High yield expression of serine/threonine protein phosphatase type 5, and a fluorescent assay suitable for use in the detection of catalytic inhibitors.
Protein phosphatase type 5 (PP5) belongs to the PPP family of serine/threonine protein phosphatases and is expressed in most, if not all, human tissues. Although the physiological roles played by PP5 are not yet clear, PP5 is found in association with several proteins that influence intracellular signaling networks initiated by hormones (i.e., glucocorticoids) or cellular stress (i.e., hypoxia, oxidative stress). Recently, studies conducted with short interfering RNA and antisense oligonucleotides indicate that PP5 plays an important role in the regulation of stress-induced signaling cascades that influence both cell growth and the onset of apoptosis. Therefore, the identification of small molecule inhibitors of PP5 is desired for use in studies to further define the biological/pathological roles of PP5. Such inhibitors may also prove useful for development into novel antitumor agents. Here we describe methods to express and purify large amounts of biologically active PP5c, an inhibitor titration-based assay to determine the amount of PP5 in solution, and a fluorescent phosphatase assay that can be used to screen chemical libraries and natural extracts for the presence of catalytic inhibitors.
Ni
Li
L
Department of Biochemistry and Molecular Biology, College of Medicine, University of South Alabama, Mobile, AL 36688, USA.
Swingle
Mark S
MS
Bourgeois
Austin C
AC
Honkanen
Richard E
RE
D009597:4-Nitrophenylphosphatase / Q000378:metabolism; D002384:Catalysis; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D004926:Escherichia coli / Q000378:metabolism; D005456:Fluorescent Dyes; D006801:Humans; D006923:Hymecromone / Q000031:analogs & derivatives / Q000737:chemistry; D007202:Indicators and Reagents; D009687:Nuclear Proteins / Q000037:antagonists & inhibitors* / Q000096:biosynthesis* / Q000302:isolation & purification; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors* / Q000096:biosynthesis* / Q000302:isolation & purification; D013050:Spectrometry, Fluorescence
D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D007202:Indicators and Reagents; D009687:Nuclear Proteins; C005359:4-methylumbelliferyl phosphate; D006923:Hymecromone; D010749:Phosphoprotein Phosphatases; C089641:protein phosphatase 5; D009597:4-Nitrophenylphosphatase