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MicroRNA-155 (miRNA-155) is known to play an important role in the regulation of innate and adaptive immune responses in mammals. However, no information is available on the role of miRNA-155 in relation to type I interferon (IFN) responses in fish cells. In the present study, we found that the protein inhibitor of activated STAT 4a (PIAS4a) gene of fathead minnow (Pimephales promelas) was a target of miR-155, which was verified by the inhibitory activity of miR-155 in the expression of reporter gene harboring 3'UTR of PIAS4a of EPC cells. Furthermore, cells over-expressing miR-155 showed a significantly higher type I IFN response after polyinosinic-polycytidylic acid (poly I:C) stimulation, suggesting the targeting of PIAS4a in EPC cells by miR-155 can be a cause of the up-regulation of type I IFN, and miR-155 can act as an antiviral factor. However, as the targeting PIAS4a might not be the sole cause of the type I IFN up-regulation by miR-155, further studies on the uncovering of miR-155 target genes that are involved in type I IFN responses in fish are required.

Background Protein inhibitors of activated STAT (PIAS) proteins are regarded as negative regulators of cytokine-signaling and potent immunosuppressive proteins. However, their role in the process of organ transplant rejection has not been elucidated. Methods In the current study, we compared transcript levels of PIAS1 to 4 in the peripheral blood of renal transplant recipients who experienced transplant rejection with those having normal transplant functions. Expression of PIAS1 was significantly higher in nonrejected group compared with the rejected group among male recipients; however, differences were insignificant among female recipients. Expressions of other PIAS genes were not different between study groups. Significant pairwise correlations were found between expression levels of PIAS genes in all study subgroups. The current investigation highlights the role of PIAS1 downregulation in the evolution of graft rejection and potentiates this gene as a predictive marker for transplant fate.

Misregulation of transcription factors, including signal transducer and activator of transcription (STAT) proteins, leads to inappropriate gene expression patterns that can promote tumor initiation and progression. Under physiologic conditions, STAT signaling is stimulus-dependent and tightly regulated by endogenous inhibitors, namely suppressor of cytokine signaling (SOCS) proteins, phosphatases, and protein inhibitor of activated STAT (PIAS) proteins. However, in tumorigenesis, STAT proteins become constitutively active and promote the expression of pro-growth and pro-survival genes. Although STAT activation has been widely implicated in cancer, therapeutic STAT inhibitors are still largely absent from the clinic. This review dissects the mechanisms of action of two families of endogenous STAT inhibitors, the SOCS and PIAS families, to potentially inform the development of novel therapeutic inhibitors.

As a conserved posttranslational modification, SUMOylation has been shown to play important roles in chromatin-related biological processes including transcription. However, how the SUMOylation machinery associates with chromatin is not clear. Here, we present evidence that multiple SUMOylation machinery components, including SUMO E1 proteins SAE1 and SAE2 and the PIAS (protein inhibitor of activated STAT) family SUMO E3 ligases, are primarily associated with the nuclear matrix rather than with chromatin. We show using nuclease digestion that all PIAS family proteins maintain nuclear matrix association in the absence of chromatin. Of importance, we identify multiple histones including H3 and H2A.Z as directly interacting with PIAS1 and demonstrate that this interaction requires the PIAS1 SAP (SAF-A/B, Acinus, and PIAS) domain. We demonstrate that PIAS1 promotes SUMOylation of histones H3 and H2B in both a SAP domain- and an E3 ligase activity-dependent manner. Furthermore, we show that PIAS1 binds to heat shock-induced genes and represses their expression and that this function also requires the SAP domain. Altogether, our study reveals for the first time the nuclear matrix as the compartment most enriched in SUMO E1 and PIAS family E3 ligases. Our finding that PIAS1 interacts directly with histone proteins also suggests a molecular mechanism as to how nuclear matrix-associated PIAS1 is able to regulate transcription and other chromatin-related processes.

