Site-specific phosphorylation of phosducin in intact retina. Dynamics of phosphorylation and effects on G protein beta gamma dimer binding

• Published in: The Journal of biological chemistry Vol. 279; no. 52; pp. 54008 - 54017
• Main Authors: Lee, Bruce Y, Thulin, Craig D, Willardson, Barry M
• Format: Journal Article
• Language: English
• Published: United States 24.12.2004
• Subjects: 14-3-3 Proteins - metabolism; Adaptation, Physiological; Amino Acid Sequence; Animals; Binding Sites; Calcium - pharmacology; Cattle; Chelating Agents - pharmacology; Cyclic AMP - pharmacology; Darkness; Dimerization; Egtazic Acid - analogs & derivatives; Egtazic Acid - pharmacology; Eye Proteins - chemistry; Eye Proteins - metabolism; GTP-Binding Protein beta Subunits - metabolism; GTP-Binding Protein gamma Subunits - metabolism; GTP-Binding Protein Regulators; Kinetics; Light; Phosphoproteins - chemistry; Phosphoproteins - metabolism; Phosphorylation; Photoreceptor Cells - chemistry; Retina - metabolism; Serine - metabolism; Structure-Activity Relationship
• ISSN: 0021-9258
• DOI: 10.1074/jbc.M405669200

Phosducin (Pdc) is a G protein beta gamma dimer (G beta gamma) binding protein, highly expressed in retinal photoreceptor and pineal cells, yet whose physiological role remains elusive. Light controls the phosphorylation of Pdc in a cAMP and Ca(2+)-dependent manner, and phosphorylation in turn regulates the binding of Pdc to G(t)beta gamma or 14-3-3 proteins in vitro. To directly examine the phosphorylation of Pdc in intact retina, we prepared antibodies specific to the three principal phosphorylation sites (Ser-54, Ser-73, and Ser-106) and measured the kinetics of phosphorylation/dephosphorylation during light/dark adaptation and the subsequent effects on G(t)beta gamma binding. Ser-54 phosphorylation increased slowly (t((1/2)) approximately 90 min) during dark adaptation to approximately 70% phosphorylated and decreased rapidly (t((1/2)) approximately 2 min) during light adaptation to less than 20% phosphorylated. Ser-73 phosphorylation increased much faster during dark adaptation (t((1/2)) approximately 3 min) to approximately 50% phosphorylated and decreased more slowly during light adaptation (t((1/2)) approximately 9 min) to less than 20% phosphorylated. The Ca(2+) chelator BAPTA-AM blocked Ser-54 phosphorylation during dark adaptation but had no effect on Ser-73 phosphorylation. In contrast, Ser-106 was not phosphorylated in either the light or dark. Importantly, G beta gamma binding to Pdc was enhanced by Ca(2+) chelation and the binding kinetics closely paralleled those of Ser-54 dephosphorylation, indicating that Ser-54 phosphorylation controls G(t)beta gamma binding in vivo. These results suggest a pivotal role of Ser-54 and Ser-73 phosphorylation in determining the interactions of Pdc with its binding partners, G(t)beta gamma and 14-3-3 protein, which may regulate the light-dependent translocation of the photoreceptor G protein.