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J. Biol. Chem., Vol. 281, Issue 25, 17321-17336, June 23, 2006

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Reversible Membrane Interaction of BAD Requires two C-terminal Lipid Binding Domains in Conjunction with 14-3-3 Protein Binding^*

Mirko Hekman¹, Stefan Albert¹, Antoine Galmiche, Ulrike E. E. Rennefahrt, Jochen Fueller, Andreas Fischer, Dirk Puehringer, Stefan Wiese, and Ulf R. Rapp²

From the Institute for Medical Radiation and Cell Research and Institute for Clinical Neurobiology, University of Wuerzburg, 97078 Wuerzburg, Germany

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-X_L 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 14-3-3, -, - 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-X_L.

Received for publication, January 11, 2006 , and in revised form, March 31, 2006.

^* This work was supported by Deutsche Forschungsgemeinschaft Grants SFB 487, SFB 581, and RA 642/11 and the Scheel Foundation (Priority Program on Apoptosis). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1-S5.

¹ These two authors contributed equally to this work.

² To whom correspondence should be addressed: University of Wuerzburg, Institute for Medical Radiation and Cell Research, Versbacher Str. 5, 97078 Wuerzburg, Germany. Tel.: 49-931-201-45141; Fax: 49-931-201-45835; E-mail: rappur{at}mail.uni-wuerzburg.de.

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