Cysteine hydropersulfide (CysSSH) occurs in abundant quantities in various organisms, yet little is known about its biosynthesis and physiological functions. Extensive persulfide formation is apparent in cysteine-containing proteins in Escherichia coli and mammalian cells and is believed to result from post-translational processes involving hydrogen sulfide-related chemistry. Here we demonstrate effective CysSSH synthesis from the substrate l-cysteine, a reaction catalyzed by prokaryotic and mammalian cysteinyl-tRNA synthetases (CARSs). Targeted disruption of the genes encoding mitochondrial CARSs in mice and human cells shows that CARSs have a crucial role in endogenous CysSSH production and suggests that these enzymes serve as the principal cysteine persulfide synthases in vivo. CARSs also catalyze co-translational cysteine polysulfidation and are involved in the regulation of mitochondrial biogenesis and bioenergetics. Investigating CARS-dependent persulfide production may thus clarify aberrant redox signaling in physiological and pathophysiological conditions, and suggest therapeutic targets based on oxidative stress and mitochondrial dysfunction.

Pyruvate kinase M2 (PKM2) is an alternatively spliced variant of the pyruvate kinase gene that is preferentially expressed during embryonic development and in cancer cells. PKM2 alters the final rate-limiting step of glycolysis, resulting in the cancer-specific Warburg effect (also referred to as aerobic glycolysis). Although previous reports suggest that PKM2 functions in nonmetabolic transcriptional regulation, its significance in cancer biology remains elusive. Here we report that stimulation of epithelial-mesenchymal transition (EMT) results in the nuclear translocation of PKM2 in colon cancer cells, which is pivotal in promoting EMT. Immunoprecipitation and LC-electrospray ionized TOF MS analyses revealed that EMT stimulation causes direct interaction of PKM2 in the nucleus with TGF-β-induced factor homeobox 2 (TGIF2), a transcriptional cofactor repressor of TGF-β signaling. The binding of PKM2 with TGIF2 recruits histone deacetylase 3 to the E-cadherin promoter sequence, with subsequent deacetylation of histone H3 and suppression of E-cadherin transcription. This previously unidentified finding of the molecular interaction of PKM2 in the nucleus sheds light on the significance of PKM2 expression in cancer cells.pyruvate kinase M2 | epithelial-mesenchymal transition | colorectal cancer | invasion | transforming growth factor-β-induced factor homeobox 2 C olorectal cancer (CRC) is the second most common cancer in the world, with more than 1.2 million new cases and about 600,000 deaths annually (1). Cancerous cells exploit a cancerspecific glycolytic system known as the Warburg effect (also referred to as aerobic glycolysis), which involves rapid glucose uptake and preferential conversion to lactate, despite an abundance of oxygen (2, 3). The precise mechanism underpinning aerobic glycolysis was unclear for a long time. However, in 2008, pyruvate kinase M2 (PKM2) gained attention when its expression was shown to be required for the maintenance of aerobic glycolysis (4). PKM2 is an alternatively spliced variant of the PKM gene that regulates the final rate-limiting step of glycolysis. PKM2 is expressed during embryonic development, but it is generally not expressed in most adult tissues. However, its counterpart, PKM1, is exclusively expressed in adult tissues. PKM2 has been shown to be reactivated in tumor development (5, 6). In cancer cells, PKM2 expression allows the diversion of glycolytic flux into the pentose phosphate pathway associated with attenuated pyruvate kinase activity, thereby meeting the biosynthetic demands for rapid proliferation (3).Investigations about the nuclear function of PKM2 arose after elucidation of the PKM2 metabolic function. It was identified that in cancer cells, PKM2 can translocate into the nucleus and function as a transcriptional cofactor in response to several extracellular signals, including EGF and hypoxia, subsequently activating CYCLIN D1, C-MYC, or hypoxia-inducible factor 1α (HIF-1α) (7,8). Particularly in the hypoxic condition, PKM2 interacts with HIF-1α and partici...

