Inhibition of Dual-specificity Phosphatase 22 (DUSP22) by PRL-3 inhibitor I

Abstract

Protein phosphorylation is an essential mechanism for human health and disease, which is regulated by the coordinated activities of kinases and phosphatases. Protein phosphorylation occurs predominantly on serine, threonine, and tyrosine residues of eukaryotic proteins and plays critical roles in the regulation of physiological processes including gene expression, proliferation, differentiation, cell cycles arrest, and apoptosis. Mitogen-activated protein (MAP) kinases are critical mediators in signal transduction pathways regulating embryogenesis, cell differentiation, cell proliferation, and cell death in response to extracellular stimuli and stress. Extracellular signal-regulated kinase (ERK), p38, and c-Jun Nterminal kinase (JNK) are the major mammalian MAP kinases that are regulated by some of protein tyrosine phosphatase (PTP) family members. PTP superfamily comprises 107 enzymes. Based on the amino acid sequences of their catalytic domains, the PTPs can be grouped into four main families: (i) classical PTPs and dual-specificity phosphatases (DUSPs), (ii) tyrosine-specific low molecular weight phosphatases, (iii) the cdc25 family, and (iv) Eyes absent (EyA) protein that was recently discovered to be tyrosine-, or dual serineand tyrosine-specific protein phosphatases. DUSPs are a subfamily of mammalian class I cysteine-based PTP superfamily, which dephosphorylate both tyrosine and serine/ threonine residues. Some DUSPs have been characterized for the role in MAPK signaling pathways. Since PTPs play critical roles for cell homeostasis, chemical inhibitors that regulate DUSPs have been extensively investigated to be used as therapeutic reagents. PRL-3 inhibitor I is a rhodanine derivative and inhibits phosphatase of regenerating liver-3 (PRL-3) in vitro. PRL-3 is a nonclassical protein tyrosine phosphtase and overexpression of PRL-3 plays a role in tumorigenesis and metastasis in various types of cancer cells. PRL-3 is associated with promoting cell motility, invasion activity, and metastasis. However, the biological and mechanistic evidence for PRL-3 function has not been understood well. The half maximal inhibitory concentration (IC50) of PRL-3 inhibitor I is known to be 0.9 μM against PRL-3 (Fig. 1(a)). To investigate the role of PRL-3 inhibitor I on other PTPs, we performed in vitro phosphatase assays with purified recombinant PTPs (Table 1). We identified that dualspecificity phosphatase 22 (DUSP22) is inhibited by PRL-3 inhibitor I while other PTPs are not. DUSP22, also called JNK-stimulatory phosphatase 1 (JSP1) and JNK pathway-associated phosphatase (JKAP), positively regulates MAPK signaling pathways in contrast to most phosphatases which negatively regulate MAPK signaling pathways. Overexpression of DUSP22 in HEK 293T cells activated JNK but not p38 and ERK. Activation of JNK by DUSP22 is associated with activation of MKK4 which is an immediate JNK upstream activating kinase. In addition, a kinase inactive form of MKK4 (MKK4KR) abolished DUSP22-induced activation of JNK in COS-1 cells. Furthermore, the inactive mutant of DUSP22 failed to induce JNK activation. This suggests that DUSP22 does not directly activate JNK but supports an enzyme activity of