Inhibition of PTPN2 by PTP inhibitor V

Abstract

Protein phosphorylation is closely linked to critical pathways in numerous human diseases, which is regulated by the coordinated activities of kinases and phosphatases. Protein phosphorylation occurs predominantly on serine, threonine, and tyrosine residues of proteins and plays important roles in the regulation of physiological processes including gene expression, proliferation, differentiation, cell cycle arrest, and apoptosis. About 30% of proteins are phosphorylated in the mammalian cell. In case of eukaryotic cells, tyrosine phosphorylation occupies only 0.01-0.05% of total protein phosphorylation, while most of protein phosphorylation occurs on serine or threonine residues. However, growth factor stimulation or tumorigenesis enhances the level of tyrosine phosphorylation up to 1-2% of total protein phosphorylation. Protein phosphorylation is a reversible process that is controlled by protein kinases and protein phosphatases. In particular, protein tyrosine phosphatases (PTPs) are involved in most signaling pathways and thus play important roles in eukaryotes. PTPs superfamily comprises 107 enzymes in human genome. Based on the amino acid sequences of their catalytic domains, the PTPs can be grouped into four main families. Since PTPs play critical roles for cell homeostasis and thus diseases, chemical inhibitors that regulate PTPs have been extensively investigated to be used as therapeutic reagents. PTP inhibitor V, also known as phenyl hydrazono pyrazolone sulfate 1 (PHPS1), is 4-{N'-[3-(4-nitrophenyl)-5-oxo-1phenyl-1,5-dihydro-pyrazol-(4Z)-ylidene]-hydrazino}-benzenesulfonic acid (Fig. 1(a)). PTP inhibitor V was originally identified as a potent cell-permeable inhibitor that acts as active-site targeting, reversible, competitive inhibitor of SHP2 with the IC50 value of 2.1 μM. 5 PTP inhibitor V inhibits ECPTP, PTP1B and SHP-1 (IC50 = 5.4, 19, 30 μM, respectively). To investigate the effect of PTP inhibitor V on other PTPs, we performed in vitro phosphatase assays with purified recombinant PTPs (Table 1). We found that protein tyrosine phosphatase non-receptor type 2 (PTPN2) was inhibited by PTP inhibitor V while other PTPs were not. PTPN2 that is also called T cell protein tyrosine phosphatase (TC-PTP) belongs to class I Cys-based PTPs as the intracellular non-receptor PTPs with a high degree of sequence and structural homology within the catalytic domain. The mRNA levels of PTPN2 increase at G1 phase and return to basal level after G1 phase during cell cycle progression. 8