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- Title
Reversible protein kinase C activation in PC12 cells: effect of NGF treatment.
- Authors
Dupont, Jean‐Luc; Janoshazi, Agnes; Bellahcene, Mohamed; Mykita, Serge; De Barry, Jean
- Abstract
Abstract Although protein kinase C (PKC) is a key enzyme in the signal transduction process, there is little information on the mechanism leading to PKC activation in living cells. Using a new fluorescence imaging method, we studied this mechanism and correlated PKC conformational changes with intracellular Ca2+ concentration. PC12 cells were simultaneously loaded with Fura-2-AM and Fim-1, two fluorescent probes, which recognize Ca2+ and PKC, respectively. KCl and carbachol (an agonist to muscarinic receptors) applications induced dose-dependent increases of fluorescence for both probes. Both Ca2+ and PKC responses were observed within seconds following KCl or carbachol application, and were reversible upon stimulus withdrawal. PKC activation kinetics was slightly more rapid than the Ca2+ response after KCl application. After nerve growth factor (NGF) treatment of the cells, the amplitude of the KCl-induced PKC responses was larger indicating an increase in the activated PKC-pool in these cells. This difference between control and NGF-treated cells was not observed following carbachol application, suggesting the involvement of different PKC pools. While the Ca2+ response uniformly occurred in the cytosol, the PKC response displayed a patch pattern with higher intensities in the peripheral zone near the plasma membrane. This heterogeneous distribution of PKC activation sites was similar to the immunocytological localization of Ca2+-dependent and independent PKC isoforms, which suggested that at least several PKC isoforms interacted with intracellular elements. Upon repeated stimulation, the PKC response rapidly desensitized.
- Subjects
PROTEIN kinase C; CELLULAR signal transduction; CALCIUM ions; NERVE growth factor
- Publication
European Journal of Neuroscience, 2000, Vol 12, Issue 1, p215
- ISSN
0953-816X
- Publication type
Article
- DOI
10.1046/j.1460-9568.2000.00906.x