title: Physiological restraint of Bak by Bcl-xL is essential for cell survival.
abstract: Due to the myriad interactions between prosurvival and proapoptotic members of the Bcl-2 family of proteins, establishing the mechanisms that regulate the intrinsic apoptotic pathway has proven challenging. Mechanistic insights have primarily been gleaned from in vitro studies because genetic approaches in mammals that produce unambiguous data are difficult to design. Here we describe a mutation in mouse and human Bak that specifically disrupts its interaction with the prosurvival protein Bcl-xL Substitution of Glu75 in mBak (hBAK Q77) for leucine does not affect the three-dimensional structure of Bak or killing activity but reduces its affinity for Bcl-xL via loss of a single hydrogen bond. Using this mutant, we investigated the requirement for physical restraint of Bak by Bcl-xL in apoptotic regulation. In vitro, Bak(Q75L) cells were significantly more sensitive to various apoptotic stimuli. In vivo, loss of Bcl-xL binding to Bak led to significant defects in T-cell and blood platelet survival. Thus, we provide the first definitive in vivo evidence that prosurvival proteins maintain cellular viability by interacting with and inhibiting Bak.
authors.affiliation: The Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria 3052, Australia; Department of Medical Biology, The University of Melbourne, Parkville, Victoria 3010, Australia; Department of Chemistry and Physics, La Trobe Institute for Molecular Science, La Trobe University, Bundoora, Victoria 3086, Australia; Olivia Newton-John Cancer Research Institute, Heidelberg, Victoria 3084, Australia; School of Cancer Medicine, La Trobe University, Melbourne, Victoria 3084, Australia.
authors.forename: Erinna F
authors.initials: EF
authors.lastname: Lee
authors.affiliation: The Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria 3052, Australia; Department of Medical Biology, The University of Melbourne, Parkville, Victoria 3010, Australia;
authors.forename: Stephanie
authors.initials: S
authors.lastname: Grabow
authors.affiliation: The Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria 3052, Australia; Department of Medical Biology, The University of Melbourne, Parkville, Victoria 3010, Australia;
authors.forename: Stephane
authors.initials: S
authors.lastname: Chappaz
authors.affiliation: The Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria 3052, Australia; Department of Medical Biology, The University of Melbourne, Parkville, Victoria 3010, Australia;
authors.forename: Grant
authors.initials: G
authors.lastname: Dewson
authors.affiliation: The Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria 3052, Australia; Department of Medical Biology, The University of Melbourne, Parkville, Victoria 3010, Australia;
authors.forename: Colin
authors.initials: C
authors.lastname: Hockings
authors.affiliation: The Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria 3052, Australia; Department of Medical Biology, The University of Melbourne, Parkville, Victoria 3010, Australia;
authors.forename: Ruth M
authors.initials: RM
authors.lastname: Kluck
authors.affiliation: The Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria 3052, Australia; Department of Medical Biology, The University of Melbourne, Parkville, Victoria 3010, Australia;
authors.forename: Marlyse A
authors.initials: MA
authors.lastname: Debrincat
authors.affiliation: The Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria 3052, Australia; Department of Medical Biology, The University of Melbourne, Parkville, Victoria 3010, Australia;
authors.forename: Daniel H
authors.initials: DH
authors.lastname: Gray
authors.affiliation: The Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria 3052, Australia; Department of Medical Biology, The University of Melbourne, Parkville, Victoria 3010, Australia;
authors.forename: Matthew T
authors.initials: MT
authors.lastname: Witkowski
authors.affiliation: Olivia Newton-John Cancer Research Institute, Heidelberg, Victoria 3084, Australia; School of Cancer Medicine, La Trobe University, Melbourne, Victoria 3084, Australia.
authors.forename: Marco
authors.initials: M
authors.lastname: Evangelista
authors.affiliation: The Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria 3052, Australia;
authors.forename: Anne
authors.initials: A
authors.lastname: Pettikiriarachchi
authors.affiliation: The Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria 3052, Australia; Department of Medical Biology, The University of Melbourne, Parkville, Victoria 3010, Australia;
authors.forename: Philippe
authors.initials: P
authors.lastname: Bouillet
authors.affiliation: The Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria 3052, Australia;
authors.forename: Rachael M
authors.initials: RM
authors.lastname: Lane
authors.affiliation: The Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria 3052, Australia; Department of Medical Biology, The University of Melbourne, Parkville, Victoria 3010, Australia;
authors.forename: Peter E
authors.initials: PE
authors.lastname: Czabotar
authors.affiliation: The Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria 3052, Australia; Department of Medical Biology, The University of Melbourne, Parkville, Victoria 3010, Australia;
authors.forename: Peter M
authors.initials: PM
authors.lastname: Colman
authors.affiliation: Department of Chemistry and Physics, La Trobe Institute for Molecular Science, La Trobe University, Bundoora, Victoria 3086, Australia;
authors.forename: Brian J
authors.initials: BJ
authors.lastname: Smith
authors.affiliation: The Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria 3052, Australia; Department of Medical Biology, The University of Melbourne, Parkville, Victoria 3010, Australia;
authors.forename: Benjamin T
authors.initials: BT
authors.lastname: Kile
authors.affiliation: The Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria 3052, Australia; Department of Medical Biology, The University of Melbourne, Parkville, Victoria 3010, Australia; Department of Chemistry and Physics, La Trobe Institute for Molecular Science, La Trobe University, Bundoora, Victoria 3086, Australia; Olivia Newton-John Cancer Research Institute, Heidelberg, Victoria 3084, Australia; School of Cancer Medicine, La Trobe University, Melbourne, Victoria 3084, Australia.
authors.forename: W Douglas
authors.initials: WD
authors.lastname: Fairlie
mesh_terms: D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects / Q000235:genetics*; D001792:Blood Platelets / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D002460:Cell Line; D002470:Cell Survival / Q000235:genetics; D006801:Humans; D051379:Mice; D008810:Mice, Inbred C57BL; D009154:Mutation; D011485:Protein Binding; D011487:Protein Conformation; D000072417:Protein Domains / Q000235:genetics; D013601:T-Lymphocytes / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D050998:bcl-2 Homologous Antagonist-Killer Protein / Q000235:genetics / Q000378:metabolism*; D051020:bcl-X Protein / Q000378:metabolism*
chemical_list: D000970:Antineoplastic Agents; D050998:bcl-2 Homologous Antagonist-Killer Protein; D051020:bcl-X Protein
keywords: BH3; Bak; Bcl-2; Bcl-xL; apoptosis