In vivo Effect of NSC-95397 as a DUSP14 Inhibitor

Abstract

Dual-specificity phosphatases (DUSPs) as a heterogeneous group of protein phosphatases belong to a subclass of PTP (protein tyrosine phosphatase) family and dephosphorylate both phosphotyrosine and phosphoserine/phosphothreonine residues. DUSPs play as major modulators of critical signal transduction pathways that are deregulated in many diseases. Particularly, mitogen-activated protein kinase phosphatases (MKPs) have function as negative regulators of mitogen-activated protein kinase (MAPK)-mediated signaling in many cellular processes. DUSP14 also known as MKP6 was first identified through a yeast two-hybrid system to identify novel proteins that interact with the T-cell costimulatory factor CD28. DUSP14 is expressed ubiquitously, but elevated expression has been observed in certain types of cells and tissues. DUSP14 is one of 19 atypical DUSPs that contain the consensus DUSP C-terminal catalytic domain but lack the N-terminal CH2 (Cdc25 homology 2) domain. The MAPK subfamily, including the c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinases (ERK), and p38, has a diverse crucial role that regulates proliferation, cell survival and death, differentiation, development, immune function, gene expression, and other intracellular events. Several PTPs are able to function as oncoproteins or tumor suppressors by dephosphorylating specific MAPKs and modifying the pathway. DUSP14 dephosphorylates and inactivates MAPKs. NSC-95397 (2,3-bis-[2-hydroxyethylsulfanyl]-[1,4]naphthoquinone) was originally reported to inhibit Cdc25 family of DUSPs, including Cdc25A, B, and C and VH1-related dual-specificity phosphatase or PTP1B. In recent experiments, we showed that NSC-95397 effectively and specifically down-regulates DUSP14 phosphatase activity in vitro. In this study, we show that NSC-95397 regulates MAPK signaling in vivo through inhibition of DUSP14. We first examined whether the inhibitory function of NSC-95397 on DUSP14 regulates the phosphorylation of MAPKs. It has been reported that DUSP14 can dephosphorylate phospho-ERK, JNK, and p38 in vitro. However, in Tcells expressing the catalytically inactive mutant DUSP14 (C111S), only the phosphorylation of ERK and JNK increased whereas that of p38 remains unchanged, suggesting that ERK and JNK are the valid targets of DUSP14 in vivo. In this study, we used JNK as a substrate for DUSP14 since DUSP14 dephosphorylates active phospho-JNK both in vitro and in vivo. After incubation of recombinant phosphoJNK with DUSP14 in the presence of various concentrations of NSC-95397, samples were examined by immunoblotting with phospho-JNK and JNK antibodies (Fig. 1). NSC-95397 prevents in vitro JNK dephosphorylation by DUSP14. To investigate whether NSC-95397 negatively regulates DUSP14 phosphatase activity in vivo, HEK 293 cells were transiently transfected with FLAG-tagged DUSP14 WT or catalytically inactive mutant (D80A) expression plasmid. Transfected HEK 293 cells were pretreated with 0-100 μM NSC-95397 for 3 h before cells were lysed with PTP lysis buffer. DUSP14 was immunoprecipitated from cell lysates using anti-FLAG M2-agarose. Phosphatase activity of immunoprecipitated DUSP14 was then determined using 3O-methylfluorescein phosphate (OMFP) as a substrate as described previously. As shown in Figure 2, NSC-95397 inhibits DUSP14 phosphatase activity in vivo. The results indicate that NSC-95397 effectively penetrates the cells and inhibits DUSP14 activity. We further examined whether NSC-95397 inhibited DUSP14 activity against endogenous JNK in cells. HEK 293 cells were transfected with FLAG-tagged DUSP14 WT or catalytically inactive mutant (D80A) expression plasmid, pretreated with NSC-95397 for 3 h, and then stimulated with H2O2 to phosphorylate JNK. The levels of phospho-JNK were determined using immunoblotting analysis. As shown Figure 3(a), DUSP14 WT-mediated dephosphorylation of JNK was significantly inhibited by NSC-95397, whereas DUSP14 (D80A) mutant showed no effect on phosphorylated JNK levels. The results suggest that NSC-95397 effectively inhibits DUSP14 to protect endogenous phosphoJNK.