Phorbol ester- and light-induced endogenous phosphorylation of rat rod outer-segment proteins

We have previously described the presence of a C-kinase in bovine retinal rod outer segments (ROS) (Kapoor and Chader, 1984). In this study, we have labeled rat retinas with freshly neutralized radiolabeled sodium phosphate (32P or 33P) by intravitreal injection and compared the phosphorylation patterns of ROS proteins induced by light and specific activators of the C-kinase phosphorylation system. Except for light treatment, all procedures were carried out in complete darkness using an infrared image converter. Incubation of 33P-labeled retinas in light for 5 min resulted in the phosphorylation of rhodopsin, 80-, 65-, 47-, 44-, and 15,000 MW proteins of crude ROS. Incubation of 33P-labeled retinas with 0.5 microM 12-O-tetradecanoylphorbol-13-acetate (TPA) resulted in the phosphorylation of several proteins including those at 80-, 65-, 47-, 44-, and 15,000 MW in crude ROS. ROS prepared in complete darkness did not exhibit any phosphorylation of proteins whereas ROS prepared in red light exhibited variable low phosphorylation of 80-, 47-, 44- and 15,000 MW proteins. 1-oleoyl-2-acetylglycerol (OAG) at 500 micrograms ml-1 caused the phosphorylation of the same proteins as observed with TPA. TPA (0.5-500 microM) and OAG (150-500 micrograms ml-1) did not induce rhodopsin phosphorylation. When purified ROS were prepared from 33P-pre-labeled retinas, the complete darkness control did not exhibit phosphorylation of any proteins. TPA, however, induced the phosphorylation of 80- and 65,000 MW proteins and light induced the phosphorylation of 80-, 65,000 MW proteins as well as opsin monomer and dimer. Affinity chromatography of phosphorylated ROS proteins on con A-Sepharose revealed that TPA does not induce rhodopsin phosphorylation whereas light does. Since light and TPA induced the phosphorylation of 80- and 65,000 MW proteins in ROS, it is possible to suggest at least a partial linkage of light- and C-kinase-mediated effects in situ.