Biophysical characterization of phosducin/14-3-3 protein complex
L. Rezabkova1,2, M. Kacirova1,2, P. Herman3, J. Vecer3, M. Sulc4, T. Obsil1,2
2Institute of Physiology, Academy of Sciences of the Czech Republic, 14220 Prague 4, Czech Republic
of Mathematics and Physics,
Phosducin (Pd) is a regulator of G-protein-mediated signaling that is especially abundant in photoreceptors and pineal gland but expressed in other tissues as well . In photoreceptors, Pd is phosphorylated in dark-adapted retina and undergoes dephosphorylation in response to light. Dephosphorylated Pd binds Gβγ, sequestering and translocating it away from disk membrane, which blocks the interaction between Gβγ and effectors or reasociation with Gα subunit [2, 3]. When Pd is phosphorylated at Ser54 and Ser73 it binds the multifunctional 14-3-3 protein. 14-3-3 proteins are a family of acidic regulatory proteins that function as molecular scaffolds by modulating the structure of their binding partners. The biological meaning of the interaction between 14-3-3 and Pd could be to sequester Pd from Gβγ subunit or to protect phosporylated Pd from degradation [4, 5].
To elucidate the mechanism of 14-3-3 protein-dependent regulation of phosducin function, we performed biophysical characterization of Pd and Pd/14-3-3ζ protein complex. We have prepared Pd/14-3-3 complex in vitro. Nondenaturating electrophoresis was used to verify that the formation of Pd/14-3-3 complex is phosporyalation dependent. Analytical ultracentrifugation was used to study the oligomerization state of phosducin and the stochiometry of Pd/14-3-3 complex. Various methods of fluorescence spectroscopy have been employed to characterize conformational changes of Pd induced by 14-3-3 protein binding.
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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.