Isoform-specific differences in the potencies of murine protein kinase inhibitors are due to nonconserved amino-terminal residues

Abstract

We provide here a detailed characterization of two isoforms of the protein kinase inhibitor (PKI) protein of cAMP-dependent protein kinase that have dramatically different inhibition constants. Murine PKIβ1 possesses a 32- fold higher K(i) than murine PKIα as determined by Henderson analysis. This finding led to the investigation of C subunit·PKI interactions involving nonconserved regions in the carboxyl and amino termini of marine PKIα and PKIβ1. Chimeric cDNAs coding for amino acid sequences from both PKI isoforms were constructed and expressed in bacteria. Surprisingly, exchanging the carboxyl-terminal two-thirds of PKIα and PKIβ1 has relatively little effect on the inhibition constants of the two isoforms. Similarly, introducing amino acid residues corresponding to a β-turn region of PKIα into PKIβ1 fails to lower PKIβ1 inhibition constants. However, introducing the amino-terminal α-helical region of PKIα into PKIβ1 reduces the K(i) and IC50 of PKIβ1 to values identical with full length PKIα. Site-directed mutagenesis of specific residues within this region implicates the presence of a tyrosine at position 7 in PKIα as a major contributor to its enhanced inhibitory potency. The results of this study suggest that variations in C subunit·PKI interactions within an amino-terminal α-helix provide a major mechanism for altering the inhibitory properties of PKI isoforms.