Michaud N R, Fabian J R, Mathes K D, Morrison D K
Molecular Mechanisms of Carcinogenesis Laboratory, National Cancer Institute-Frederick Cancer Research and Development Center, Maryland 21702-1201, USA.
Mol Cell Biol. 1995 Jun;15(6):3390-7. doi: 10.1128/MCB.15.6.3390.
Recent reports have demonstrated the in vivo association of Raf-1 with members of the 14-3-3 protein family. To address the significance of the Raf-1-14-3-3 interaction, we investigated the enzymatic activity and biological function of Raf-1 in the presence and absence of associated 14-3-3. The interaction between these two molecules was disrupted in vivo and in vitro with a combination of molecular and biochemical techniques. Biochemical studies demonstrated that the enzymatic activities of Raf-1 were equivalent in the presence and absence of 14-3-3. Furthermore, mixing of purified Raf-1 and 14-3-3 in vitro was not sufficient to activate Raf-1. With a molecular approach, Cys-165 and Cys-168 as well as Ser-259 were identified as residues of Raf-1 required for the interaction with 14-3-3. Cys-165 and Cys-168 are located within the conserved cysteine-rich region of the CR1 domain, and Ser-259 is a conserved site of serine phosphorylation found within the CR2 domain. Mutation of either Cys-165 and Cys-168 or Ser-259 prevented the stable interaction of Raf-1 with 14-3-3 in vivo. Consistent with the model in which a site of serine phosphorylation is involved in the Raf-1-14-3-3 interaction, dephosphorylated Raf-1 was unable to associate with 14-3-3 in vitro. Phosphorylation may represent a general mechanism mediating 14-3-3 binding, because dephosphorylation of the Bcr kinase (known to interact with 14-3-3) also eliminated its association with 14-3-3. Finally, mutant Raf-1 proteins unable to stably interact with 14-3-3 exhibited enhanced enzymatic activity in human 293 cells and Xenopus oocytes and were biologically activated, as demonstrated by their ability to induced meiotic maturation of Xenopus oocytes. However, in contrast to wild-type Raf-1, activation of these mutants was independent of Ras. Our results therefore indicate that interaction with 14-3-3 is not essential for Raf-1 function.
最近的报告显示，Raf-1在体内与14-3-3蛋白家族成员有关联。为了探究Raf-1与14-3-3相互作用的意义，我们研究了在有或没有相关14-3-3存在的情况下Raf-1的酶活性和生物学功能。利用分子和生化技术相结合的方法，在体内和体外破坏了这两种分子之间的相互作用。生化研究表明，无论有无14-3-3，Raf-1的酶活性都是相同的。此外，在体外将纯化的Raf-1和14-3-3混合不足以激活Raf-1。通过分子方法，确定Cys-165、Cys-168以及Ser-259是Raf-1与14-3-3相互作用所需的残基。Cys-165和Cys-168位于CR1结构域保守的富含半胱氨酸区域内，Ser-259是在CR2结构域中发现的丝氨酸磷酸化保守位点。Cys-165和Cys-168或Ser-259的突变阻止了Raf-1在体内与14-3-3的稳定相互作用。与丝氨酸磷酸化位点参与Raf-1与...
