Protein S-nitrosylation mediated by cellular nitric oxide (NO) plays a primary role in executing biological functions in cGMP-independent NO signaling. Although S-nitrosylation appears similar to Cys oxidation induced by reactive oxygen species, the molecular mechanism and biological consequence remain unclear. We investigated the structural process of S-nitrosylation of protein-tyrosine phosphatase 1B (PTP1B). We treated PTP1B with various NO donors, including S-nitrosothiol reagents and compound-releasing NO radicals, to produce site-specific Cys S-nitrosylation identified using advanced mass spectrometry (MS) techniques. Quantitative MS showed that the active site Cys-215 was the primary residue susceptible to S-nitrosylation. The crystal structure of NO donor-reacted PTP1B at 2.6 A resolution revealed that the S-NO state at Cys-215 had no discernible irreversibly oxidized forms, whereas other Cys residues remained in their free thiol states. We further demonstrated that S-nitrosylation of the Cys-215 residue protected PTP1B from subsequent H(2)O(2)-induced irreversible oxidation. Increasing the level of cellular NO by pretreating cells with an NO donor or by activating ectopically expressed NO synthase inhibited reactive oxygen species-induced irreversible oxidation of endogenous PTP1B. These findings suggest that S-nitrosylation might prevent PTPs from permanent inactivation caused by oxidative stress.

An increased expression of Yes-associated protein (YAP1) has been shown to promote tumorigenesis in many cancer types including colon. However, the role of YAP1 in promoting colon tumorigenesis remains unclear. Here, we demonstrate that YAP1 expression is associated with M2 tumor-associated macrophage polarization and the generation of colon cancer stem-like cells. YAP1 downregulation by gene silencing or a phytochemical, ovatodiolide, not only suppresses colon cancer tumorigenesis but also prevents M2 TAM polarization. Human monocytic cells, THP-1, and colon cancer cell lines, HCT116 and DLD-1, were co-cultured to mimic the interactions between tumor and its microenvironment. M2 polarization of the THP-1 cells were examined using both flow cytometry and q-PCR technique. The inhibition of YAP1 signaling was achieved by gene-silencing technique or ovatodiolide. The molecular consequences of YAP1 inhibition was demonstrated via colony formation, migration, and colon-sphere formation assays. 5-FU and ovatodiolide were used in drug combination studies. Xenograft and syngeneic mouse models were used to investigate the role of YAP1 in colon tumorigenesis and TAM generation. An increased YAP1 expression was found to be associated with a poor prognosis in patients with colon cancer using bioinformatics approach. We showed an increased YAP1 expression in the colon spheres, and colon cancer cells co-cultured with M2 TAMs. YAP1-silencing led to the concomitant decreased expression of major oncogenic pathways including Kras, mTOR, β-catenin, and M2-promoting IL-4 and tumor-promoting IL-6 cytokines. TAM co-cultured colon spheres showed a significantly higher tumor-initiating ability in vivo. Ovatodiolide treatment alone and in combination with 5-FU significantly suppressed in vivo tumorigenesis and less TAM infiltration in CT26 syngeneic mouse model. We have identified the dual function of YAP1 where its suppression not only inhibited tumorigenesis but also prevented the generation of cancer stem-like cells and M2 TAM polarization. Ovatodiolide treatment suppressed YAP1 oncogenic pathways to inhibit colon tumorigenesis and M2 TAM generation both in vitro and in vivo. Ovatodiolide should be considered for its potential for adjuvant therapeutic development.

The oxidation and inactivation of protein tyrosine phosphatases is one mechanism by which reactive oxygen species influence tyrosine phosphorylation‐dependent signaling events and exert their biological functions. In the present study, we determined the redox status of endogenous protein tyrosine phosphatases in HepG2 and A431 human cancer cells, in which reactive oxygen species are produced constitutively. We used mass spectrometry to assess the state of oxidation of the catalytic cysteine residue of endogenous PTP1B and show that this residue underwent both reversible and irreversible oxidation to high stoichiometry in response to intrinsic reactive oxygen species production. In addition, our data show that the oxidation of PTP1B is specific to the active site Cys, with the other Cys residues in the protein remaining in a reduced state. Treatment of these cells with diphenyleniodonium, an inhibitor of NADPH oxidases, decreased reactive oxygen species levels. This resulted in inhibition of protein tyrosine phosphatase oxidation, concomitant with decreased tyrosine phosphorylation of cellular proteins and inhibition of anchorage‐independent cell growth. Therefore, our data also suggest that the high level of intrinsic reactive oxygen species may contribute to the transformed phenotype of HepG2 and A431 cells via constitutive inactivation of cellular protein tyrosine phosphatases.

