Synthetic fragments of protamines as model substrates for rat liver cyclic AMP-dependent protein kinase

Abstract

Nine synthetic peptides reproducing either exactly or with suitable substitutions three phosphorylatable sites of the protamines thynnine Z1 and galline have been prepared and tested as phosphate acceptors for the rat liver cyclic AMP-dependent protein kinase (type I). The most significant results obtained can be summarized as follows: 1. The hexapeptide: Arg-Arg-Ser-Thr-Val-Ala gives two phpsphorylated products, containing only Ser-P and both Ser-P and Thr-P, respectively. The relative amount of the di-phosphorylated derivative is not dependent on the incubation time but rather on the concentration of the substrates. 2. Both the replacements of ornithine for the two N-terminal arginines of glutamic acid for Val 5 in the above peptide completely prevent phosphorylation, without conferring any inhibitory activity to the modified peptides, thus supporting the view that the N-terminal guanido groups and the C-terminal hydrophobic residue(s) are both required for the binding at the catalytic site rather than for the subsequent transphosphorylation reaction. 3. The replacement of alanine for Ser 3 gives rise to a peptide whose Thr 4 residue is still phosphorylated with an efficiency comparable to that of the unmodified peptide. The di-substituted peptide: Arg-Arg-Ala-Ser-Val-Ala however exhibits a dramatically lower K m value indicating that serine is a much better target than threonine whenever it is not adjacent to the N-terminal arginine couple. 4. The importance of the distance between the target residue and the N-terminal basic determinants is also evidenced by the phosphorylation of the dodecapeptide: Pro-(Arg) 5-Ser-Ser-Arg-Pro-Val-Arg exhibiting a K m value about 20-times higher than that of salmine A1, whose phosphorylation site is comprised within an identical amino acid sequence, including however three rather than two adjacent serine residues. 5. The tetradecapeptide: (Arg) 4-Tyr-Gly-Ser-(Arg) 6-Tyr is completely unaffected by the kinase though a very similar site is found phosphorylated in native iridines, probably through a cyclic AMP-independent mechanism.