Studies of Cell Signaling mechanism of the 14-3-3 Protein Family

14-3-3蛋白家族在多种细胞信号转导过程中发挥作用，包括单胺合成、胞吐和细胞周期调节，但该蛋白家族活性的结构要求在很大程度上是未知的。本研究利用在大肠杆菌中表达的14-3-3ETA亚型的一系列截断突变体，证明了COOH末端区域，特别是在171-213氨基酸中限制的区域，与Ras-MAP激酶信号通路中涉及的原癌基因产物Raf-1和BCR特异结合。该限制性区域被称为14-3-3 box-1，是物种间高度保守的结构区之一，也被认为是与磷酸化色氨酸和酪氨酸羟基酶相互作用的部位，这两种酶是单胺生物合成途径中的限速酶。为了研究Box-1是否可能与其他靶蛋白结合，将融合到谷胱甘肽-S转移酶上的eta异构体或其缺失的突变体与大鼠脑干提取物混合，在磷酸化条件下与[γ-1；32&gt；P]三磷酸腺苷和钙调蛋白共同孵育，用谷胱甘肽-琼脂糖沉淀法回收蛋白复合体，用十二烷基硫酸钠-凝胶电泳法和放射自显影技术分析。本实验检测到许多磷酸化蛋白与ETA蛋白共沉淀，但与box-1缺失突变体共沉淀。这些结果表明，Box-1可能是许多靶蛋白和酶的共同结合部位，包括Raf-1、BCR、色氨酸和酪氨酸羟化酶。

The 14-3-3 protein family plays a role in a wide variety of cell signaling processes including monoamine synthesis, exocytosis, and cell cycle regulation, but the structural requirements for the activity of this protein family are largely unknown. This research demonstrates, using a series of truncation mutants of the 14-3-3 eta isoform expressed in Escherichia coli, that the COOH-terminal region, especially restricted in amino acids 171-213, binds specifically with Raf-1 and Bcr, the proto-oncogene products involved in the Ras-MAP kinase signaling pathway. This restricted region, termed 14-3-3 box-1, is one of the structural regions whose sequence is highly conserved among species, and is also identified as the site of interaction with phosphorylated tryptophan and tyrosine hydroxylases, the rate-limiting enzymes in the pathway of monoamine biosynthesis. To examine whether box-1 might bind with other target proteins, the eta isoform fused to glutathion-S-transferase or its mutant which lacks box-1 was mixed with rat brainstem extracts, incubated under phosphorylation conditions in the presence of [gamma-^<32>P] ATP and calmodulin, and the protein complex recovered by precipitation with glutathione-Sepharose was analyzed by SDS-gel electrophoresis and autoradiography. This experiment detected many phosphoproteins co-precipitated with the eta protein, but with the box-1-deletion mutant. These results suggest that box-1 may serve as a common binding site for many target proteins and enzymes including Raf-1, Bcr, and tryptophan and tyrosine hydroxylases.