title: Reversible membrane interaction of BAD requires two C-terminal lipid binding domains in conjunction with 14-3-3 protein binding.
abstract: BAD is a Bcl-2 homology domain 3 (BH3)-only proapoptotic member of the Bcl-2 protein family that is regulated by phosphorylation in response to survival factors. Binding of BAD to mitochondria is thought to be exclusively mediated by its BH3 domain. We show here that BAD binds to lipids with high affinities, predominantly to negatively charged phospholipids, such as phosphatidylserine, phosphatidic acid, and cardiolipin, as well as to cholesterol-rich liposomes. Two lipid binding domains (LBD1 and LBD2) with different binding preferences were identified, both located in the C-terminal part of the BAD protein. BAD facilitates membrane translocation of Bcl-XL in a process that requires LBD2. Integrity of LBD1 and LBD2 is also required for proapoptotic activity in vivo. Phosphorylation of BAD does not affect membrane binding but renders BAD susceptible to membrane extraction by 14-3-3 proteins. BAD can be removed efficiently by 14-3-3zeta, -eta, -tau and lesxs efficiently by other 14-3-3 isoforms. The assembled BAD.14-3-3 complex exhibited high affinity for cholesterol-rich liposomes but low affinity for mitochondrial membranes. We conclude that BAD is a membrane-associated protein that has the hallmarks of a receptor rather than a ligand. Lipid binding is essential for the proapoptotic function of BAD in vivo. The data support a model in which BAD shuttles in a phosphorylation-dependent manner between mitochondria and other membranes and where 14-3-3 is a key regulator of this relocation. The dynamic interaction of BAD with membranes is tied to activation and membrane translocation of Bcl-XL.
authors.affiliation: Institute for Medical Radiation and Cell Research, University of Wuerzburg, 97078 Wuerzburg, Germany.
authors.forename: Mirko
authors.initials: M
authors.lastname: Hekman
authors.affiliation: Institute for Medical Radiation and Cell Research, University of Wuerzburg, 97078 Wuerzburg, Germany.
authors.forename: Stefan
authors.initials: S
authors.lastname: Albert
authors.affiliation: Institute for Medical Radiation and Cell Research, University of Wuerzburg, 97078 Wuerzburg, Germany.
authors.forename: Antoine
authors.initials: A
authors.lastname: Galmiche
authors.affiliation: Institute for Medical Radiation and Cell Research, University of Wuerzburg, 97078 Wuerzburg, Germany.
authors.forename: Ulrike E E
authors.initials: UEE
authors.lastname: Rennefahrt
authors.affiliation: Institute for Medical Radiation and Cell Research, University of Wuerzburg, 97078 Wuerzburg, Germany.
authors.forename: Jochen
authors.initials: J
authors.lastname: Fueller
authors.affiliation: Institute for Medical Radiation and Cell Research, University of Wuerzburg, 97078 Wuerzburg, Germany.
authors.forename: Andreas
authors.initials: A
authors.lastname: Fischer
authors.affiliation: Institute for Clinical Neurobiology, University of Wuerzburg, 97078 Wuerzburg, Germany.
authors.forename: Dirk
authors.initials: D
authors.lastname: Puehringer
authors.affiliation: Institute for Clinical Neurobiology, University of Wuerzburg, 97078 Wuerzburg, Germany.
authors.forename: Stefan
authors.initials: S
authors.lastname: Wiese
authors.affiliation: Institute for Medical Radiation and Cell Research, University of Wuerzburg, 97078 Wuerzburg, Germany. Electronic address: rappur@mail.uni-wuerzburg.de.
authors.forename: Ulf R
authors.initials: UR
authors.lastname: Rapp
mesh_terms: D048948:14-3-3 Proteins / Q000737:chemistry*; D000818:Animals; D017209:Apoptosis; D002462:Cell Membrane / Q000378:metabolism; D006801:Humans; D051379:Mice; D008928:Mitochondria / Q000378:metabolism; D041681:NIH 3T3 Cells; D016716:PC12 Cells; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D051381:Rats; D051025:bcl-Associated Death Protein / Q000378:metabolism / Q000502:physiology*; D051020:bcl-X Protein / Q000737:chemistry
chemical_list: D048948:14-3-3 Proteins; D051025:bcl-Associated Death Protein; D051020:bcl-X Protein