title: Protein kinase Cbeta modulates ligand-induced cell surface death receptor accumulation: a mechanistic basis for enzastaurin-death ligand synergy.
abstract: Although treatment with the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) is known to protect a subset of cells from induction of apoptosis by death ligands such as Fas ligand and tumor necrosis factor-alpha-related apoptosis-inducing ligand, the mechanism of this protection is unknown. This study demonstrated that protection in short term apoptosis assays and long term proliferation assays was maximal when Jurkat or HL-60 human leukemia cells were treated with 2-5 nm PMA. Immunoblotting demonstrated that multiple PKC isoforms, including PKCalpha, PKCbeta, PKCepsilon, and PKC, translocated from the cytosol to a membrane-bound fraction at these PMA concentrations. When the ability of short hairpin RNA (shRNA) constructs that specifically down-regulated each of these isoforms was examined, PKCbeta shRNA uniquely reversed PMA-induced protection against cell death. The PKCbeta-selective small molecule inhibitor enzastaurin had a similar effect. Although mass spectrometry suggested that Fas is phosphorylated on a number of serines and threonines, mutation of these sites individually or collectively had no effect on Fas-mediated death signaling or PMA protection. Further experiments demonstrated that PMA diminished ligand-induced cell surface accumulation of Fas and DR5, and PKCbeta shRNA or enzastaurin reversed this effect. Moreover, enzastaurin sensitized a variety of human tumor cell lines and clinical acute myelogenous leukemia isolates, which express abundant PKCbeta, to tumor necrosis factor-alpha related apoptosis-inducing ligand-induced death in the absence of PMA. Collectively, these results identify a specific PKC isoform that modulates death receptor-mediated cytotoxicity as well as a small molecule inhibitor that mitigates the inhibitory effects of PKC activation on ligand-induced death receptor trafficking and cell death.
authors.affiliation: Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic College of Medicine, Rochester, Minnesota 55905, USA.
authors.forename: Xue Wei
authors.initials: XW
authors.lastname: Meng
authors.forename: Michael P
authors.initials: MP
authors.lastname: Heldebrant
authors.forename: Karen S
authors.initials: KS
authors.lastname: Flatten
authors.forename: David A
authors.initials: DA
authors.lastname: Loegering
authors.forename: Haiming
authors.initials: H
authors.lastname: Dai
authors.forename: Paula A
authors.initials: PA
authors.lastname: Schneider
authors.forename: Timothy S
authors.initials: TS
authors.lastname: Gomez
authors.forename: Kevin L
authors.initials: KL
authors.lastname: Peterson
authors.forename: Sergey A
authors.initials: SA
authors.lastname: Trushin
authors.forename: Allan D
authors.initials: AD
authors.lastname: Hess
authors.forename: B Douglas
authors.initials: BD
authors.lastname: Smith
authors.forename: Judith E
authors.initials: JE
authors.lastname: Karp
authors.forename: Daniel D
authors.initials: DD
authors.lastname: Billadeau
authors.forename: Scott H
authors.initials: SH
authors.lastname: Kaufmann
mesh_terms: D017209:Apoptosis / Q000187:drug effects*; D002273:Carcinogens / Q000494:pharmacology; D049109:Cell Proliferation / Q000187:drug effects*; D020536:Enzyme Activators / Q000494:pharmacology; D053222:Fas Ligand Protein / Q000494:pharmacology; D018922:HL-60 Cells; D006801:Humans; D007211:Indoles / Q000494:pharmacology*; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000378:metabolism; D019169:Jurkat Cells; D010766:Phosphorylation / Q000187:drug effects; D011493:Protein Kinase C / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D064546:Protein Kinase C beta; D021381:Protein Transport / Q000187:drug effects; D053220:Receptors, TNF-Related Apoptosis-Inducing Ligand / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D019014:fas Receptor / Q000378:metabolism*
chemical_list: D002273:Carcinogens; D020536:Enzyme Activators; D053222:Fas Ligand Protein; D007211:Indoles; D007527:Isoenzymes; D053220:Receptors, TNF-Related Apoptosis-Inducing Ligand; D014409:Tumor Necrosis Factor-alpha; D019014:fas Receptor; D011493:Protein Kinase C; D064546:Protein Kinase C beta; D013755:Tetradecanoylphorbol Acetate; C504878:enzastaurin