The phosphorylation state of phosducin determines its ability to block transducin subunit interactions and inhibit transducin binding to activated rhodopsin
Yoshida, T.; Willardson, B.M.; Wilkins, J.F.; Jensen, G.J.; Thornton, B.D.; Bitensky, M.W.
Journal of Biological Chemistry 269(39): 24050-24057
Heterotrimeric GTP-binding proteins (G-proteins) serve many different signal transduction pathways. Phosducin, a 28-kDa phosphoprotein, is expressed in a variety of mammalian cell types and blocks activation of several classes of G-proteins. Phosphorylation of phosducin by cyclic AMP-dependent protein kinase prevents phosducin-mediated inhibition of G-protein GTPase activity (Bauer, P. H., Muller, S., Puzicha, M., Pippig, S., Obermaier, B., Helmreich, E. J. M., and Lohse, M. J. (1992) Nature 358, 73-76). In retinal rods, phosducin inhibits transducin (Gt) activation by binding its beta-gamma subunits. While rod phosducin is phosphorylated in the dark and dephosphorylated after illumination (Lee, R.H., Brown, B. M., and Lolley, R. N. (1984) Biochemistry 23, 1972-1977), the significance of these reactions is still unclear. The data presented here permit a more precise characterization of phosducin function and the consequences of its phosphorylation. Dephosphophosducin blocked binding of the G-t-alpha-1 subunit to activated rhodopsin in the presence of stoichiometric amounts of G-t-beta-gamma, whereas phosphophosducin did not. Surprisingly, the binding affinity of phosphophosducin for G-t-beta-gamma was not significantly reduced compared with the binding affinity of dephosphophosducin. However, the association of phosducin with G-t-beta-gamma in a size exclusion column matrix was dependent on the phosphorylation state of phosducin. Moreover, the ability of phosducin to compete with G-ta for binding to G-t-beta-gamma was also dependent on the phosphorylation state of phosducin. No interaction was found between phosducin and G-t-alpha. These data indicate that phosducin decreases rod responsiveness by binding to the beta-gamma subunits of G-t and preventing their interaction with G-t-alpha, thereby inhibiting G-t-alpha activation by the activated receptor. Moreover, phosphorylation of phosducin blocks its ability to compete with G-t-alpha for binding to G-t-beta-gamma.