TY - JOUR
T1 - Protein kinase Cβ modulates ligand-induced cell surface death receptor accumulation
T2 - A mechanistic basis for enzastaurin-death ligand synergy
AU - Meng, Xue Wei
AU - Heldebrant, Michael P.
AU - Flatten, Karen S.
AU - Loegering, David A.
AU - Dai, Haiming
AU - Schneider, Paul A.
AU - Gomez, Timothy S.
AU - Peterson, Kevin L.
AU - Trushin, Sergey A.
AU - Hess, Allan D.
AU - Smith, B. Douglas
AU - Karp, Judith E.
AU - Billadeau, Daniel D.
AU - Kaufmann, Scott H.
PY - 2010
Y1 - 2010
N2 - 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-α-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 PKCα, PKCβ, PK∈, and PKCθ, 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, PKCβ shRNA uniquely reversed PMA-induced protection against cell death. The PKCβ-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 PKCβ 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 PKCβ, to tumor necrosis factor-α 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.
AB - 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-α-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 PKCα, PKCβ, PK∈, and PKCθ, 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, PKCβ shRNA uniquely reversed PMA-induced protection against cell death. The PKCβ-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 PKCβ 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 PKCβ, to tumor necrosis factor-α 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.
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U2 - 10.1074/jbc.M109.057638
DO - 10.1074/jbc.M109.057638
M3 - Article
C2 - 19887445
AN - SCOPUS:74049110147
SN - 0021-9258
VL - 285
SP - 888
EP - 902
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 2
ER -