title: Proteolytic degradation of protein kinase C in the phorbol ester-induced interleukin-2 secreting thymoma cells.
abstract: Effects of phorbol 12-myristate 13-acetate (PMA) on the fate of protein kinase C in two mouse thymoma cell lines, which are either responsive (EL4) or unresponsive (IEL4) to PMA-induced interleukin-2 (IL-2) production, were investigated with polyclonal antibodies raised against rat brain enzyme. These antibodies immunoprecipitated completely the protein kinase C from both cell lines and detected mainly an 82-kDa protein by immunoblot analysis of the crude homogenates as well as the partially purified kinase preparations. PMA elicited a time- and dose-dependent redistribution of protein kinase C from cytosol to the particulate fraction and proteolytic degradation of the kinase from both cell lines. The dose of PMA required for half-maximum protein kinase C translocation and degradation was at least five times lower for EL4 than for IEL4. In the presence of 16 nM PMA the rates of protein kinase C translocation and degradation were faster in EL4 than in IEL4, and the half-lives of protein kinase C in EL4 and IEL4 were less than 5 min and greater than 2 h, respectively. Analysis of the tryptic fragments of the immunoprecipitated enzyme, previously phosphorylated in the presence of [gamma-32P]ATP, revealed minor structural differences between the protein kinase C from these two cell lines. In neither cell line did the PMA-induced degradation of protein kinase C result in an accumulation of the Ca2+/phospholipid-independent kinase (catalytic unit) as analyzed by immunoblotting and gel filtration chromatography. Thus, activation of protein kinase C through the proteolytic conversion to the effector-independent catalytic unit plays little role in IL-2 production. The role of protein kinase C translocation and degradation in the PMA-induced responses in EL4 cells is unknown. However, IL-2 production in EL4 cells was reduced when PMA-induced degradation of protein kinase C was retarded by exogenously added protease inhibitors.
authors.affiliation: Section on Metabolic Regulation, National Institute of Child Health and Human Development, Bethesda, Maryland 20892.
authors.forename: F L
authors.initials: FL
authors.lastname: Huang
authors.forename: P K
authors.initials: PK
authors.lastname: Arora
authors.forename: E E
authors.initials: EE
authors.lastname: Hanna
authors.forename: K P
authors.initials: KP
authors.lastname: Huang
mesh_terms: D000818:Animals; D002460:Cell Line / Q000187:drug effects; D003600:Cytosol / Q000201:enzymology; D006868:Hydrolysis; D015151:Immunoblotting; D007376:Interleukin-2 / Q000378:metabolism*; D051379:Mice; D010446:Peptide Fragments / Q000032:analysis; D010449:Peptide Mapping; D010766:Phosphorylation; D011233:Precipitin Tests; D011480:Protease Inhibitors / Q000494:pharmacology; D011493:Protein Kinase C / Q000302:isolation & purification* / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D013945:Thymoma / Q000201:enzymology* / Q000378:metabolism
chemical_list: D007376:Interleukin-2; D010446:Peptide Fragments; D011480:Protease Inhibitors; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate