Articles pubs.acs.org/acschemicalbiology
Interrogating Endogenous Protein Phosphatase Activity with Rationally Designed Chemosensors Jon R. Beck,† Antoneal Lawrence,‡ Amar S. Tung,‡ Edward N. Harris,# and Cliff I. Stains*,† †
Department of Chemistry, University of Nebraska-Lincoln, Lincoln, Nebraska 68588, United States Department of Biochemistry, University of Nebraska-Lincoln, Lincoln, Nebraska 68588, United States ‡ Department of Chemistry, Lincoln University, Lincoln University, Pennsylvania 19352, United States #
S Supporting Information *
ABSTRACT: We introduce a versatile approach for repurposing protein kinase chemosensors, containing the phosphorylationsensitive sulfonamido-oxine fluorophore termed Sox, for the specific determination of endogenous protein phosphatase activity from whole cell lysates and tissue homogenates. As a demonstration of this approach, we design and evaluate a direct chemosensor for protein tyrosine phosphatase-1B (PTP1B), an established signaling node in human disease. The optimal sensor design is capable of detecting as little as 6 pM (12 pg) fulllength recombinant PTP1B and is remarkably selective for PTP1B among a panel of highly homologous tyrosine phosphatases. Coupling this robust activity probe with the specificity of antibodies allowed for the temporal analysis of endogenous PTP1B activity dynamics in lysates generated from HepG2 cells after stimulation with insulin. Lastly, we leveraged this assay format to profile PTP1B activity perturbations in a rat model of nonalcoholic fatty liver disease (NAFLD), providing direct evidence for elevated PTP1B catalytic activity in this disease state. Given the modular nature of this assay, we anticipate that this approach will have broad utility in monitoring phosphatase activity dynamics in human disease states.
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for NAFLD and is also utilized as a proxy to infer increased catalytic activity which is thought to contribute to the insulin resistance phenotype observed in this disease.17,18 However, chemical approaches capable of routinely assessing relevant amounts of PTP1B activity in human disease models have been lacking, leading to the common implementation of indirect proxies to estimate enzymatic function. To address this issue, we set out to interrogate endogenous PTP1B activity in a dietinduced rat model of NAFLD by developing a sensitive, straightforward activity assay to directly monitor PTP1B function. The catalytic activity of protein tyrosine phosphatases is modulated, in part, by post-translational modification of the catalytic cysteine.19−21 As a consequence, a variety of techniques to investigate modifications to, and reactivity of, the catalytic cysteine have been described.22−24 One such example is activity-based protein profiling (ABPP) probes that form a covalent bond with the active site cysteine or adjacent nucleophiles within the PTP active site.23,25−27 These probes represent a powerful approach to rapidly assess the reactivity of the PTP active site cysteine. However, these probes are not true substrates for PTPs, and therefore, labeling with these probes may not always reflect catalytic activity. For example, ABPP probes have been shown to react equally well with catalytically inactive PTPs as compared to wild-type enzymes.23 Moreover,
rotein phosphatases catalyze the dephosphorylation of protein substrates, acting in opposition to protein kinases in order to maintain the integrity of the phosphoproteome. For many years, protein phosphatases have been viewed as housekeeping enzymes, counteracting the activity of protein kinases1 which have established roles in human disease. More recently, there has been a renaissance in protein phosphatase research that has greatly aided our understanding of the phosphoproteome and the complexity that surrounds its regulation. For example, the human genome encodes 105 protein tyrosine phosphatases (PTPs), implying a possible level of fidelity in signaling on par with the 90 protein tyrosine kinases (PTKs).2−4 The first protein tyrosine phosphatase described in the literature, PTP1B,5,6 has now been established as a node in a variety of cellular signaling pathways including insulin signaling, through dephosphorylation of the insulin receptor tyrosine kinase (IRTK).7 In addition, increasing evidence suggests that dysregulation of PTP1B can play a significant role in a variety of human disease states8−11 such as hepatocellular carcinoma (HCC)12 and the metabolic disorder known as NAFLD. 13 NAFLD is characterized as the accumulation of >5% fat by weight in the liver. Due to the current obesity epidemic in the U.S., it is estimated that 20− 30% of the U.S. population is afflicted with NAFLD.13,14 Furthermore, there is accumulating evidence that NAFLD patients have a higher risk for developing nonviral HCC.15 Indeed, this correlation may explain, in part, the rise in HCC cases since 1975.16 Overexpression of PTP1B in both animal models and clinically derived tissues can be used as a biomarker © 2015 American Chemical Society
