14-3-3 PROTEIN CHANGES CONFORMATION OF NUCLEAR LOCALIZATION SEQUENCEOF FORKHEAD TRANSCRIPTION FACTOR FOXO4

14-3-3 PROTEIN CHANGES CONFORMATION OF NUCLEAR LOCALIZATION SEQUENCE OF FORKHEAD TRANSCRIPTION FACTOR FOXO4

V. Obsilova², J. Vecer³, P. Herman³, A. Pabianova^1,2, M. Sulc⁴, J. Teisinger², E. Boura^1,2, and T. Obsil^1,2

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;

³Institute of Physics, Faculty of Mathematics and Physics, Charles University, 121 16 Prague;

⁴Institute of Microbiology, Academy of Sciences of the Czech Republic, 142 20 Prague.

The 14-3-3 proteins are a family of regulatory signaling molecules that interact with other proteins in a phosphorylation-dependent manner. 14-3-3 proteins are thought to play a direct role in the regulation of subcellular localization of FoxO forkhead transcription factors. It has been suggested that the interaction with the 14-3-3 protein affects FoxO binding to the target DNA and interferes with the function of nuclear localization sequence (NLS). Masking or obscuring of NLS could inhibit interaction between FoxO factors and nuclear importing machinery and thus shift the equilibrium of FoxO localization toward the cytoplasm. According to our best knowledge, there is no experimental evidence showing a direct interaction between the 14-3-3 protein and NLS of FoxO. Therefore, the main goal of this work was to investigate whether the phosphorylation by protein kinase B, the 14-3-3 protein, and DNA binding affect the structure of FoxO4 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. Our results also suggest that the DNA-binding domain of FoxO4 remains relatively mobile while bound to the 14-3-3 protein [1].

1. V. Obsilova, J. Vecer, P. Herman, A. Pabianova, M. Sulc, J. Teisinger, E. Boura, T. Obsil, Biochemistry, 44 (2005) 11608-11617.

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.