Structural studies of 14-3-3/phosducin complex


M. Kacirova1,2, L. Rezabkova1,2, P. Herman3, J. Vecer3, M. Sulc4, T. Obsil1,2


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

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

3Faculty of Mathematics and Physics, Inst. Physics, Charles University in Prague; 12116 Prague, Czech Republic

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


Phosducin (Pd) is an important protein, which is involved in eyesight sense biochemistry. It regulates perception of sensitivity of light. Pd is expressed in photoreceptors, especially in rods, but it is found in pineal gland and other tissues as well. It is involved in G-protein signaling that allows sight [1, 2].

Dephosphorylation of Pd is induced by light and causes its binding to Gβγ complex. The transport of the resulting Pd/Gβγ complex from the outer rod disk membrane then induces the decrease in G-protein signaling. Therefore, the dephosphorylation of Pd protects the signaling pathway against saturation in the presence of very intense light. In the dark-adapted retina Pd is predominantly fosforylated at Ser54 and Ser73 [3]. Phosforylated Pd doesn´t bind Gβγ but binds multifunctional 14-3-3 protein. This interaction increases the G-protein signaling and thus the sensitivity towards the light [4].

14-3-3 proteins are family of acidic regulatory proteins that are expressed in all eukaryotic cells. They bind to other proteins in a phosphorylation-dependent manner and affects the structure of their binding partners [5]. Interaction between 14-3-3 and phosphorylated Pd probably enables sequestering Pd from Gβγ subunit or protecting phosphorylated Pd from degradation [2].

To elucidate the mechanism of 14-3-3-dependent regulation of Pd function, we performed several biophysical studies of Pd/14-3-3 complex. We have prepared Pd/14-3-3ζ complex in vitro. Nondenaturating electrophoresis was used to verify that the formation of Pd/14-3-3 complex is phosphorylation dependent. Analytical ultracentrifugation was used to estimate the complex stoichiometry. Conformational changes of Pd, induced by 14-3-3 protein binding, were studied using fluorescence spectroscopy techniques.


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This work was funded by Grant P305/11/0708 of the Czech Science Foundation, by Research Project MSM0021620857 and Centre of Neurosciences LC554 of the Ministry of Education, Youth, and Sports of the Czech Republic, by Grant 28510 of the Grant Agency of the Charles University and by Research Project AV0Z50110509 of the Academy of Sciences of the Czech Republic.