Underphosphorylated BAD interacts with diverse antiapoptotic Bcl-2 family proteins to regulate apoptosis.
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.
Bae
J
J
Division of Reproductive Biology, Department of Gynecology and Obstetrics, Stanford University School of Medicine, Stanford, CA 94305-5317, USA.
Hsu
S Y
SY
Leo
C P
CP
Zell
K
K
Hsueh
A J
AJ
D048948:14-3-3 Proteins; D000595:Amino Acid Sequence; D000818:Animals; D017209:Apoptosis; D001665:Binding Sites; D016466:CHO Cells; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism* / Q000502:physiology*; D017124:Conserved Sequence; D006224:Cricetinae; D004926:Escherichia coli / Q000235:genetics; D019143:Evolution, Molecular; D006801:Humans; D008969:Molecular Sequence Data; D010766:Phosphorylation; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D014162:Transfection; D020798:Two-Hybrid System Techniques; D014446:Tyrosine 3-Monooxygenase / Q000378:metabolism; D015003:Yeasts / Q000235:genetics; D029961:Zebrafish Proteins; D051025:bcl-Associated Death Protein
D048948:14-3-3 Proteins; C494819:BAD protein, human; D002352:Carrier Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D029961:Zebrafish Proteins; C494818:badb protein, zebrafish; D051025:bcl-Associated Death Protein; D014446:Tyrosine 3-Monooxygenase