Identification of protein kinase C phosphorylation sites on bovine rhodopsin.
The protein kinase C phosphorylation sites on bovine rhodopsin were identified using proteolytic, phosphoamino acid, mass spectrometric, and peptide sequencing analyses. Tryptic removal of the 9 carboxyl-terminal residues of rhodopsin revealed that a major fraction of the phosphates incorporated by protein kinase C are in a region containing Ser334, Thr335, and Thr336. Phosphoamino acid analysis of the tryptic product established that Ser334 accounts for approximately 65% of the phosphorylation in this region. Analysis of the endoproteinase Asp-N-generated carboxyl terminus of rhodopsin by mass spectrometry and peptide sequencing revealed that Ser338 is also a primary phosphorylation site, with minor phosphorylation of Ser343. Quantitation of high pressure liquid chromatography-separated phosphopeptides, taken together with phosphoamino acid analysis of the tryptic product, revealed that Ser334 and Ser338 were phosphorylated equally and each accounted for approximately 35% of the total phosphorylation; Thr335/336 accounted for just under 20% of the phosphorylation, and Ser343 accounted for 10%. Thus, the primary protein kinase C sites are Ser334 and Ser338, with minor phosphorylation of Thr335/336 and Ser343. Ser334 and Ser338 have recently been identified as the primary sites of phosphorylation of rhodopsin in vivo (Ohguro, H., Van Hooser, J. P., Milam, A. H., and Palczewski, K. (1995) J. Biol. Chem. 270, 14259-14262). Of these sites, only Ser338 is a significant substrate for rhodopsin kinase in vitro. Identification of Ser334 as a primary protein kinase C target in vitro is consistent with protein kinase C modulating the phosphorylation of this site in vivo.
Greene
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NM
Department of Pharmacology, University of California at San Diego, La Jolla, California 92093-0640, USA.
Williams
D S
DS
Newton
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AC
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D005136:Eye Proteins; D051606:G-Protein-Coupled Receptor Kinase 1; D007527:Isoenzymes / Q000378:metabolism; D007700:Kinetics; D008961:Models, Structural; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D051571:Protein Kinase C-alpha; D011494:Protein Kinases / Q000302:isolation & purification / Q000378:metabolism*; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012160:Retina / Q000201:enzymology*; D012243:Rhodopsin / Q000737:chemistry* / Q000378:metabolism*; D012694:Serine; D018411:Spodoptera; D013379:Substrate Specificity; D013912:Threonine; D014162:Transfection
D005136:Eye Proteins; D007527:Isoenzymes; D011994:Recombinant Proteins; D013912:Threonine; D012694:Serine; D012243:Rhodopsin; D011494:Protein Kinases; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha; D051606:G-Protein-Coupled Receptor Kinase 1