Structure of the 14-3-3/FOXO4 complex derived from the fluorescence spectroscopy data

T. Obsil^1,2

¹Faculty of Science, Charles University in Prague, Hlavova 8, 12843 Prague 2, Czech Republic

²Institute of Physiology, Academy of Sciences of the Czech Republic, Videnska 1083, 14220 Prague 4, Czech Republic

obsil@natur.cuni.cz

The FOXO class of forkhead transcription factors consists of four members (FOXO1, FOXO3, FOXO4, and FOXO6) that play a central role in cell cycle control, differentiation, metabolism control, stress response, and apoptosis [1]. Transcriptional activity of FOXO proteins is regulated through the insulin-phosphatidylinositol 3-kinase-AKT/protein kinase B (PKB) signaling pathway. The AKT/PKB-mediated phosphorylation triggers phosphorylation of additional sites by casein kinase-1 and dual specificity tyrosine-regulated kinase-1A and induces FOXO binding to the 14-3-3 protein. The role of 14-3-3 proteins in the regulation of FOXO forkhead transcription factors is at least 2-fold. First, the 14-3-3 binding inhibits the interaction between the FOXO and the target DNA. Second, the 14-3-3 proteins prevent nuclear reimport of FOXO factors by masking their nuclear localization signal [2]. The exact mechanisms of these processes are still unclear, mainly due to the lack of structural data. We used fluorescence spectroscopy to investigate the mechanism of the 14-3-3 protein-dependent inhibition of FOXO4 DNA-binding properties. Time-resolved fluorescence measurements revealed that the 14-3-3 binding affects fluorescence properties of 5-(((acetylamino)ethyl)amino) naphthalene-1-sulfonic acid moiety attached at four sites within the forkhead domain of FOXO4 that represent important parts of the DNA binding interface. Observed changes in 5-(((acetylamino)ethyl)amino) naphthalene-1-sulfonic acid fluorescence strongly suggest physical contacts between the 14-3-3 protein and labeled parts of the FOXO4 DNA binding interface. The 14-3-3 protein binding, however, does not cause any dramatic conformational change of FOXO4 as documented by the results of tryptophan fluorescence experiments. To build a realistic model of the 14-3-3/FOXO4 complex, we measured six distances between 14-3-3 and FOXO4 using Förster resonance energy transfer time-resolved fluorescence experiments. The model of the complex suggests that the forkhead domain of FOXO4 is docked within the central channel of the 14-3-3 protein dimer, consistent with our hypothesis that 14-3-3 masks the DNA binding interface of FOXO4 [3].

References

1. A. van der Horst, B.M.T. Burgering, Nat. Rev. Mol. Cell Biol., 8, (2007), 440.

2. T. Obsil, V. Obsilova, Oncogene, 27, (2008), 2263.

3. J. Silhan, P. Vacha, P. Strnadova, J. Vecer, P. Herman, M. Sulc, J. Teisinger, V. Obsilova, T. Obsil, J. Biol. Chem., 284, (2009), 19349.

Acknowledgements

This work was supported by Grant Agency of the Academy of Sciences of the Czech Republic Grant IAA501110801; Grant Agency of the Czech Republic Grant 204/06/0565; Ministry of Education, Youth, and Sports of the Czech Republic Research Projects MSM0021620857 and MSM0021620835 and Centre of Neurosciences LC554; and Academy of Sciences of the Czech Republic Research Project AV0Z50110509.