Highlights d PKM1 promotes tumor growth cell intrinsically in some contexts d PKM1 activates glucose catabolism without interfering with biosynthetic pathways d PKM1-dependent autophagy/mitophagy contributes to malignancy d Expression of PKM1, but not PKM2, is sufficient to support SCLC cell proliferation

Polycomb group (PcG) proteins are key regulators of stem-cell and cancer biology. They mainly act as repressors of differentiation and tumor-suppressor genes. One key silencing step involves the trimethylation of histone H3 on Lys27 (H3K27) by EZH2, a core component of the Polycomb Repressive Complex 2 (PRC2). The mechanism underlying the initial recruitment of mammalian PRC2 complexes is not well understood. Here, we show that NIPP1, a regulator of protein Ser/Thr phosphatase-1 (PP1), forms a complex with PP1 and PRC2 components on chromatin. The knockdown of NIPP1 or PP1 reduced the association of EZH2 with a subset of its target genes, whereas the overexpression of NIPP1 resulted in a retargeting of EZH2 from fully repressed to partially active PcG targets. However, the expression of a PP1-binding mutant of NIPP1 (NIPP1m) did not cause a redistribution of EZH2. Moreover, mapping of the chromatin binding sites with the DamID technique revealed that NIPP1 was associated with multiple PcG target genes, including the Homeobox A cluster, whereas NIPP1m showed a deficient binding at these loci. We propose that NIPP1 associates with a subset of PcG targets in a PP1-dependent manner and thereby contributes to the recruitment of the PRC2 complex.

Protein phosphatase type 1 (PP1), together with protein phosphatase 2A (PP2A), is a major eukaryotic serine/threonine protein phosphatase involved in regulation of numerous cell functions. Although the roles of PP2A have been studied extensively using okadaic acid, a well known inhibitor of PP2A, biological analysis of PP1 has remained restricted because of lack of a specific inhibitor. Recently we reported that tautomycetin (TC) is a highly specific inhibitor of PP1. To elucidate the biological effects of TC, we demonstrated in preliminary experiments that treatment of COS-7 cells with 5 M TC for 5 h inhibits endogenous PP1 by more than 90% without affecting PP2A activity. Therefore, using TC as a specific PP1 inhibitor, the biological effect of PP1 on MAPK signaling was examined. First, we found that inhibition of PP1 in COS-7 cells by TC specifically suppresses activation of ERK, among three MAPK kinases (ERK, JNK, and p38). TC-mediated inhibition of PP1 also suppressed activation of Raf-1, resulting in the inactivation of the MEK-ERK pathway. To examine the role of PP1 in regulation of Raf-1, we overexpressed the PP1 catalytic subunit (PP1C) in COS-7 cells and found that PP1C enhanced activation of Raf-1 activity, whereas phosphatase-dead PP1C blocked Raf-1 activation. Furthermore, a physical interaction between PP1C and Raf-1 was also observed. These data strongly suggest that PP1 positively regulates Raf-1 in vivo.Protein phosphatases regulate numerous cellular functions and signal transduction pathways in cooperation with protein kinases (1, 2). Protein phosphatase types 1 and 2A, known as PP1 1 and PP2A, are two of four major protein serine/threonine phosphatases (PPs) that regulate diverse cellular events such as cell division, transcription, translation, muscle contraction, glycogen synthesis, and neuronal signaling (3-5).Okadaic acid (OA), a polyether fatty acid from the marine black sponge Halichondria okadai, was first identified as a small molecular weight inhibitor of PP and has been studied extensively (6). More than 40 compounds that inhibit PP1 as well as PP2A have been identified. Using these natural compounds, numerous experiments have been performed to analyze the roles of PPs in various cellular events (6, 7). The IC 50 values of such phosphatase inhibitors are almost identical for PP1 and PP2A, with the exception of compounds such as OA, TF-23A, and fostriecin (8 -10). PP2A is selectively inhibited by OA, TF-23A, and fostriecin, and this selectivity has made it possible to analyze PP2A function in living cells. However, no known inhibitor inhibits PP1 specifically. Oikawa et al. (11) reported the total chemical synthesis of tautomycin (TM), a small molecular weight PP inhibitor originally isolated from Streptomyces spiroverticillatus. Using the synthesized TM and related compounds, we previously examined the structure-function relationship of TM and found that the left-and right-hand moieties of TM are required for inhibition of PP and induction of apoptosis, respectively (12). We also re...