(AP) is a kind of inflammatory misorder existing in pancreas. Non-coding RNAs (ncRNAs) have been reported to play important roles in development of AP. The current study was designed to explore the role of circular RNA zinc finger protein 644 (circRNA circ_ZFP644) in caerulein-induced AR42J cells. AP model in vitro was established by exposure of rat pancreatic acinar AR42J cells to caerulein. Amylase activity was measured using a kit. Enzyme-linked immunosorbent assay (ELISA) was performed to examine the levels of several inflammatory factors. The expression of circ_ZFP644, microRNA (miR)-106b and protein inhibitor of activated STAT 3 (Pias3) was detected by quantitative real-time PCR (qRT-PCR) or western blot assay. And flow cytometry was employed to monitor cell apoptosis. Western blot assay was also conducted to analyze the expression of apoptosis-related proteins. The association among circ_ZFP644, miR-106b and Pias3 was validated by dual-luciferase reporter assay. Caerulein treatment activated amylase activity and promoted the secretion of inflammatory cytokines in AR42J cells. Circ_ZFP644 and Pias3 were downregulated, but miR-106b was upregulated in caerulein-induced AR42J cells. Enforced expression of circ_ZFP644 or miR-106b inhibition could reduce amylase activity and inflammatory cytokine secretion, while promote apoptosis in caerulein-induced AR42J cells, which was almost reversed by Pias3 knockdown. Circ_ZFP644 targeted miR-106b to upregulate Pias3 expression. Circ_ZFP644 might exert its anti-inflammation and pro-apoptosis roles in caerulein-induced AR42J cells by regulating miR-106b/Pias3 axis.

Hepatocellular carcinoma (HCC) is one of the lethal and leading types of cancer threatening the globe with a high mortality rate. STAT3 is an oncogenic transcription factor that is aberrantly activated in several human malignancies including HCC. Many STAT3-driven genes control cell proliferation and survival, apoptotic resistance, cell cycle progression, metastasis, and chemotherapeutic resistance. STAT3 signaling is regulated by endogenous modulators such as protein tyrosine phosphatase (PTP), suppressor of cytokine signaling (SOCS), protein inhibitor of activated STAT (PIAS), and various long noncoding RNAs (lncRNAs). Interestingly, lncRNAs have been reported to exhibit oncogenic and tumor suppressor functions, and these effects are mediated through diverse molecular mechanisms including sponging of microRNAs (miRs), transcription activation/inhibition, and epigenetic modifications. In this article, we have discussed the possible role of STAT3 signaling in hepatocarcinogenesis and various mechanisms by which lncRNAs impart their oncogenic or tumor suppressive action by modulating the STAT3 pathway in HCC.

The development of STAT protein-specific inhibitors has been the focus of a number of drug discovery programs. STAT activation occurs through phosphorylation at the STAT SH2 domain, resulting in dimerization, translocation to the nucleus, and transcription of proliferative genes. Due to the functional significance of the SH2 domain in mediating multiple components of the STAT signalling cascade, many libraries of inhibitors have been designed to target the SH2 domain. This has triggered the requirement for effective high-throughput screening platforms for analyzing binding by larger chemical libraries to STAT proteins. Herein, we present strategies for the development of a high-throughput thermal denaturation-based assay for identifying STAT inhibitors as well as high-yielding recombinant expression and purification of untagged STAT1, STAT3, and STAT5 proteins. This assay reports changes in the fluorescence of a labelled peptide bound to the STAT protein as a function of increasing temperature. STAT inhibitors which displace the labelled peptide elicit a change in the melt profile, which is quantitatively determined as a change in the area under the curve. This assay offers an alternative, but complimentary, high-throughput screening strategy for identifying new inhibitors of STAT proteins as well as characterizing further, the mode of inhibition by existing libraries of compounds.

