Protein kinase Cβ modulates ligand-induced cell surface death receptor accumulation

A mechanistic basis for enzastaurin-death ligand synergy

Xue Wei Meng, Michael P. Heldebrant, Karen S. Flatten, David A. Loegering, Haiming Dai, Paul A. Schneider, Timothy S. Gomez, Kevin L. Peterson, Sergey A. Trushin, Allan D. Hess, B. Douglas Smith, Judith E. Karp, Daniel D Billadeau, Scott H Kaufmann

Research output: Contribution to journalArticle

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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-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.

Original languageEnglish (US)
Pages (from-to)888-902
Number of pages15
JournalJournal of Biological Chemistry
Volume285
Issue number2
DOIs
StatePublished - 2010

Fingerprint

Death Domain Receptors
Cell Surface Receptors
Protein Kinase C
Cell Death
Ligands
Acetates
Apoptosis
Small Interfering RNA
Protein Isoforms
Cell death
Tumor Necrosis Factor-alpha
Assays
enzastaurin
Fas Ligand Protein
Threonine
phorbol-12-myristate
Tumor Cell Line
Molecules
Immunoblotting
Acute Myeloid Leukemia

ASJC Scopus subject areas

  • Biochemistry
  • Cell Biology
  • Molecular Biology
  • Medicine(all)

Cite this

Protein kinase Cβ modulates ligand-induced cell surface death receptor accumulation : A mechanistic basis for enzastaurin-death ligand synergy. / Meng, Xue Wei; Heldebrant, Michael P.; Flatten, Karen S.; Loegering, David A.; Dai, Haiming; Schneider, Paul A.; Gomez, Timothy S.; Peterson, Kevin L.; Trushin, Sergey A.; Hess, Allan D.; Smith, B. Douglas; Karp, Judith E.; Billadeau, Daniel D; Kaufmann, Scott H.

In: Journal of Biological Chemistry, Vol. 285, No. 2, 2010, p. 888-902.

Research output: Contribution to journalArticle

Meng, XW, Heldebrant, MP, Flatten, KS, Loegering, DA, Dai, H, Schneider, PA, Gomez, TS, Peterson, KL, Trushin, SA, Hess, AD, Smith, BD, Karp, JE, Billadeau, DD & Kaufmann, SH 2010, 'Protein kinase Cβ modulates ligand-induced cell surface death receptor accumulation: A mechanistic basis for enzastaurin-death ligand synergy', Journal of Biological Chemistry, vol. 285, no. 2, pp. 888-902. https://doi.org/10.1074/jbc.M109.057638
Meng, Xue Wei ; Heldebrant, Michael P. ; Flatten, Karen S. ; Loegering, David A. ; Dai, Haiming ; Schneider, Paul A. ; Gomez, Timothy S. ; Peterson, Kevin L. ; Trushin, Sergey A. ; Hess, Allan D. ; Smith, B. Douglas ; Karp, Judith E. ; Billadeau, Daniel D ; Kaufmann, Scott H. / Protein kinase Cβ modulates ligand-induced cell surface death receptor accumulation : A mechanistic basis for enzastaurin-death ligand synergy. In: Journal of Biological Chemistry. 2010 ; Vol. 285, No. 2. pp. 888-902.
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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-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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AU - Gomez, Timothy S.

AU - Peterson, Kevin L.

AU - Trushin, Sergey A.

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