Development of fluorescence-based selective assays for serine/threonine and tyrosine phosphatases.
A number of aromatic substrates were evaluated for their ability to detect tyrosine phosphatase and serine/threonine phosphatase activity. Results demonstrated that the fluorinated coumarin DiFMUP is the most sensitive substrate for detecting LAR and PP-2A activity. Using this substrate, selective high-throughput screening assays for serine/threonine and tyrosine phosphatases were developed. Specific inhibitor cocktails were added to each assay to limit the activity of other phosphatases. LAR, CD-45, and PTP-1B all rapidly hydrolyze DiFMUP in the tyrosine phosphatase assay. The activity of non-tyrosine phosphatases is less than 6% of the LAR activity. PP-1 and PP-2A are highly active in the serine/threonine phosphatase assay. Inhibition of LAR and PP-2A in these assays is demonstrated using known inhibitors. Both of these assays are sensitive, robust, kinetic assays that can be used to quantify enzyme activity.
Pastula
Christina
C
Biomics Department, Molecular Probes, Inc., 29851 Willow Creek Rd, Eugene, Oregon, 97402, USA. christina.pastula@probes.com
Johnson
Iain
I
Beechem
Joseph M
JM
Patton
Wayne F
WF
D000818:Animals; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D006923:Hymecromone / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D019169:Jurkat Cells; D007700:Kinetics; D010749:Phosphoprotein Phosphatases / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000378:metabolism; D017027:Protein Tyrosine Phosphatases / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000378:metabolism; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence
C119285:6,8-difluoro-4-methylumbelliferyl phosphate; D004791:Enzyme Inhibitors; D006923:Hymecromone; D010749:Phosphoprotein Phosphatases; D017027:Protein Tyrosine Phosphatases