Nutrient restriction, also known as caloric restriction, has been extensively examined for its positive impact on lifespan, immune system boost, and aging. In addition, nutrient restriction is implicated in decreasing cancer initiation and progression. Given the phenotypic changes associated with nutrient restriction, we hypothesized significant protein expression alterations must be associated with caloric restriction. To compare the molecular and phenotypic changes caused by glucose restriction and fetal bovine serum (FBS) restriction there is need for an efficient model system. We establish three dimensional cell culture models, known as spheroids, in the HCT 116 colorectal cancer cell line as a high throughput model for studying the proteomic changes associated with nutrient restriction. Flow cytometry was used to assess apoptosis and autophagy levels in the spheroids under nutrient restriction. Isobaric tags for relative and absolute quantification (iTRAQ) and liquid chromatography tandem mass spectrometry were used to determine differential protein abundances between the nutrient restriction conditions. We identified specific proteins that have implications in cancer progression and metastasis that are differentially regulated by restriction of either glucose or serum. These proteins include the up regulation of sirtuin 1 (SIRT1) and protein inhibitor of activated STAT 1 (PIAS1) and down regulation of multi-drug resistance protein (MRP1) and Zinc finger and BTB domain-containing protein 7A (ZBTB7). The results indicate nutrient restriction causes lower apoptotic and higher autophagy rates in HCT 116 spheroids. In addition, proteins shown to be differentially regulated by both glucose and serum restriction were similarly regulated.

Parkin-interacting substrate (PARIS), a member of the family of Krüppel-associated box (KRAB)-containing zinc-finger transcription factors, is a substrate of the ubiquitin E3 ligase parkin. PARIS represses the expression of peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α), although the underlying mechanisms remain largely unknown. In the present study, we demonstrate that PARIS can be SUMOylated, and its SUMOylation plays a role in the repression of PGC-1a promoter activity. Protein inhibitor of activated STAT y (PIASy) was identified as an interacting protein of PARIS and shown to enhance its SUMOylation. PIASy repressed PGC-1a promoter activity, and this effect was attenuated by PARIS in a manner dependent on its SUMOylation status. Co-expression of SUMO-1 with PIASy completely repressed PGC-1a promoter activity independently of PARIS expression. PARIS-mediated PGC-1a promoter repression depended on the activity of histone deacetylases (HDAC), whereas PIASy repressed the PGC-1a promoter in an HDAC-independent manner. Taken together, these results suggest that PARIS and PIASy modulate PGC-1a gene transcription through distinct molecular mechanisms.

The signal transducers and activators of the transcription (STAT) family play an important role in regulatory and cellular functions by regulating the expression of a variety of genes, including cytokines and growth factors. In the present study, a Pinctada fucata STAT protein, termed PfSTAT, was described. The deduced amino acid sequence of PfSTAT contains the conserved STAT_bind domain and the SH2 domain, and the additional Bin/Amphiphysin/Rvs (BAR) domain, but does not have STAT_alpha and STAT_int domains. Multiple sequence alignments revealed that PfSTAT showed relatively low identity with vertebrate and other invertebrate STATs, and phylogenetic analysis indicated that the evolution of STAT may have been more complex and ancient. Gene expression analysis revealed that PfSTAT is involved in the immune response to polyinosinic-polycytidylic acid (poly I:C) stimulation and in the nucleus insertion operation. This study contributes to a better understanding of PfSTAT in protecting the pearl oyster from disease or injury caused by grafting.

Protein inhibitor of activated STAT (PIAS) plays a critical role in the feedback modulation of various signaling pathways as a negative regulator. To further understand the role of PIAS in immune and stress response in mud crab ( Scylla paramamosain ), in the present study, a full-length cDNA of PIAS from S. paramamosain (SpPIAS) was cloned for the first time, which contained an open reading frame of 2364 bp encoding a polypeptide of 788 amino acids. Quantitative reverse transcription PCR (qRT-PCR) was used to analyze the tissue distribution of PIAS in mud crab. The results revealed that SpPIAS was expressed in hemocytes, heart, hepatopancreas, gills, stomach, intestines, muscle, connective tissue and gonad of S. paramamosain , and the higher expression levels were found in connective tissue and gonad. The expression levels of SpPIAS in hemocytes were up-regulated after challenge with Staphylococcus aureus , Vibrio harveyi , and white spot syndrome virus (WSSV) for 2–24 h, which suggested that SpPIAS was involved in the pathogen-resistant activities of mud crab. Furthermore, the RING-like domain (RLD) and Serine/Threonine rich repeat C-terminal domain (S/T) of SpPIAS were expressed by prokaryotic expression system and purified by glutathione Sepharose 4B chromatography. This study contributed to define the biological characteristics of SpPAIS and further demonstrate the critical role of SpPAIS in vivo during the pathogen infection.

