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-de- phostatin 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 ex- pressed 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.