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 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.