The development of the follicle and competent oocyte is highly coordinated, requiring interplay among several systems. These implicate endocrine, immune, and metabolic signals, intrafollicular paracrine factors from theca, mural, and cumulus granulosa cells, and the oocyte itself. Granulosa cells play a key role in their interaction. COV434 is one of the few human granulosa cell lines that can be used as an in vitro model for ovarian research. We aimed to evaluate the possible activation of the Janus kinase/signal transducers and activators of transcription (JAK/STAT) signaling pathway by IL-6-type cytokines leukemia inhibitory factor (LIF) and oncostatin M (OSM) in COV434 cells. Expression of GP130 (glycoprotein 130), STAT3 (signal transducer and activators of transcription 3), PIAS3 (protein inhibitor of activated STAT 3), and SOCS3 (suppressor of cytokine signaling 3) genes after stimulation with LIF or OSM was assessed using RT-qPCR (real-time PCR). GP130 transcripts were significantly upregulated after incubation with LIF or OSM for 24h. Expression of the STAT3 gene was stimulated only after incubation with LIF, but not OSM. SOCS3 showed significant upregulation for all time periods and the levels of PIAS3 were initially down- and after 24h upregulated. Furthermore, the major signaling components of the JAK/STAT pathway, GP130 and STAT3, and the kinase activation patterns of STAT3, were examined at protein level. We found constitutive protein expression for GP130, STAT3, pSTAT3(ser727) and upregulation of pSTAT3(tyr705) by LIF and OSM. Our results demonstrate the activation of the JAK/STAT pathway by LIF and OSM in human granulosa cells.

The Janus Kinase/Signal Transducers and Activators of Transcription (JAK/STAT) signaling pathway is one of a limited number of signaling cascades that is extensively utilized for many developmental and homeostatic functions. The JAK/STAT pathway is evolutionarily conserved from insects to mammals, with homologous transduction machinery in each. Yet the mammalian pathway is composed of multiple members for each family of proteins, while flies have only a single homologue of most pathway components. This simplicity and the abundance of genetic, biochemical, and developmental tools make Drosophila an attractive model to investigate this important signaling pathway. This review provides a basic description of the Drosophila JAK/STAT cascade and summarizes currently available reagents and tools to study and manipulate the pathway.

Background & Aims Constitutive activation of NF-κB is an important event involved in chronic inflammation in hepatocellular carcinoma (HCC). CPAP, which plays important roles in centrosomal functions, was previously identified as the transcriptional co-activator of NF-κB. However, the molecular mechanism is unclear. The goal of this study was to investigate the role of CPAP in activating the NF-κB pathway in HCC. Methods SK-Hep1, HuH7, HepG2, HepG2X, Hep3B, and Hep3BX cells with CPAP overexpression or CPAP siRNA were used to evaluate activation of NF-κB under TNF-α stimulation by reporter assay, RT-PCR, Q-PCR, and Western blot analysis. In vivo SUMO modification of CPAP was demonstrated by an in situ PLA assay. Human HCC tissues were used to perform Q-PCR, Western blot, and IHC. Results CPAP siRNA abolished the interaction between IKKβ and NF-κB, whereas overexpression of CPAP enhanced this interaction and finally led to augmented NF-κB activation by increasing the phosphorylation of NF-κB. CPAP could enter nuclei by associating with NF-κB. Furthermore, CPAP was SUMO-1 modified upon TNF-α stimulus, and this is essential for its NF-κB co-activator activity. SUMO-1-deficient CPAP mutant lost its NF-κB co-activator activity and failed to enter nuclei. Importantly, SUMOylated CPAP could synergistically increase the HBx-induced NF-κB activity. Conclusions CPAP is essential for the recruitment of the IKK complex to inactivated NF-κB upon TNF-α treatment. Expression of CPAP was positively correlated with a poor prognosis in HBV-HCC. CPAP has the potential to serve as a therapeutic target for inflammation and inflammation-related diseases.

