Selective association of protein kinase C with 14-3-3 zeta in neuronally differentiated PC12 Cells. Stimulatory and inhibitory effect of 14-3-3 zeta in vivo.
The 14-3-3 protein is a family of highly conserved acidic proteins found in a wide range of eukaryotes from yeast to mammals. 14-3-3 acts as an adapter protein and interacts with signaling molecules including protein kinase C (PKC). Although 14-3-3 zeta was originally characterized as an endogenous PKC inhibitor, it was reported to activate PKC in vitro, but the in vivo regulation of PKC by 14-3-3 is still not well understood. To examine the regulation of PKC by 14-3-3 in the cell, we have generated a sub-cell line, PC12-B3, that stably expresses FLAG epitope-tagged 14-3-3 zeta isoform in PC12 cells. Here we show that PKC-alpha and PKC-epsilon become associated with 14-3-3 zeta when the cells are neuronally differentiated by nerve growth factor. We found that the immunoprecipitate by anti-FLAG antibody contains constitutive and autonomous Ca(2+)-independent non-classical PKC activity. In contrast, the FLAG immunoprecipitate has no Ca(2+)-dependent classical PKC activity despite the fact that PKC-alpha is present in the FLAG immunoprecipitate from differentiated PC12-B3 cells. Our results show that the association with 14-3-3 zeta has distinct effects on classical PKC and non-classical PKC activity.
Gannon-Murakami
Laura
L
Department of Biology, University of Vermont, Burlington, Vermont 05405, USA.
Murakami
Kentaro
K
D048948:14-3-3 Proteins; D000818:Animals; D002454:Cell Differentiation; D007527:Isoenzymes / Q000378:metabolism; D020932:Nerve Growth Factor / Q000494:pharmacology; D009842:Oligopeptides; D016716:PC12 Cells; D010455:Peptides / Q000378:metabolism*; D011233:Precipitin Tests; D011493:Protein Kinase C / Q000378:metabolism*; D051381:Rats; D014446:Tyrosine 3-Monooxygenase / Q000378:metabolism*
D048948:14-3-3 Proteins; D007527:Isoenzymes; D009842:Oligopeptides; D010455:Peptides; D020932:Nerve Growth Factor; C085706:FLAG peptide; D014446:Tyrosine 3-Monooxygenase; D011493:Protein Kinase C