Received: July 2, 2015 Accepted: November 18, 2015 Published: November 18, 2015 284
DOI: 10.1021/acschembio.5b00506 ACS Chem. Biol. 2016, 11, 284−290
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the chemical instability and potential off-target reactivity of ABPP probes has limited their widespread use.25 Alternatively, there have been relatively few examples of selective phosphatase assays capable of directly quantifying phosphatase catalytic activity in biological samples in a target specific manner.25 One notable exception is the peptide-based probes described by the Barrios laboratory, which leverage a phosphocoumaryl amino propionic acid (pCAP) reporter.28 Peptide substrates incorporating pCAP undergo an increase in fluorescence upon dephosphoryaltion of pCAP by a target PTP. Recently, cell-permeable pCAP-based probes that were preferentially dephosphoarylated by CD45 have been utilized for high-throughput inhibitor screening by flow cytometry.29 Lawrence and co-workers have shown that the activity of the highly promiscuous tyrosine phosphatase known as YOP, from Y. enterocolitica, can be monitored in vitro through a turn-off fluorescence approach using peptide substrates containing a pyrene chromophore proximal to the site of dephosphorylation.30 Lastly, FRET has been employed to monitor the interaction between PTP1B and a peptide substrate in living cells.31 Although these direct activity probes are powerful, additional work is needed in order to define a generalizable platform capable of sensitively and specifically monitoring endogenous phosphatase activity in biological samples. We hypothesized that the extensive efforts to develop direct activity assays for protein kinases32 could be repurposed to afford analogous direct activity assays for protein phosphatases. In particular, the phosphorylation-sensitive Sox fluorophore can be covalently appended to an engineered cysteine residue in a peptide substrate and utilized to report tyrosine or serine/ threonine kinase activity via chelation-enhanced fluorescence (CHEF) in response to phosphorylation.33−41 Sox-based assays provide a real-time readout of kinase activity and can be utilized to interrogate kinase signaling perturbations in biologically relevant samples, such as cell lysates and tissue homogenates.36,42 Building upon this work, we set out to investigate whether the Sox fluorophore could be employed in a complementary manner in order to monitor the dephosphorylation of a synthetic peptide substrate, producing a decrease in fluorescence signal over time (Figure 1a).
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RESULTS AND DISCUSSION
Given its role in human disease, we chose to develop a Soxbased probe for PTP1B as a proof-of-principle to test our hypothesis. We based the probe sequence on the autophosphorylation site of anaplastic lymphoma kinase (ALK), a known native substrate of PTP1B ( 12 7 4 ARDIYRASYYRKG1286).43 We initially synthesized three derivatives of the above sequence and incorporated the Sox fluorophore by on-resin alkylation of a cysteine residue placed proximal to the site of dephosphorylation. Tyrosine residues at positions other than the site of dephosphorylation were mutated to phenylalanine so that potential cross reactivity with PTKs in subsequent lysate experiments could be avoided. We termed these three constructs PTP1Btide-pS1 (ARDIpYRA-CSoxFFRKG), PTP1Btide-pS2 (ARDIpYR-CSox-SFFRKG), and PTP1Btide-pS3 (ARDIpYR-CSox-FFRKG). Additionally, we synthesized the corresponding nonphosphorylated peptides, PTP1Btide-S1 (ARDIYRA-CSox-FFRKG), PTP1Btide-S2 (ARDIYR-CSox-SFFRKG) and PTP1Btide-S3 (ARDIYR-CSoxFFRKG) to act as positive controls. Each peptide was purified by reverse-phase HPLC (Figure S1) and identities were confirmed by ESI-MS (Table S1). In order to determine the optimal assay conditions for these potential PTP1B probes, we measured the dissociation constants (KD) for Mg2+ for each peptide (Figure S2) and used these values in order to define optimal Mg2+ concentrations for assay reactions with each peptide.40 We found that the optimal concentration of Mg2+ was 25 mM for PTP1Btide-pS1, 9 mM for PTP1Btide-pS2, and 7 mM for PTP1Btide-pS3 with fold fluorescence increases of 3.4, 6.4, and 4.6 respectively (Table S2). We next endeavored to determine whether these probes would act as substrates for recombinant PTP1B. Gratifyingly, we observed a decrease in the fluorescence of each probe in the presence of full-length recombinant PTP1B (Figure S3). Kinetic parameters for each sensor were obtained by varying