REGULATION BY MOLECULAR INTERFERENCE: HOW 14-3-3 PROTEIN CONTROLS ACTIVITY OF FORKHEAD TRANSCRIPTION FACTOR FOXO4
1Department Physical and Macromolecular Chemistry, Faculty of Science, Charles University, 128 43 Prague;
2Institute of Physiology, Academy of Sciences of the Czech Republic, 142 20 Prague.
The 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. FOXO transcription factors are promising candidates to serve as molecular links between longevity and tumor suppression. These factors are major substrates of the protein kinase B (Akt). In the presence of insulin and growth factors, FOXO proteins are relocalized from the nucleus to the cytoplasm and degraded via the ubiquitin-proteasome pathway. In the absence of growth factors, FOXO proteins translocate to the nucleus and upregulate a series of target genes, thereby promoting cell cycle arrest, stress resistance, or apoptosis [1,2].
Under conditions of low protein kinase B (PKB) activity (in the absence of growth factors), FOXO proteins are predominantly nuclear and the rate of import exceeds the rate of export. This shift in equilibrium probably occurs because the binding of FOXO to DNA anchors FOXO within the nucleus. Following the activation of PKB (PKB-regulated shuttling) by insulin–phosphatidylinositol-3-kinase (PI3K) signalling, PKB translocates to the nucleus. A 14-3-3 protein devoid of ligand enters the nucleus passively. PKB phosphorylates FOXO, probably on multiple sites, and PKB-mediated phosphorylation induces the binding of 14-3-3 protein to the FOXO proteins in the nucleus, which results in the release of the FOXO protein from the DNA. Following translocation to the cytosol, the bound 14-3-3 protein prevents re-entry into the nucleus by masking the NLS and inhibiting importin binding. This results in an equilibrium shift towards the cytosol.
Main goal of our research was to elucidate the molecular mechanism of 14-3-3 protein-dependent regulation of FOXO4 function. We have shown that phosphorylation of FOXO4 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 14-3-3zeta form a complex with 1:2 molar stoichiometry and a K(D) of less than 30 nM. 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 FOXO4 binding to its target DNA . We have also investigated whether the phosphorylation by protein kinase B, the 14-3-3 protein, and DNA binding affect the structure of FOXO4 nuclear localization sequence (NLS). We have used site-directed labeling of FOXO4 NLS with the extrinsic fluorophore 1,5-IAEDANS in conjunction with steady-state and time-resolved fluorescence spectroscopy to study conformational changes of FOXO4 NLS in vitro. Our data show that the 14-3-3 protein binding significantly changes the environment around AEDANS-labeled NLS and reduces its flexibility. On the other hand, the phosphorylation itself and the binding of double-stranded DNA have a small effect on the structure of this region .
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This work has been funded by Grant No. 204/06/0565 of the Grant Agency of the Czech Republic, by Centre of Neurosciences LC554 of the Ministry of Education, Youth, and Sports of the Czech Republic, and by Research Project AVOZ50110509.