Site-specific phosphorylation of phosducin in intact retina. Dynamics of phosphorylation and effects on G protein beta gamma dimer binding.
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.
Lee
Bruce Y
BY
Department of Chemistry and Biochemistry, Brigham Young University, Provo, Utah 84602, USA.
Thulin
Craig D
CD
Willardson
Barry M
BM
D048948:14-3-3 Proteins / Q000378:metabolism; D000222:Adaptation, Physiological; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000494:pharmacology; D002417:Cattle; D002614:Chelating Agents / Q000494:pharmacology; D000242:Cyclic AMP / Q000494:pharmacology; D003624:Darkness; D019281:Dimerization; D004533:Egtazic Acid / Q000031:analogs & derivatives* / Q000494:pharmacology; D005136:Eye Proteins / Q000737:chemistry / Q000378:metabolism*; D020555:GTP-Binding Protein Regulators; D044387:GTP-Binding Protein beta Subunits / Q000378:metabolism*; D044388:GTP-Binding Protein gamma Subunits / Q000378:metabolism*; D007700:Kinetics; D008027:Light; D010750:Phosphoproteins / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation; D010786:Photoreceptor Cells / Q000737:chemistry; D012160:Retina / Q000378:metabolism*; D012694:Serine / Q000378:metabolism; D013329:Structure-Activity Relationship
D048948:14-3-3 Proteins; D002614:Chelating Agents; D005136:Eye Proteins; D020555:GTP-Binding Protein Regulators; D044387:GTP-Binding Protein beta Subunits; D044388:GTP-Binding Protein gamma Subunits; D010750:Phosphoproteins; C065631:phosducin; C070379:1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid acetoxymethyl ester; D012694:Serine; D004533:Egtazic Acid; D000242:Cyclic AMP; D002118:Calcium