Inhibition of Dual-Specificity Phosphatase 26 by Ethyl-3,4-Dephostatin: Ethyl-3,4-Dephostatin As a Multiphosphatase Inhibitor

ORIGINAL ARTICLES

College of Pharmacy, Chung-Ang University, Seoul, Republic of Korea

Inhibition of dual-specificity phosphatase 26 by ethyl-3,4-dephostatin: Ethyl-3,4-dephostatin as a multiphosphatase inhibitor

HUIYUN SEO, SAYEON CHO

Received September 30, 2015, accepted November 6, 2015 Sayeon Cho, Ph.D., College of Pharmacy, Chung-Ang University, Seoul 156-756, Republic of Korea [email protected] Pharmazie 71: 196–200 (2016) doi: 10.1691/ph.2016.5803

Protein tyrosine phosphatases (PTPs) regulate protein function by dephosphorylating phosphorylated proteins in many signaling cascades and some of them have been targets for drug development against many human diseases. There have been many reports that some chemical inhibitors could regulate particular phosphatases. However, there was no extensive study on specificity of inhibitors towardss phosphatases. We investigated the effects of ethyl-3,4-dephostatin, a potent inhibitor of five PTPs including PTP-1B and Src homology-2-containing protein tyrosine phosphatase-1 (SHP-1), on thirteen other PTPs using in vitro phosphatase assays. Of them, dual-specificity protein phosphatase 26 (DUSP26), which inhibits mitogen-activated protein kinase (MAPK) and p53 tumor suppressor and is known to be overexpressed in anaplastic thyroid carcinoma, was inhibited by ethyl- 3,4-dephostatin in a concentration-dependent manner. Kinetic studies with ethyl-3,4-dephostatin and DUSP26 revealed competitive inhibition, suggesting that ethyl-3,4-dephostatin binds to the catalytic site of DUSP26 like other substrate PTPs. Moreover, ethyl-3,4-dephostatin protects DUSP26-mediated dephosphorylation of p38, a member of the MAPK family, and p53. Taken together, these results suggest that ethyl-3,4-dephostatin functions as a multiphosphatase inhibitor and is useful as a therapeutic agent for cancers overexpressing DUSP26.

