HEAT-STABLE INHIBITOR PROTEIN-DERIVED PEPTIDE SUBSTRATE-ANALOGS - PHOSPHORYLATION BY CAMP-DEPENDENT AND CGMP-DEPENDENT PROTEIN-KINASES

The phosphorylation of substrate peptides derived from PKI, the heat-stable inhibitor protein of the cAMP-dependent protein kinase (PKA), has been studied with both PKA and the cGMP-dependent protein kinase (PKG) using a variety of substitution and deletion analogs. On the basis of K-m, k(cat) and k(cat)/K-m values, (Ser(21))PKIalpha(14-22)amide (numbering based upon native PKIalpha is the most effective peptide substrate yet discovered for either kinase, although other peptides, while phosphorylated considerably less efficiently by PKG, are more specific. Although the inhibitory peptide corresponding to this sequence (i.e., with an Ala at position 21) is a much more potent inhibitor of PKA than of PKG (similar to 250-fold), PKG actually exhibits a 60% higher k(cat) than does PKA with the (Ser(21))PKIalpha(14-22)amide substrate peptide, with only a 20-fold higher K-m value. The two key PKI residues within this peptide which were found to be essential for substrate activity with both kinases were Arg(18) (P-3) and Ile(22) (P+1). The Arg(19) (P-2) residue, which contributes significantly to both PKI-based peptide inhibitors and substrates of PKA, was only a more minor contributor to PKG substrate efficacy. Of particular note, the Phe(10) (P-11) residue, which contributes very substantially to high affinity binding of both PKI and longer PKI peptide inhibitors, neither positively nor negatively affects the kinetics of either PKA or PKG with PKI-based substrates. A Phe(10) equivalent residue is not present in the linear sequence of any natural PKA substrate, and it would appear that the PKA hydrophobic binding pocket that recognizes Phe(10) may serve to uniquely contribute to the specificity of the interaction of PKI with PKA. Despite there being clear overall similarities in kinetics between PKI-based peptides acting as substrates for either PKA or PKG, as would be expected given the high degree of homology between the two protein kinases, several subtle kinetic differences suggest some variance between the catalytic processes in each, and these differences are likely correlated with key amino acid substitutions in the catalytic sites of the two kinases.