Synthetic peptides reproducing the site phosphorylated by cAMP‐dependent protein kinase in protein phosphatase inhibitor‐1

Abstract

The hexapeptide (Arg)₂‐Pro‐Thr‐Pro‐Ala (T1) and octapeptide (Arg)₂‐Pro‐Thr‐Pro‐Ala (T5), reproducing the phosphorylatable site of protein phosphatase inhibitor‐1, a physiological target of cAMP‐dependent protein kinase, and five related peptides were synthesized by the method in solution. The phosphorylation rates of such peptides by cAMP‐dependent protein kinase and their kinetic parameters have been determined and compared with those of the hexapeptide (Arg)₂‐Ala‐Ser‐Val‐Ala, reproducing the phosphorylatable site of rat liver pyruvate kinase. The results obtained show that both the presence of threonine instead of serine and the adjacent C‐terminal proline represent highly unfavourable factors seriously impairing the protein kinase reaction by both increasing K_m and depressing V. On the other hand the N‐terminal proline is compatible with high phosphorylation rates and the row of four rather than two consecutive arginines improves the phosphorylation efficiency by lowering tenfold the K_m, without affecting the V. The extension of the hexapeptide T 1 on its C‐terminal side to give the derivative (Arg) ₂‐Pro‐Thr‐Pro‐Ala‐Thr‐Val‐Ala has no significant effect on the kinetic parameters. Moreover no relationship between the phosphorylation efficiency and the predicted secondary structures around the target residue could be evidenced. Therefore the local structural features of the phosphorylatable site of inhibitor‐1 cannot fully account for the fast phosphorylation of this regulatory protein. Other factors must optimize the protein kinase reaction.