Structural and enzymatic aspects of rhodopsin phosphorylation.
Photoactivated rhodopsin (Rho*) is phosphorylated near the C terminus at multiple sites, predominantly at Ser334, Ser338, and Ser343. We systematically examined the sites of phosphorylation upon flash activation of Rho in rod outer segment (ROS) homogenates. Addition of an inhibitory antibody against rhodopsin kinase (RK) lowered phosphorylation at Ser334, Ser338, and Ser343, without changing the ratio between phosphorylation sites. In contrast, no effect of protein kinase C was detected after stimulation (by a phorbol ester), inhibition (with H7), or reconstitution of protein kinase C with purified ROS membranes. The stoichiometry and the ratio between different phosphorylation sites in purified Rho were also reproduced using RK, purified to apparent homogeneity from ROS or from an insect cell expression system. Thus, we conclude that light-dependent phosphorylation of Rho is mediated primarily by RK. Depalmitoylation of Rho at Cys322 and Cys323 altered the conformation of the C terminus of Rho, as observed by phosphorylation by casein kinase I, but did not affect phosphorylation by RK. The sites of phosphorylation were influenced, however, by the presence of four conserved amino acids at the C terminus of Rho. The accumulation of phosphorylated Ser334 observed in vivo could result from slower dephosphorylation of this site as compared with dephosphorylation of Ser338 and Ser343. These data provide a molecular mechanism for the site-specific phosphorylation of Rho observed in vivo.
Pubmed ID: 8617805 RIS Download
1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine | Amino Acid Sequence | Animals | Cattle | Cell Line | Chromatography, High Pressure Liquid | Enzyme Inhibitors | Eye Proteins | G-Protein-Coupled Receptor Kinase 1 | Insects | Isoquinolines | Kinetics | Light | Mass Spectrometry | Mice | Palmitic Acid | Palmitic Acids | Peptide Fragments | Phosphopeptides | Phosphoprotein Phosphatases | Phosphorus Radioisotopes | Phosphorylation | Piperazines | Protein Kinases | Recombinant Proteins | Rhodopsin | Rod Cell Outer Segment | Serine | Tetradecanoylphorbol Acetate | Transfection