Conformational changes of FoxO4-DNA binding domain upon binding to 14-3-3 protein

 

J. Silhan1,2, E. Boura1,2, T. Obsil1,2

 

1 Department of Physical and Macromolecular Chemistry, Faculty of Science, Charles University; 12843 Prague, Czech Republic

2 Institute of Physiology, Academy of Sciences of the Czech Republic; 14220 Prague, Czech Republic

 

14-3-3 proteins are abundant regulatory molecules expressed among all eukaryotes. Their function is represented by protien-protein interactions and thus regulation of the function of the binding partners. Seven isoforms of 14-3-3 proteins have been found in human and over 200 target proteins is currently known to interact with some of these isoforms (e.g. Cdk2, p21CIP1, p27Kip1, p53, MDM2, BAD, A20, TERT, ExoS, Raf-1) [1,2]. 14-3-3 proteins preferentially bind the conserved motif RXY/FXpSXP in phosphorylation dependent manner [5]. Phosphorylation-independent binding has been observed to be important in several cellular processes [3]. Versatile 14-3-3 proteins action is involved in cell signaling events, enzymatic or binding activity of bound ligands and localization within the cell.Despite a large number of the X-ray structures of 14-3-3 proteins and their shorted ligands the exact mechanisms of 14-3-3 action remains unclear. It seems to have distinct ways of action such as hindrance of binding site for other component of the cell followed by e.g. sequestration, inhibition of phosphorylation or degradation [4]. On the other hand 14-3-3 binding of the ligand causes significant conformation changes that leads to altered function of the ligand. 14-3-3 proteins are preferentially homodimeric and thus contain two binding. It is thought that rigid character of 14-3-3 proteinís structure may facilitate conformation change and was proposed that 14-3-3 proteins act like molecular anvil when bind ligand by both binding sites. The molecular anvil theory says that by the binding of two sites of ligand by one molecule of 14-3-3 may induce greater changes of the ligand structure and modify its function (e.g. enzymatic or binding affinity) [4].

Protein FoxO4 (Afx) is member of the family of forkhead transcription factors regulating life span and apoptosis. DNA binding domain (DBD) is responsible for interaction between FoxO4 and DNA. PKB-induced phosphorylation of FoxO4 at threonine and serine residues (28T, 198S) generates two 14-3-3 protein binding sites. 14-3-3/FoxO4 protein complex is subsequently exported from the nucleus via 14-3-3 hindrance of the nuclear localization sequence (NLS) [6]. It is also known than phosphorylation and further 14-3-3 binding to FoxO4 disrupt FoxO4/DNA complex [7].

To illustrate the molecular anvil theory of 14-3-3 proteins we have investigated conformational changes of FoxO4-DBD caused by 14-3-3 protein binding to one or both binding sites. We have constructed mutants of 14-3-3 protein without any tryptofan residue and mutants of FoxO4 containing two tryptofan residues within the DBD. Fluorescence anisotropy decays of single and double phosphorylated FoxO4 mutants complexed with 14-3-3 protein were measured to reveal differences of mobility DBD.

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Acknowledgements

This work was funded by Grant 204/06/0565 of the Grant Agency of the Czech Republic, by Grant KJB500110601 of the Grant Agency of the Academy of Sciences of the Czech Republic, by Research Project MSM0021620835 and Centre of Neurosciences LC554 of the Ministry of Education, Youth, and Sports of the Czech Republic, and by Research Project AV0Z50110509 of the Academy of Sciences of the Czech Republic.