ISOFORM-SPECIFIC DIFFERENCES IN THE POTENCIES OF MURINE PROTEIN-KINASE INHIBITORS ARE DUE TO NONCONSERVED AMINO-TERMINAL RESIDUES

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 PKI beta 1 possesses a 32-fold higher K-i than murine PKI alpha 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 murine PKI alpha and PHI beta 1. 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 PKI alpha and PKI beta 1 has relatively little effect on the inhibition constants of the two isoforms. Similarly, introducing amino acid residues corresponding to a beta-turn region of PKI alpha into PKI beta 1 fails to lower PKI beta 1 inhibition constants. However, introducing the amino-terminal alpha-helical region of PKI alpha into PKI beta 1 reduces the K-i and IC50 of PKI beta 1 to values identical with full length PKl alpha. Site-directed mutagenesis of specific residues within this region implicates the presence of a tyrosine at position 7 in PKI alpha 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 alpha-helix provide a major mechanism for altering the inhibitory properties of PKI isoforms.