Abstract
Selective Association of Protein Kinase C with 14-3-3 ζ in Neuronally Differentiated PC12 Cells: STIMULATORY AND INHIBITORY EFFECT OF 14-3-3 ζ IN VIVO
Highlights
291 Ϯ 18 237 Ϯ 33 321 Ϯ 64 251 Ϯ 46and -⑀ subtypes are the protein kinase C (PKC) isoforms highly enriched in the growth cones in differentiating SH-SY-5Y cells
Because PKC-␦ and -⑀ are the only non-classical PKCs that become associated or increase association with 143-3 in differentiated PC12-B3 cells, we attempted to determine whether nPKC activity found in the FLAG immunoprecipitate from differentiated cells is sensitive to arachidonic acid
We have examined whether 14-3-3 interacts with PKC in vivo in neuronal cells using PC12 and its derivative cell line B3 that stably expresses epitope-tagged 14-3-3
Summary
14-3-3 cDNA—14-3-3 cDNA isolated from rat hippocampal cDNA library (pB5BN7, Ref. 27) and subcloned in mammalian expression vector pcDNA3 (clone pc1412) was used for the study. Establishment of PC12 Sub-cell Line, Which Stably Expresses Epitope-tagged 14-3-3 —For transfection, 1.4 ϫ 105 cells were plated in a 60-mm collagen-coated dish and grown in the serum containing RPMI 1640 media overnight. Cells were washed three times with 5 ml of phosphate-buffered saline and incubated in 5 ml of OptiMEM (Invitrogen) for 45 min. OptiMEM was added for a final volume of 10 ml and further incubated for 30 h After this transfection, the medium was replaced with RPMI 1640 with 10% horse serum and 5% fetal bovine serum, and the cells were maintained for 1 week. PKC Assay for Immunoprecipitates—The activity of PKC of the immunoprecipitates with FLAG antibody from differentiated or undifferentiated cells was measured using two PKC assay systems that were optimized for Ca2ϩ-dependent cPKC activity and for Ca2ϩ-independent non-classical novel and atypical PKC activities (hereafter cPKC and nPKC assay, respectively). After a 20 min incubation at 30 °C, protein A-Sepharose beads in the reaction mixture was centrifuged by pulse spin, 30 l of the supernatant was spotted onto phosphocellulose paper, and the radioactivity was counted after the wash
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