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Originally published In Press as doi:10.1074/jbc.M408852200 on December 21, 2004
J. Biol. Chem., Vol. 280, Issue 9, 7917-7924, March 4, 2005
Epidermal and Hepatocyte Growth Factors, but Not Keratinocyte Growth Factor, Modulate Protein Kinase C Translocation to the Plasma Membrane through 15(S)-Hydroxyeicosatetraenoic Acid Synthesis*
Guru Dutt Sharma,
Paulo Ottino,
Nicolas G. Bazan, and
Haydee E. P. Bazan
From the
Department of Ophthalmology and Neuroscience Center of Excellence, Louisiana State University Health Sciences Center School of Medicine, New Orleans, Louisiana 70112
Activation of protein kinase C (PKC) involves its recruitment to the membrane, where it interacts with its activator(s). We expressed PKC 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), PKC translocated to the plasma membrane. Keratinocyte growth factor did not stimulate PKC 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 PKC 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 PKC. PD153035, a specific inhibitor of tyrosine kinase activity of the EGF receptor, completely blocked PKC translocation induced by EGF. PD98059, a specific MEK inhibitor, significantly inhibited EGF- and HGF-mediated PKC translocation, which was reversed by addition of 15(S)-HETE. Phosphorylation of ERK1/2 by EGF was followed by phosphorylation of cytosolic phospholipase A2 (cPLA2), and blocking ERK1/2 inhibited cPLA2 activation. Immunofluorescence demonstrated translocation of p-cPLA2 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 [3H]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 PKC to the membrane and elucidate a mechanism that plays a regulatory role in cell proliferation crucial to corneal wound healing.
Received for publication, August 3, 2004
, and in revised form, November 30, 2004.
* This work was supported by United States Public Health Service Grants R01 EY06635 and R01 EY04928 from the NEI, National Institutes of Health and by the Neurobiotechnology Program of Louisiana. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The on-line version of this article (available at http://www.jbc.org) contains a supplemental video.
 To whom correspondence should be addressed: Dept. of Ophthalmology and Neuroscience Center of Excellence, LSU Health Sciences Center, 2020 Gravier St., Suite D, New Orleans, LA 70112. Tel.: 504-599-0877; Fax: 504-568-5801; E-mail: hbazan1{at}lsuhsc.edu.
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Copyright © 2005 by the American Society for Biochemistry and Molecular Biology.
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