Inhibition of Dual-specificity phosphatase 14 (DUSP14) by Ethyl-3,4-dephostatin

Abstract

Most cellular functions derived from signal transduction pathways require protein phosphorylation. Protein phosphorylation is processed by opposing activities of protein kinases and protein phosphatases. Dual-specificity phosphatases (DUSPs) are a subclass of protein tyrosine phosphatases (PTP) families that comprises 107 genes in human genome and can dephosphorylate both phosphotyrosine and phosphoserine/phosphothreonine residues on substrates. Some DUSPs have been reported as key regulators for inactivating mitogen-activated protein kinases (MAP kinase). The three major subfamilies of MAPK in mammalian cells are p38, extracellular signal regulated kinase (ERK), c-Jun N-terminal kinase (JNK). Most PTPs are related to various cellular biological processes and thus modulation of their enzymatic activities is important in regulating disease susceptibility. Therefore, specific chemical compounds that regulate the activity of PTPs may have potent therapeutic approaches for diseases. Ethyl-3,4-dephostatin is known to inhibit PTP-1B and Src homology-2-containing protein tyrosine phosphatase-1 (SHP-1) selectively (Fig. 1). Since ethyl-3,4-dephostatin might inhibit other phosphatases, we performed in vitro phosphatase assays with recombinant PTPs in the presence of ethyl-3,4-dephostatin. DUSP14 was one of a few PTPs found to be strongly inhibited by ethyl-3,4-dephostatin. When DUSP14 was treated with increasing concentrations of ethyl-3,4-dephostatin, its DUSP14 activity was decreased by ethyl-3,4-dephostatin in a dose-dependent manner. When an inhibition curve was plotted for DUSP14, the half maximal inhibitory concentration (IC50) was about 9.7 ± 0.02 μM (Fig. 2(a)). Km value was determined to 14.46 ± 4.05 μM. We then performed kinetic studies based on the Michaelis-Menten equation to examine the DUSP14 inhibitory mechanism by ethyl-3,4-dephostatin. Analysis of the mode of inhibition indicated a competitive inhibition with Ki of 7.91 μM (Fig. 2(b)), suggesting that ethyl-3,4dephostatin inhibits the activity of DUSP14 through binding to the catalytic site. It has been suggested that DUSP14 dephosphorylates phospho-ERK, phospho-JNK, and phospho-p38, suggesting that DUSP14 behaves as a general MAPK phosphatase. To examine that ethyl-3,4-dephostatin could function as a DUSP14 inhibitor and thus activate JNK in cells, we measured the in vivo phosphorylation level of JNK. HEK 293 cells were transfected with FLAG-DUSP14 expression plasmid, pretreated with or without ethyl-3,4-dephostatin for 3 h, and then stimulated with H2O2 to phosphorylate JNK. Levels of phospho-JNK were determined with immunoblotting analysis. Ethyl-3,4-dephostatin activates JNK kinase activity by inhibiting DUSP14 in vivo (Fig. 3). These results suggest that ethyl-3,4-dephostatin recovers DUSP14-suppressed JNK activity by inhibiting DUSP14. In conclusion, we found that ethyl-3,4-dephostatin acts as an effective inhibitor of DUSP14. DUSP14 has been reported to a negative regulator of CD28 signaling through dephosphorylation of MAPKs in T cells. We found that ethyl-3,4-dephostatin inhibits DUSP14 activity and thus recovers JNK activity. Therefore, our study suggests that