Structural characterization of phosphoprotein phosducin and its interaction with 14-3-3

Miroslava Kacirova1,2, Jiri Novacek3, Alan Kadek4, Petr Man4, Veronika Obsilova2, and Tomas Obsil1,2

1Faculty of Science, Charles University in Prague, 12843 Prague, Czech Republic
2Institute of Physiology, Czech Academy of Sciences, 14220 Prague, Czech Republic
3Masaryk University, CEITEC Central European Institute of Technology, 60177 Brno, Czech Rep.
4Institute of Microbiology, Czech Academy of Sciences, 14220 Prague, Czech Rep.


Phosducin (Pdc), a highly conserved phosphoprotein involved in the regulation of retinal phototransduction cascade, transcriptional control, and the modulation of blood pressure, is controlled in the phosphorylation-dependent manner including the binding to the 14-3-3 protein. However, the molecular mechanism of this regulation is largely unknown [1-3]. Here, the solution structure of Pdc and its interaction with the 14-3-3 protein were investigated using small angle X-ray scattering, circular dichroism, quenching of tryptophan fluorescence, analytical ultracentrifugation, hydrogen-deuterium exchange coupled to mass spectrometry and nuclear magnetic resonance. We show that the 14-3-3 protein interacts with and sterically occludes both the N- and C-terminal Gtβγ binding interfaces of phosphorylated Pdc, thus providing a mechanistic explanation for the 14-3-3-depedent inhibition of Pdc function. The 14-3-3 protein dimer interacts with Pdc using surfaces both inside and outside its central channel. The N-terminal domain of Pdc, where both phosphorylation sites and the 14-3-3 binding motifs are located, is intrinsically disordered protein which remains likely highly flexible when bound to 14-3-3 indicating the fuzzy-like character of this complex. In addition, it has been speculated that the 14-3-3 protein binding decreases the rate of Pdc dephosphorylation after a light stimulus by virtue of its interaction with phosphorylated Ser54 and Ser73, thus lengthening the time that Pdc remains phosphorylated after a light exposure. Pdc is dephosphorylated in vivo by protein phosphatases 1 (PP1) and 2A (PP2A). Indeed, our dephosphorylation experiments with PP1 revealed that the 14-3-3 protein does slow down the dephosphorylation of doubly phosphorylated Pdc in vitro.


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3. L. Rezabkova, M. Kacirova, M. Sulc, P. Herman, J. Vecer, M. Stepanek, V. Obsilova, T. Obsil, Biophysical J. 103, (2012), 1960-1969.


This work was supported by the Czech Science Foundation (Project P305/11/0708), Grant Agency of Charles University in Prague (Project 793913); and Academy of Sciences of the Czech Republic (Research Projects RVO: 67985823 of the Institute of Physiology).