We have recently isolated two cDNAs encoding two forms of transmembrane and cytosolic protein tyrosine phosphatase 1 (PTP1). In this study, the 5 H end of the rat PTP1 gene was isolated and characterized. Transmembrane PTP1 (PTP1M) and cytosolic PTP1 (PTP1C) were encoded by a single gene. 5 H RACE analysis and RNase protection assay showed that the mRNA of each PTP1 isoform was transcribed from different promoters. The putative promoter regions of two alternative first exons lacked a TATA box, but contained potential recognition sites for several transcription factors. Reverse transcription PCR analysis revealed that PTP1C mRNA was up-regulated during interleukin 6-induced differentiation of murine leukemia M1 cells, whereas PTP1M mRNA was downregulated. With the use of luciferase as a reporter gene, the promoter activities of the 5 H -flanking regions were examined during phorbol myristate acetate-induced differentiation of HL-60 cells. In the differentiated HL-60 cells, the activity of the PTP1C promoter, but not that of PTP1M, was dramatically elevated. Furthermore, we found that PTP1C mRNA is highly expressed in mouse peritoneal macrophages and enhanced during activation by lipopolysaccharide. These results suggest that the different promoters control expression of PTP1 isoforms during the differentiation and/or activation of macrophages.

Pre-mRNA splicing entails reversible phosphorylation of spliceosomal proteins. Recent work has revealed essential roles for Ser/Thr phosphatases, such as protein phosphatase-1 (PP1), in splicing, but how these phosphatases are regulated is largely unknown. We show that nuclear inhibitor of PP1 (NIPP1), a major PP1 interactor in the vertebrate nucleus, recruits PP1 to Sap155 (spliceosome-associated protein 155), an essential component of U2 small nuclear ribonucleoprotein particles, and promotes Sap155 dephosphorylation. C-terminally truncated NIPP1 (NIPP1-⌬C) formed a hyper-active holoenzyme with PP1, rendering PP1 minimally phosphorylated on an inhibitory site. Forced expression of NIPP1-WT and -⌬C resulted in slight and severe decreases in Sap155 hyperphosphorylation, respectively, and the latter was accompanied with inhibition of splicing. PP1 overexpression produced similar effects, whereas small interfering RNA-mediated NIPP1 knockdown enhanced Sap155 hyperphosphorylation upon okadaic acid treatment. NIPP1 did not inhibit but rather stimulated Sap155 dephosphorylation by PP1 in vitro through facilitating Sap155/PP1 interaction. Further analysis revealed that NIPP1 specifically recognizes hyperphosphorylated Sap155 thorough its Forkhead-associated domain and dissociates from Sap155 after dephosphorylation by associated PP1. Thus NIPP1 works as a molecular sensor for PP1 to recognize phosphorylated Sap155.Pre-mRNA splicing is an essential step for expression of most genes in metazoans. Intron excision from a nascent transcript is achieved by pre-mRNA splicing catalyzed by the spliceosome, a macromolecular complex consisting of five small nuclear ribonucleoprotein particles (snRNPs) 4 and a large number of nonsnRNP proteins. Spliceosome assembly is an ordered process that includes stepwise recruitment of U1, U2, U5, and U4/6 snRNPs on a pre-mRNA and sequential formation of complex E 3 A/B 3 B* 3 C. The activated B* spliceosome catalyzes step I of splicing, whereas the C complex catalyzes step II. During and after splicing, spliceosome components dissociate and are recycled for further rounds of splicing. Spliceosome assembly/disassembly and splicing catalysis are thought to be regulated in part by reversible phosphorylation of spliceosomal proteins (1-3).U2 snRNP includes U2 snRNA and two heteromeric protein complexes, Sf3a and Sf3b. Sap155, also known as Sf3b1 or Sf3b155, is a component of the Sf3b and becomes hyperphosphorylated concomitant with or just after the first catalytic step of splicing in vitro (4). A recent study reveals that Sf3a/b proteins are destabilized and dissociate from the RNP core of the activated spliceosome during the transition from the B to C complex (5). Although Sf3a and Sf3b are essential early in the splicing reaction, they are apparently not required for the second catalytic step. Currently, it is not known what triggers exchange of proteins during spliceosome transitions. Shi et al. (6) reported that the protein Ser/Thr phosphatase (PPase) type 1 (PP1) and/or type 2A (PP2A) ar...