Liver X receptors (LXRs) are nuclear receptors that function to modulate lipid metabolism as well as immune and inflammatory responses. Upon activation by their ligands, LXRs up-regulate a spectrum of gene transcription programs involved in cholesterol and fatty acid homeostasis. However, the mechanisms by which LXR-mediated transcriptional activation is regulated remain incompletely understood. Here, we show that PIAS1, a member of the protein inhibitor of the activated STAT family of proteins with small ubiquitin-like modifier (SUMO) E3 ligase activity, acts to suppress LXR ligand-dependent transcriptional activation of the lipogenic program in hepatocytes. We found that liver mRNA expression levels of Pias1 and Pias3 were inversely associated with those of genes involved in lipogenesis in mouse models with diet-induced or genetic obesity. Overexpression of PIAS1 in primary hepatocytes resulted in a reduction of LXR ligand-induced fatty acid synthesis and suppression of the expression of lipogenic genes, including Srebp1c and Fas. Moreover, PIAS1 was able to interact with LXRβ and repress its transcriptional activity upon ligand stimulation, which did not require PIAS1-promoted SUMO modification of LXRβ. In addition, PIAS1 could also interact with PGC-1β and attenuate its association with LXRβ, blunting the ability of PGC-1β to co-activate LXRβ. Importantly, PIAS1 impaired LXRβ binding to its target DNA sequence. Taken together, our results suggest that PIAS1 may serve as a lipogenic regulator by negatively modulating LXRs in a SUMOylation-independent manner.

Signal transducer and activator of transcription (STAT) proteins are key signaling molecules in response to cytokines and in regulating T cell biology. However, there are contradicting reports on whether STAT is involved in T-cell antigen receptor (TCR) signaling. To better define the role of STAT in TCR signaling, we activated the CD4/CD8-associated Lck kinase by co-crosslinking TCR and CD4/CD8 co-receptors in human peripheral blood T cells. Sequential STAT1, STAT3 and STAT5 activation was observed 1 hour after TCR stimulation suggesting that STAT proteins are not the immediate targets in the TCR complex. We further identified interferon-gamma as the key cytokine in STAT1 activation upon TCR engagement. In contrast to transient STAT activation in cytokine response, this autocrine/paracrine-induced STAT activation was sustained. It correlated with the absence of two suppressor of cytokine signaling (SOCS) proteins, SOCS3 and cytokine-inducible SH2 containing protein, that are negative feedback regulators of STAT signaling. Moreover, enforced expression of SOCS3 inhibited tyrosine phosphorylation of zeta-associated protein kinase of 70 kD in TCR-stimulated human Jurkat T cells. This is the first report demonstrating delayed and prolonged STAT activation coordinated with the loss of SOCS expression in human primary T cells after co-crosslinking of TCR and CD4/CD8 co-receptors.

Since its discovery in the early 1990s, the Janus kinase (JAK)-signal transducer and activator of transcription (STAT) signaling pathway has been found to play key roles in regulating many key cellular processes such as survival, proliferation, and differentiation. There are seven known mammalian STAT family members: STAT1, 2, 3, 4, 5a, 5b, and 6. In the liver, activation of these STAT proteins is critical for anti-viral defense against hepatitis viral infection and for controlling injury, repair, inflammation, and tumorigenesis. The identification of functions for these STAT proteins has increased our understanding of liver disease pathophysiology and treatments, while also suggesting new therapeutic modalities for managing liver disease.

