Protein Kinase C Contains a Pseudosubstrate Prototope in Its Regulatory Domain

Abstract

The regulatory domain of protein kinase C contains an amino acid sequence between residues 19 and 36 that resembles a substrate phosphorylation site in its distribution of basic residue recognition determinants. The corresponding synthetic peptide (Arg 19 -Phe-Ala-Arg-Lys-Gly- Ala 25 -Leu-Arg-Gln-Lys-Asn-Val-His-Glu-Val-Lys-Asn 36 ) acts as a potent substrate antagonist with an inhibitory constant of 147 ± 9 n M . It is a specific inhibitor of protein kinase C and inhibits both autophosphorylation and protein substrate phosphorylation. Substitution of Ala 25 with serine transforms the pseudosubstrate into a potent substrate. These results demonstrate that the conserved region of the regulatory domain (residues 19 to 36) of protein kinase C has the secondary structural features of a pseudosubstrate and may be responsible for maintaining the enzyme in the inactive form in the absence of allosteric activators such as phospholipids.