title: Single-point mutations of a lysine residue change function of Bax and Bcl-xL expressed in Bax- and Bak-less mouse embryonic fibroblasts: novel insights into the molecular mechanisms of Bax-induced apoptosis.
abstract: Members of the Bcl-2 family play key roles as proapoptotic (e.g., Bax) and antiapoptotic (e.g., Bcl-x(L)) regulators of programmed cell death. We previously identified the mitochondrial potassium channel Kv1.3 as a novel target of Bax. Incubating Kv1.3-positive isolated mitochondria with Bax triggered apoptotic events, whereas Kv1.3-deficient mitochondria were resistant to this stimulus. Mutation of Bax at lysine 128 (BaxK128E) abrogated its effects on Kv1.3 and the induction of apoptotic changes in mitochondria. These data indicate a toxin-like action of Bax on Kv1.3 to trigger at least some of the mitochondrial changes typical for apoptosis. To gain insight into the mechanism of Bax-Kv1.3 interaction, we mutated Glu158 of Bcl-x(L) (corresponding to K128 in Bax) to lysine. This substitution turned Bcl-x(L) proapoptotic. Transfection of double knockout (Bax(-/-)/Bak(-/-)) mouse embryonic fibroblasts (DKO MEFs) with either wild-type Bax, BaxK128E, or Bcl-x(L)E158K showed that apoptosis induced by various stimuli was defective in DKO MEFs and BaxK128E-transfected cells, but was recovered upon transfection with Bcl-xLE158K or wild-type Bax. Both wild-type Bax and BaxK128E can form similar ion-conducting pores upon incorporation into planar lipid bilayers. Our results point to a physiologically relevant interaction of Bax with Kv1.3 and further indicate a crucial role of a distinct lysine in determining the proapoptotic character of Bcl2-family proteins.
authors.affiliation: Department of Biology, University of Padova, Padova, Italy. ildi@civ.bio.unipd.it
authors.forename: I
authors.initials: I
authors.lastname: Szabò
authors.forename: M
authors.initials: M
authors.lastname: Soddemann
authors.forename: L
authors.initials: L
authors.lastname: Leanza
authors.forename: M
authors.initials: M
authors.lastname: Zoratti
authors.forename: E
authors.initials: E
authors.lastname: Gulbins
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D017209:Apoptosis / Q000235:genetics*; D045304:Cytochromes c / Q000378:metabolism; D004622:Embryo, Mammalian / Q000166:cytology; D005347:Fibroblasts / Q000166:cytology* / Q000378:metabolism; D006801:Humans; D015640:Ion Channel Gating; D019169:Jurkat Cells; D051664:Kv1.3 Potassium Channel / Q000378:metabolism; D008051:Lipid Bilayers / Q000378:metabolism; D008239:Lysine / Q000235:genetics*; D053078:Membrane Potential, Mitochondrial; D051379:Mice; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D017354:Point Mutation / Q000235:genetics*; D020836:Protein Structure, Quaternary; D017386:Sequence Homology, Amino Acid; D050998:bcl-2 Homologous Antagonist-Killer Protein / Q000737:chemistry / Q000172:deficiency* / Q000378:metabolism; D051028:bcl-2-Associated X Protein / Q000737:chemistry / Q000172:deficiency / Q000235:genetics* / Q000378:metabolism; D051020:bcl-X Protein / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
chemical_list: D051664:Kv1.3 Potassium Channel; D008051:Lipid Bilayers; D050505:Mutant Proteins; D050998:bcl-2 Homologous Antagonist-Killer Protein; D051028:bcl-2-Associated X Protein; D051020:bcl-X Protein; D045304:Cytochromes c; D008239:Lysine