Reversible membrane interaction of BAD requires two C-terminal lipid binding domains in conjunction with 14-3-3 protein binding.
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.
Hekman
Mirko
M
Institute for Medical Radiation and Cell Research, University of Wuerzburg, 97078 Wuerzburg, Germany.
Albert
Stefan
S
Institute for Medical Radiation and Cell Research, University of Wuerzburg, 97078 Wuerzburg, Germany.
Galmiche
Antoine
A
Institute for Medical Radiation and Cell Research, University of Wuerzburg, 97078 Wuerzburg, Germany.
Rennefahrt
Ulrike E E
UEE
Institute for Medical Radiation and Cell Research, University of Wuerzburg, 97078 Wuerzburg, Germany.
Fueller
Jochen
J
Institute for Medical Radiation and Cell Research, University of Wuerzburg, 97078 Wuerzburg, Germany.
Fischer
Andreas
A
Institute for Medical Radiation and Cell Research, University of Wuerzburg, 97078 Wuerzburg, Germany.
Puehringer
Dirk
D
Institute for Clinical Neurobiology, University of Wuerzburg, 97078 Wuerzburg, Germany.
Wiese
Stefan
S
Institute for Clinical Neurobiology, University of Wuerzburg, 97078 Wuerzburg, Germany.
Rapp
Ulf R
UR
Institute for Medical Radiation and Cell Research, University of Wuerzburg, 97078 Wuerzburg, Germany. Electronic address: rappur@mail.uni-wuerzburg.de.
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
D048948:14-3-3 Proteins; D051025:bcl-Associated Death Protein; D051020:bcl-X Protein