Pseudomonas exotoxin A-mediated apoptosis is Bak dependent and preceded by the degradation of Mcl-1.
Pseudomonas exotoxin A (PE) is a bacterial toxin that arrests protein synthesis and induces apoptosis. Here, we utilized mouse embryo fibroblasts (MEFs) deficient in Bak and Bax to determine the roles of these proteins in cell death induced by PE. PE induced a rapid and dose-dependent induction of apoptosis in wild-type (WT) and Bax knockout (Bax(-/-)) MEFs but failed in Bak knockout (Bak(-/-)) and Bax/Bak double-knockout (DKO) MEFs. Also a loss of mitochondrial membrane potential was observed in WT and Bax(-/-) MEFs, but not in Bak(-/-) or in DKO MEFs, indicating an effect of PE on mitochondrial permeability. PE-mediated inhibition of protein synthesis was identical in all 4 cell lines, indicating that differences in killing were due to steps after the ADP-ribosylation of EF2. Mcl-1, but not Bcl-x(L), was rapidly degraded after PE treatment, consistent with a role for Mcl-1 in the PE death pathway. Bak was associated with Mcl-1 and Bcl-x(L) in MEFs and uncoupled from suppressed complexes after PE treatment. Overexpression of Mcl-1 and Bcl-x(L) inhibited PE-induced MEF death. Our data suggest that Bak is the preferential mediator of PE-mediated apoptosis and that the rapid degradation of Mcl-1 unleashes Bak to activate apoptosis.
Du
Xing
X
Laboratory of Molecular Biology, National Cancer Institute, 37 Convent Drive, Bethesda, MD 20892-4264, USA.
Youle
Richard J
RJ
FitzGerald
David J
DJ
Pastan
Ira
I
D036002:ADP Ribose Transferases / Q000633:toxicity*; D000818:Animals; D017209:Apoptosis / Q000187:drug effects* / Q000502:physiology*; D001427:Bacterial Toxins / Q000633:toxicity*; D002478:Cells, Cultured; D005098:Exotoxins / Q000633:toxicity*; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D051379:Mice; D018345:Mice, Knockout; D064549:Myeloid Cell Leukemia Sequence 1 Protein; D011500:Protein Synthesis Inhibitors / Q000633:toxicity; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism*; D037521:Virulence Factors / Q000633:toxicity*; D050998:bcl-2 Homologous Antagonist-Killer Protein / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D051028:bcl-2-Associated X Protein / Q000172:deficiency / Q000235:genetics / Q000378:metabolism; D051020:bcl-X Protein / Q000378:metabolism; D000097668:Pseudomonas aeruginosa Exotoxin A
D036002:ADP Ribose Transferases; D001427:Bacterial Toxins; D005098:Exotoxins; D064549:Myeloid Cell Leukemia Sequence 1 Protein; D011500:Protein Synthesis Inhibitors; D019253:Proto-Oncogene Proteins c-bcl-2; D037521:Virulence Factors; D050998:bcl-2 Homologous Antagonist-Killer Protein; D051028:bcl-2-Associated X Protein; D051020:bcl-X Protein; D000097668:Pseudomonas aeruginosa Exotoxin A; C494773:Bak1 protein, mouse; C494827:Bax protein, mouse; C494812:Bcl2l1 protein, mouse; C579094:Mcl1 protein, mouse
J Clin Oncol. 2001 Jan 15;19(2):376-88
11208829
Cell Death Differ. 2005 Jul;12(7):713-23
15861188
Genes Dev. 2003 Jun 15;17(12):1475-86
12783855
J Biol Chem. 2006 Apr 14;281(15):10153-63
16478725
Adv Exp Med Biol. 2008;615:47-79
18437891
Genes Dev. 2001 Jun 15;15(12):1481-6
11410528
Mol Cell. 2005 Feb 4;17(3):393-403
15694340
Cancer Res. 2005 Jun 15;65(12):5399-407
15958589
Genes Dev. 2007 Apr 15;21(8):929-41
17403773
Science. 2001 Apr 27;292(5517):727-30
11326099
Cell. 2002 Jan 25;108(2):153-64
11832206
Mol Cancer Ther. 2008 Jul;7(7):2224-32
18645031
Apoptosis. 2007 May;12(5):803-13
17294081
EMBO J. 2003 Jul 15;22(14):3580-90
12853473
Cell Death Differ. 2008 Sep;15(9):1396-407
18437160
N Engl J Med. 2001 Jul 26;345(4):241-7
11474661
Genes Dev. 2005 Jun 1;19(11):1294-305
15901672
Int J Cancer. 2004 Nov 10;112(3):475-83
15382075
J Biol Chem. 1998 Apr 24;273(17):10777-83
9553144
Oncogene. 2007 Feb 26;26(9):1324-37
17322918
J Biol Chem. 2007 Mar 2;282(9):6192-200
17200126
J Cell Biol. 2007 Nov 19;179(4):701-15
18025305
Curr Opin Cell Biol. 2006 Dec;18(6):685-9
17046225
Clin Cancer Res. 2003 Feb;9(2):861-5
12576460
Blood. 2002 Mar 15;99(6):1885-93
11877256
Apoptosis. 2004 Jan;9(1):19-25
14739595
Science. 2007 Feb 9;315(5813):856-9
17289999
Clin Cancer Res. 1997 Mar;3(3):347-55
9815691
Int J Cancer. 2000 Jul 1;87(1):86-94
10861457
Science. 2000 Nov 3;290(5493):989-92
11062132
Genes Dev. 2003 Dec 1;17(23):2922-32
14633975
J Cell Biol. 2006 Aug 14;174(4):509-19
16893972
Nat Rev Cancer. 2008 Feb;8(2):121-32
18202696
Annu Rev Med. 2007;58:221-37
17059365
Nat Rev Cancer. 2006 Jul;6(7):559-65
16794638
Trends Cell Biol. 2008 Apr;18(4):157-64
18314333
Oncogene. 2005 Jun 9;24(25):4052-64
15806163
J Biol Chem. 2004 Aug 27;279(35):37201-7
15205454
J Clin Oncol. 2005 Sep 20;23(27):6719-29
16061911
EMBO J. 2007 Feb 7;26(3):825-34
17235284
Cancer Cell. 2008 Jun;13(6):472-82
18538731
Oncogene. 2004 Oct 28;23(50):8320-32
15467752
Biochemistry. 1998 Dec 1;37(48):16934-42
9836586