title: p14(ARF)-induced apoptosis in p53 protein-deficient cells is mediated by BH3-only protein-independent derepression of Bak protein through down-regulation of Mcl-1 and Bcl-xL proteins.
abstract: The p14(ARF) tumor suppressor plays a central role in regulating cell cycle arrest and apoptosis. We reported previously that p14(ARF) is capable of triggering apoptosis in a p53-independent manner. However, the mechanism remained unclear. Here we demonstrate that the p53-independent activation of the mitochondrial apoptosis pathway by p14(ARF) is primarily mediated by the pro-apoptotic Bax-homolog Bak. Expression of p14(ARF) exclusively triggers a N-terminal conformational switch of Bak, but not Bax, which allows for mitochondrial permeability shift, release of cytochrome c, activation of caspases, and subsequent fragmentation of genomic DNA. Although forced expression of Bak markedly sensitizes toward p14(ARF)-induced apoptosis, re-expression of Bax has no effect. Vice versa, knockdown of Bak by RNA interference attenuates p14(ARF)-induced apoptosis, whereas down-regulation of Bax has no effect. Bak activation coincides with a prominent, caspase-independent deprivation of the endogenous Bak inhibitors Mcl-1 and Bcl-x(L). In turn, mitochondrial apoptosis is fully blocked by overexpression of either Mcl-1 or Bcl-x(L). Taken together, these data indicate that in the absence of functional p53 and Bax, p14(ARF) triggers mitochondrial apoptosis signaling by activating Bak, which is facilitated by down-regulating anti-apoptotic Mcl-1 and Bcl-x(L). Moreover, our data suggest that the simultaneous inhibition of two central endogenous Bak inhibitors, i.e. Mcl-1 and Bcl-x(L), may be sufficient to activate mitochondrial apoptosis in the absence of BH3-only protein regulation.
authors.affiliation: Experimental and Clinical Research Center (ECRC), Charité-Universitätsmedizin Berlin, Campus Berlin Buch, D13125, Berlin, Germany; Max-Delbrück-Center for Molecular Medicine, D13125, Berlin, Germany.
authors.forename: Annika
authors.initials: A
authors.lastname: Müer
authors.affiliation: Experimental and Clinical Research Center (ECRC), Charité-Universitätsmedizin Berlin, Campus Berlin Buch, D13125, Berlin, Germany; Max-Delbrück-Center for Molecular Medicine, D13125, Berlin, Germany.
authors.forename: Tim
authors.initials: T
authors.lastname: Overkamp
authors.affiliation: Experimental and Clinical Research Center (ECRC), Charité-Universitätsmedizin Berlin, Campus Berlin Buch, D13125, Berlin, Germany; Max-Delbrück-Center for Molecular Medicine, D13125, Berlin, Germany.
authors.forename: Bernd
authors.initials: B
authors.lastname: Gillissen
authors.affiliation: Experimental and Clinical Research Center (ECRC), Charité-Universitätsmedizin Berlin, Campus Berlin Buch, D13125, Berlin, Germany; Max-Delbrück-Center for Molecular Medicine, D13125, Berlin, Germany.
authors.forename: Antje
authors.initials: A
authors.lastname: Richter
authors.affiliation: Department of Hematology, Oncology and Tumor Immunology, Charité-Universitätsmedizin Berlin, Campus Virchow-Klinikum, 13353 Berlin, Germany.
authors.forename: Thomas
authors.initials: T
authors.lastname: Pretzsch
authors.affiliation: Experimental and Clinical Research Center (ECRC), Charité-Universitätsmedizin Berlin, Campus Berlin Buch, D13125, Berlin, Germany; Max-Delbrück-Center for Molecular Medicine, D13125, Berlin, Germany.
authors.forename: Ana
authors.initials: A
authors.lastname: Milojkovic
authors.affiliation: Experimental and Clinical Research Center (ECRC), Charité-Universitätsmedizin Berlin, Campus Berlin Buch, D13125, Berlin, Germany; Max-Delbrück-Center for Molecular Medicine, D13125, Berlin, Germany; Department of Hematology, Oncology and Tumor Immunology, Charité-Universitätsmedizin Berlin, Campus Virchow-Klinikum, 13353 Berlin, Germany.
authors.forename: Bernd
authors.initials: B
authors.lastname: Dörken
authors.affiliation: Experimental and Clinical Research Center (ECRC), Charité-Universitätsmedizin Berlin, Campus Berlin Buch, D13125, Berlin, Germany; Max-Delbrück-Center for Molecular Medicine, D13125, Berlin, Germany; Department of Hematology, Oncology and Tumor Immunology, Charité-Universitätsmedizin Berlin, Campus Virchow-Klinikum, 13353 Berlin, Germany. Electronic address: pdaniel@mdc-berlin.de.
authors.forename: Peter T
authors.initials: PT
authors.lastname: Daniel
authors.affiliation: Experimental and Clinical Research Center (ECRC), Charité-Universitätsmedizin Berlin, Campus Berlin Buch, D13125, Berlin, Germany; Max-Delbrück-Center for Molecular Medicine, D13125, Berlin, Germany; Department of Hematology, Oncology and Tumor Immunology, Charité-Universitätsmedizin Berlin, Campus Virchow-Klinikum, 13353 Berlin, Germany.
authors.forename: Philipp
authors.initials: P
authors.lastname: Hemmati
mesh_terms: D017209:Apoptosis; D051023:BH3 Interacting Domain Death Agonist Protein / Q000235:genetics / Q000378:metabolism*; D045744:Cell Line, Tumor; D015536:Down-Regulation / Q000502:physiology*; D006801:Humans; D008928:Mitochondria / Q000235:genetics / Q000378:metabolism; D064549:Myeloid Cell Leukemia Sequence 1 Protein; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology; D025803:Tumor Suppressor Protein p14ARF / Q000235:genetics / Q000378:metabolism*; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism*; D050998:bcl-2 Homologous Antagonist-Killer Protein / Q000235:genetics / Q000378:metabolism*; D051020:bcl-X Protein / Q000235:genetics / Q000378:metabolism*
chemical_list: C494772:BAK1 protein, human; C494811:BCL2L1 protein, human; D051023:BH3 Interacting Domain Death Agonist Protein; D064549:Myeloid Cell Leukemia Sequence 1 Protein; D019253:Proto-Oncogene Proteins c-bcl-2; C495901:TP53 protein, human; D025803:Tumor Suppressor Protein p14ARF; D016159:Tumor Suppressor Protein p53; D050998:bcl-2 Homologous Antagonist-Killer Protein; D051020:bcl-X Protein