Protein engineering of protein kinase A catalytic subunits results in the acquisition of novel inhibitor sensitivity.
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 Calpha or Cbeta 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 IC(50) 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, Cbeta 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 Cbeta 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.
Niswender
Colleen M
CM
Department of Pharmacology, University of Washington, Seattle, Washington 98195-7750, USA.
Ishihara
R Wesley
RW
Judge
Luke M
LM
Zhang
Chao
C
Shokat
Kevan M
KM
McKnight
G Stanley
GS
D000409:Alanine / Q000737:chemistry; D000595:Amino Acid Sequence; D000818:Animals; D002384:Catalysis; D020134:Catalytic Domain; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005998:Glycine / Q000737:chemistry; D006801:Humans; D020128:Inhibitory Concentration 50; D007532:Isoleucine / Q000737:chemistry; D007546:Isoquinolines / Q000494:pharmacology; D007930:Leucine / Q000737:chemistry; D008239:Lysine / Q000737:chemistry; D008715:Methionine / Q000737:chemistry; D051379:Mice; D008956:Models, Chemical; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D015202:Protein Engineering; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary; D015398:Signal Transduction; D013449:Sulfonamides; D014158:Transcription, Genetic; D014162:Transfection; D014407:Tumor Cells, Cultured
D004791:Enzyme Inhibitors; D007546:Isoquinolines; D020033:Protein Isoforms; D013449:Sulfonamides; D007532:Isoleucine; D008715:Methionine; D017868:Cyclic AMP-Dependent Protein Kinases; D007930:Leucine; D008239:Lysine; C063509:N-(2-(4-bromocinnamylamino)ethyl)-5-isoquinolinesulfonamide; D000409:Alanine; D005998:Glycine