Inter- and intra-molecular phosopho-transfers catalysed by rhodopsin kinase

Pullen, Nicholas (1994) Inter- and intra-molecular phosopho-transfers catalysed by rhodopsin kinase. University of Southampton, Doctoral Thesis.

Record type: Thesis (Doctoral)

Abstract

Rhodopsin kinase is a member of a novel group of protein kinases, those that are coupled to a G-protein cascade. It specifically transfers phosphate groups from γATP to the C-terminus of the light-activated form of rhodopsin, Rho*, of the rod outer segment. Synthetic peptides, derived from this C-terminal region, were substrates for phosphorylation when reconstituted with rhodopsin and purified rhodopsin kinase and bleached. The site-specificity and relative propensity of particular residues to undergo phosphorylation was analysed and quantified. The data support a kinase-mediated reaction which begins with the modification of Ser 343 and Ser 338, followed by Thr 336, Thr 342 and either Thr 340 or Ser 334. The study emphasised that a small, uncharged amino acid at the P+4 position with respect to the phosphorylation site, designated the P site, was preferred by the kinase. This constraint precluded the modification of certain residues due to the occupancy of a phosphorylated residue or Lys 339 at the P+4 site. Three phosphopeptides based on the sequence of the last 12 amino acids of rhodopsin were synthesised and used to probe the Rho* dependent phosphorylation reaction catalysed by rhodopsin kinase. These peptides were not substrates for further phosphorylation, but were inhibitors, such that the affinity of a diphosphorylated peptide (³³⁷VS[PO₃H₂]KTETS[PO₃H₂]QVAPA³⁴⁸) for the kinase was higher than a mono-phosphorylated derivative (³³⁷VSKTETS[PO₃H₂]QVAPA³⁴⁸) which in turn was higher than the non-phosphorylated counterpart. The data support a co-operative mechanism of kinase-mediated phosphorylation.

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