THE PHOSPHORYLATION STATE OF PHOSDUCIN DETERMINES ITS ABILITY TO BLOCK TRANSDUCIN SUBUNIT INTERACTIONS AND INHIBIT TRANSDUCIN BINDING TO ACTIVATED RHODOPSIN

Heterotrimeric GTP-binding proteins (G-proteins) serve many different signal transduction pathways. Phosducin, a 28-kDa phosphoprotein, is e xpressed 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 o f G-protein GTPase activity (Bauer, P. H., Muller, S., Puzicha, M., Pi ppig, S., Obermaier, B., Helmreich, E. J. M., and Lohse, M. J. (1992) Nature 358, 73-76). In retinal rods, phosducin inhibits transducin (G( t)) 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, 1 972-1977), the significance of these reactions is still unclear. The d ata presented here permit a more precise characterization of phosducin function and the consequences of its phosphorylation. Dephosphophosdu cin blocked binding of the G(t) alpha(1) subunit to activated rhodopsi n in the presence of stoichiometric amounts of G(t) beta gamma, wherea s phosphophosducin did not. Surprisingly, the binding affinity of phos phophosducin for G(t) beta gamma was not significantly reduced compare d with the binding affinity of dephosphophosducin. However, the associ ation of phosducin with G(t) beta gamma in a size exclusion column mat rix was dependent on the phosphorylation state of phosducin. Moreover, the ability of phosducin to compete with G(t) alpha for binding to G( t) beta gamma was also dependent on the phosphorylation state of phosd ucin. 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 wi th G(t) alpha, thereby inhibiting G(t) alpha activation by the activat ed receptor. Moreover phosphorylation of phosducin blocks its ability to compete with G(t) alpha for binding to G(t) beta gamma.