Journal of Biological Chemistry
Volume 277, Issue 32, 9 August 2002, Pages 28916-28922
Journal home page for Journal of Biological Chemistry

ENZYME CATALYSIS AND REGULATION
Protein Engineering of Protein Kinase A Catalytic Subunits Results in the Acquisition of Novel Inhibitor Sensitivity*

https://doi.org/10.1074/jbc.M203327200Get rights and content
Under a Creative Commons license
open access

Analysis of the role of specific protein kinases in signal transduction networks has relied heavily on ATP analog inhibitors. Currently used agents, however, often do not distinguish between kinase family members. Genetic approaches can also be used to inactivate a specific kinase, but these techniques do not afford the rapid kinetics possible with pharmacological inhibitors. To circumvent this problem, modification of the structure of a particular protein kinase can be performed to engineer a drug-target interaction of choice. We have used this method to create protein kinase A (PKA) catalytic subunits with modifications that confer sensitivity to novel ATP analog inhibitors. Mutation of methionine 120 to alanine or glycine in either the Cα or Cβ subunits of PKA induces sensitivity to a series of C-3 derivatized pyrazolo[3,4-d]pyrimidine-based inhibitors. Modification of threonine 183 enhances this inhibitor sensitivity. The IC50 values in cell culture of the most broadly effective agent, 1-NM, ranged from 25 to 200 nm depending upon the combination of modified amino acids and were significantly higher than the potencies observed with H-89. Despite their high sequence conservation, Cβ enzymes with inhibitor-sensitive amino acids at position 120 showed a substantial loss of overall catalytic activity when used to induce reporter gene transcription in transfected cells. Conversion of position 46 (lysine to isoleucine) rescued the ability of position 120 mutated Cβ enzymes to induce gene transcription. Application of this combined genetic and pharmacological approach should allow analysis of the specific roles of PKA isoforms in cell culture and in vivo.

Cited by (0)

Published, JBC Papers in Press, May 28, 2002, DOI 10.1074/jbc.M203327200

*

This work was supported by National Institutes of Health Grants F32 DK10005-03 (to C. M. N.), 5T32 GM0266 (to L. M. J.), AI44009 (to K. M. S.), and GM32875 (to G. S. M.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.