1. Introduction DUSPs are able to control the activation of MAPKs by dephos- Most cellular signal transductions in mammalian cells are regulated phorylating residues located on the activation loop of MAPKs, by several post-translational modifications, including phosphory- thus modifying the cellular signal (Stoker 2005). Modulation of lation, acetylation, methylation, ubiquitination, hydroxylation, and PTP activity is involved in regulating various cellular biological nitration (Hunter 2000). From among these, protein phosphory- functions and disease susceptibility (Fischer et al. 1991). There- lation regulates various essential cellular processes such as gene fore, chemical compounds that regulate the activity of PTPs may expression, cell growth, proliferation, differentiation, cell cycle be useful in the treatment of diseases such as cancer, inflammation, arrest, and apoptosis (Ciesla et al. 2011). Protein phosphorylation and diabetes. However, development of specific therapeutic PTP occurs on serine, threonine, or tyrosine residues and is regulated inhibitors is a critical challenge since over 100 PTPs paly diverse by protein kinases and protein phosphatases (Ciesla et al. 2011; roles by acting on a broad range of protein targets and the overall Franklin and Kraft 1997; Schlessinger 2000). Among the protein active site pockets of PTPs are known to be shallower than those of phosphatases, the protein tyrosine phosphatase (PTP) superfamily kinases. In addition, knowledge of the selectivity of PTP inhibitors is composed of over 100 members divided into four main fami- for their target phosphatases will provide important information lies based on the amino acid sequences of their catalytic domains. for the prediction of the effects of inhibitors in vivo. The four families include the class I cysteine (Cys)-based PTPs, Ethyl-3,4-dephostatin is known to inhibit PTP-1B and Src homol- the class II Cys-based PTPs, the class III Cys-based PTPs, and ogy-2-containing protein tyrosine phosphatase-1 (SHP-1) (Suzuki the aspartic acid-based PTPs (Patterson et al. 2009). Dual-speci- et al. 2001). In previous reports, we showed that DUSP14, ficity phosphatases (DUSPs) are a heterogeneous group of protein DUSP22, and protein tyrosine phosphatase non-receptor type 2 phosphatases that belong to a subclass of the class I Cys-based (PTPN2) were inhibited by ethyl-3,4-dephostatin in a competitive PTPs. DUSPs dephosphorylate both phospho-tyrosine and serine/ manner (Seo and Cho 2011a, b; Seo et al. 2011). We have screened threonine residues. for additional targets of ethyl-3,4-dephostatin by in vitro phospha- Protein phosphorylation and dephosphorylation lead to confor- tase assays with thirteen human PTPs. Dual-specificity phospha- mational changes in target proteins and then regulate their protein tase 26 (DUSP26) was identified as a novel target of ethyl-3,4- activity. The mitogen-activated protein kinase (MAPK) pathway is dephostatin. DUSP26, also known as mitogen-activated protein a protein phosphorylation-mediated signal transduction pathway kinase phophatase-8, is a member of atypical DUSPs, first char- that transmits signals from extracellular stimuli to genes in the acterized using a yeast two-hybrid screen by Mivechi’s research nucleus (Johnson and Lapadat 2002). The major subfamilies of group in 2005 (Hu and Mivechi 2006). DUSP26 overexpression MAPK are c-Jun N-terminal kinase (JNK), extracellular signal-reg- enhances colony formation in anaplastic thyroid carcinoma by ulated kinase (ERK), and p38 (Schaeffer and Weber 1999). Several inhibiting p38 kinase, while knock-down of DUSP26 inhibits cell proliferation and induces apoptosis (Yu et al. 2007). In addition, DUSP26 physically interacts with and functionally inhibits p53, Abbreviations: PTP, protein tyrosine phosphatase; DUSP, which results in reduction of p53 activity and its down-stream dual-specificity protein phosphatase; SHP-1, Src homolo- targets that are induced in response to genotoxic stress (Shang et gy-2-containing protein tyrosine phosphatase-1; MAPK, mito- al. 2010). In this study, we show that ethyl-3,4-dephostatin specifi- gen-activated protein kinase cally inhibits DUSP26 phosphatase activity and thus regulates p53 and p38, substrates of DUSP26. 196 Pharmazie 71 (2016) ORIGINAL ARTICLES

Fig. 1: Inhibitory effect of ethyl-3,4-dephostatin on DUSP26 and kinetic analysis of DUSP26 inhibition. (A) Chemical structure of ethyl-3,4-dephostatin. (B) DUSP26 was incubated with various concentrations (0, 5, 10, 15, or 20 μM) of ethyl-3,4-dephostatin at 37 ˚C for 30 min. Fluorescence emission from the product was measured. (C) Lineweaver-Burk plots of DUSP26 were generated from the data. Each experiment was performed in triplicate.

