Abstract
Phenyl vinyl sulfone (PVS) and phenyl vinyl sulfonate (PVSN) inactivate protein tyrosine phosphatases (PTPs) by mimicking the phosphotyrosine structure and providing a Michael addition acceptor for the active-site cysteine residue of PTPs, thus forming covalent adducts between PVS (or PVSN) and PTPs. We developed a specific antiserum against PVS. This antiserum can be used in general antibody-based assays such as immunoblotting, immunofluorescence staining, and immunoprecipitation. Target identification through immunoprecipitation and mass spectrometry analysis reveals potential targets of PVS, mostly proteins with reactive cysteine residues or low-pKa cysteine residues that are prone to reversible redox modifications. Target identification of PVSN has been conducted because the anti-PVS antiserum can also recognize PVSN. Among the targets, protein arginine methyltransferase 1 (PRMT1), inosine-5′-monophosphate dehydrogenase 1, vimentin, and glutathione reductase (GR) were further confirmed by immunoprecipitation followed by immunoblotting. In addition, PVSN and Bay11-7082 inhibited GR activity, and PVS, PVSN, and Bay 11-7082 inhibited PRMT1 activity in in vitro assays. In addition, treatment of PVSN, Bay11-7082, or Bay 11-7085 in cultured HeLa cells can cause the quick decline in the levels of protein asymmetric dimethylarginine. These results indicate that the similar moiety among PVS, PVSN, Bay 11-7082, and Bay 11-7085 can be the key structure of lead compounds of PRMT1. Therefore, we expect to use this approach in the identification of potential targets of other covalent drugs.
Highlights
Protein tyrosine phosphorylation, dynamically controlled by the activities of protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs), is critical in the regulation of cell proliferation, differentiation, metabolism, and survival
Based on the observations that Phenyl vinyl sulfone (PVS) and phenyl vinyl sulfonate (PVSN) were PTP covalent inhibitors [22], we attempted to develop an antiserum against PVS and use the antiserum in the identification of PVS-tagged proteins through immunoblotting and immunoprecipitation
HeLa cells were treated with various concentrations of PVS for 1 h and the cell lysate was examined by SDS/PAGE and immunoblotting using the antisera against phosphotyrosine and PVS (Figure 1)
Summary
Dynamically controlled by the activities of protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs), is critical in the regulation of cell proliferation, differentiation, metabolism, and survival. The classical PTPs possess an active-site site motif HCX5R, in which the cysteine sulfhydryl group deprotonates due to its low pKa and functions as a nucleophile for the enzymatic catalysis [2,3]. The low pKa property of the catalytic cysteine residue renders PTPs susceptible to oxidation and transient inactivation by reactive oxygen species (ROS). Similar modification of the catalytic cysteine residue has been shown for PTPN11 (SHP2) in PDGF signaling [5], PTPN1 and PTPN2 (TC-PTP) in insulin signaling [6], and PTPN6 (SHP1) in B cell receptor signaling [7,8]. SHP-1, SHP-2, and PTP1B are prone to oxidation c 2018 The Author(s).
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