Ethyl-3,4-dephostatin Inhibits Dual-specificity phosphatase 22 (DUSP22) Activity

Protein phosphorylation is one of the major protein modification processes that play critical roles in a variety of biological processes, including gene expression, proliferation, differentiation, cell cycle arrest, and apoptosis. Protein phosphorylation usually occurs in serine, threonine, or tyrosine residues and is processed by protein kinases. Of those protein kinases, extracellular signal-regulated kinase (ERK), p38, and c-Jun N-terminal kinase (JNK) that are members of mitogen-activated protein kinases (MAPK) family, have diverse roles in cell proliferation, cell survival, cell death, differentiation, development, immune function, gene expression, and other intracellular events. Therefore, regulation of those MAPK family members results in diverse outcome of cell fate. Many of protein tyrosine phosphatases (PTPs) control the activation of MAPKs through dephosphorylation of phosphoserine, phosphothreonine, or phosphotyrosine residues that are located on the activation loop of MAPKs. The human genome contains 107 PTP genes. Dualspecificity phosphatases (DUSPs) as a heterogeneous group of protein phosphatases belong to a subclass of PTP families and dephosphorylate both phosphotyrosine and phosphoserine/phosphothreonine residues. Many studies have demonstrated that the modulation of PTP enzymatic activity is involved in regulating diverse cellular biological functions and disease susceptibility. Thus, chemical compounds that regulate the activity of PTPs may have ability to be used as therapeutic reagents for diseases such as cancer, inflammation, and diabetes. Ethyl-3,4-dephostatin is a stable synthetic analog of dephostatin and was originally identified as a potent inhibitor of PTP-1B and Src homology region 2 (SH2) domain-containing phosphatase 1 (SHP-1) (Fig. 1). Since ethyl-3,4dephostatin might regulate other phosphatases, we screened PTPs by performing in vitro phosphatase assays to identify phosphatases that are inhibited by ethyl-3,4-dephostatin. We found that DUSP22 activity was inhibited by ethyl-3,4dephostatin. DUSP22 was treated with various concentrations of ethyl3,4-dephostatin and then its phosphatase activity was measured. DUSP22 activity was decreased by ethyl-3,4dephostatin in a dose-dependent manner. An inhibition curve was plotted for DUSP22 and the half maximal inhibitory concentration (IC50) was obtained. This result suggests that ethyl-3,4-dephostatin inhibits DUSP22 with IC50 of 3.06 ± 0.07 μM (Fig. 2(a)). In subsequent experiments, kinetic analyses based on the Michaelis-Menten equation were performed with ethyl-3,4dephostatin and DUSP22 to provide experimental evidences for the mechanism of DUSP22 catalysis and for binding of