Fischer Andreas, Baljuls Angela, Reinders Joerg, Nekhoroshkova Elena, Sibilski Claudia, Metz Renate, Albert Stefan, Rajalingam Krishnaraj, Hekman Mirko, Rapp Ulf R
Bayerisches Krebsforschungszentrum, University of Wuerzburg, 97078 Wuerzburg, Germany.
Institute of Functional Genomics, University of Regensburg, 93053 Regensburg, Germany.
J Biol Chem. 2009 Jan 30;284(5):3183-3194. doi: 10.1074/jbc.M804795200. Epub 2008 Dec 2.
Mammalian 14-3-3 proteins play a crucial role in the activation process of RAF kinases. However, little is known about the selectivity of the mammalian 14-3-3 isoforms with respect to RAF association and activation. Using mass spectrometry, we analyzed the composition of the 14-3-3 isoforms attached to RAF kinases and found that B-RAF associates in vivo with 14-3-3 at much higher diversity than A- and C-RAF. We also examined in vitro binding of purified mammalian 14-3-3 proteins to RAF kinases using surface plasmon resonance techniques. While B- and C-RAF exhibited binding to all seven 14-3-3 isoforms, A-RAF bound with considerably lower affinities to epsilon, tau, and sigma 14-3-3. These findings indicate that 14-3-3 proteins associate with RAF isoforms in a pronounced isoform-specific manner. Because 14-3-3 proteins appear in dimeric forms, we addressed the question of whether both homo- and heterodimeric forms of 14-3-3 proteins participate in RAF signaling. For that purpose, the budding yeast Saccharomyces cerevisiae, possessing only two 14-3-3 isoforms (BMH1 and BMH2), served as testing system. By deletion of the single BMH2 gene, we found that both homo- and heterodimeric forms of 14-3-3 can participate in RAF activation. Furthermore, we show that A-, B-, and C-RAF activity is differentially regulated by its C-terminal and internal 14-3-3 binding domain. Finally, prohibitin, a scaffold protein that affects C-RAF activation in a stimulatory manner, proved to interfere with the internal 14-3-3 binding site in C-RAF. Together, our results shed more light on the complex mechanism of RAF activation, particularly with respect to activation steps that are mediated by 14-3-3 proteins and prohibitin.
哺乳动物的14-3-3蛋白在RAF激酶的激活过程中发挥着关键作用。然而，关于哺乳动物14-3-3亚型在与RAF结合及激活方面的选择性，我们所知甚少。利用质谱分析，我们分析了与RAF激酶相连的14-3-3亚型的组成，发现B-RAF在体内与14-3-3的结合多样性远高于A-RAF和C-RAF。我们还使用表面等离子体共振技术检测了纯化的哺乳动物14-3-3蛋白与RAF激酶的体外结合情况。虽然B-RAF和C-RAF与所有七种14-3-3亚型均表现出结合，但A-RAF与ε、τ和σ 14-3-3的结合亲和力明显较低。这些发现表明，14-3-3蛋白以显著的亚型特异性方式与RAF亚型结合。由于14-3-3蛋白以二聚体形式存在，我们探讨了14-3-3蛋白的同型二聚体和异型二聚体形式是否都参与RAF信号传导的问题。为此，仅拥有两种14-3-3亚型（BMH1和BMH2）的芽殖酵母酿酒酵母用作测试系统。通过缺失单个BMH2基因，我们发现14-3-3的同型二聚体和异型二聚体形式均可参与RAF激活。此外，我们表明A-、B-和C-RAF的活性受到其C末端和内部14-3-3结合域的差异调节。最后，prohibitin是一种以刺激方式影响C-RAF激活的支架蛋白，事实证明它会干扰C-RAF中的内部14-3-3结合位点。总之，我们的研究结果进一步揭示了RAF激活的复杂机制，特别是在由14-3-3蛋白和prohibitin介导的激活步骤方面。
