WT p53, but not tumor-derived mutants, bind to Bcl2 via the DNA binding domain and induce mitochondrial permeabilization
WT p53, but not tumor-derived mutants, bind to Bcl2 via the DNA binding domain and induce mitochondrial permeabilization
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DOI：
10.1074/jbc.m507611200
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发表时间：
2006-03-31
影响因子：
4.8
通讯作者：
Moll, UM
中科院分区：
文献类型：
--
作者：
Tomita, Y;Marchenko, N;Moll, UM
p53在细胞应激中诱导细胞凋亡是其最保守的功能，对p53肿瘤抑制至关重要。我们最近报道，p53直接诱导BH 1，2，3效应蛋白巴克的寡聚化，导致外线粒体膜透化（OMMP）与凋亡激活蛋白的释放。p53实现OMMP的一个重要机制是通过与抗凋亡BclXL蛋白形成抑制复合物。与此相反，p53与Bcl 2同源物的复合物尚未被研究。在这里，我们进行了详细的表征p53-Bcl 2的相互作用，使用结构，生物物理和突变分析。我们已经确定了p53 DNA结合结构域作为Bcl 2的结合界面，使用溶液NMR。通过表面等离子体共振分析（BIAcore）测定p53-Bcl 2复合物的亲和力，以具有主导组分K-D 535 +/-24 nM。此外，与野生型p53相反，p53的内源性错义突变体不能与人癌细胞中的内源性Bcl 2形成复合物。在功能上，这些突变体在介导OMMP中全部完全或强烈受损，如通过从分离的线粒体释放细胞色素c所测量的。这些数据暗示p53-Bcl 2复合物有助于直接线粒体p53凋亡途径，并进一步支持p53的DNA结合结构域是双重功能结构域的概念，介导其反式激活功能和直接线粒体凋亡功能。
The induction of apoptosis by p53 in response to cellular stress is its most conserved function and crucial for p53 tumor suppression. We recently reported that p53 directly induces oligomerization of the BH1,2,3 effector protein Bak, leading to outer mitochondrial membrane permeabilization (OMMP) with release of apoptotic activator proteins. One important mechanism by which p53 achieves OMMP is by forming an inhibitory complex with the antiapoptotic BclXL protein. In contrast, the p53 complex with the Bcl2 homolog has not been interrogated. Here we have undertaken a detailed characterization of the p53-Bcl2 interaction using structural, biophysical, and mutational analyses. We have identified the p53 DNA binding domain as the binding interface for Bcl2 using solution NMR. The affinity of the p53-Bcl2 complex was determined by surface plasmon resonance analysis (BIAcore) to have a dominant component K-D 535 +/- 24 nM. Moreover, in contrast to wild type p53, endogenous missense mutants of p53 are unable to form complexes with endogenous Bcl2 in human cancer cells. Functionally, these mutants are all completely or strongly compromised in mediating OMMP, as measured by cytochrome c release from isolated mitochondria. These data implicate p53-Bcl2 complexes in contributing to the direct mitochondrial p53 pathway of apoptosis and further support the notion that the DNA binding domain of p53 is a dual function domain, mediating both its transactivation function and its direct mitochondrial apoptotic function.