2. Investigations and results Protein tyrosine Classification IC50 (μM) References 2.1. Effect of ethyl-3,4-dephostatin on DUSP26 phosphatase (n = 3) Ethyl-3,4-dephostatin is a stable synthetic analog of dephostatin PTPN2 NRPTPs 6.5 ± 0.54 (Seo and Cho that inhibits PTP-1B and SHP-1 (Fig. 1A) (Suzuki et al. 2001). 2011a) Since PTP-1B and SHP-1 are related to diabetes and cancer LAR Transmembrane >100 (μg/ml) * (Suzuki et al. 2001) (Kundu et al. 2010; Zhang and Lee 2003), respectively, ethyl-3,4- Classical PTPs dephostatin might be useful for the treatment of those diseases. CD45 Transmembrane 28.5 (μg/ml) * (Suzuki et al. 2001) However, the selectivity of ethyl-3,4-dephostatin towardss phos- Classical PTPs phatases should be determined before it is considered as a thera- DUSP3 Atypical DUSPs >200 This study peutic reagent. We previously reported that DUSP14, DUSP22, and PTPN2, which DUSP6 MKPs >200 This study play roles in MAPK signaling (Jeffrey et al. 2007), were regulated DUSP7 MKPs >50 This study by ethyl-3,4-dephostatin (Seo and Cho 2011a, b; Seo et al. 2011). We tested additional thirteen recombinant human PTPs with in vitro DUSP8 MKPs >50 This study phosphatase assays to determine the effect of ethyl-3,4-dephostatin DUSP13B Atypical DUSPs >200 This study on the regulation of PTPs. An inhibition curve was plotted for each DUSP18 Atypical DUSPs >100 This study PTP and IC50 values were calculated. As shown in the Table, we found that DUSP26 was inhibited by ethyl-3,4-dephostatin. DUSP23 Atypical DUSPs >200 This study PTPN6 NRPTPs >50 This study Table: Inhibition of PTPs by ethyl-3,4-dephostatin in vitro PTPN7 NRPTPs >100 This study PTPN20 NRPTPs >50 This study Protein tyrosine Classification IC50 (μM) References phosphatase (n = 3) SSH3 Slinghots >50 This study DUSP26 Atypical DUSPs 6.8 ± 0.41 This study ACP1 Class II Cys-Based >200 This study PTP-1B NRPTPs 0.58 (μg/ml) * (Suzuki et al. 2001) PTPs SHP-1 NRPTPs 0.96 (μg/ml) * (Suzuki et al. 2001) * The values were obtained from the experimental conditions that were different from ours. DUSP14 Atypical DUSPs 9.7 ± 0.02 (Seo and Cho 2011b) Each experiment was performed in triplicate. In vitro PTP assay was processed as described in Experimental. Data are presented DUSP22 Atypical DUSPs 3.06 ± 0.07 (Seo et al. 2011) as mean ± SEM.

Pharmazie 71 (2016) 197 ORIGINAL ARTICLES

The inhibitory activity of ethyl-3,4-dephostatin on DUSP26 was concentration-dependent as shown in Fig. 1B. Ethyl-3,4-dephostatin inhibits DUSP26 with an IC50 of 6.8±0.4 μM. The Line- weaver-Burk plot showed that the Ki was 5.9 μM (Fig. 1C). We found that ethyl-3,4-dephostatin acts as a competitive inhibitor of DUSP26, suggesting that ethyl-3,4-dephostatin suppresses the activity of DUSP26 through binding to the catalytic site.

2.2. Effect of ethyl-3,4-dephostatin on DUSP26 expressed in mammalian cells To confirm the inhibitory effect of ethyl-3,4-dephostatin on DUSP26 in mammalian cells, HEK 293 cells transfected with FLAG-DUSP26 were treated with various concentrations of ethyl- 3,4-dephostatin. Phosphatase activity was measured after immu- noprecipitation of DUSP26 from cell lysates with anti-FLAG M2 agarose beads. As with the in vitro phosphatase assay, this result showed that ethyl-3,4-dephostatin inhibits the phosphatase activity of DUSP26 expressed in mammalian cells (Fig. 2).

Fig. 3: Ethyl-3,4-dephostatin inhibits the action of DUSP26 on p38 in vitro and in cells. (A) DUSP26 (1 μg) was pre-mixed with ethyl-3,4-dephostatin (0, 50, or 100 μM) and then incubated with active p38. Phosphorylation levels of p38 were determined by immunoblotting analysis. Relative phosphorylation levels of p38 were normalized to the expression levels of the corresponding total p38 and presented as fold increase. Data are representative of three independent experiments. (B) Transfected HEK 293 cells were pretreated with various concentrations of ethyl-3,4-dephostatin (0, 50, and 100 μM) for 3 h and then