We engineered and expressed both a wild-type and mutant cytosolic isoform of PTPε (PTPεC) in murine M1 leukemic cells, which can be induced to growth arrest and monocytic differentiation by interleukin-6 (IL-6) and leukemia inhibitory factor (LIF). Forced expression of PTPεC inhibited IL-6-and LIF-induced monocytic differentiation and apoptosis in M1 cells, whereas expression of PTPεM, a transmembrane isoform of PTPε, did not. PTPεC expression resulted in lower levels of IL-6-induced tyrosine phosphorylation of Jak1, Tyk2, gp130 and Stat3 compared to parent cells. In M1 transfectants expressing an inactive mutant of PTPεC, both tyrosine phosphorylation and apoptosis induced by IL-6 and LIF were potentiated rather than inhibited. These results suggest an important role for PTPεC in negative regulation of IL-6and LIF-induced Jak-STAT signaling. cytokines. The Jak-STAT pathway mediates many of these functions and constitutes an important signaling pathway. Binding of cytokines to their receptors activates a family of transcription factors, designated STAT(s), through phosphorylation of a specific tyrosine residue by receptor-associated Jak tyrosine kinases (1-3). Interleukin (IL)-6, a typical multifunctional cytokine, induces both differentiation of normal B lymphocytes and production of acute phase protein in hepatocytes. IL-6 stimulates growth in cells such as multiple myelomas and B cell plasmacytomas/hybridomas, while it inhibits growth in several myeloid leukemia cells. Murine M1 myeloid leukemia cells, for example, undergo cell cycle arrest and monocytic differentiation following IL-6 stimulation and have been used as a model system for studying IL-6-signaling (4-9). The receptor for IL-6 is composed of an IL-6-specific receptor α-chain subunit, gp80, and a signal transducing chain, gp130. gp130 is a shared signaling moiety for cytokine receptors of the IL-6 family, including IL-6, leukemia inhibitory factor (LIF), oncostatin M, IL-11, ciliary neurotrophic factor and cardiotrophin 1 (10-15). In the Jak family, Jak1, Jak2 and Tyk2 associate constitutively with gp130 and are activated by IL-6 family cytokines, while Jak1 is thought to play an essential role in the signaling (16-18). Activated Jak(s), in turn, induces tyrosine phosphorylation of the heart, lung and testis (39, 42). We and others have shown that expression of PTPεC, but not PTPεM, is up-regulated during monocytic differentiation of several cell lines (39, 42). To elucidate roles of PTPεC in IL-6 signaling, we engineered and over-expressed wild-type and a dominant-negative form of PTPεC in M1 cells and analysed the biological consequences of ectopic expression. Our findings identified an isoform-specific role for PTPεC in inhibition of Jak-STAT signaling elicited by cytokines belonging to the IL-6 family. EXPERIMENTAL PROCEDURES Materials. Human recombinant IL-6 was kindly provided by Ajinomoto Co. (Yokohama, Japan). Murine LIF was obtained from Gibco-BRL. FCS was purchased from Intergen. Cell culture. All cells were cultured in RPMI1640 medium con...