Protein kinase A phosphorylates retinal phosducin on serine 73 in situ.
Photoreceptors of vertebrate retinas contain a 33,000-dalton phosphoprotein, phosducin, which complexes with the beta, gamma subunits of the photoreceptor G-protein (guanine nucleotide-binding protein), transducin. In situ, the retinal content of phosphorylated phosducin is modulated by light in conjunction with light-triggered changes in intracellular cyclic nucleotide concentration. In vitro, phosducin is phosphorylated by either exogenous or endogenous protein kinase A. 32P-Labeled rat retina phosducin was isolated by immunoprecipitation either after phosphorylation by protein kinase A in the presence of [gamma-32P]ATP or after incubation of retinas in darkness with 32Pi. In either case, phosphoamino acid analysis showed that greater than 98% of 32P was linked to serine, with less than 2% to threonine. Two-dimensional peptide mapping showed that [32P]phosphoserine was associated with the same characteristic set of tryptic peptides. Furthermore, Cleveland peptide analysis using four different proteases showed that either sample exhibited identical patterns of phosphopeptides which were characteristic of the protease used. Identical phosphopeptide maps were also obtained from 32P-labeled bovine retina phosducin, indicating that the serine phosphorylation site for protein kinase A is conserved between rat and bovine. Edman degradation of phosphopeptides derived from 32P-labeled bovine phosducin showed that radioactive phosphate was incorporated into serine residue 73 which is located within a consensus phosphorylation sequence for protein kinase A (-R-K-M-S73(P)-). These observations are uniformly in agreement with protein kinase A being the endogenous kinase that phosphorylates phosducin in vivo.
Lee
R H
RH
Department of Anatomy and Cell Biology, University of California, Los Angeles 90024.
Brown
B M
BM
Lolley
R N
RN
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D015153:Blotting, Western; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D003488:Cyanogen Bromide; D005136:Eye Proteins / Q000302:isolation & purification / Q000378:metabolism*; D020555:GTP-Binding Protein Regulators; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000302:isolation & purification; D010748:Phosphopeptides / Q000302:isolation & purification; D010750:Phosphoproteins / Q000302:isolation & purification / Q000378:metabolism*; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D012160:Retina / Q000378:metabolism*; D013379:Substrate Specificity
D000911:Antibodies, Monoclonal; D005136:Eye Proteins; D020555:GTP-Binding Protein Regulators; D010446:Peptide Fragments; D010748:Phosphopeptides; D010750:Phosphoproteins; D010761:Phosphorus Radioisotopes; C065631:phosducin; D000255:Adenosine Triphosphate; D011494:Protein Kinases; D003488:Cyanogen Bromide