Assaying Bcr-Abl kinase activity and inhibition in whole cell extracts by phosphorylation of substrates immobilized on agarose beads.
There is a current and increasing demand for simple, robust, nonradioactive assays of protein tyrosine kinase activity with applications for clinical diagnosis and high-throughput screening of potential molecularly targeted therapeutic agents. One significant challenge is to detect and measure the activity of specific kinases with key roles in cell signaling as an approach to distinguish normal cells from cancer cells and as a means of evaluating targeted drug efficacy and resistance in cancer cells. Here, we describe a method in which kinase substrates fused to glutathione-S-transferase and immobilized on glutathione agarose beads are phosphorylated, eluted, and then assayed to detect kinase activity. The activity of recombinant, purified c-Abl kinase or Bcr-Abl kinase in whole cell extracts can be detected with equivalent specificity, sensitivity, and reproducibility. Similarly, inhibition of recombinant c-Abl or Bcr-Abl in cells or cell extracts by imatinib mesylate and other Bcr-Abl targeted kinase inhibitors is readily assayed. This simple kinase assay is sufficiently straightforward and robust for use in clinical laboratories and is potentially adaptable to high-throughput assay formats.
Wu
Ding
D
Center for Molecular Oncology and Department of Molecular Genetics and Cell Biology, University of Chicago, IL 60637, USA.
Nair-Gill
Evan
E
Sher
Dorie A
DA
Parker
Laurie L
LL
Campbell
Jennifer M
JM
Siddiqui
Mariah
M
Stock
Wendy
W
Kron
Stephen J
SJ
D000595:Amino Acid Sequence; D000818:Animals; D001549:Benzamides; D002457:Cell Extracts / Q000737:chemistry; D002478:Cells, Cultured; D016044:Fusion Proteins, bcr-abl; D005982:Glutathione Transferase / Q000737:chemistry; D000068877:Imatinib Mesylate; D051379:Mice; D008863:Microspheres; D008969:Molecular Sequence Data; D010766:Phosphorylation; D010879:Piperazines / Q000494:pharmacology; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011505:Protein-Tyrosine Kinases / Q000032:analysis* / Q000037:antagonists & inhibitors; D011743:Pyrimidines / Q000494:pharmacology; D012685:Sepharose / Q000737:chemistry*
D001549:Benzamides; D002457:Cell Extracts; D010879:Piperazines; D047428:Protein Kinase Inhibitors; D011743:Pyrimidines; D000068877:Imatinib Mesylate; D012685:Sepharose; D005982:Glutathione Transferase; D011505:Protein-Tyrosine Kinases; D016044:Fusion Proteins, bcr-abl