Interactions among members of the Bcl-2 protein family analyzed with a yeast two-hybrid system.
Interactions of the Bcl-2 protein with itself and other members of the Bcl-2 family, including Bcl-X-L, Bcl-X-S, Mcl-1, and Bax, were explored with a yeast two-hybrid system. Fusion proteins were created by linking Bcl-2 family proteins to a LexA DNA-binding domain or a B42 trans-activation domain. Protein-protein interactions were examined by expression of these fusion proteins in Saccharomyces cerevisiae having a lacZ (beta-galactosidase) gene under control of a LexA-dependent operator. This approach gave evidence for Bcl-2 protein homodimerization. Bcl-2 also interacted with Bcl-X-L and Mcl-1 and with the dominant inhibitors Bax and Bcl-X-S. Bcl-X-L displayed the same pattern of combinatorial interactions with Bcl-2 family proteins as Bcl-2. Use of deletion mutants of Bcl-2 suggested that Bcl-2 homodimerization involves interactions between two distinct regions within the Bcl-2 protein, since a LexA protein containing Bcl-2 amino acids 83-218 mediated functional interactions with a B42 fusion protein containing Bcl-2 amino acids 1-81 but did not complement a B42 fusion protein containing Bcl-2 amino acids 83-218. In contrast to LexA/Bcl-2 fusion proteins, expression of a LexA/Bax protein was lethal to yeast. This cytotoxicity could be abrogated by B42 fusion proteins containing Bcl-2, Bcl-X-L, or Mcl-1 but not those containing Bcl-X-S (an alternatively spliced form of Bcl-X that lacks a well-conserved 63-amino acid region). The findings suggest a model whereby Bax and Bcl-X-S differentially regulate Bcl-2 function, and indicate that requirements for Bcl-2/Bax heterodimerization may be different from those for Bcl-2/Bcl-2 homodimerization.
Sato
T
T
La Jolla Cancer Research Foundation, CA 92037.
Hanada
M
M
Bodrug
S
S
Irie
S
S
Iwama
N
N
Boise
L H
LH
Thompson
C B
CB
Golemis
E
E
Fong
L
L
Wang
H G
HG
D001426:Bacterial Proteins / Q000378:metabolism; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000378:metabolism*; D046911:Macromolecular Substances; D008961:Models, Structural; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D016133:Polymerase Chain Reaction / Q000379:methods; D011487:Protein Conformation; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000096:biosynthesis / Q000378:metabolism*; D019253:Proto-Oncogene Proteins c-bcl-2; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D017384:Sequence Deletion; D012697:Serine Endopeptidases; D000099160:LexA Repressor Protein
D001426:Bacterial Proteins; D017931:DNA Primers; D004268:DNA-Binding Proteins; D046911:Macromolecular Substances; D011505:Protein-Tyrosine Kinases; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D011993:Recombinant Fusion Proteins; D012697:Serine Endopeptidases; D000099160:LexA Repressor Protein