Structural determinants controlling 14-3-3 recruitment to the endocytic adaptor Numb and dissociation of the Numb•α-adaptin complex
Structural determinants controlling 14-3-3 recruitment to the endocytic adaptor Numb and dissociation of the Numb•α-adaptin complex
复制标题
控制 14-3-3 募集到内吞适配器 Numb 和 Numb 中心点 α-适应素复合物解离的结构决定因素
DOI：
10.1074/jbc.ra117.000897
复制
发表时间：
2018-03-16
影响因子：
4.8
通讯作者：
Wen, Wenyu
中科院分区：
文献类型：
--
作者：
Chen, Xing;Liu, Ziheng;Wen, Wenyu
跨膜的货物运输有助于建立、维持和重组不同的细胞区室，是许多代谢过程的基础。货物选择性内吞接头Numb通过将货物连接到网格蛋白接头α -适应蛋白参与依赖网格蛋白的内吞作用。Numb在Ser（265）和Ser（284）上的磷酸化募集了14-3-3调节蛋白，伴随着Numb从α -适应蛋白的解离和Numb从皮质膜转移到细胞质。然而，Numb- α -适应蛋白相互作用的分子机制及其通过Numb磷酸化和14-3-3招募的调控机制仍然知之甚少。通过对Numb.14-3-3复合物的生化和结构分析发现，Numb与14-3-3的有效相互作用需要在Ser（265）和Ser（284）上磷酸化。我们还发现，Numb中Ser（265）周围的RQFRF基序与典型的c端DPF基序一起作用，这是Numb与α -adaptin相互作用所必需的，从而与α -adaptin形成稳定的复合物。值得注意的是，我们提供的证据表明，磷酸化诱导的14-3-3与Numb的结合直接与α -适应蛋白与Numb的结合竞争。我们的发现提示了一种控制Numb与α -适应素或14-3-3动态组装的潜在机制。α -适应蛋白对Numb的这种双位点识别可能对其他α -适应蛋白靶标有影响。我们提出，在Numb中新发现的Ser（265）周围的α -适应蛋白结合位点是Numb动态解离的触发机制。alpha-adaptin复杂。
Traffic of cargo across membranes helps establish, maintain, and reorganize distinct cellular compartments and is fundamental to many metabolic processes. The cargo-selective endocytic adaptor Numb participates in clathrin-dependent endocytosis by attaching cargoes to the clathrin adaptor alpha-adaptin. The phosphorylation of Numb at Ser(265) and Ser(284) recruits the regulatory protein 14-3-3, accompanied by the dissociation of Numb from alpha-adaptin and Numb's translocation from the cortical membrane to the cytosol. However, the molecular mechanisms underlying the Numb-alpha-adaptin interaction and its regulation by Numb phosphorylation and 14-3-3 recruitment remain poorly understood. Here, biochemical and structural analyses of the Numb.14-3-3 complex revealed that Numb phosphorylation at both Ser(265) and Ser(284) is required for Numb's efficient interaction with 14-3-3. We also discovered that an RQFRF motif surrounding Ser(265) in Numb functions together with the canonical C-terminal DPF motif, required for Numb's interaction with alpha-adaptin, to form a stable complex with alpha-adaptin. Of note, we provide evidence that the phosphorylation-induced binding of 14-3-3 to Numb directly competes with the binding of alpha-adaptin to Numb. Our findings suggest a potential mechanism governing the dynamic assembly of Numb with alpha-adaptin or 14-3-3. This dual-site recognition of Numb by alpha-adaptin may have implications for other alpha-adaptin targets. We propose that the newly identified alpha-adaptin-binding site surrounding Ser(265) in Numb functions as a triggering mechanism for the dynamic dissociation of the Numb.alpha-adaptin complex.