Underphosphorylated BAD interacts with diverse antiapoptotic Bcl-2 family proteins to regulate apoptosis

• Published in: Apoptosis (London) 2001-10, Vol.6 (5), p.319-330
• Main Authors: Bae, J, Hsu, S Y, Leo, C P, Zell, K, Hsueh, A J
• Format: Article
• Language: English
• Subjects: 14-3-3 Proteins; Amino Acid Sequence; Animals; Apoptosis; bcl-Associated Death Protein; Binding Sites; Carrier Proteins - genetics; Carrier Proteins - metabolism; Carrier Proteins - physiology; CHO Cells; Conserved Sequence; Cricetinae; Escherichia coli - genetics; Evolution, Molecular; Humans; Kinases; Molecular Sequence Data; Phosphorylation; Proteins; Proto-Oncogene Proteins c-bcl-2 - metabolism; Sequence Homology, Amino Acid; Transfection; Two-Hybrid System Techniques; Tyrosine 3-Monooxygenase - metabolism; Yeasts; Yeasts - genetics; Zebrafish Proteins
• ISSN: 1360-8185; 1573-675X
• DOI: 10.1023/A:1011319901057

Survival factors activate kinases which, in turn, phosphorylate the proapoptotic Bcl-xl/Bcl-2-associated death promoter homolog (BAD) protein at key serine residues. Phosphorylated BAD interacts with 14-3-3 proteins, and overexpression of 14-3-3 attenuates BAD-mediated apoptosis. Although BAD is known to interact with Bcl-2, Bcl-w, and Bcl-xL, the exact relationship between BAD and anti- or proapoptotic Bcl-2 proteins has not been analyzed systematically. Using the yeast two-hybrid protein interaction assay, we found that BAD interacted negligibly with proapoptotic Bcl-2 proteins. Even though wild type BAD only interacted with selected numbers of antiapoptotic proteins, underphosphorylated mutant BAD interacted with all antiapoptotic Bcl-2 proteins tested (Bcl-2, Bcl-w, Bcl-xL, Bfl-1/A1, Mcl-1, Ced-9, and BHRF-1). Using nonphosphorylated recombinant BAD expressed in bacteria, direct interactions between BAD and diverse antiapoptotic Bcl-2 members were also observed. Furthermore, apoptosis induced by BAD was blocked by coexpression with Bcl-2, Bcl-w, and Bfl-1. Comparison of BAD orthologs from zebrafish to human indicated the conservation of a 14-3-3 binding site and the BH3 domain during evolution. Thus, highly conserved BAD interacts with diverse antiapoptotic Bcl-2 members to regulate apoptosis.