the concentration of the sensor; rates of product formation were calculated as described in the Supporting Information. Comparisons of the kinetic parameters for each peptide are given in Figure 1b. These results reinforce the increased catalytic efficiency of peptide substrates for PTPs compared to generic substrates such as p-nitrophenyl phosphate (pNPP, kcat/KM = 7.9 × 103 M−1 s−1).44 Interestingly, all three peptides demonstrated substrate inhibition at high concentrations (Figure S4). We determined that by assaying at sub KM concentrations of substrate, we could avoid any appreciable inhibition, underscoring the need to fully characterize peptidebased phosphatase activity sensors. On the basis of catalytic efficiency, we elected to proceed with PTP1Btide-pS3 as the optimal substrate and sought to define the limit of detection for PTP1B using PTP1Btide-pS3. By varying the concentrations of recombinant PTP1B in the assay, we determined that we could reliably report on recombinant PTP1B concentrations as low as 6 pM or 12 pg in vitro (Figure 2a). This represents a significant improvement over the detection limit of pNPP (3 ng PTP1B). Furthermore, assay results were highly reproducible with a Z′factor of 0.7 using 100 pM PTP1B. Encouraged by the robust nature of this sensor, we evaluated the potential of using this assay format to report on PTP1B inhibition. To accomplish this, we assayed PTP1B activity against varying concentrations of sodium orthovanadate, a known PTP inhibitor. We found that the IC50 of sodium orthovanadate for PTP1B in this assay format was 31 nM (Figure 2b), consistent with the previously
Figure 1. Sox-based protein phosphatase sensors. (a) In the presence of Mg2+, the Sox chromophore undergoes CHEF when proximal to a phosphoryl-amino. Phosphatase activity removes the phosphate group from the peptide probe and greatly reduces the affinity for Mg2+, leading to a decrease in fluorescence. (b) Kinetic parameters for PTP1B chemosensor designs. 285
DOI: 10.1021/acschembio.5b00506 ACS Chem. Biol. 2016, 11, 284−290
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that are disfavored in highly efficient substrates. For example, basic residues are well-known to be disfavored in highly efficient PTP1B substrates (PTP1Btide-pS3 contains four basic amino acids, see Figure 1). Our results indicate that selectivity between PTP1B and TCPTP may be more readily achieved by utilizing nonoptimized PTP1B peptide substrates with reduced catalytic efficiency (Figure 1b). Taken together, these data reinforce the idea that PTPs are capable of selective recognition of peptide substrates at a level that is comparable to PTKs.53 While encouraged by the results of this experiment, we suspected that PTPs in a biological sample that were not represented in this panel might be capable of dephosphorylating the sensor, in a similar manner to off-target effects observed with protein kinases and peptide-based chemosensors.35,36,40 To test this hypothesis, we generated lysates from MEF PTP1B+/+ and MEF PTP1B−/− cell lines.7 Importantly, MEF PTP1B−/− cell lysates allow for the assessment of probe specificity in a biological context as they contain all endogenous phosphatases found in MEF PTP1B+/+ cells except PTP1B. Studies with these lysates showed that PTP1Btide-pS3 was dephosphorylated to a greater extent in MEF PTP1B+/+ lysates; however, a significant amount of unknown off-target activity was observed in MEF PTP1B−/− lysates (Figure S10). Previous protein kinase chemosensors have relied on inhibitors to suppress off-target activity, enabling the analysis of protein kinase activity in unfractionated lysates.35,36,40,42 However, compared to the significant advances in kinase inhibitors,54−57 there remains a paucity of highly selective PTP inhibitors. Consequently, we turned to antibody enrichment19 in order to overcome off-target effects (Figure 3a). To develop this pulldown assay in a controlled context, we verified that the nonselective enzyme YOP could dephosphorylate PTP1BtidepS3 (Figure S11). Next we optimized the pull-down assay using a PTP1B-specific antibody to selectively immobilize PTP1B on
Figure 2. Characterization of PTP1Btide-pS3 with recombinant phosphatases. (a) Dephosphorylation of PTP1Btide-pS3 (10 μM) in the presence of decreasing amounts of recombinant PTP1B. The limit of detection was calculated as described in the Supporting Information. (b) A dose-dependent inhibition of PTP1Btide-pS3 (10 μM) dephosphorylation by recombinant PTP1B (1 nM) is observed in the presence of increasing concentrations of sodium orthovanadate. (c) Dephosphorylation of PTP1Btide-pS3 (10 μM) by a panel of closely related phosphatases (5 nM each). Error bars represent the standard deviation of triplicate experiments. The absolute values of reaction slopes are shown.
