Reversible protein kinase C activation in PC12 cells: effect of NGF treatment.
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.
Dupont
J L
JL
Laboratoire de Neurobiologie Cellulaire, UPR 9009 CNRS, 5 rue B. Pascal, F-67084 Strasbourg Cedex, France.
Janoshazi
A
A
Bellahcene
M
M
Mykita
S
S
de Barry
J
J
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002217:Carbachol / Q000494:pharmacology; D004789:Enzyme Activation; D005452:Fluoresceins; D005456:Fluorescent Dyes; D016257:Fura-2 / Q000031:analogs & derivatives; D007150:Immunohistochemistry; D007211:Indoles; D007527:Isoenzymes / Q000378:metabolism*; D007700:Kinetics; D008856:Microscopy, Fluorescence; D009414:Nerve Growth Factors / Q000494:pharmacology*; D016716:PC12 Cells; D011189:Potassium Chloride / Q000494:pharmacology; D011493:Protein Kinase C / Q000378:metabolism*; D064546:Protein Kinase C beta; D051571:Protein Kinase C-alpha; D051381:Rats; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D005452:Fluoresceins; D005456:Fluorescent Dyes; D007211:Indoles; D007527:Isoenzymes; D009414:Nerve Growth Factors; C049925:fura-2-am; C082335:fim 1; D011189:Potassium Chloride; D002217:Carbachol; D011493:Protein Kinase C; D064546:Protein Kinase C beta; D051571:Protein Kinase C-alpha; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium; D016257:Fura-2