EGF-induced ERK phosphorylation independent of PKC isozymes in human corneal epithelial cells.
OBJECTIVE
To investigate the role of protein kinase C (PKC) isozymes in epithelial growth factor (EGF)-induced activation of extracellular signal-regulated kinase (ERK) and cell proliferation in cultured human corneal epithelial cells.


METHODS
Simian virus (SV)40 stably transfected human corneal epithelial (THCE) cells were cultured in keratinocyte growth medium. PKC isozymes and phosphorylation of ERK in THCE cells were assessed by Western blot analysis. Translocation of the PKC isozyme was determined by subcellular fractionation followed by Western blot analysis. Cell proliferation was measured by incorporation of [(3)H]-thymidine into DNA.


RESULTS
Six PKC isozymes-PKC-alpha, -betaI, -betaII, -delta, - epsilon, and - micro -were found in THCE cells. Phorbol 12-myristate 13-acetate (PMA) caused PKC-alpha, -betaI, and - epsilon, initially present in the cytoplasm, to be translocated to the membrane and nuclear subcellular fractions and PKC-delta to be depleted from the cytoskeleton. The PKC inhibitor GF109203X inhibited PMA-induced, but not basal or EGF-induced, phosphorylation of ERK, whereas the EGF receptor inhibitor tyrphostin AG1478 blocked basal and EGF-, but not PMA-, induced phosphorylation of ERK. Depletion of PMA-sensitive PKC isozymes including PKC-alpha, -betaI, -betaII, -delta, and - epsilon, inhibited PMA-, but not EGF-, induced phosphorylation of ERK. Depletion of these PKC isozymes blocked PMA-, but not EGF-, induced cell proliferation.


CONCLUSIONS
Although activation of PKC by PMA results in activation of ERK, EGF-induced phosphorylation of ERK and/or cell proliferation is independent of the conventional and novel isozymes PKC-alpha, -betaI, -betaII, -delta, and - epsilon in human corneal epithelial cells.
Xu
Ke-Ping
KP
Department of Cellular Biology and Anatomy, Medical College of Georgia, Augusta 30912, USA.
Dartt
Darlene A
DA
Yu
Fu-Shin X
FS
D002455:Cell Division; D002461:Cell Line, Transformed; D004247:DNA / Q000096:biosynthesis; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004815:Epidermal Growth Factor / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D004847:Epithelial Cells / Q000187:drug effects / Q000201:enzymology; D019573:Epithelium, Corneal / Q000187:drug effects* / Q000201:enzymology; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D004791:Enzyme Inhibitors; D007527:Isoenzymes; D004815:Epidermal Growth Factor; D004247:DNA; D011493:Protein Kinase C; D020928:Mitogen-Activated Protein Kinases; D013755:Tetradecanoylphorbol Acetate