Overexpression of ∊-Protein Kinase C Enhances Nerve Growth Factor-induced Phosphorylation of Mitogen-activated Protein Kinases and Neurite Outgrowth

Bhupinder Hundle,Thomas Mcmahon,Jahan Dadgar,Robert O Messing

doi:10.1074/jbc.270.50.30134

Abstract

Protein kinase C (PKC) activation enhances neurite outgrowth in several cell lines and primary neurons. The PKC isozymes that mediate this response are unknown. One clue to their identity has come from studies using PC12 cells treated with ethanol. In these cells, ethanol increases levels of delta-PKC and epsilon-PKC and markedly enhances nerve growth factor (NGF)-induced neurite outgrowth and activation of mitogen-activated protein (MAP) kinases by a PKC-dependent mechanism. Since these findings suggest that delta-PKC or epsilon-PKC can promote neural differentiation, we studied neurite outgrowth in stably transfected PC12 cell lines that overexpress these isozymes. Overexpression of epsilon-PKC markedly increased NGF-induced neurite outgrowth. This effect was blocked by down-regulating PKC or by treating cells with the PKC inhibitor GF 109203X. In addition, overexpression of epsilon-PKC enhanced NGF-induced phosphorylation of MAP kinases. In contrast, overexpression of delta-PKC did not alter responses to NGF. These results demonstrate that epsilon-PKC promotes NGF-induced neurite outgrowth by enhancing NGF signal transduction. These findings suggest a role for epsilon-PKC in neural differentiation and plasticity.

Highlights

Protein kinase C (PKC)1 is a multigene family of phospholipid-dependent, serine-threonine kinases central to many signal transduction pathways
Localization of ␦-PKC and ⑀-PKC in Differentiating PC12 Cells—We examined the subcellular localization of ␦- and ⑀-PKC in PC12 cells by immunofluorescence microscopy
Our findings provide the first evidence that ⑀-PKC regulates nerve growth factor (NGF) signaling and neurite outgrowth

Summary

EXPERIMENTAL PROCEDURES

Materials—NGF 2.5 was a gift from Dr W. After aspiration of the blocking solution, cells were incubated at 4 °C for 24 – 48 h with anti-␦-PKC or anti-⑀-PKC antibody (1 ␮g/ml) in PBS containing 2 mg/ml fatty acid-free bovine serum albumin and 0.1% (v/v) Triton X-100. Generation of Overexpressing Cell Lines—A 2.7-kilobase XhoI fragment that contains the full-length mouse ⑀-PKC cDNA was excised from the vector pMT2 ⑀-PKC and cloned into the NotI site of pRc/RSV using a NotI linker to obtain pR⑀. Cells were incubated for 15 min on ice and transferred into 40 ml of culture medium prewarmed to 37 °C Twenty ml of this cell suspension were plated onto poly-L-ornithine-coated 150-mm tissue culture plates and incubated at 37 °C in a humidified atmosphere of 10% CO2 and 90% air for 48 h. Values obtained were divided by values measured in corresponding fractions from control cells to determine the fold increase in ␦- or ⑀-PKC activity in transfected clones. Differences between means were analyzed by ANOVA and where p Ͻ 0.05, the significance of differences between means was evaluated by the Scheffe F-test

RESULTS

22 Ϯ 1 25 Ϯ 2 25 Ϯ 2 19 Ϯ 1 18 Ϯ 1 51 Ϯ 5a 44 Ϯ 3a

DISCUSSION