Inhibition of DUSP13A Activity by PTP Inhibitor V

Abstract

Protein phosphorylation is regulated by two groups of enzymes, protein kinases and protein phosphatases. Regulation of phosphorylation has been a target for development of therapeutic reagents for many human diseases. Most protein phosphatases are members of protein tyrosine phosphatases (PTPs). The human genome encodes 107 PTPs, which are classified into four families: (i) class I cysteine-based PTPs, contains 2 classical PTPs and VH1-like PTPs, (ii) class II cysteine-based PTPs; tyrosine-specific low molecular weight phosphatases (LMPTP), (iii) class III cysteine-based PTPs containing cell division cycle25 (CDC25) homology (CH2) domain, (iv) transcription factors Eyes absent (EYA) proteins as members of aspartic acid based PTPs.1 DUSPs (dual-specificity phosphatases) are members of VH-1 like PTPs and dephosphorylate both phosphotyrosine and phosphoserine/threonine residues. Sixty one members of DUSPs are known in the human genome.2 Many DUSPs have been found to play roles in the regulation of mitogenic signal transduction for extracellular stimulation and to collaborate with protein kinases to regulate cell proliferation, differentiation, and apoptosis.3 Among DUSPs, DUSP13 was known to be located in human chromosome 10q22.2 and classified as small atypical DUSP.4, 5 DUSP13 encodes DUSP13A/MDSP (muscle-restricted DUSP) and DUSP13B/ TMDP (testis-and skeletal muscle-specific DUSP) due to alternative splicing of the ORF.3,6 DUSP13B is encoded by the downstream ORF. These two proteins share 42% identity in amino acid sequences. DUSP13A had no significant inhibitory effect on AP-1-dependent gene expression.7 Our recent report showed that DUSP13A is involved in the activation of apoptosis signal-regulating kinase 1 (ASK1), but the phosphatase activity of DUSP13A was not necessary to activate ASK1. 8PTP Inhibitor V is 4-(N'-(3-(4-Nitrophenyl)-5-oxo-1-phenyl-1,5-dihydro-pyrazol-(4Z)-ylidene)-hydrazino)-benzenesulfonic acid (Fig. 1).9 PTP Inhibitor V is known to inhibit SHP-2-dependent cellular functions and selectively penetrates into the substrate-binding site of SHP-2. The half maximal inhibitory concentration (IC 50 ) of PTP Inhibitor V is 2.1 µM against SHP-2.10 PTP Inhibitor V also inhibits ECPTP, PTP1B, SHP-1, mycobacterium MptpA (IC 50 = 5.4, 19, 30, and 39 µM, respectively). 10To study the action of PTP inhibitor V on other PTPs, we performed in vitro phosphatase assays with purified recombinant PTPs (Table 1). We found that DUSP13A was inhibited by PTP inhibitor V while other PTPs were not. We then examined inhibition profile of PTP inhibitor V on DUSP13A. When DUSP13A were treated with various concentrations of PTP inhibitor V, DUSP13A phosphatase activities were decreased by the inhibitor in a dose-dependent manner. The IC 50 of DUSP13A was 3.64 ± 0.57 µM as determined by the Prism 3.0 program (GraphPad Software) ( Fig. 2(a)). In the next step, kinetic analyses based on the Michaelis-Menten equation were carried out. The LineweaverBurk showed that the K m value of DUSP13A for OMFP was 104.1 µM and K i value...