stimulated with H2O2 (1 mM, 1 h). Cell lysates were analyzed by immunoblotting with appropriate antibodies. Relative phosphorylation levels of p38 were normalized to the expression levels of the corresponding total p38 and presented as fold increase. Data are representative of three independent experiments. Fig. 2: Inhibition of DUSP26 expressed in mammalian cells by ethyl-3,4-dephostatin in vitro. HEK 293 cells were transfected with 3 μg of FLAG-DUSP26 expression 2.4. Ethyl-3,4-dephostatin inhibits DUSP26-mediated plasmid for 48 h. After HEK 293 cells were treated with various concentrations (0, 50, or 100 μM) of ethyl-3,4-dephostatin for 3 h, cells were harvested dephosphorylation of phospho-Ser-37 of p53 tumor sup- and immunoprecipitated with anti-FLAG M2 agarose. In vitro PTP assay was pressor protein processed as described in Experimental. The results represent the mean data from three independent experiments. *p < 0.01 versus the FLAG-DUSP26 To further determine the functional effect of ethyl-3,4-dephostatin expression plasmid control (Student’s t-test). on DUSP26-mediated cellular responses, we tested the effect of ethyl-3,4-dephostatin on DUSP26-mediated p53 dephosphoryla- 2.3. Ethyl-3,4-dephostatin inhibits the action of DUSP26 tion in 5-fluorouracil (5-FU)-stimulated cells. It has been suggested on p38 that DUSP26 is a novel p53 phosphatase that dephosphorylates p-Ser-20 and p-Ser-37 of p53 (Shang et al. 2010). In this assay, We next examined whether ethyl-3,4-dephostatin influences the we used an anti-p-p53 (S37) antibody to investigate whether ethyl- phosphorylation of DUSP26 substrates in mammalian cells. Since 3,4-dephostatin regulates dephosphorylation of phospho-Ser-37 by Yu et al. (2007) reported that DUSP26 dephosphorylates and inhibiting DUSP26 in human colon cancer cells (HCT 116). Ethyl- inhibits phosphorylated p38, we used recombinant phosphory- 3,4-dephostatin significantly enhanced p53 phosphorylation at the lated p38 as a substrate for DUSP26 to determine whether ethyl- Ser-37 residue in 5-FU-stimulated HCT 116 cells (Fig. 4). This 3,4-dephostatin regulates p38 activation by inhibiting DUSP26 result indicates that ethyl-3,4-dephostatin blocks DUSP26-medi- activity in vitro. After treatment of active p38 with DUSP26 in ated p53 dephosphorylation by inhibiting DUSP26 activity. the presence of various concentrations of ethyl-3,4-dephostatin, samples were subjected to immunoblotting with phospho (p)-p38 and total p38 antibodies. As shown in Fig. 3A, ethyl-3,4-de- 3. Discussion phostatin significantly inhibited p38 dephosphorylation by inhib- We showed that ethyl-3,4-dephostatin effectively inhibits the iting DUSP26 phosphatase activity in vitro. We further examined activity of DUSP26 both in vitro and in cells in a competitive whether ethyl-3,4-dephostatin regulates DUSP26-mediated p38 manner. Ethyl-3,4-dephostatin was first identified as an inhibitor inhibition in cells. After HEK 293 cells were transfected with or of PTP-1B and SHP-1 and later found to be a competitive inhibitor without FLAG-tagged DUSP26 plasmids, transfected cells were of DUSP14, DUSP22, and PTPN2 (Table). Taken together, these treated with various concentrations of ethyl-3,4-dephostatin for results suggest that those PTPs inhibited by ethyl-3,4-dephostatin 3 h and then the endogenous levels of p-p38 were determined with have similar environments at the active sites. PTP-1B, SHP-1, immunoblotting analysis. As shown in Fig. 3B, DUSP26-mediated DUSP14, DUSP22, and PTPN2 belong to Class I Cys-based PTPs dephosphorylation of p38 was significantly inhibited by ethyl-3,4- that includes 99 PTPs (Alonso et al. 2004). Class I Cys-based PTPs dephostatin. These results suggest that ethyl-3,4-dephostatin effec- can be further classified into classical PTPs or DUSPs on the basis tively protects DUSP26-mediated p38 inactivation by inhibiting of the similarity of the catalytic domains. Classical PTPs can be DUSP26 activity. divided into transmembrane receptor-like enzymes (RPTPs) and 198 Pharmazie 71 (2016) ORIGINAL ARTICLES

4. Experimental 4.1. Reagents and antibodies Antibodies specific for anti-phospho-p38 (Thr-180/Tyr-182), anti-phospho-p53 (pSer-37), and His antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-p38 antibody was from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Active p38 proteins were from Upstate Biotechnology (Lake Placid, NY, USA). Ethyl-3,4-dephostatin, 3-O-methylfluorescein phosphate (OMFP), anti- FLAG M2 antibody, and anti-FLAG M2 agarose beads were from Sigma-Aldrich (St. Louis, MO, USA).

