INTERACTIONS AMONG MEMBERS OF THE BCL-2 PROTEIN FAMILY ANALYZED WITH A YEAST 2-HYBRID SYSTEM
INTERACTIONS AMONG MEMBERS OF THE BCL-2 PROTEIN FAMILY ANALYZED WITH A YEAST 2-HYBRID SYSTEM
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DOI：
10.1073/pnas.91.20.9238
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发表时间：
1994-09-27
影响因子：
11.1
通讯作者：
REED, JC
中科院分区：
文献类型：
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作者：
SATO, T;HANADA, M;REED, JC
利用酵母双杂交系统，研究了Bcl-2蛋白与自身以及Bcl-X-L、Bcl-X-S、Mcl-1和Bax等Bcl-2家族成员的相互作用。通过将Bcl-2家族蛋白与LexA DNA结合域或B42反式激活域连接，形成融合蛋白。通过在具有lacZ （β -半乳糖苷酶）基因的酿酒酵母中表达这些融合蛋白来检测蛋白-蛋白相互作用，该基因受LexA依赖算子的控制。该方法为Bcl-2蛋白的同型二聚化提供了证据。Bcl-2还与Bcl-X-L和Mcl-1以及主要抑制剂Bax和Bcl-X-S相互作用。Bcl-X-L与Bcl-2家族蛋白的组合相互作用模式与Bcl-2相同。对Bcl-2缺失突变体的研究表明，Bcl-2同源二聚化涉及Bcl-2蛋白内两个不同区域之间的相互作用，因为含有Bcl-2氨基酸83-218的LexA蛋白介导了与含有Bcl-2氨基酸1-81的B42融合蛋白的功能相互作用，但不补充含有Bcl-2氨基酸83-218的B42融合蛋白。与LexA/Bcl-2融合蛋白相比，LexA/Bax蛋白的表达对酵母具有致死性。含有Bcl-2、Bcl-X- l或Mcl-1的B42融合蛋白可以消除这种细胞毒性，但含有Bcl-X- s （Bcl-X的一种选择性剪接形式，缺乏一个保守的63个氨基酸区域）的融合蛋白则不能消除这种细胞毒性。这些发现提示了Bax和Bcl-X-S调控Bcl-2功能的差异模型，并表明Bcl-2/Bax异源二聚化的需求可能与Bcl-2/Bcl-2同源二聚化的需求不同。
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 mag be different from those for Bcl-2/Bcl-2 homodimerization.