The phosphorylation of a synthetic peptide, corresponding to the C-terminal 11 amino acids of bovine rhodopsin (MI, residues 338 -348), was studied under different conditions. The peptide was only phosphorylated in the presence of photoactivated rhodopsin. Using the same protocol, 12 other peptides, mapping in the rhodopsin C-terminal, were screened for their effectiveness as substrates for rhodopsin kinase. It was found that the peptides became poorer substrates with increasing length, and the best substrates comprised the most C-terminal9 -12 amino acids as opposed to other parts of the C-terminus. It was noted that the absence of the two-terminal residues Pro347 and Ala348 impaired peptide phosphorylation.The effect of the decay of metarhodopsin I1 on the phosphorylation of rhodopsin and the peptides was determined, and it was found that the rhodopsin and peptide phosphorylations decayed with half times of approximately 33 min and 28 min, respectively.The sites of phosphorylation on the peptides were determined and in all cases the phosphorylation was found to be predominantly on serine residues. Only the 11-residue peptide (MI, residues 338-348) contained significant threonine phosphorylation, which was about 25% that on serine residues. Cumulatively, the results suggest that Ser343 is the preferred site of phosphorylation in vitro.The reason for the poor substrate effectiveness of the larger peptides was examined by competitive experiments in which it was shown that a poorly phosphorylated larger peptide successfully inhibited the phosphorylation of a 'good' peptide substrate. The studies above support a mechanism for rhodopsin kinase that we have termed the 'kinase-activation hypothesis'. This requires that the kinase exists in an inactive form and is activated only after binding to photoactivated rhodopsin. [18, 191 and rhodopsin kinase [20-221. This kinase, in vitro, acts specifically on several serine and threonine residues in the C-terminal domain of bleached rhodopsin [23-261. This dual role of Rho* in the transduction and termination processes is illustrated in Scheme 1.With respect to the mechanism of light-dependent phosphorylation, it was originally assumed that the C-terminal domain of rhodopsin is inaccessible to the kinase but becomes Correspondence to M. Akhtar,

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].

Background: Vaccinia virus gene B1R encodes a serine/threonine protein kinase. In vitro this protein kinase phosphorylates ribosomal proteins Sa and S2 and vaccinia virus protein H5R, proteins that become phosphorylated during infection. Nothing is known about the sites phosphorylated on these proteins or the general substrate specificity of the kinase. The work described is the first to address these questions. Results:Vaccinia virus protein H5R was phosphorylated by the B1R protein kinase in vitro, digested with V8 protease, and phosphopeptides separated by HPLC. The N-terminal sequence of one radioactively labelled phosphopeptide was determined and found to correspond to residues 81-87 of the protein, with Thr-84 and Thr-85 being phosphorylated. A synthetic peptide based on this region of the protein was shown to be a substrate for the B1R protein kinase, and the extent of phosphorylation was substantially decreased if either Thr residue was replaced by an Ala. Conclusions:We have identified the first phosphorylation site for the vaccinia virus B1R protein kinase. This gives important information about the substrate-specificity of the enzyme, which differs from that of other known protein kinases. It remains to be seen whether the same site is phosphorylated in vivo.

Background: Vaccinia virus gene B1R encodes a serine/threonine protein kinase. In vitro this protein kinase phosphorylates ribosomal proteins Sa and S2 and vaccinia virus protein H5R, proteins that become phosphorylated during infection. Nothing is known about the sites phosphorylated on these proteins or the general substrate specificity of the kinase. The work described is the first to address these questions. Results:Vaccinia virus protein H5R was phosphorylated by the B1R protein kinase in vitro, digested with V8 protease, and phosphopeptides separated by HPLC. The N-terminal sequence of one radioactively labelled phosphopeptide was determined and found to correspond to residues 81-87 of the protein, with Thr-84 and Thr-85 being phosphorylated. A synthetic peptide based on this region of the protein was shown to be a substrate for the B1R protein kinase, and the extent of phosphorylation was substantially decreased if either Thr residue was replaced by an Ala. Conclusions:We have identified the first phosphorylation site for the vaccinia virus B1R protein kinase. This gives important information about the substrate-specificity of the enzyme, which differs from that of other known protein kinases. It remains to be seen whether the same site is phosphorylated in vivo.