Use of pseudosubstrate affinity to measure active protein kinase A.
Traditional cAMP-dependent protein kinase (also known as protein kinase A [PKA]) assays, which are based on substrate phosphorylation, often have high background activity from other kinases, thereby limiting sensitivity and making it difficult to detect low levels of active PKA in cell lysates. Therefore, a better technique that measures active PKA in crude cell lysates undoubtedly is necessary. We developed an efficient and sensitive assay to compare active PKA levels based on binding of the active PKA catalytic subunit to its pseudosubstrate domain inhibitor (PKI) fused with glutathione S-transferase (GST-PKI). This pseudosubstrate affinity assay can detect variations in the active PKA levels in the presence of common inducers of PKA activity such as forskolin and prostaglandins. It has resolution to detect a concentration-dependent curve of active PKA in a linear range, and it also has sensitivity to detect up to 2.5 ng of active enzyme. An observed change in the binding affinity between PKA and PKI in the presence of the PKA inhibitor N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H89) shows that this assay can be successfully used to measure how active PKA is affected by specific inhibitors. We conclude that this method is a simple, inexpensive, and nonhazardous method to compare active PKA levels with high sensitivity and specificity with negligible background.
Paulucci-Holthauzen
Adriana A
AA
Department of Surgery and Sealy Center for Cancer Cell Biology, University of Texas Medical Branch, Galveston, TX 77555, USA.
O'Connor
Kathleen L
KL
D000595:Amino Acid Sequence; D001665:Binding Sites; D020134:Catalytic Domain; D002462:Cell Membrane / Q000378:metabolism*; D002478:Cells, Cultured; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism*; D004789:Enzyme Activation; D005982:Glutathione Transferase / Q000378:metabolism; D007546:Isoquinolines / Q000494:pharmacology; D008969:Molecular Sequence Data; D047428:Protein Kinase Inhibitors / Q000494:pharmacology*; D013379:Substrate Specificity; D013449:Sulfonamides / Q000494:pharmacology
D007546:Isoquinolines; D047428:Protein Kinase Inhibitors; D013449:Sulfonamides; D005982:Glutathione Transferase; D017868:Cyclic AMP-Dependent Protein Kinases