ErbB4 is a receptor tyrosine kinase implicated in the development and homeostasis of the heart, central nervous system, and mammary gland. Cleavable isoforms of ErbB4 release a soluble intracellular domain (ICD) that can translocate to the nucleus and function as a transcriptional coregulator. In search of regulatory mechanisms of ErbB4 ICD function, we identified PIAS3 as a novel interaction partner of ErbB4 ICD. In keeping with the small ubiquitin-like modifier (SUMO) E3 ligase function of protein inhibitor of activated STAT (PIAS) proteins, we showed that the ErbB4 ICD is modified by SUMO, and that PIAS3 stimulates the SUMOylation. Upon overexpression of PIAS3, the ErbB4 ICD generated from the full-length receptor accumulated into the nucleus in a manner that was dependent on the functional nuclear localization signal of ErbB4. In the nucleus, ErbB4 colocalized with PIAS3 and SUMO-1 in promyelocytic leukemia nuclear bodies, nuclear domains involved in regulation of transcription. Accordingly, PIAS3 overexpression had an effect on the transcriptional coregulatory activity of ErbB4, repressing its ability to coactivate transcription with Yes-associated protein. Finally, knockdown of PIAS3 with siRNA partially rescued the inhibitory effect of the ErbB4 ICD on differentiation of MDA-MB-468 breast cancer and HC11 mammary epithelial cells. Our findings illustrate that PIAS3 is a novel regulator of ErbB4 receptor tyrosine kinase, controlling its nuclear sequestration and function.

Janus kinase 1/signal transducers and activators of transcription 3 (JAK1/STAT3) pathway is one of the recognized oncogenic signaling pathways that frequently overactivated in a variety of human tumors. Despite rapid progress in elucidating the molecular mechanisms of activation of JAK/STAT pathway, the processes that regulate JAK/STAT deactivation need to be further clarified. Here we demonstrate that CUE domain-containing 2 (CUEDC2) inhibits cytokine-induced phosphorylation of JAK1 and STAT3 and the subsequent STAT3 transcriptional activity. Further analysis by a yeast two-hybrid assay showed that CUEDC2 could engage in a specific interaction with a key JAK/STAT inhibitor, SOCS3 (suppressors of cytokine signaling 3). The interaction between CUEDC2 and SOCS3 is required for the inhibitory effect of CUEDC2 on JAK1 and STAT3 activity. Additionally, we found CUEDC2 functions collaboratively with SOCS3 to inhibit JAK1/STAT3 signaling by increasing SOCS3 stability via enhancing its association with Elongin C. Therefore, our findings revealed a new biological activity for CUEDC2 as the regulator of JAK1/STAT3 signaling and paved the way to a better understanding of the mechanisms by which SOCS3 has been linked to suppression of the JAK/STAT pathway.

Metastasis tumor antigen 1 (MTA1), a component of the Mi-2·nucleosome remodeling and deacetylase complex, plays a crucial role in gene transcription, but the mechanism involved remains largely unknown. Here, we report that MTA1 is a substrate for small ubiquitin-related modifier 2/3 (SUMO2/3) in vivo. Protein inhibitor of activated STAT (PIAS) proteins enhance SUMOylation of MTA1 and may participate in paralog-selective SUMOylation, whereas sentrin/SUMO-specific protease 1 (SENP1) and 2 may act as deSUMOylation enzymes for MTA1. Moreover, MTA1 contains a functional SUMO-interacting motif (SIM) at its C terminus, and SIM is required for the efficient SUMOylation of MTA1. SUMO conjugation on Lys-509, which is located within the SUMO consensus site, together with SIM synergistically regulates the co-repressor activity of MTA1 on PS2 transcription, probably by recruiting HDAC2 onto the PS2 promoter. Interestingly, MTA1 may up-regulate the expression of SUMO2 via interaction with RNA polymerase II and SP1 at the SUMO2 promoter. These findings not only provide novel mechanistic insights into the regulation of the transcriptional repressor function of MTA1 by SUMOylation and SIM but also uncover a potential function of MTA1 in modulating the SUMOylation pathway.

