Mapping the interactions between 14-3-3 protein and forkhead transcription factor FOXO4

 

P. Vacha1,2, J. Silhan1,2, J. Vecer3, P. Herman3, M. Sulc4, V. Obsilova2, T. Obsil1,2

 

1Faculty of Science, Charles University, 12843 Prague, Czech Republic

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

3Faculty of Mathematics and Physics, Institute of Physics, Charles University; 12116 Prague, Czech Republic

4Institute of Microbiology, Academy of Sciences of the Czech Republic; 14220 Prague, Czech Republic

krmelec@volny.cz

 

The forkhead family of transcription factors shares a highly conserved 100-amino-acid large DNA binding (DBD) or FOX (Forkhead box) domain. The FOX proteins display large functional diversity and play a wide range of roles in a number of physiological and pathological processes [1]. Among the forkhead family, the FOXO class 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. Transcriptional activity of FOXO proteins is regulated through insulin–phosphatidylinositol 3-kinase–AKT/protein kinase B (PI3K–AKT/PKB) signaling pathway. The AKT/PKB-mediated phosphorylation triggers phosphorylation of additional sites by other kinases and induces FOXO binding to 14-3-3 protein. This in turn both promotes the nuclear export of the resulting complex and inhibits the nuclear import of FOXO likely by interfering with the function of its nuclear localization signal (NLS) [2]. In addition to phosphorylation, the function of FOXO proteins is further controlled by other types of posttranslational modifications including acetylation and ubiquitination [3].

The role of 14-3-3 proteins in the regulation of FOXO forkhead transcription factors is at least twofold. 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 NLS. The exact mechanisms of these processes are still unclear, mainly due to the lack of structural data. In this work, we have used fluorescence spectroscopy techniques to investigate the mechanism of 14-3-3 protein-dependent inhibition of FOXO4 DNAbinding properties. We have labeled four different sites within the forkhead domain of FOXO4 (N-terminal region, C-terminal wing W2, and both sides of helix H3) with the extrinsic fluorophore 1,5-IAEDANS and used methods of time-resolved fluorescence spectroscopy to study interaction between FOXO4 and 14-3-3 protein. Our results suggest that 14-3-3 protein physically interacts with all four tested regions of forkhead domain that represent important parts of its DNA-binding interface. Such interactions likely mask the DNA-binding interface thus blocking the FOXO4 binding to the target DNA. In addition, time-resolved tryptophan fluorescence measurements indicate no significant 14-3-3 protein binding-induced conformational change within the forkhead domain of FOXO4. Thus the 14-3-3 protein functions as a “molecular hood” that covers the DNA-binding interface of FOXO4 and blocks its interaction with the target DNA.  

 

References

1.     D. Weigel, H. Jackle, Cell, 63, (1990), 455.

2.     V. Obsilova, J. Vecer, P. Herman, A. Pabianova, M. Sulc, J. Teisinger, E. Boura, T. Obsil, Biochemistry, 44, (2005), 11608.

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

 

Acknowledgements.

This work was funded by Grant IAA501110801 of the Grant Agency of the Academy of Sciences of the Czech Republic, by Research Project MSM0021620857 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.