A cell-permeable, activity-based probe for protein and lipid kinases.
Protein and lipid kinases are two important classes of biomedically relevant enzymes. The expression and activity of many kinases are known to be dysregulated in a variety of diseases, and proteomic tools that can assess the presence and activity of these enzymes are likely to be useful for their evaluation. Because many of the mechanisms by which protein kinases can become unregulated involve post-translational modifications or changes in protein localization, they can only be detected by examining protein activity, sometimes within the context of the living cell. Wortmannin is a steroid-derived fungal metabolite that covalently inhibits both protein and lipid kinases. Here we describe the synthesis of three wortmannin derivatives, biotin-wortmannin, BODIPY-wortmannin, and tetramethylrhodamine-wortmannin. We demonstrate that these reagents exhibit reactivity similarly as wortmannin and react with members of the phosphatidylinositol 3-kinase and PI3-kinase related kinase families in cellular lysates. Moreover, in some cases these reagents can differentiate between the active and inactive forms of the enzyme, indicating that they are activity-based probes. The reagents also exhibit complementary properties. The biotin-wortmannin reagent is effective in the isolation of labeled proteins; all three can be used for protein labeling, and BODIPY-wortmannin is cell-permeable and can be used to label proteins within cells.
Yee
Muh-Ching
MC
Department of Molecular Pharmacology, Stanford University, Stanford, CA 94305-5441, USA.
Fas
Stefanie C
SC
Stohlmeyer
Michelle M
MM
Wandless
Thomas J
TJ
Cimprich
Karlene A
KA
D000730:Androstadienes / Q000737:chemistry* / Q000494:pharmacology; D001671:Biochemistry / Q000379:methods*; D001710:Biotin / Q000737:chemistry; D001896:Boron Compounds / Q000737:chemistry; D002460:Cell Line; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005456:Fluorescent Dyes / Q000494:pharmacology; D005982:Glutathione Transferase / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D020128:Inhibitory Concentration 50; D008055:Lipids / Q000737:chemistry*; D008956:Models, Chemical; D019869:Phosphatidylinositol 3-Kinases / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D012235:Rhodamines / Q000737:chemistry; D013997:Time Factors; D014162:Transfection; D000077191:Wortmannin
C095489:4,4-difluoro-4-bora-3a,4a-diaza-s-indacene; D000730:Androstadienes; D001896:Boron Compounds; D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D008055:Lipids; D012235:Rhodamines; C005358:tetramethylrhodamine; D001710:Biotin; D005982:Glutathione Transferase; D019869:Phosphatidylinositol 3-Kinases; D000077191:Wortmannin