Aims: Distinctive states of redox-dependent cysteine (Cys) modifications are known to regulate signaling homeostasis under various pathophysiological conditions, including myocardial injury or protection in response to ischemic stress. Recent evidence further implicates a dynamic interplay among these modified forms following changes in cellular redox environment. However, a precise delineation of multiplexed Cys modifications in a cellular context remains technically challenging. To this end, we have now developed a mass spectrometry (MS)-based quantitative approach using a set of novel iodoacetyl-based Cys-reactive isobaric tags (irreversible isobaric iodoacetyl Cys-reactive tandem mass tag [iodoTMT]) endowed with unique irreversible Cys-reactivities. Results: We have established a sequential iodoTMT-switch procedure coupled with efficient immunoenrichment and advanced shotgun liquid chromatography-MS/MS analysis. This workflow allows us to differentially quantify the multiple redox-modified forms of a Cys site in the original cellular context. In one single analysis, we have identified over 260 Cys sites showing quantitative differences in multiplexed redox modifications from the total lysates of H9c2 cardiomyocytes experiencing hypoxia in the absence and presence of S-nitrosoglutathione (GSNO), indicative of a distinct pattern of individual susceptibility to S-nitrosylation or S-glutathionylation. Among those most significantly affected are proteins functionally implicated in hypoxic damage from which we showed that GSNO would protect. Innovation: We demonstrate for the first time how quantitative analysis of various Cys-redox modifications occurring in biological samples can be performed precisely and simultaneously at proteomic levels. Conclusion: We have not only developed a new approach to map global Cys-redoxomic regulation in vivo , but also provided new evidences implicating Cys-redox modifications of key molecules in NO-mediated ischemic cardioprotection. Antioxid. Redox Signal. 20, 1365–1381.

Mitogen-activated protein kinases (MAPKs) are important mediators that integrate signaling from upstream pathways in response to various environmental cues. In order to control appropriate gene expression through phosphorylation of transcription factors, the activity of MAPKs must be tightly regulated by the actions coordinated between protein kinases and phosphatases. In this study, we explore the underlying mechanism through which the oxidative stress-activated c-Jun N-terminal kinases (JNKs), members of MAPKs, are regulated by dual specificity phosphatases (DUSPs). DUSPs are a group of enzymes that belong to the superfamily of protein-tyrosine phosphatases. They are able to recognize phospho-Ser/Thr and phospho-Tyr residues in substrates. Using quantitative real time PCR, we found that stimulation of human embryonic kidney 293T cells with H(2)O(2) or xanthine/xanthine oxidase led to inducible expression of multiple DUSPs. We used RNA interference to characterize the functional role of these DUSPs and found rapid and transient induction of DUSP1 and DUSP10 to be essential for determining the appropriate magnitude of JNK activation in response to oxidative stress. The transcription factor ATF2, which is phosphorylated and activated by JNK, is a critical mediator for inducible expression of DUSP1 and DUSP10 in this signaling pathway. We further demonstrated that DUSP4 and DUSP16, both showing significant late phase induction, dephosphorylate JNK effectively, causing the down-regulation of the signaling cascade. Thus, this study provides new insights into the role of several DUSPs that coordinate with each other to control the magnitude and duration of JNK activity in response to oxidative stress.

NO synthesis is a prerequisite for proper insulin sensitivity in insulin-targeted tissues; however, the molecular basis for this process remains unclear. Using a gain-of-function model of endothelial nitric-oxide synthase (eNOS)-transfected COS-7 cells, we have shown a critical role of NO in insulin responsiveness, as evidenced by an NO-dependent increase of tyrosine phosphorylation levels of the insulin receptor and its downstream effectors insulin receptor substrate-1 and PKB/AKT. We hypothesized that NO-induced inactivation of endogenous protein-tyrosine phosphatases (PTPs) would enhance insulin receptor-mediated signaling. To test this hypothesis, we devised a new method of the PTP labeling using a cysteine sulfhydryl-reacted probe. Under the acidic conditions employed in this study, the probe recognized the reduced and active forms but not the S-nitrosylated and inactive forms of endogenous PTPs. Our data suggest that phosphatases SHP-1, SHP-2, and PTP1B, but not TC-PTP, are likely S-nitrosylated at the active site cysteine residue concomitantly with a burst of NO production in signaling response to insulin stimulation. These results were further confirmed by phosphatase activity assays. We investigated further the role of NO as a regulator of insulin signaling by RNA interference that ablates endogenous eNOS expression in endothelial MS-1 cells. We have shown that eNOS-dependent NO production is essential for the activation of insulin signaling. Our findings demonstrate that NO mediates enhancement of insulin responsiveness via the inhibition of insulin receptor phosphatases.