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 PKI beta 1 possesses a 32-fold higher Ki 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 PKI 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 Ki and IC50 of PKI beta 1 to values identical with full length PKI 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.
Highlights
From the Weuroscience Program, §Mental Health Research Institute and §Department of Biological Chemistry, University of Michigan, Ann Arbor, Michigan 48109
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
The results ofthis study suggest that variations in C subunit·PKI interactions within an amino-terminal a-helix provide a major mechanism for altering the inhibitory properties of PKI isoforms
Summary
Vol 270, No 13, Issue of March 31, pp. 7227-7232, 1995 Printed in U.S.A. Isoform-specific Differences in the Potencies of Murine Protein Kinase Inhibitors Are Due to Nonconserved Amino-terminal Residues*. From the Weuroscience Program, §Mental Health Research Institute and §Department of Biological Chemistry, University of Michigan, Ann Arbor, Michigan 48109. The costs of publication of this article were defrayed in part by the payment of page charges. To probe the structural cause of this difference in inhibitory potency, we produced recombinant PKI proteins that contained selected amino acid sequences from both murine PKIa and PKIf3l These chimeric PKIs were subjected to kinetic analysis. Results from this study suggest that amino acid changes within the amino-terminal amphipathic a-helix of murine PKIa can fully account for the difference in inhibitory potency between the two murine PKI isoforms Together, these experiments constitute the first mutational analysis of a PKIf3 protein, and the first investigation of the structural basis for a difference in PKI isoform function
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