Phosphorylation of rhodopsin by protein kinase C in vitro.
Calium/phospholipid-dependent protein kinase (protein kinase C) was purified from bovine retinae rod outer segments (ROS). In the presence of 0.1-2 microM calcium protein kinase C binds tightly to ROS and phosphorylates rhodopsin in the absence or presence of illumination. This property of protein kinase C contrasts with that of rhodopsin kinase, which in vitro phosphorylates only bleached rhodopsin. Peptide maps of rhodopsin phosphorylated by protein kinase C or rhodopsin kinase were compared using limited Staphylococcus aureus V8 protease digestion or complete tryptic digestion. Phosphorylation sites map to serine and threonine residues on the cytoplasmic carboxylterminal domain of rhodopsin for both kinases. The functional consequence of protein kinase C phosphorylation of rhodopsin was a reduced ability to stimulate the light-dependent rhodopsin activation of [35S]guanosine 5'-O-(thiotriphosphate) binding to transducin, the GTP-binding regulatory protein present in ROS. Properties of the calcium-stimulated interaction of protein kinase C with membranes and in vitro phosphorylation of intrinsic proteins are discussed based upon the findings.
D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002417:Cattle; D004591:Electrophoresis, Polyacrylamide Gel; D019204:GTP-Binding Proteins / Q000378:metabolism; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D007700:Kinetics; D008565:Membrane Proteins / Q000378:metabolism; D010446:Peptide Fragments / Q000032:analysis; D010766:Phosphorylation; D010786:Photoreceptor Cells / Q000201:enzymology*; D006656:Protamine Kinase / Q000378:metabolism; D011485:Protein Binding; D011493:Protein Kinase C / Q000302:isolation & purification / Q000378:metabolism*; D012168:Retinal Pigments / Q000378:metabolism*; D012243:Rhodopsin / Q000378:metabolism*; D012374:Rod Cell Outer Segment / Q000201:enzymology*; D013873:Thionucleotides / Q000378:metabolism; D015293:Transducin
D008565:Membrane Proteins; D010446:Peptide Fragments; D012168:Retinal Pigments; D013873:Thionucleotides; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate; D012243:Rhodopsin; D006656:Protamine Kinase; D011493:Protein Kinase C; D019204:GTP-Binding Proteins; D015293:Transducin; D002118:Calcium