COOPERATIVITY DURING MULTIPLE PHOSPHORYLATIONS CATALYZED BY RHODOPSINKINASE - SUPPORTING EVIDENCE USING SYNTHETIC PHOSPHOPEPTIDES

Rhodopsin kinase is a key component in the shutdown of visual transduc tion. The phosphorylation of rhodopsin's C-terminus was evaluated usin g synthetic peptides derived from the last 12 amino acids (337-348) as substrates and their phosphorylated counterparts as inhibitors. It wa s found that synthetic peptides were phosphorylated at the serine resi due corresponding to Ser-343 in the primary sequence of bovine rhodops in. The phosphopeptides were prepared by incorporating into the peptid e chain a trityl-protected serine derivative at the site destined to c ontain 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 t he kinase compared with their nonphosphorylated counterparts; for the peptides corresponding to residues 337-348 of rhodopsin the affinity i ncreased in the order VSKTETSQVAPA < VSKTETS [PO3H2] QVAPA < VS-[PO3H2 ]KTETS[PO3H2]QVAPA. The results are interpreted to support the coopera tivity hypotheses proposed previously [Wilden, U., & Kuhn, H. (1982) B iochemistry 21, 3014-3022; Aton, B. R., Litman, B. J., & Jackson, M. L . (1984) Biochemistry 23, 1737-1741].