Design and Synthesis of AX7574: A Microcystin-Derived ...

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A Microcystin-Derived Phosphatase Probe Bioconjugate Chem., Vol. 15, No. 4, 2004 795Figure 2. Mass spectrometric (MS) structural identification of AX7574. Shown are some of the observed fragmentation patternsfrom the two major parent ions observed during the MS/MS analysis of the doubly charged parent ion peak. Dual letter fragmentmasses (vide infra) are derived from a counterclockwise trace along the cyclic peptide backbone of AX7574 from the designated pointof fragmentation to the next indicated point of fragmentation: ea - H 2O ) 592, ga + NH 2-H 2O ) 521, fb ) 842, fb - CO ) 814, dc) ih ) 971, gc ) ai ) 786, hc ) 703, ic ) ag ) 574, ad ) 390, af ) 503, ah ) 657, jh ) 674, hj - H 2O ) 408.Downloaded by WUHAN UNIV on October 25, 2009 | http://pubs.acs.orgPublication Date (Web): June 18, 2004 | doi: 10.1021/bc0499580Figure 3. Kinetics of AX7574 labeling of PP-1. PP-1 (25 nM)was labeled with AX7574 (0.5 µM). At the indicated timepoints,sample aliquots were quenched. The quenched samples wereelectrophoresed, the gel was scanned, and the fraction of PP-1labeled was determined as described in the ExperimentalProcedures. The curve drawn is the best fit of the data to a singleexponential.Figure 4. Inhibition of AX7574 labeling of PP-1 with phosphataseinhibitors. PP-1 (25 nM) was labeled with AX7574 (0.5µM) (A) in the absence of phosphatase inhibitors or in thepresence of (B) microcystin-LR, (C) calyculin A, (D) proteinphosphatase inhibitor 2, and (E) fostriecen at 100 nM each. Anadditional AX7574 (0.5 µM) labeling experiment using denaturedPP-1 (25 nM, F) is also shown. Labeling reactions werequenched after 3 h. The samples were electrophoresed, the gelwas scanned, and the scanned image was quantified as describedin the Experimental Procedures.PP-1 (see Figure 4). In the first set of experiments,inhibition of AX7574 labeling with known inhibitors ofFigure 5. PP-1 IC 50 determination for microcystin (b, IC 50 )0.3 nM) and AX7574 (O, IC 50 ) 4.0 nM). The curves drawn arethe best fit of the data to a variation of the Hill equation (seeExperimental Procedures section).PP-1 was tested. The inhibitors selected included microcystin,calyculin A (PP-1 IC 50 ) 1.2 nM) (29), and proteinphosphatase inhibitor 2 (PP-1 IC 50 ) 1 nM) (42). Theseparticular inhibitors were selected because they interactwith phosphatases through diverse mechanisms. Forexample, microcystin is an irreversible inhibitor, and bothcalyculin A and protein phosphatase inhibitor 2 arereversible ones. Furthermore, while microcystin andcalyculin A are small molecules, protein phosphataseinhibitor 2 is a protein macromolecule (MW ) 31 kDa).In addition, fostriecin, a weak inhibitor of PP-1 (PP-1 IC 50) 131 µM, PP-2A IC 50 ) 3.2 nM), was included as anegative control. PP-1 (25 nM) was preincubated for 10min with microcystin (100 nM), calyculin A (100 nM),protein phosphatase inhibitor 2 (100 nM), or fostriecin(100 nM) or allowed to stand for 10 min in the absenceof added phosphatase inhibitors. Afterward, AX7574 (0.5µM) was added to each sample and the labeling reactionstopped after 3 h using DTT-containing sample buffer.When compared to the same labeling reaction done inthe absence of phosphatase inhibitors, significantly lesslabeling with AX7574 was observed in the presence ofmicrocystin, calyculin A, or protein phosphatase inhibitor2. In the case of microcystin and calyculin A, the resultsindicate that AX7574 competes for the active site wherethese inhibitors are known to bind. As expected, the PP-2A specific inhibitor, fostriecin, did not cause a significantdecrease in labeling of PP-1 by AX7574.
796 Bioconjugate Chem., Vol. 15, No. 4, 2004 Shreder et al.Downloaded by WUHAN UNIV on October 25, 2009 | http://pubs.acs.orgPublication Date (Web): June 18, 2004 | doi: 10.1021/bc0499580Figure 6. AX7574 can detect serine/threonine phosphatases in a proteome. (A) Soluble proteome derived from Jurkat cellspreincubated with or without microcystin (1 µM) or calyculin a (1 µM) for 10 min and then treated with AX7574 (0.5 µM) for 60 min.Reactions were quenched with standard 2× SDS/PAGE loading buffer (reducing), separated from unreacted AX7574 using SDS-PAGE, and visualized in-gel using a flatbed laser-induced fluorescence scanner. (B) Coomassie blue stained gel from Figure 6Aconfirming that all three lanes contained approximately equal amounts of protein. (C) AX7574-labeled proteins identified using MSprotein identification.Figure 7. AX7574 can detect the decrease of serine/threonine protein phosphatase activities in calyculin A-treated Jurkat T cells.(A) Jurkat cells treated with calyculin A (25 nM) for 60 min or control DMSO, and soluble proteome derived from these cells weretreated with AX7574 (0.5 µM) for 60 min. Reactions were quenched with standard 2× SDS/PAGE loading buffer (reducing), separatedby SDS/PAGE, and visualized in-gel using a flatbed laser-induced fluorescence scanner. (B) Side-trace analysis of the gel data fromFigure 7A. (C) The scanned gel from A was transferred to nitrocellulose and immunoblotted with anti-PP1 and anti-PP2A.A second kind of experiment was carried out toexamine whether the labeling of PP-1 by AX7574 requiredPP-1 to retain its native tertiary structure (seeFigure 4). To address this question, PP-1 (25 nM) wasfirst denatured by heating the protein at 65 °C for 10min. The subsequent addition of AX7574 (0.5 µM) resultedin no significant labeling of PP-1. This resultindicates that the recognition and subsequent attachmentof AX7574 to PP-1 does in fact require the nativestructure of PP-1.To investigate the impact of TAMRA conjugation onthe affinity of the microcystin scaffold for PP-1, the IC 50values of both microcystin-LR and AX7574 for PP-1 weredetermined and compared (see Figure 5). Both compoundsat various concentrations (0-30 nM) were incubatedwith PP-1 (5 nM) for 10 min. Postincubation,p-nitrophenyl phosphate (pNPP, 20 mM) was added andthe degree of substrate hydrolysis measured spectroscopicallyat 450 nm. For each inhibitor, A 450 was plottedversus inhibitor concentration, and the points were fitto a variation of the Hill equation (43) to determine IC 50values. Such an analysis yielded IC 50 values of 0.3 and4.0 nM for microcystin and AX7574, respectively. Bycomparison, a similar pNPP-based determination yieldeda PP-1 IC 50 value of 0.25 nM for microcystin-LR (44). Asevidenced by these values, TAMRA conjugation had onlya modest impact on binding and the resulting probeAX7574 remained a potent PP-1 inhibitor.To test whether AX7574 would react with serine/threonine protein phosphatases in a complex proteome,we incubated soluble fractions of Jurkat cells with thisprobe (0.5 µM) for 60 min. Protein separation of the
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