reported IC50 of 17 nM.45 Taken together, these results indicate that this assay platform could be utilized for high-throughput screening of protein phosphatase inhibitors. To determine if PTP1Btide-pS3 could be used to selectively report on PTP1B activity, we assessed whether this probe could act as a substrate for highly homologous PTPs.2 Accordingly, we assayed four closely related PTPs (TCPTP, SHP1, SHP2, and PTP−PEST) with PTP1Btide-pS3 (Figure 2c). Although each enzyme in the panel was active and able to dephosphorylate the nonspecific phosphotyrosine mimics pNPP and DiFMUP (Figures S5 and S6), none of the offtarget phosphatases that we assayed showed appreciable activity toward PTP1Btide-pS3 under these conditions. The selectivity between PTP1B and TCPTP appears to be an intrinsic property of this peptide sequence as the selectivity of the substrate sequence for PTP1B compared to TCPTP was preserved upon replacement of CSox with Ala (Figure S7). These results are particularly startling given that selectivity between PTP1B and TCPTP has thus far been difficult to achieve.46 For example, both PTP1B and TCPTP are capable of dephosphorylating a peptide sequence derived from the epidermal growth factor receptor (Figure S8), further demonstrating that both PTP1B and TCPTP are active. Comparison of kinetic parameters revealed that PTP1BtidepS3 was an 8-fold more efficient substrate for PTP1B than TCPTP (Figure S9). Extensive in vitro screening of peptide libraries has defined the amino acid preference of PTP1B around the site of dephosphorylation,46−52 resulting in the rational design of highly efficient peptide substrates (kcat/KM > 1 × 107 M−1 s−1).47,49 In this context, it is interesting to note that the PTP1Btide-pS3 sequence contains numerous residues
Figure 3. Direct assessment of enriched PTP1B activity from cell lysates. (a) Protein G coupled sepharose beads are preincubated with an anti-PTP1B antibody. The resulting bead/antibody complexes are then added to a heterogeneous mixture containing multiple phosphatases (left). Following incubation the beads are washed, added to reaction mixtures containing PTP1Btide-pS3, and assayed for PTP1B activity (right). (b) Recombinant PTP1B (2.5 nM) and/or YOP (10.1 nM) were added to pull-downs using a PTP1B-specific antibody as indicated. Beads were assayed with PTP1Btide-pS3 (10 μM). (c) HeLa cell lysate (500 μg total protein) was incubated with a ̈ antibody. Recovered bead-bound phosphatase PTP1B-specific or naive was assayed using PTP1Btide-pS3 (10 μM). Error bars represent the standard deviation of triplicate experiments. The absolute values of reaction slopes are shown. 286
DOI: 10.1021/acschembio.5b00506 ACS Chem. Biol. 2016, 11, 284−290
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ACS Chemical Biology protein G coupled beads. We then showed that PTP1B could be selectively isolated from solutions containing YOP and that the recovered PTP1B activity could be assayed using PTP1Btide-pS3 (Figure 3b). As expected, we observed no off-target activity in pull-downs containing TCPTP (Figure S12). Satisfied with this in vitro proof-of-concept, we next proceeded to apply this pull-down assay to HeLa cell lysates ̈ antibody as a control for off-target activity (Figure using a naive 3c). We clearly observed dephosphorylation in pull-downs containing the PTP1B antibody, while pull-downs using the ̈ antibody did not show any appreciable activity toward naive PTP1Btide-pS3. These experiments provide definitive evidence for the specificity of this assay approach in the presence of endogenous phosphatases. Although measuring the activity of immunoprecipitated PTP1B has been demonstrated previously with generic substrates, such as pNPP,58 the use of the