Abstract
We have studied nerve growth factor (NGF)-induced differentiation of PC12 cells to identify PKC isozymes important for neuronal differentiation. Previous work showed that tumor-promoting phorbol esters and ethanol enhance NGF-induced mitogen-activated protein (MAP) kinase activation and neurite outgrowth by a PKC-dependent mechanism. Ethanol also increases expression of PKCdelta and PKCepsilon, suggesting that one these isozymes regulates responses to NGF. To examine this possibility, we established PC12 cell lines that express a fragment encoding the first variable domain of PKCepsilon (amino acids 2-144), which acts as an isozyme-specific inhibitor of PKCepsilon in cardiac myocytes. Phorbol ester-stimulated translocation of PKCepsilon was markedly reduced in these PC12 cell lines. In addition, phorbol ester and ethanol did not enhance NGF-induced MAP kinase activation or neurite outgrowth in these cells. In contrast, phorbol ester and ethanol increased neurite outgrowth and MAP kinase phosphorylation in cells expressing a fragment derived from the first variable domain of PKCdelta. These results demonstrate that PKCepsilon mediates enhancement of NGF-induced signaling and neurite outgrowth by phorbol esters and ethanol in PC12 cells.
Highlights
Protein kinase C (PKC)1 is a multigene family of phospholipiddependent, serine-threonine kinases that plays a central role in cell growth and differentiation
We found that overexpression of PKC⑀, but not of PKC␦, enhances nerve growth factor (NGF)-induced mitogen-activated protein (MAP) kinase activation and neurite outgrowth [27]
The current results identify PKC⑀ as the PKC isozyme responsible for enhancement of NGF responses by phorbol esters and ethanol in PC12 cells
Summary
Materials—NGF (2.5 S) was purchased from Collaborative Research (Bedford, MA). Geneticin (G418), laminin, and poly-L-ornithine (30 –70 kDa) were purchased from Sigma. Cells were washed three times in PBS, and immunoreactivity was detected using fluorescein-conjugated goat anti-rabbit IgG as described previously [27]. Blots were incubated overnight at 4 °C with 1 g/ml of anti-phospho-MAP kinase antibody in buffer B containing 0.05% Tween 20 and 5% bovine serum albumin (incubation buffer) They were washed three times for 5 min with 15 ml of blocking buffer and incubated with goat antirabbit alkaline phosphatase-conjugated antibody (1:2000 dilution) in incubation buffer for 1 h at 25 °C. Samples of supernatant and pellet suspension derived from 100 g of crude homogenate were separated by SDS-polyacrylamide gel electrophoresis using 10% gels and analyzed for PKC␦ and PKC⑀ immunoreactivity by Western analysis as described previously [27]. Where p Ͻ 0.05, the significance of differences between means was evaluated by the Scheffe F-test or the Newman Keuls test
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