The protein inhibitor of activated STAT (PIAS) family proteins regulates innate immune responses by controlling transcription induced by Toll-like receptor, RIG-I-like receptor signaling, and JAK/STAT pathways. Here, we show that PIASy negatively regulates type I interferon (IFN) transcription. Virus infection led to enhanced type I IFN induction in PIASy null cells, and conversely PIASy overexpression reduced IFN transcription. A mutation in the LXXLL motif of the SAP domain abolished inhibition of IFN-stimulated gene expression but did not affect virus or Toll-like receptor/RIG-I-like receptor-stimulated IFN transcription, indicating that PIASy employs distinct mechanisms to inhibit virus-induced and IFN-stimulated transcription. SUMO E3 activity was not required for PIASy inhibition of IFN transcription; however, PIASy relied on the SUMO modification mechanism to inhibit IFN transcription, because the activity of the SUMO-interacting motif was required for inhibition, and knockdown of SUMO E2 enzyme UBC9 decreased inhibitory activity of PIASy. Our results demonstrate that PIASy negatively regulates both IFN transcription and IFN-stimulated gene expression through multiple mechanisms utilizing the function of different domains.

The signal transducers and activators of transcription (STAT) proteins are a small family of signaling proteins that are crucial for cytokine and growth factor receptor-mediated signaling in various blood cell types. Despite their central role in immune and hematopoietic cellular regulation, there are relatively few options for monitoring receptor-mediated JAK/STAT signaling events in a cell-based format, without the need for cellular transfections or labor intensive methodology. Indeed, traditional methods such as the Western blot or ELISA remain a standard method for determining the phosphorylation status of endogenous STAT proteins. Here we present data for the rapid detection of endogenous receptor-mediated phosphorylation of multiple STAT proteins using the bead-based AlphaScreen SureFire technology. With three different cell lines (human acute monocytic leukemia THP-1 cells, human erythroleukemic TF-1 cells, and human T lymphocytic Jurkat cells), we have optimized a rapid and homogeneous methodology for monitoring endogenous, receptor-mediated signaling via STAT 1, STAT 3, or STAT 5 phosphorylation, in response to several agonists. These assays, which can be tailored for both standard research applications or high-throughput drug screening applications, afford quantitative data for receptor-mediated signaling mechanisms in an endogenous, cellular environment.

The PIAS proteins (protein inhibitor of activated STAT) were originally identified as inhibitors of the JAK-STAT pathway. Subsequently, their roles on transcriptional regulation have been identified in modulation of the androgen receptor (AR) and other nuclear hormone receptor-mediated actions. Zimp7, also named Zmiz2, is a novel PIAS-like protein and functions as a transcriptional co-activator. In this study, we demonstrate an interaction between Zimp7 and PIAS proteins with higher preference for PIAS3. A modified mammalian one-hybrid assay showed that the NH(2)-terminal proline-rich domain of Zimp7 and the region spanning amino acids 321-486 of PIAS3 were the primary interaction segments. The interaction between Zimp7 and PIAS3 proteins was further confirmed by in vitro protein pull-down and co-immunoprecipitation assays with both exogenous and endogenous proteins. Expression of exogenous PIAS3 further enhances Zimp7-mediated augmentation of AR transcription. Knockdown of the endogenous PIAS3 protein using a specific PIAS3 small hairpin RNA reduced the augmentation of Zimp7 on AR-mediated transcription. Co-localization of Zimp7 and PIAS3 proteins was observed in the nuclei of cells by immunostaining. Exogenous PIAS3 expression enhances the stability of the Zimp7 protein. Using chromatin immunoprecipitation assays, we showed that PIAS3 is involved in the AR- and Zimp7-formed protein complex(es) in the AR downstream target promoter to facilitate androgen-induced transcription. Finally, we further demonstrated that loss of Zimp7 significantly impaired PIAS3-mediated enhancement on AR activity in mouse Zimp7 null (zimp7(-/-)) embryonic fibroblasts. Taken together, these results demonstrate a novel interaction between PIAS and PIAS-like proteins and elucidate a novel regulatory mechanism for PIAS proteins in AR-mediated transcription.