peptidebased substrate described in this work has several significant advantages. First, the improved sensitivity of this substrate reduces the amount of sample required, providing an important improvement for the routine analysis of endogenous PTP1B activity as opposed to employing indirect proxies. Second, the use of a selective substrate with enriched enzyme samples obtained from immunoprecipitation increases the reliability of the assay by decreasing potential off-target signal from enzymes nonspecifically bound to the bead complexes. Third, the utilization of a real-time readout of enzymatic activity allows for kinetic measurements that are not readily achievable using endpoint assays. In the long term, the development of improved phosphatase inhibitors59 may provide a means to selectively monitor phosphatase activity in unfractionated samples in a similar manner to protein kinases.40 Nonetheless, we propose that the assay described herein has significant advantages over generic phosphatase probes and represents an important step forward in the development of selective phosphatase assays for use in unfractionated cell lysates. With this assay in hand, we wanted to determine whether temporal changes in PTP1B activity upon activation of the insulin signaling pathway in liver cells could be observed. Previous work has shown that PTP1B undergoes rapid oxidation upon insulin stimulation in HepG2 cells, leading to decreased activity and productive insulin signaling by alleviating the negative regulation of IRTK by PTP1B.58 Importantly, the previous assessment of PTP1B activity required the use of an anaerobic chamber for cell lysate preparation and subsequent analysis of immunoprecipitated PTP1B activity using pNPP. We hypothesized that the pull-down-based assay presented herein may provide a more straightforward approach to the analysis of PTP1B catalytic activity by eliminating the need for specialized equipment. Accordingly, we stimulated HepG2 cells with insulin and prepared lysates at different time points using a lysis buffer containing catalase and superoxide dismutase in order to prevent spurious oxidation of PTP1B upon cell lysis.19,60 We also verified that the activity of these enzymes did not influence PTP1B catalytic activity (Figure S13). The resulting lysates were then assessed for PTP1B activity using the pull-down assay format with PTP1Btide-pS3 (Figure 3a). From these experiments, we observed 33 and 41% decreases in PTP1B catalytic activity at 2 and 5 min after insulin stimulation, respectively (Figure 4a). Moreover, this decrease in PTP1B catalytic activity was not due to a change in the total amount of PTP1B present in the lysate (Figure 4a, inset). These data correlate with the previously observed decreases of 54 and 71% at 2 and 5 min, respectively.58 Differences in the observed
Figure 4. Endogenous PTP1B activity in insulin-stimulated HepG2 cells and livers from a rat model of NAFLD. (a) Pulled-down PTP1B activity was quantified in HepG2 lysates (500 μg total protein) after insulin stimulation (100 nM) using PTP1Btide-pS3 (10 μM). The inset shows a Western blot of PTP1B at the indicated time points. Error bars represent the standard deviation of triplicate experiments. (b) Pulled-down PTP1B activity from control (C) and NFALD (high fat, HF) rat liver homogenates (1000 μg total protein). Activity assays were performed with PTP1Btide-pS3 (10 μM). The inset shows a representative Western blot of PTP1B expression in liver tissues (see Figure S14 for a blot containing all samples). Error bars represent the standard deviation from duplicate assays run on liver tissue homogenates from 3 control and 3 high fat animals. All reaction slopes are shown as absolute values. *P ≤ 0.03.
