Structural and biophysical characterization of the PI4KB:14-3-3 protein complex

Dominika Chalupska and Evzen Boura

Institute of Organic Chemistry and Biochemistry AS CR, v.v.i., Flemingovo nam. 2., 166 10, Prague 6, Czech Republic

Phosphatidylinositol 4-kinase IIIβ (PI4KB) produces the Golgi pool of phosphatidylinositol 4-phosphate (PI4P). PI4P serves as a signaling molecule, and, additionally, as a precursor for higher phosphoinositides. PI4KB is a soluble cytoplasmic enzyme with no membrane binding properties and is recruited to the membranes by the Golgi resident ACBD3 protein [1]. However, the regulation of PI4KB activity is not understood in detail. Another protein that has been reported to control the PI4KB enzymatic activity in the cell is the 14-3-3 protein [2]. 14-3-3 proteins bind PI4KB upon its phosphorylation by protein kinase D, however, the structural basis of PI4KB regulation by 14-3-3 proteins is unknown. Here, we characterized the PI4KB:14-3-3 protein complex biophysically and structurally. Using analytical ultracentrifugation we discovered that the PI4KB:14-3-3 protein complex is tight and is formed with 2:2 stoichiometry. To reveal the structure of the PI4KB:14-3-3 protein complex, we utilized small angle X-ray scattering (SAXS) in combination with molecular dynamics simulations. Our structural analysis revealed that the PI4KB:14-3-3 protein complex is flexible but mostly within the disordered regions connecting the 14-3-3 binding site of the PI4KB with the rest of the PI4KB enzyme. In each of our model structures the PI4KB active site is well accessible, which predicts no effect of 14-3-3 binding on the activity of PI4KB. To confirm these results, we measured enzymatic activity of PI4KB in vitro and we did not observe any activation of PI4KB by 14-3-3 proteins. However, using protease degradation assay, we discovered that 14-3-3 proteins are able to protect PI4KB from degradation. The biological role of binding of 14-3-3 proteins to PI4KB is not clear, but we hypothesize that 14-3-3 proteins regulate PI4P production via stabilization of PI4KB in the intracellular environment.

 

1.              Klima, M., Toth, D.J., Hexnerova, R., Baumlova, A., Chalupska, D., Tykvart, J.,Rezabkova, L., Sengupta, N., Man, P., Dubankova, A., Humpolickova, J., Nencka, R., Veverka, V., Balla, T., Boura, E., 2016. Structural insights and in vitro reconstitution of membrane targeting and activation of human PI4KB by the ACBD3 protein. Sci.Rep. 6, 23641.

2.              Hausser, A., Link, G., Hoene, M., Russo, C., Selchow, O., Pfizenmaier, K., 2006. Phospho-specific binding of 14-3-3 proteins to phosphatidylinositol 4-kinase III beta protects from dephosphorylation and stabilizes lipid kinase activity. J. Cell Sci. 119,3613–3621.

 

The project was supported by Project InterBioMed LO1302 from Ministry of Education of the Czech Republic.