Cooperativity during multiple phosphorylations catalyzed by rhodopsin kinase: supporting evidence using synthetic phosphopeptides.
Rhodopsin kinase is a key component in the shutdown of visual transduction. The phosphorylation of rhodopsin's C-terminus was evaluated using synthetic peptides derived from the last 12 amino acids (337-348) as substrates and their phosphorylated counterparts as inhibitors. It was found that synthetic peptides were phosphorylated at the serine residue corresponding to Ser-343 in the primary sequence of bovine rhodopsin. The phosphopeptides were prepared by incorporating into the peptide chain a trityl-protected serine derivative at the site destined to contain the phosphoryl group. The trityl group was selectively released with 20% (v/v) dichloroacetic acid; the free hydroxyl group was then phosphitylated with di-tert-butyl N,N-diethylphosphoramidite, and the resulting phosphite derivative was oxidized with m-chloroperoxybenzoic acid. The phosphopeptides were found to have a greater affinity for the kinase compared with their nonphosphorylated counterparts; for the peptides corresponding to residues 337-348 of rhodopsin the affinity increased in the order VSKTETSQVAPA < VSKTETS[PO3H2]QVAPA < VS[PO3H2]KTETS[PO3H2]QVAPA. The results are interpreted to support the cooperativity hypotheses proposed previously [Wilden, U., & Kühn, H. (1982) Biochemistry 21, 3014-3022; Aton, B. R., Litman, B. J., & Jackson, M. L. (1984) Biochemistry 23, 1737-1741].
Pullen
N
N
Department of Biochemistry, University of Southampton, U.K.
Brown
N G
NG
Sharma
R P
RP
Akhtar
M
M
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D005136:Eye Proteins; D051606:G-Protein-Coupled Receptor Kinase 1; D007202:Indicators and Reagents; D007700:Kinetics; D008969:Molecular Sequence Data; D010748:Phosphopeptides / Q000138:chemical synthesis / Q000302:isolation & purification / Q000378:metabolism*; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D012243:Rhodopsin / Q000378:metabolism; D012374:Rod Cell Outer Segment / Q000378:metabolism*; D013379:Substrate Specificity
D005136:Eye Proteins; D007202:Indicators and Reagents; D010748:Phosphopeptides; D012243:Rhodopsin; D011494:Protein Kinases; D051606:G-Protein-Coupled Receptor Kinase 1