Wang H, Zhang L, Liddington R, Fu H
Department of Pharmacology, Emory University School of Medicine, Atlanta, Georgia 30322, USA.
J Biol Chem. 1998 Jun 26;273(26):16297-304. doi: 10.1074/jbc.273.26.16297.
14-3-3 proteins bind to a diverse group of regulatory molecules such as Raf-1, Cbl, and c-Bcr that are involved in signal transduction pathways. The crystal structure of 14-3-3zeta reveals a conserved amphipathic groove that may mediate the association of 14-3-3 with diverse ligands. Consistently, mutations on the charged surface of the groove (Lys-49, Arg-56, and Arg-60) decrease the binding of 14-3-3zeta to the ligands tested (Zhang, L., Wang, H., Liu, D., Liddington, R., and Fu, H. (1997) J. Biol. Chem. 272, 13717-13724). Here we report that mutations that altered the hydrophobic property of the groove, V176D, L216D, L220D, and L227D, disrupted the interaction of 14-3-3zeta with Raf-1 kinase. The reduced binding of the 14-3-3zeta mutants to Raf-1 was apparently not because of gross structural changes in the mutants as judged by their ability to form dimers, by partial proteolysis profiles, and by circular dichroism analysis. These hydrophobic residues appeared to be required for the binding of 14-3-3zeta to distinct activation states of Raf-1 because mutations V176D, L216D, L220D, and L227D reduced the interaction of 14-3-3zeta with Raf-1 from both phorbol 12-myristate 13-acetate-stimulated and unstimulated Jurkat T cells. These same mutations also disrupted the association of 14-3-3zeta with other regulatory molecules such as Cbl and c-Bcr, suggesting that the hydrophobic surface of the amphipathic groove represents part of a binding site shared by a number of 14-3-3-associated proteins. The conservation of the hydrophobic residues Val-176, Leu-216, Leu-220, and Leu-227 among known 14-3-3 family members implies their general importance in ligand binding.
14-3-3蛋白可与多种参与信号转导途径的调节分子结合，如Raf-1、Cbl和c-Bcr。14-3-3ζ的晶体结构显示出一个保守的两性离子凹槽，该凹槽可能介导14-3-3与多种配体的结合。与此一致的是，凹槽带电表面（Lys-49、Arg-56和Arg-60）上的突变会降低14-3-3ζ与所测试配体的结合（Zhang, L., Wang, H., Liu, D., Liddington, R., and Fu, H. (1997) J. Biol. Chem. 272, 13717 - 13724）。在此我们报告，改变凹槽疏水性质的突变V176D、L216D、L220D和L227D破坏了14-3-3ζ与Raf-1激酶的相互作用。从14-3-3ζ突变体形成二聚体的能力、部分蛋白酶解图谱以及圆二色性分析判断，14-3-3ζ突变体与Raf-1结合的减少显然不是由于突变体的整体结构变化。这些疏水残基似乎是14-3-3ζ与Raf-1不同激活状态结合所必需的，因为突变V176D、L216D、L220D和L227D降低了14-3-3ζ与佛波醇12-肉豆蔻酸酯13-乙酸酯刺激和未刺激的Jurkat T细胞中Raf-1的相互作用。这些相同的突变也破坏了14-3-3ζ与其他调节分子如Cbl和c-Bcr的结合，这表明两性离子凹槽的疏水表面代表了许多与14-3-3相关蛋白共享的结合位点部分。已知14-3-3家族成员中疏水残基Val-176、Leu-216、Leu-220和Leu-227的保守性意味着它们在配体结合中具有普遍重要性。