absolute decreases in PTP1B activity may be attributable to differences in sample handling or off-target effects from the nonspecific pNPP substrate. Importantly, the assay format presented herein does not require the use of specialized equipment. To further demonstrate the utility of this assay platform, we chose to investigate alterations in PTP1B activity in NAFLD. Using livers collected from rats (n = 3) fed a high fat or control diet,61 we observed a 90% increase in PTP1B catalytic activity in the high fat (NAFLD) liver tissue (Figure 4b). Additionally, we determined that this increase in PTP1B activity could be attributed, in part, to an average 58% increase in PTP1B expression in NAFLD animals (Figure 4b inset and Figure S14). Assays performed utilizing the nonspecific DiFMUP substrate did not yield a statistically significant increase in phosphatase activity in NAFLD tissues (Figure S15). We attribute this difference in assay performance to the increased selectivity of PTP1Btide-pS3, as off-target phosphatases present in enriched samples could bias data obtained using DiFMUP. For example, DiFMUP is a substrate for numerous PTPs as well as for serine/threonine phosphatases.62,63 Importantly, previous studies have also utilized Western blotting to demonstrate increases in PTP1B expression in the livers of C57BL/6 mice fed a high fat diet.17 This observed increase in PTP1B expression has been used to infer increased PTP1B catalytic activity in NAFLD. Coupled with an observed inhibition of IRTK phosphorylation upon insulin stimulation, these data provide a mechanistic explanation for the insulin resistance phenotype observed in NAFLD. The data presented herein provide direct evidence for increased catalytic activity of PTP1B in NAFLD, suggesting that PTP1B inhibitors could reverse insulin resistance in this disease state.64 Taken together, these data highlight the ability of this approach to investigate the fundamental biochemistry of disease states without the use of proxies. In conclusion, we have developed a robust assay platform for the interrogation of protein phosphatase activity in heterogeneous biological samples. Using PTP1B as a test case, we demonstrate the ability to specifically monitor enriched 287
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37 °C. After the cells had reached 90% confluency, the media was removed, and the cells were washed three times with ice cold PBS (Life Technologies, 10010023) and lysed by scraping with the indicated lysis buffer (100 μL per dish). The lysates were then collected and centrifuged at 15 000 rcf for 5 min at 4 °C. Soluble protein was removed, aliquoted, flash frozen, and stored at −80 °C. Total protein concentrations were determined using the Bio-Rad Assay (Bio-Rad, 500-0006). Insulin-Stimulated HepG2 Lysate Pull-Down Assay. AntiPTP1B antibody (4 μL, Millipore MABS197) was incubated for 1 h (rotating at 4 °C) with 6 μL protein G sepharose beads (prewashed three times with Nondenaturing/Nonreducing Lysis Buffer) in 50 μL of Nondenaturing/Nonreducing Lysis Buffer. The beads were then washed three times (50 μL) with Nondenaturing/Nonreducing Lysis Buffer and were incubated (final volume 50 μL) for 1 h at 4 °C with 500 μg total protein from HepG2 (ATCC, HB-8065) cell lysates prepared from cells that had been serum starved (12 h) and then stimulated with 100 nM insulin (Sigma, I9278) for 0, 2, and 5 min and subsequently lysed with Nondenaturing/Nonreducing lysis buffer. The bead/antibody/PTP1B complexes were then assayed as described in the “HeLa Lysate Pull-down Assay” section in the Supporting Information except that beads were resuspended in Nondenaturing/ Nonreducing Lysis Buffer. NAFLD Tissue Homogenate Pull-Down Assay. Anti-PTP1B antibody/protein G beads (prepared as described in the section Insulin-Stimulated HepG2 Lysate Pull-Down Assay) were incubated for 1 h at 4 °C with 1000 μg total protein from NAFLD and control rat liver homogenates that had been homogenized with Nondenaturing/Nonreducing lysis buffer.36 NAFLD was confirmed in high fat animals by assessing the amount of triglyceride (≥5% of tissue weight) in the liver (data not shown). Rat livers were harvested from 14 week old Wistar Rats (Charles River) fed either control, 12% calories from fat (TestDiet, 58G7), or high fat, 60% calories from fat (TestDiet, 58G9), diets for 8−9 weeks. Upon completion of the diet, the animals were sacrificed and livers were excised as previously described.61 Final animal weights were between 450 and 500 g for both diets. All animal handling procedures were carried out in accordance with the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) IACUC protocol #956. The bead/antibody/PTP1B complexes were assayed as described in the “HeLa Lysate Pull-down Assay” section of the Supporting Information except that beads were resuspended in 10 μL Nondenaturing/ Nonreducing Lysis Buffer and 5 μL of beads were used to initiate reactions.
