Rhodopsin kinase autophosphorylation. Characterization of site-specific mutations.
Upon illumination, rhodopsin kinase (RK) phosphorylates the visual pigment rhodopsin, which is thought to partially terminate the biochemical events that follow photon absorption. RK enzymology was explored by mutagenesis of the residues Ser488, Thr489 (major autophosphorylation sites), and Lys491 (a distal residue). We found the following to be true. (i) Double mutations at residues Ser488 and Thr489 to Ala or Asp decrease autophosphorylation to substoichiometrical levels, while single mutations at either residue independently reduce autophosphorylation by half. (ii) Phosphorylation of residue Ser488 influences the affinity of RK for heparin-Sepharose only moderately, whereas Thr489 and Lys491 are important for this interaction. RK K491A does not phosphorylate acidic peptides, suggesting that this residue participates in substrate binding. (iii) Mutations in the autophosphorylation region affect the Km for ATP, suggesting that this region is involved in binding of ATP to the catalytic site. (iv) RK mutants S488A or S488D and RK S488A and T489A have an increased ability to phosphorylate Rho in the dark. (v) Mutations at the autophosphorylation region change the initial site of phosphorylation on photolyzed rhodopsin (Rho*), implying that this region may regulate selectivity of the site of phosphorylation.
Palczewski
K
K
Department of Ophthalmology, School of Medicine, University of Washington, Seattle 98195, USA.
Ohguro
H
H
Premont
R T
RT
Inglese
J
J
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002846:Chromatography, Affinity; D005136:Eye Proteins; D051606:G-Protein-Coupled Receptor Kinase 1; D007700:Kinetics; D008027:Light; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D017354:Point Mutation; D011494:Protein Kinases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012374:Rod Cell Outer Segment / Q000201:enzymology*; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D005136:Eye Proteins; D011994:Recombinant Proteins; D011494:Protein Kinases; D051606:G-Protein-Coupled Receptor Kinase 1