KINETICS AND LOCALIZATION OF THE PHOSPHORYLATION OF RHODOPSIN BY PROTEIN-KINASE-C

Protein kinase C isolated from retina catalyzes the stoichiometric pho sphorylation of bovine rhodopsin, Enzymological studies using receptor in rod outer segment membranes stripped of peripheral proteins reveal that the phosphorylation is independent of receptor conformation or l iganded state; the half-time for phosphorylation of unbleached (dark-a dapted) rhodopsin, bleached (light-activated) rhodopsin, and opsin (ch romophore removed) is the same. The phosphorylation by protein kinase C is Ca2+ and lipid regulated; the K-m for Ca2+ decreases with increas ing concentrations of membrane, consistent with known properties of Ca 2+-regulated protein kinase Cs. The K-m for ATP is 27 mu M, with an op timal concentration for MgCl2 of approximately 1 mM. The phosphorylati on of rhodopsin by protein kinase C is inhibited by the protein kinase C-selective inhibitor sangivamycin. Proteolysis by Asp-N reveals that all the protein kinase C phosphorylation sites are on the carboxyl te rminus of the receptor. Cleavage with trypsin indicates that Ser(338), the primary phosphorylation site of rhodopsin kinase, is not phosphor ylated significantly; rather, the primary phosphorylation site of prot ein kinase C is on the membrane proximal half of the carboxyl terminus . The protein kinase C catalyzed phosphorylation of rhodopsin is analo gous to the ligand-independent phosphorylation of other G protein-coup led receptors that is catalyzed by second messenger-regulated kinases.