phosphatase activity from cell lysates and tissue homogenates. We show that temporal activity dynamics of phosphatases can be assessed using this straightforward approach and provide direct evidence for increased PTP1B catalytic activity in NAFLD. Nonetheless, this assay format is not without limitations. For example, in the absence of an anaerobic chamber we cannot fully rule out the oxidation of PTP1B during sample preparation. Therefore, quantification of the total amount of PTP1B activity in biological samples using this approach is not recommended. Introduction of CSox into a peptide substrate close to the site of dephosphorylation, generally at the ±2 or 3 position, may significantly alter catalytic efficiency and/or selectivity. Although our detection limit for PTP1B is similar to those observed for Sox-based kinase sensors,35,38,39,41 there is a possibility that monitoring the decrease in fluorescence of certain substrates may result in reduced assay sensitivity. Keeping these potential limitations in mind, this method clearly allows one to compare the relative amounts of PTP1B activity in biological samples (Figure 4), enabling the investigation of PTP1B signaling dynamics in model systems. The generalizable nature of this assay format allows for the potential application across classical PTPs and, in certain cases, with serine/threonine phosphatases (e.g., calcineurin).65 In addition, the work described herein provides an important proof-of-principle for the development of selective chemosensors for PTPs, similar to those described for protein kinases.35,36,38−42 We envision that this will lay the groundwork for the development of selective phosphatase chemosensors capable of directly monitoring enzymatic activity in unfractionated samples, providing improved insights into the fundamental biochemistry of this underappreciated class of signaling molecules.
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METHODS
Methods for assays containing endogenous PTP1B are given below. See the Supporting Information for details concerning sensor synthesis and characterization, assays with recombinant enzymes, and control experiments with endogenous PTP1B. General Reagents and Procedures. All assays were conducted in 384-well, white, low-volume plates (Corning, 3824) in a total volume of 40 μL. Fluorescence was monitored with a Bio-Tek Synergy H1 microplate reader (λex = 360 nm, λem = 485 nm) at 30 °C. Following the determination of the optimal concentration of Mg2+ and optimal sensor construct based of kinetic parameters, all subsequent assays were conducted with 7 mM MgCl2 and 10 μM PTP1Btide-pS3. All results are from data collected in triplicate unless otherwise noted. Recombinant Enzyme Assay Buffer. 50 mM Tris-HCl (pH = 7.5 at 22 °C), 2 mM EGTA, 1 mM DTT, and 0.01% Brij-35 (v/v). Lysate Assay Buffer. 50 mM Tris-HCl (pH = 7.5 at 22 °C), 2 mM EGTA, 0.01% Brij-35 (v/v), and 7 mM MgCl2. Nonreducing Wash Buffer. 50 mM Tris-HCl (pH = 7.5 at 22 °C), 150 mM NaCl, 1% Triton X-100 (v/v), and 2 mM EGTA. Nondenaturing Lysis Buffer. 50 mM Tris-HCl (pH = 7.5 at 22 °C), 150 mM NaCl, 1 mM DTT, 2 mM EGTA, 1% Triton X-100 (v/v), and 1% (v/v) Protease Inhibitor Cocktail (Life Tech, 78430). Nondenaturing/Nonreducing Lysis Buffer. 50 mM Tris-HCl (pH = 7.5 at 22 °C), 150 mM NaCl, 1% Triton X-100 (v/v), 1% (v/v) Protease Inhibitor Cocktail (Life Tech, 78430), 2 mM EGTA, 100 μg mL−1 catalase (Millipore, 219261), and 100 μg mL−1 superoxide dismutase (Millipore, 574594). General Cell Culture and Lysate Preparation Procedures. MEF, HeLa, and HepG2 cells were grown in DMEM (Life Technologies, 11965) supplemented with 10% FBS (v/v) and Pen/ Strep (Life Technologies, 15140) on 150 mm Polystyrene Dishes (Corning, 430599) in a humidified environment with 5% CO2 in air at
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acschembio.5b00506. Supplementary methods as well as supporting figures and tables (PDF)
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We thank M. Tremblay for providing MEF PTP1B+/+ and MEF PTP1B−/− cell lines and the Nebraska Center for Mass Spectrometry. Funding for this work was provided by the Proposed Center for Integrated Biomolecular Communication (CIBC), the Nebraska Research Initiative, and the Department 288
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ACS Chemical Biology of Chemistry at the University of Nebraska − Lincoln. A. Lawrence was supported by an NSF REU grant (1156560).
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