Two 14-3-3 binding motifs are required for stable association of forkhead transcription factor FOXO4 with 14-3-3 proteins and inhibition of DNA binding
Two 14-3-3 binding motifs are required for stable association of forkhead transcription factor FOXO4 with 14-3-3 proteins and inhibition of DNA binding
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DOI：
10.1021/bi0352724
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发表时间：
2003-12-30
期刊：
影响因子：
2.9
通讯作者：
Dyda, F
中科院分区：
文献类型：
--
作者：
Obsil, T;Ghirlando, R;Dyda, F
d14-3-3 蛋白是二聚体调节蛋白家族，参与许多重要的生物学过程。 14-3-3 蛋白质的共同特征是它们能够以磷酸化依赖性方式与其他蛋白质结合。通过这些结合相互作用，14-3-3 蛋白充当分子支架，调节其伴侣的生物功能。 14-3-3 蛋白识别含有磷酸化丝氨酸或苏氨酸残基的短基序。在这项研究中，我们定量表征了叉头转录因子 FOXO4 14-3-3 与其靶 DNA（胰岛素反应元件）之间的体外相互作用。蛋白激酶 B 在 Thr-28 和 Ser-193 处磷酸化 FOXO4（残基 11-213），产生两个 14-3-3 结合基序。分析凝胶过滤和沉降平衡实验表明，双磷酸化的 FOXO4 和 14-3-3 形成摩尔化学计量比为 1:2 且 K-D 小于 30 nM 的复合物。相比之下，单磷酸化的 FOXO4 突变体以显着较低的亲和力结合 14-3-3zeta，同时保留结合 DNA 的能力。 14-3-3 在 FOXO4/DNA 复合物分解中的积极作用通过以下事实得到证明：在 14-3-3 存在的情况下，需要两个磷酸化的 14-3-3 结合基序才能完全抑制 FOX04 与其靶 DNA 的结合。
dThe 14-3-3 proteins, a family of dimeric regulatory proteins, are involved in many biologically important processes. The common feature of 14-3-3 proteins is their ability to bind to other proteins in a phosphorylation-dependent manner. Through these binding interactions, 14-3-3 proteins work as molecular scaffolds, modulating the biological functions of their partners. 14-3-3 proteins recognize short motifs containing a phosphorylated serine or threonine residue. In this study, we have quantitatively characterized the in vitro interactions among 14-3-3, the Forkhead transcription factor FOXO4, and its target DNA, the insulin response element. Phosphorylation of FOXO4 (residues 11-213) by protein kinase B at Thr-28 and Ser-193 creates two 14-3-3 binding motifs. Analytical gel filtration and sedimentation equilibrium experiments indicate that doubly phosphorylated FOXO4 and 14-3-3 form a complex with 1:2 molar stoichiometry and a K-D of less than 30 nM. In contrast, singly phosphorylated FOXO4 mutants bind 14-3-3zeta with significantly lower affinity while retaining the ability to bind DNA. An active role for 14-3-3 in the disassembly of the FOXO4/DNA complex is demonstrated by the fact that, in the presence of 14-3-3, two phosphorylated 14-3-3 binding motifs are needed for the complete inhibition of FOX04 binding to its target DNA.