Identification of the autophosphorylation sites in rhodopsin kinase.
Rhodopsin kinase (RK) is a second-messenger-independent protein kinase that is involved in deactivation of photolyzed rhodopsin (Rho*). We have developed a significantly improved method for isolation of RK based on the specific interactions of phosphorylated forms of the enzyme with heparin-Sepharose. Conversion of the dephosphorylated form of RK to the fully phosphorylated enzyme leads to specific elution of the kinase from the resin. Limited proteolysis of RK with endoproteinase Asp-N removes the phosphorylation sites. Peptides containing the autophosphorylation sites were isolated by reverse-phase high performance liquid chromatography and analyzed by Edman degradation and tandem mass spectrometry. The derived amino acid sequence of the peptide containing the major autophosphorylation site yielded the following sequence: DVGAFS488T489VKGVAFEK, where Ser488 and Thr489 are phosphorylated. Additionally, a minor autophosphorylation site was identified at Ser21. A 15-residue peptide (DVGAFSTVKGVAFEK) encompassing the major autophosphorylation site was synthesized and used for phosphorylation and inhibition studies. In contrast to many other protein kinases, the low catalytic activity of RK toward its autophosphorylation site peptide and the poor inhibitory properties of this peptide suggest unique properties of this member of the family of G protein-coupled receptor kinases.
Palczewski
K
K
R. S. Dow Neurological Sciences Institute, Good Samaritan Hospital and Medical Center, Portland, Oregon 97209.
Buczyłko
J
J
Van Hooser
P
P
Carr
S A
SA
Huddleston
M J
MJ
Crabb
J W
JW
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D005136:Eye Proteins; D051606:G-Protein-Coupled Receptor Kinase 1; D006868:Hydrolysis; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000235:genetics; D010449:Peptide Mapping; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D012374:Rod Cell Outer Segment / Q000201:enzymology; D014357:Trypsin
D005136:Eye Proteins; D010446:Peptide Fragments; D011494:Protein Kinases; D051606:G-Protein-Coupled Receptor Kinase 1; D014357:Trypsin