Structural basis of the 14-3-3 protein-dependent regulation of fosducin

Miroslava Kacirova^1,2, Lenka Rezabkova^1,2, Miroslav Sulc¹, Veronika Obsilova² and Tomas Obsil^1,2

¹Faculty of Science, Charles University in Prague, 12843 Prague, Czech Republic
²Institute of Physiology, Academy of Science of Czech Republic, 14220 Prague, Czech Rep.
mirkakacirova@seznam.cz

Phosducin (Pdc), a regulatory and highly conserved phosphoprotein, plays an important role in the regulation of G protein signaling by modulating the amount of G_tabgheterotrimer through the competition with the G_ta subunit for binding to the G_tbg complex [1]. Besides its well-established role in the regulation of G protein signaling, Pdc is also involved in the transcriptional control and the modulation of blood pressure. The function of Pdc is regulated through its phosphorylation and the binding to the regulatory 14-3-3 protein [2]. The 14-3-3 proteins are scaffolding molecules that regulate the function of other proteins through a number of different mechanisms. The exact role of the 14-3-3 protein in the regulation of Pdc is, however, still elusive. In this work, we performed a biophysical analysis of the Pdc:14-3-3z complex. First, the analytical ultracentrifugation was used to study the binding affinity and the stoichiometry of the complex. Our data show that Pdc phosphorylated simultaneously at Ser54 and Ser73 form a stable complex with the 14-3-3z protein while unphosphorylated or singly phosphorylated forms of Pdc show no significant binding to 14-3-3z. The time-resolved emission spectroscopy of tryptophan and dansyl fluorescence was then used to probe the structural changes of Pdc molecule induced by both the phosphorylation by itself and the 14-3-3 protein binding. The time-resolved tryptophan fluorescence intensity and anisotropy decay measurements show significant structural alterations in the region surrounding Trp29 at the N-terminus of Pdc molecule upon the 14-3-3 protein binding. The time-resolved fluorescence measurements of Pdc labeled at five different positions by 5-(((acetylamino)ethyl)amino) naphthalene-1-sulfonic acid (AEDANS) revealed that the 14-3-3 binding induces significant structural changes in both the N- and C-terminal domains of Pdc. In addition, our data also indicates that the simultaneous phosphorylation at both Ser54 and Ser73 by itself significantly affects the structure of Pdc molecule.

1. R. Gaudet, A. Bohm, P. B. Sigler, Cell 87, (1996), 577-588.

2. B. Y. Lee, C. D. Thulin, B. M. Willardson, J. Biol. Chem. 279, (2004), 54008-54017.

This work was funded by Grant P305/11/0708 of the Czech Science Foundation, by Research Project MSM0021620857 and by Research Project AV0Z50110509 of the Academy of Sciences of the Czech Republic.