The chaperone protein 14-3-3 interacts with 3BP2/SH3BP2 and regulates its adapter function
The chaperone protein 14-3-3 interacts with 3BP2/SH3BP2 and regulates its adapter function
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DOI：
10.1074/jbc.m209509200
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发表时间：
2003-02-28
影响因子：
4.8
通讯作者：
Deckert, M
中科院分区：
文献类型：
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作者：
Foucault, I;Liu, YC;Deckert, M
免疫受体对淋巴细胞的刺激是通过激活多条信号通路导致细胞因子基因转录而实现的。接头蛋白是关键的信号成分，可以通过组装多分子信号复合体来整合多条通路。我们之前已经证明，细胞质适配器3BP2(也称为SH3BP2)通过激活依赖于RAS和钙调神经磷酸酶的途径促进T细胞中NFAT/AP-1的转录活性。然而，3BP2/SH3BP2调控细胞信号和激活的分子机制仍鲜有报道。在这项研究中，结合酵母双杂交分析和生化方法，我们提出了3BP2和伴侣蛋白14-3-3之间存在物理相互作用的证据。这种相互作用在酵母和哺乳动物细胞中被直接和有成分地检测到。佛波醇酯、过钒酸盐和Forskolin/异丁基甲基黄嘌呤刺激增强了这种相互作用，并促进了丝氨酸/苏氨酸激酶组成活性突变体的共表达，包括蛋白激酶C。我们发现，碱性磷酸酶对3BP2的去磷酸化破坏了它与14-3-3的相互作用，3BP2在体外是纯化的蛋白激酶C的底物，这表明14-3-3结合需要上游酶对3BP2的磷酸化。利用3BP2的缺失突变体，将两个14-3-3结合区定位到3BP2的两个富含脯氨酸的结构域(残基201240和270-310)。这些结构域包含两个14-3-3共识结合基序。我们鉴定了3BP2的Ser(225)和Ser(277)残基是与14-3-3家族蛋白相互作用所必需的，最适的3BP2丝氨酸磷酸化，然后是3BP2依赖的功能。事实上，不能与14-3-3结合的3BP2突变蛋白显示出刺激NFAT转录活性的能力增强，这表明14-3-3与3BP2结合负调控淋巴细胞中的3BP2接头功能。
Lymphocyte stimulation by immunoreceptors is achieved through the activation of multiple signaling pathways leading to cytokine gene transcription. Adapter proteins are critical signaling components that can integrate multiple pathways by allowing the assembly of multimolecular signaling complexes. We previously showed that the cytoplasmic adapter 3BP2 (also known as SH3BP2) promotes NFAT/AP-1 transcriptional activities in T cells through the activation of Ras- and calcineurin-dependent pathways. However, the molecular mechanisms by which 3BP2/SH3BP2 regulates cell signaling and activation remain poorly documented. In this study, using a combination of yeast two-hybrid analysis and biochemical approaches, we present evidence for a physical interaction between 3BP2 and the chaperone protein 14-3-3. This interaction was direct and constitutively detected in yeast and in mammalian cells. Phorbol ester, pervanadate, and forskolin/isobutylmethylxanthine stimulations enhanced this interaction, as well as co-expression of constitutive active mutants of serine/threonine kinases, including protein kinase C. We found that dephosphorylation of 3BP2 by alkaline phosphatase disrupted its interaction with 14-3-3 and that 3BP2 was a substrate of purified protein kinase C in vitro, suggesting that the phosphorylation of 3BP2 by upstream kinases was required for 14-3-3 binding. Using deletion mutants of 3BP2, two 14-3-3 binding domains were mapped to two proline-rich (residues 201240 and 270-310) domains of 3BP2. These domains were shown to contain two 14-3-3 consensus binding motifs. We identified residues Ser(225) and Ser(277) of 3BP2 as being essential for interaction with 14-3-3 family proteins, optimal 3BP2 serine phosphorylation, and then for 3BP2-dependent function. Indeed, a 3BP2 mutant protein incapable of binding 14-3-3 showed increased capacity to stimulate NFAT transcriptional activities, suggesting that 14-3-3 binding to 3BP2 negatively regulates 3BP2 adapter function in lymphocytes.