Interference of BAD (Bcl-xL/Bcl-2-associated death promoter)-induced apoptosis in mammalian cells by 14-3-3 isoforms and P11
Interference of BAD (Bcl-xL/Bcl-2-associated death promoter)-induced apoptosis in mammalian cells by 14-3-3 isoforms and P11
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DOI：
10.1210/me.11.12.1858
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发表时间：
1997-11-01
影响因子：
--
通讯作者：
Hsueh, AJW
中科院分区：
文献类型：
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作者：
Hsu, SY;Kaipia, A;Hsueh, AJW
不同类型细胞的凋亡和存活都受到激素的控制，但细胞内调节细胞死亡的机制尚不清楚。Bcl-2/Ced-9蛋白家族包含保守的Bcl-2同源区域，介导对增强或抑制细胞凋亡重要的原生或异源二聚体的形成。与Bcl-2家族的大多数其他成员不同，BAD （Bcl-xL/Bcl-5相关死亡启动子）是一种死亡增强子，它没有针对线粒体外膜和核膜的c端跨膜结构域。我们假设BAD除了结合Bcl-xL和Bcl-2外，还可能与Bcl-2家族外的蛋白相互作用。利用酵母双杂交系统在卵巢融合cDNA文库中寻找bad结合蛋白，我们发现了多个编码14-3-3不同亚型的cDNA克隆，14-3-3是一组在信号转导和细胞周期进程中必需的进化保守蛋白。BAD在一个（S137A）而另一个（S113A）中发生点突变，在多种14-3-3相互作用蛋白中发现的推定结合位点使BAD与14-3-3相互作用失效，但不影响BAD与Bcl-2的相互作用。由于S137A BAD突变体可能类似于BAD的欠磷酸化形式，我们使用该突变体在酵母双杂交系统中筛选其他BAD相互作用蛋白。P11是一种神经生长因子诱导的神经突延伸因子，是钙结合S-100蛋白家族的成员，它与突变体BAD有很强的相互作用，但与野生型蛋白的相互作用较弱。在中国仓鼠卵巢（CHO）细胞中，野生型BAD或其突变体的短暂表达增加了凋亡细胞的死亡，这可以通过与杆状病毒衍生的半胱氨酸蛋白酶抑制剂P35共转染来阻断。与14-3-3共转染可抑制野生型或S113A突变体BAD诱导的细胞凋亡，而不能结合14-3-3的S137A突变体则不能。此外，P11共转染降低了野生型BAD和S137A突变体的促凋亡作用。14-3-3和P11在体外也能与BAD直接结合。这些结果提示14-3-3和P11可能作为bad结合蛋白抑制其凋亡活性。由于14-3-3蛋白家族可与Raf-1激酶、蛋白激酶C、磷脂酰肌醇3激酶等关键信号蛋白相互作用，而P11是神经元存活因子、神经生长因子诱导的早期应答基因，因此本研究提示BAD在激素信号调节的不同信号转导通路之间的沟通和Bcl-2家族成员控制的细胞凋亡机制中发挥重要作用。
Apoptosis and survival of diverse cell types are under hormonal control, but intracellular mechanisms regulating cell death are unclear. The Bcl-2/Ced-9 family of proteins contains conserved Bcl-2 homology regions that mediate the formation of home-or heterodimers important for enhancing or suppressing apoptosis. Unlike most other members of the Bcl-2 family, BAD (Bcl-xL/Bcl-5 associated death promoter), a death enhancer, has no C-terminal transmembrane domain for targeting to the outer mitochondrial membrane and nuclear envelope. We hypothesized that BAD, in addition to binding Bcl-xL and Bcl-2, may interact with proteins outside the Bcl-2 family. Using the yeast two-hybrid system to search for BAD-binding proteins in an ovarian fusion cDNA library, we identified multiple cDNA clones encoding different isoforms of 14-3-3, a group of evolutionally conserved proteins essential for signal transduction and cell cycle progression. Point mutation of BAD in one (S137A), but not the other (S113A), putative binding site found in diverse 14-3-3 interacting proteins abolished the interaction between BAD and 14-3-3 without affecting interactions between BAD and Bcl-2. Because the S137A BAD mutant presumably resembles an underphosphorylated form of BAD, we used this mutant to screen for additional BAD-interacting proteins in the yeast two-hybrid system. P11, a nerve growth factor-induced neurite extension factor and member of the calcium-binding S-100 protein family, interacted strongly with the mutant BAD but less effectively with the wild type protein. In Chinese hamster ovary (CHO) cells, transient expression of wild type BAD or its mutants increased apoptotic cell death, which was blocked by cotransfection with the baculovirus-derived cysteine protease inhibitor, P35. Cotransfection with 14-3-3 suppressed apoptosis induced by wild type or the S113A mutant BAD but not by the S137A mutant incapable of binding 14-3-3. Furthermore, cotransfection with P11 attenuated the proapoptotic effect of both wild type BAD and the S137A mutant. For both 14-3-3 and P11, direct binding to BAD was also demonstrated in vitro. These results suggest that both 14-3-3 and P11 may function as BAD-binding proteins to dampen its apoptotic activity. Because the 14-3-3 family of proteins could interact with key signaling proteins including Raf-1 kinase, protein kinase C, and phosphatidyl inositol 3 kinase, whereas P11 is an early response gene induced by the neuronal survival factor, nerve growth factor, the present findings suggest that BAD plays an important role in mediating communication between different signal transduction pathways regulated by hormonal signals and the apoptotic mechanism controlled by Bcl-2 family members.