4.2. Cell culture and transfection Human embryonic kidney (HEK) 293 and human colon carcinoma (HCT) 116 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen, Carlsbad, CA, USA) supplemented with 10 % fetal bovine serum (FBS, Invitrogen,

Carlsbad, CA, USA) and penicillin/streptomycin at 37 ˚C in the presence of 5 % CO2. For transient transfection, 1.4 x 106 cells were plated in each 60-mm cell culture plate, grown overnight, and transfected with DNA using OmicsFect™ (Omicsbio, Taipei City, Taiwan). Fig. 4: Ethyl-3,4-dephostatin inhibits the action of DUSP26 on p53 phosphorylation at Ser-37 residue. Transfected HCT 116 cells were pretreated with various concentrations of 4.3. Plasmid construction ethyl-3,4-dephostatin (0, 50, and 100 μM) for 3 h and then stimulated with pcDNA3.1 plasmid (Invitrogen, Carlsbad, CA, USA) with FLAG-tagged DUSP26 5-FU (200 μg/ml, 3 h). Cell lysates were analyzed by immunoblotting with was constructed. For protein expression in E. coli, pET-28a (+) plasmid (Novagen, appropriate antibodies. Relative phosphorylation levels of p53 (S37) were normalized to the expression levels of the corresponding p53 and presented Darmstadt, Germany) with His-tagged DUSP26 was constructed. as fold increase. Data are representative of three independent experiments. 4.4. Purifi cation of the six-His-tagged proteins pET-28a (+) plasmids with PTP expression were constructed and transformed into BL21 (DE3)-RIL E. coli. Recombinant proteins were induced with 1 mM isopro- the intracellular non-receptor PTPs (NRPTPs). DUSPs are much pyl-β-d-thiogalactopyranoside at 20 ˚C for 16 h. Cells were harvested and then lysed more various and can be comprised of six subgroups, including by sonication in 50 mM Tris–HCl (pH 8.0), 300 mM NaCl, 20 mM imidazole, 1 % mitogen-activated protein kinase phosphatases (MKPs), atypical IGEPAL CA-630, and 1 mM phenylmethylsulphonyl fluoride (PMSF). The lysates were centrifuged at 13,000 rpm for 30 min at 4 ˚C. The supernatant was applied by DUSPs, slingshots, and other DUSPs phosphatases (Alonso et al. gravity flow to a column of Ni–NTA resin (PEPTRON, Daejeon, Korea). The resin 2004). DUSP26 is an atypical DUSP of Class I Cys-based PTPs was washed with 20 mM Tris–HCl (pH 8.0), 300 mM NaCl, and 20 mM imidazole that have evolved from a common ancestor, based on their similar and then eluted with 20 mM Tris–HCl (pH 8.0), 300 mM NaCl, and 250 mM imid- structural folds for classical PTPs, DUSPs, and other VH1-like azole. The eluted proteins were dialyzed overnight against 20 mM Tris–HCl, 150 mM phosphatases. All PTPs have a highly conserved active site motif NaCl, 20 % glycerol, and 0.5 mM PMSF before storage at -80 ˚C.

