. Upon 10 min of stimulation with epidermal and hepatocyte growth factors (EGF and HGF), PKCalpha translocated to the plasma membrane. Keratinocyte growth factor did not stimulate PKCalpha translocation up to 1 h after stimulation. Pretreatment with the 15-lipoxygenase metabolite, 15(S)-hydroxyeicosatetraenoic acid (15(S)-HETE), followed by EGF or HGF, produced faster translocation of PKCalpha detectable at 2 min. However, the same concentration of 15(S)-HETE alone did not stimulate translocation. 15(S)-Hydroperoxyeicosatetraenoic acid and 5(S)-HETE did not affect growth factor-induced translocation of PKCalpha. PD153035, a specific inhibitor of tyrosine kinase activity of the EGF receptor, completely blocked PKCalpha translocation induced by EGF. PD98059, a specific MEK inhibitor, significantly inhibited EGF- and HGF-mediated PKCalpha translocation, which was reversed by addition of 15(S)-HETE. Phosphorylation of ERK1/2 by EGF was followed by phosphorylation of cytosolic phospholipase A(2) (cPLA(2)), and blocking ERK1/2 inhibited cPLA(2) activation. Immunofluorescence demonstrated translocation of p-cPLA(2) to plasma and nuclear membranes as early as 2 min. This may further increase arachidonic acid release from membrane phospholipid pools and increase the intracellular pool of HETEs. In fact, in cells prelabeled with [(3)H]arachidonic acid, EGF stimulated synthesis of 15(S)-HETE in the cytosolic fraction. 15(S)-HETE also reversed the effect of LOX inhibitor on EGF-mediated cell proliferation. Our results indicate that 15(S)-HETE is an intracellular second messenger that facilitates translocation of PKCalpha to the membrane and elucidate a mechanism that plays a regulatory role in cell proliferation crucial to corneal wound healing.
Sharma
Guru Dutt
GD
Department of Ophthalmology and Neuroscience Center of Excellence, Louisiana State University Health Sciences Center School of Medicine, New Orleans, Louisiana 70112, USA.
Ottino
Paulo
P
Bazan
Nicolas G
NG
Bazan
Haydee E P
HE
D001093:Arachidonate 15-Lipoxygenase / Q000378:metabolism; D015153:Blotting, Western; D002462:Cell Membrane / Q000378:metabolism; D049109:Cell Proliferation; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D003315:Cornea / Q000378:metabolism; D003600:Cytosol / Q000201:enzymology; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004815:Epidermal Growth Factor / Q000502:physiology*; D004847:Epithelial Cells / Q000166:cytology; D051523:Fibroblast Growth Factor 7; D005346:Fibroblast Growth Factors / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D017228:Hepatocyte Growth Factor / Q000502:physiology*; D006801:Humans; D006893:Hydroxyeicosatetraenoic Acids / Q000737:chemistry; D007700:Kinetics; D008084:Lipoxygenase / Q000378:metabolism; D020930:MAP Kinase Kinase Kinases / Q000378:metabolism; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D051571:Protein Kinase C-alpha; D021381:Protein Transport; D011799:Quinazolines / Q000494:pharmacology; D015398:Signal Transduction; D013997:Time Factors; D014162:Transfection; D014945:Wound Healing
D004791:Enzyme Inhibitors; C496453:FGF7 protein, human; D006893:Hydroxyeicosatetraenoic Acids; D011799:Quinazolines; D051523:Fibroblast Growth Factor 7; D049452:Green Fluorescent Proteins; D005346:Fibroblast Growth Factors; D004815:Epidermal Growth Factor; D017228:Hepatocyte Growth Factor; D008084:Lipoxygenase; D001093:Arachidonate 15-Lipoxygenase; C496628:PRKCA protein, human; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha; D020930:MAP Kinase Kinase Kinases; C088860:4-((3-bromophenyl)amino)-6,7-dimethoxyquinazoline