Protein-acrylamide copolymer hydrogels for array-based detection of tyrosine kinase activity from cell lysates.
We describe the development of an array-based assay for the molecular level detection of tyrosine kinase activity directly from cellular extracts. Glutathione S-transferase-Crkl (GST-Crkl) fusion proteins are covalently immobilized into polyacrylamide gel pads via copolymerization of acrylic monomer and acrylic-functionalized GST-Crkl protein constructs on a polyacrylamide surface. The resulting hydrogels resist nonspecific protein adsorption, permitting quantitative and reproducible determination of Abl tyrosine kinase activity and inhibition, even in the presence of a complex cell lysate mixture. Half-maximal inhibition (IC50) values for imatinib mesylate inhibition of GST-Crkl (SH3) phosphorylation by v-Abl in a purified system and Bcr-Abl within a K562 cell lysate were determined to be 1.5 and 20 microM, respectively. Additionally, the protein-acrylamide copolymer arrays detected CML cell levels as low as 15% in a background of Bcr-Abl- leukemic cells and provided the framework for the parallel evaluation of six tyrosine kinase inhibitors. Such a system may have direct application to the detection and treatment of cancers resulting from upregulated tyrosine kinase activity, such as chronic myeloid leukemia (CML). These findings also establish a basis for screening tyrosine kinase inhibitors and provide a framework on which protein-protein interactions in other complex systems can be studied.
Brueggemeier
Shawn B
SB
Department of Chemical and Biological Engineering, University of Wisconsin-Madison, 1415 Engineering Drive, Madison, Wisconsin 53706, USA.
Wu
Ding
D
Kron
Stephen J
SJ
Palecek
Sean P
SP
D000178:Acrylamides / Q000737:chemistry*; D000180:Acrylic Resins / Q000737:chemistry; D000327:Adsorption; D001549:Benzamides; D001672:Biocompatible Materials / Q000737:chemistry; D001704:Biopolymers / Q000737:chemistry; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005982:Glutathione Transferase / Q000378:metabolism; D018922:HL-60 Cells; D006801:Humans; D020100:Hydrogels / Q000737:chemistry; D000068877:Imatinib Mesylate; D020128:Inhibitory Concentration 50; D020014:K562 Cells; D015464:Leukemia, Myelogenous, Chronic, BCR-ABL Positive / Q000188:drug therapy; D008956:Models, Chemical; D010766:Phosphorylation; D010879:Piperazines / Q000494:pharmacology; D011107:Polylysine / Q000737:chemistry; D011108:Polymers / Q000737:chemistry; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D011506:Proteins / Q000737:chemistry; D016315:Proto-Oncogene Proteins c-abl / Q000378:metabolism; D011743:Pyrimidines / Q000494:pharmacology; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D000099166:Polyacrylamides
D000178:Acrylamides; D000180:Acrylic Resins; D001549:Benzamides; D001672:Biocompatible Materials; D001704:Biopolymers; D004791:Enzyme Inhibitors; D005982:Glutathione Transferase; D020100:Hydrogels; D000068877:Imatinib Mesylate; D010879:Piperazines; D011107:Polylysine; D011108:Polymers; D011505:Protein-Tyrosine Kinases; D011506:Proteins; D016315:Proto-Oncogene Proteins c-abl; D011743:Pyrimidines; D011993:Recombinant Fusion Proteins; D000099166:Polyacrylamides