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

Abstract

Activation of protein kinase C (PKC) involves its recruitment to the membrane, where it interacts with its activator(s). Protein kinase C (PKC)1 is a multifunctional family of serine/ threonine protein kinases with 12 different isoforms, whose activities are dependent on Ca 2ϩ , lipid second messengers, and/or protein activators and regulators (1, 2). Among them, four classical PKC isoforms (␣, ␤I, ␤II, and ␥) require Ca 2ϩ , diacylglycerol, and phosphatidylserine for their activation. The novel PKC isoforms (␦, ⑀, , , and ) require diacylglycerol and phosphatidylserine for activation but are independent of Ca 2ϩ . The atypical PKC isoforms (, , and ) are activated by phospholipids. These isoenzymes play crucial roles as transducers of various extracellular receptor-originated signals by hormones, neurotransmitters, and growth factors triggering cell proliferation, differentiation, cytoskeletal alterations, and gene expression (1, 3). They also contribute to cellular signaling through cross-talk with other signaling cascades.Upon various physiologic stimuli, PKCs display differential patterns of subcellular localization (4, 5). The movement of PKC is a prerequisite for its effective response to physiologic activators, such as membrane lipids, and allows it to gain access to its specific substrates and to be selective in their response. The time course and duration of PKC relocation may determine the eventual signal response. Previous studies from our laboratory showed that rabbit corneal epithelium expresses PKC␣, ␥, ⑀, , and , that PKC␣ is activated after corneal injury, and that inhibition of its expression delays epithelial wound healing (6, 7). We previously proposed that PKC␣ plays a role in corneal epithelial proliferation (7). However, the transduction mechanisms underlying PKC␣ activation in cornea are still poorly understood.Growth factors such as EGF, HGF, and KGF increase in response to corneal epithelial injury (8) and are important in homeostasis and wound repair (9 -11). Earlier studies showed that PKC␣ activation requires changes in subcellular localization after phorbol ester (12-O-tetradecanoylphorbol-13-acetate) stimulation (12). However, information about relocalization of PKC␣ as a function of time upon growth factor stimulation is relatively scant.

Specifying protein kinase C functions in melanoma

“…Several growth factor ⁄ receptor pairs mitogenic for melanocytes, including HGF ⁄ c-Met and Endothelin-1 ⁄ ET(B)-R, lead to PKC activation, and in fact chronic treatment with the PKC agonist 12-O-tetradecanoylphobol-13-acetate (TPA) is mitogenic for normal melanocytes (Arita et al, 1992;Eisinger et al, 1983;Eisinger and Marko, 1982;Hirobe, 2001;Imokawa et al, 1997;Pittelkow and Shipley, 1989;Sharma et al, 2005Sharma et al, , 2007Swope et al, 1995). The stimulation of phosphatidylinositol metabolism by growth factors mitogenic in melanoma promotes the generation of phosphatidylinositol, 4,5-bisphosphate, which can be cleaved to generate the PKC activator DAG and inositol 1,4,5-trisphosphate (Nishizuka, 1992).…”

Protein kinase C alpha and epsilon differentially modulate hepatocyte growth factor-induced epithelial proliferation and migration

“…PKCα increases its activity after epithelial damage, and inhibition of its expression delays wound closure (Chandrasekher et al, 1998;Lin and Bazan, 1992). More recently we have shown that epidermal growth factor (EGF) and HGF can induce translocation (activation) of PKCα to the cell membrane while KGF does not (Sharma et al, 2005). This selective activation points to a specific function of PKCα upon stimulation with growth factors.…”

“…In previous studies we had shown that in HCE cells HGF induced translocation (and activation) of PKCα to the plasma membrane while KGF does not, suggesting that different signaling pathways are activated by these two paracrine growth factors (Sharma et al, 2005). To examine the kinetics of translocation of PKCε to the plasma membrane upon KGF and HGF stimulation, we performed experiments using live-cell imaging to track real-time movement of PKCε-GFP.…”

“…Transient transfections were performed in HCE cells with PKCε-GFP (1 μg DNA/dish) using FuGENE 6 transfection reagent (ratio 3:1, FuGENE: DNA) as described (Sharma et al, 2005). The transfected cells were incubated in KGM for an additional 24 h to allow the expression of GFP-tagged PKCs.…”

“…Cells were stimulated with growth factors (HGF or KGF [20 ng/ml]) and images were recorded prior to treatment (control, t = 0 min) at 60-second intervals up to 5 min, and at 5-min intervals up to 30 min as previously described (Sharma et al, 2005). The medium contained HEPES and the pH was maintained constant during the time of the recording.…”