Identification of Protein Kinase C Phosphorylation Sites on Bovine Rhodopsin

• Published in: The Journal of biological chemistry 1997-04, Vol.272 (16), p.10341-10344
• Main Authors: Greene, N M, Williams, D S, Newton, A C
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; Cattle; Eye Proteins; G-Protein-Coupled Receptor Kinase 1; Isoenzymes - metabolism; Kinetics; Models, Structural; Molecular Sequence Data; Phosphorylation; Protein Kinase C - metabolism; Protein Kinase C-alpha; Protein Kinases - isolation & purification; Protein Kinases - metabolism; Protein Structure, Secondary; Recombinant Proteins - isolation & purification; Recombinant Proteins - metabolism; Retina - enzymology; Rhodopsin - chemistry; Rhodopsin - metabolism; Serine; Spodoptera; Substrate Specificity; Threonine; Transfection
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.272.16.10341

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 Ser 334 , Thr 335 , and Thr 336 . Phosphoamino acid analysis of the tryptic product established that Ser 334 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 Ser 338 is also a primary phosphorylation site, with minor phosphorylation of Ser 343 . Quantitation of high pressure liquid chromatography-separated phosphopeptides, taken together with phosphoamino acid analysis of the tryptic product, revealed that Ser 334 and Ser 338 were phosphorylated equally and each accounted for approximately 35% of the total phosphorylation; Thr 335/336 accounted for just under 20% of the phosphorylation, and Ser 343 accounted for 10%. Thus, the primary protein kinase C sites are Ser 334 and Ser 338 , with minor phosphorylation of Thr 335/336 and Ser 343 . Ser 334 and Ser 338 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 Ser 338 is a significant substrate for rhodopsin kinase in vitro . Identification of Ser 334 as a primary protein kinase C target in vitro is consistent with protein kinase C modulating the phosphorylation of this site in vivo .