HCX5R (PTP signature motif) and thus possess a common catalytic mechanism (Barford et al. 1998). Since only a few of these PTPs 4.5. In vitro phosphatase assays and kinetic analysis were inhibited by ethyl-3,4-dephostatin in a competitive inhibition Phosphatase activities were measured using OMFP as a substrate at concentra- pattern, their environments at the active sites might be critical for tions that varied according to the Km of each enzyme in a 96-well microtiter plate binding to ethyl-3,4-dephostatin. In this study, although most PTPs assay based on methods described previously (Tierno et al. 2007). Ethyl-3,4-de- tested here belong to the same class of PTPs (Class I Cys-based phostatin and OMFP were solubilized in DMSO. All reactions were performed at a final concentration of 1 % DMSO. The final incubation mixture (150 μl) was PTPs), they seem to be differentially regulated by ethyl-3,4-de- optimized for enzyme activity and was composed of 30 mM Tris–HCl (pH 7.0), phostatin, suggesting that other amino acids as well as conserved 75 mM NaCl, 1 mM ethylenediaminetetraacetic acid (EDTA), 0.1 mM dithiothreitol amino acids at the active sites are involved in enzyme catalysis. (DTT), 0.33 % bovine serum albumin (BSA), and 100 nM of each PTP. Reactions The study of PTP inhibitors with selectivity towards PTPs will were initiated by addition of OMFP and incubated for 30 min at 37 ˚C. Fluorescence emission from the product was measured with a multiwall plate reader (Synergy provide crucial information to predict the effects of the inhibitor in H1; excitation filter, 485 nm; emission filter, 535 nm). The reaction was linear over cells and clinical researches. However, most known PTP inhibitors the experimental time period and was directly proportional to both enzyme and bind to active sites of target PTPs for regulation and the active-site substrate concentration. The half-maximal inhibition constant (IC50) was defined pockets of PTPs are shallow in general, which implies that it might as the concentration of inhibitor that caused a 50 % decrease in the PTP activity. be difficult to create a highly selective PTP inhibitor derived from Half-maximal inhibition constants and best curve fit for Lineweaver-Burk plots were determined by using the curve fitting program PRISM 3.0 (GraphPad Soft- active site conformation. In addition, the PTP inhibitor selectivity ware, San Diego, CA). might provide useful data in obtaining dynamic structural information around active sites of target PTPs that are not available from current protein structure databases. As shown in the Table, current 4.6. Inhibition study classification of PTPs does not inform any inhibitor selectivity and The inhibition constant (Ki) to DUSP26 phosphatase for the inhibitor was determined it implies that there might be novel features that can be used for by measuring the initial rates at several OMFP concentrations for each fixed concentration of the inhibitor. The data were fitted to the following equation to obtain the classification based on PTP inhibitor selectivity. inhibition constant for reversible competitive inhibitors. The slopes obtained were In summary, we found that ethyl-3,4-dephostatin inhibits DUSP26 replotted against the inhibitor concentrations. The Ki value was obtained from the activity to enhance the activities of p38 and p53. Since p38 and p53 slopes of these plots (Shi et al. 2007). 1 / V = K (1 + [I] / K ) V [S] + 1 / V act as critical regulators in apoptosis and carcinogenesis, modula- m i max max V, velocity; I, inhibition; S, substrate; V , maximal velocity; K , Michaelis constant; tion of DUSP26 by ethyl-3,4-dephostatin might provide important max m K , inhibitory constant information on how DUSP26-mediated tumorigenesis and cell i growth can be regulated. However, among the target PTPs of ethyl- 3,4-dephostatin, PTPN2 expression is associated with Crohn’s 4.7. Effect of ethyl-3,4-dephostatin on DUSP26 expressed in mamma- disease (Yu et al. 2012), whereas PTP-1B and SHP-1 are related lian cells to diabetes and cancer, respectively (Kundu et al. 2010; Zhang and HEK 293 cells were transfected with the FLAG-DUSP26 expression plasmid. After Lee 2003). Since ethyl-3,4-dephostatin with multiphosphatase 48 h, cells were washed twice with phosphate-buffered saline (PBS) and lysed in inhibitor activity might induce multiple effects if it is used in vivo, PTP lysis buffer (1 % IGEPAL CA-630, 0.5 % Triton X-100, 150 mM NaCl, 20 mM it is of importance to investigate the effects of the chemical on each Tris-HCl (pH 8.0), 1 mM EDTA, 1 % glycerol, 1 mM PMSF, and 1 μg /ml aprotinin) for 30 min at 4 ˚C. Cleared cell lysates from centrifugation were mixed with washed phosphatase. Furthermore, accumulated results from selectivity FLAG M2-agarose and incubated for 16 h at 4 ˚C using a rotating device. After incu- studies with an inhibitor will provide critical information on how bation, FLAG M2-agarose was washed three times with PTP lysis buffer and their the inhibitor influences overall cellular networks in vivo. phosphatase activities were measured. Pharmazie 71 (2016) 199 ORIGINAL ARTICLES

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