PROTEINS OF THE 14-3-3-FAMILY ASSOCIATE WITH RAF AND CONTRIBUTE TO ITS ACTIVATION
PROTEINS OF THE 14-3-3-FAMILY ASSOCIATE WITH RAF AND CONTRIBUTE TO ITS ACTIVATION
复制标题
DOI：
10.1101/sqb.1994.059.01.023
复制
发表时间：
1994-01-01
期刊：
影响因子：
--
通讯作者：
RUGGIERI, R
中科院分区：
文献类型：
--
作者：
FREED, E;MCCORMICK, F;RUGGIERI, R
使用与Gal 4激活结构域融合的cDNA文库，将与Gal 4 DNA结合结构域融合的全长人c-Raf-1（Raf）用作筛选Raf结合蛋白的靶标（Fields和Song 1989; Chien等1991）。鉴定了10个与Raf特异性相互作用的克隆。它们不与APC片段反应（图1B），也不与单独的Gal 4 DNA结合结构域反应。三个阳性克隆编码14-3-3家族的成员，其中两个是14-3-3/3的不同分离株（Leffers等，1993），一个是14-3-3（Zupan等，1992; Fu等，1993）。在另外的对照实验中，14-3-3蛋白在双杂交系统中显示不与Ras（Ser-186）或p53相互作用（数据未显示）。使用由Raf蛋白的三个区域中的每一个组成的蛋白质杂交体来确定与这些蛋白质相互作用的Raf区域。结果总结见图1。Ras（Ser-186）融合体与全长Raf和包括CR 1的氨基末端区域相互作用，但不与包含CR2的中间区域相互作用，也不与羧基末端激酶结构域（CR 3）相互作用（图1）。两个14-3-3蛋白也与Raf的氨基末端结构域相互作用，并且14-3-3/3显示与Raf的中间部分和与羧基末端激酶结构域类似的相互作用。虽然在β-半乳糖苷酶试验中几乎检测不到14-3-3-”与Raf中间部分和羧基末端激酶结构域的相互作用，但使用缺乏组氨酸（-His）的培养基上更灵敏的生长测量，它们是明显的（图1B）。一般来说，14-3-3/3与Raf的相互作用比14-3-3~强。还应注意，双杂交系统中相互作用的两个不同读数导致14-3-3与Raf蛋白的不同部分之间相互作用的相对强度的不同印象。这种差异可能是由于编码含有CR 1的Raf的氨基末端部分的构建体的表达似乎对细胞有毒，并且可能选择性地影响在缺乏组氨酸的培养基上的细胞生长的读数。总之，这两种14-3-3蛋白质都显示出与我们研究的所有三种Raf片段的一些相互作用。考虑到这些Raf片段之间存在一些重叠，我们得出结论，Raf的至少两个不同区域似乎参与了与14-3-3蛋白的相互作用：氨基末端调控区的一部分，以及含有激酶结构域的羧基末端区域。在体外试验中，14-3-3/3不与Ras p21竞争结合Raf的Ras结合结构域（Vojtek等，1993）（图2）。因此，与14-3-3蛋白相互作用的氨基末端Raf片段的区域不同于Ras结合结构域。这些结果表明，14-3-3不与Ras竞争结合Raf和Ras，Raf，和14-3-3蛋白可能是一个多聚体复合物在体内的一部分。
Full-length human c-Raf-1 (Raf) fused to the Gal4 DNA-binding domain was used as the target of a screen for Raf-binding proteins, using a library of cDNAs fused to the Gal4 activation domain (Fields and Song 1989; Chien et al. 1991). Ten clones were identified that interacted specifically with Raf. They did not react with a fragment of APC (Fig. 1B), nor with the Gal4 DNA-binding domain alone. Three of the positive clones encoded members of the 14-3-3 family, of which two were distinct isolates of 14-3-3/3 (Leffers et al. 1993) and one was 14-3-3 ((Zupan et al. 1992; Fu et al. 1993). In additional control experiments, the 14-3-3 proteins showed no interaction with Ras (Ser-186) nor with p53 in the two-hybrid system (data not shown). The region of Raf that interacts with these proteins was determined using protein hybrids consisting of each of three regions of the Raf protein. The results are summarized in Figure 1. A Ras (Ser-186) fusion interacted with full-length Raf and with the amino-terminal region including CR1, but not with the middle region containing CR2, nor with the carboxy-terminal kinase domain (CR3)(Fig. 1). Both of the 14-3-3 proteins also interacted with the amino-terminal domain of Raf, and 14-3-3/3 displayed a similar interaction with the middle portion of Raf and with the carboxy-terminal kinase domain. Although interactions of 14-3-3~" with both the middle portion of Raf and the carboxy-terminal kinase domain were barely detectable in the/3-galactosidase assay, they were evident using the more sensitive measure of growth on medium lacking histidine (-His)(Fig. 1B). In general, 14-3-3/3 interacted more strongly with Raf than did 14-3-3~. Note also that the two distinct readouts of interaction in the two-hybrid system resulted in different impressions of the relative strength of interaction between 14-3-3 and different portions of the Raf protein. Some of this difference may be due to the fact that expression of the construct encoding the amino-terminal portion of Raf containing CR1 appeared to be toxic to the cells and might selectively affect the readout of cell growth on medium lacking histidine. In summary, both of the 14-3-3 proteins displayed some interaction with all three of the Raf fragments we studied. Given that there is some overlap between these Raf fragments, we conclude that at least two distinct regions of Raf appear to be involved in the interaction with 14-3-3 proteins: some portion of the amino-terminal regulatory region, and the carboxy-terminal region containing the kinase domain. In an in vitro assay, 14-3-3/3 did not compete with Ras p21 for binding to the Ras binding domain (Vojtek et al. 1993) of Raf (Fig. 2). Therefore, the region of the amino-terminal Raf fragment that interacts with 14-3-3 proteins is distinct from the Ras-binding domain. These results suggest that 14-3-3 does not compete with Ras for binding to Raf and that Ras, Raf, and 14-3-3 proteins may be part of a multimeric complex in vivo.