Epidermal and Hepatocyte Growth Factors, but Not Keratinocyte Growth Factor, Modulate Protein Kinase Cα Translocation to the Plasma Membrane through 15(S)-Hydroxyeicosatetraenoic Acid Synthesis

• Published in: The Journal of biological chemistry Vol. 280; no. 9; p. 7917
• Main Authors: Guru Dutt Sharma, Paulo Ottino, Nicolas G. Bazan, Haydee E. P. Bazan
• Format: Journal Article
• Language: English
• Published: American Society for Biochemistry and Molecular Biology 04.03.2005
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M408852200

Activation of protein kinase C (PKC) involves its recruitment to the membrane, where it interacts with its activator(s). We expressed PKCα 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), PKCα translocated to the plasma membrane. Keratinocyte growth factor did not stimulate PKCα 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 PKCα 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 PKCα. PD153035, a specific inhibitor of tyrosine kinase activity of the EGF receptor, completely blocked PKCα translocation induced by EGF. PD98059, a specific MEK inhibitor, significantly inhibited EGF- and HGF-mediated PKCα 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 PKCα to the membrane and elucidate a mechanism that plays a regulatory role in cell proliferation crucial to corneal wound healing.