Members of the PIAS (protein inhibitor of activated STAT) family perform essential functions in modulating the activity of transcriptional regulators. Zimp7 and Zimp10 are two proteins that together form a subfamily of the PIAS. Like the other members of this family, they contain the zinc-binding SP-RING/Miz domain, which confers SUMO-conjugating activity. Both proteins have been shown to stimulate androgen receptor-mediated transcription. Previously, we reported that both Zimp7 and Zimp10 genes are extensively expressed and dynamically regulated in the developing mouse embryo. In this work, we investigated the expression of these genes during gonadal development. We found that their expression is sex-specific. Both genes initiate their transcription at early stages in the embryonic male gonad, reaching their peak at 13.5 days post coitum, which coincides with the process of sex-specific germ cell mitotic arrest. Zimp7 is expressed in germ cells of the embryonic gonad and the adult testis. Immunofluorescence of spermatogenic cells revealed that Zimp7 protein localizes to nuclear territories in meiotic spermatocytes, including the XY bodies. On the other hand, Zimp10 is found in somatic cells, outside the testis cords and ceases to be expressed in the adult testis.

Covalent modification by small ubiquitin-related modifiers (SUMOs) is an important means to regulate dynamic residency of transcription factors within nuclear compartments. Here, we identify a multi-C(2)H(2)-type zinc finger protein (ZNF), ZNF451, as a novel nuclear protein that can be associated with promyelocytic leukemia bodies. In keeping with its interaction with SUMO E2 conjugase Ubc9 and SUMOs, ZNF451 is covalently modified by SUMOs (sumoylated) at several, albeit nonconsensus, sites. Interestingly, noncovalent SUMO-binding activity of ZNF451 (SUMO-interacting motif) is also important for its sumoylation. SUMO modifications regulate the nuclear compartmentalization of ZNF451, since coexpression of ZNF451 with SUMO-specific proteases SENP1 or SENP2, both capable of desumoylating the protein, redistributes ZNF451 from nuclear domains to speckles and nucleoplasm. Interaction of ZNF451 with PIAS1 (protein inhibitor of activated STAT 1) is not manifested as PIAS1's E3 SUMO ligase activity towards ZNF451 but results in disintegration of ZNF451 nuclear domains and recruitment of ZNF451 to androgen receptor (AR) speckles. ZNF451 interacts weakly, but in a SUMO-1-enhanced fashion, with AR. ZNF451 does not harbor an intrinsic transcription activation function, but interestingly, ablation of endogenous ZNF451 in prostate cancer cells significantly decreases expression of several AR target genes. Thus, we suggest that ZNF451 exerts its effects via SUMO modification machinery and trafficking of transcription regulators between promyelocytic leukemia bodies and nucleoplasm.

The JAK/STAT signal transduction pathway has traditionally been viewed as a cytokine-stimulated activator of gene expression consisting of a straightforward receptor/JAK kinase/STAT transcription factor cascade. Recent studies in Drosophila, have, however consistently identified a range of chromatin-remodelling factors as regulators of in vivo JAK/STAT signalling. Now, the detailed analysis of one of these, heterochromatin protein 1 (HP1), has provided an insight into an unexpected non-canonical in vivo role for STAT. In this model, unphosphorylated STATs associate with and maintain the stability of transcriptionally repressed heterochromatin--an effect countered by the recruitment of STAT to the canonical pathway. We examine the background of this new model and its implications for JAK/STAT pathway requirements in stem cell maintenance and cancer.

Covalent attachment of small ubiquitin-like modifier (SUMO) to proteins regulates multiple processes in the eukaryotic cell. In numerous cases sumoylation is facilitated by protein inhibitor of activated STAT (PIAS) proteins, characterized by the presence of a SP-RING domain related to the RING finger of many ubiquitin E3 ligases. The importance of SP-RING relies on its capacity to bind the E2 enzyme of the pathway. Additional domains may participate in SUMO ligase function and target selection. We have studied the Arabidopsis SUMO ligase AtSIZ1, belonging to the PIAS family, and describe self-sumoylation and AtSIZ1-mediated sumoylation of the E2 enzyme AtSCE1 and GTE3, a bromodomain protein interacting with AtSIZ1. Modification of GTE3 modulates its capacity to bind acetyl-histone H3 in vitro. Interestingly, AtSIZ1, as other plant PIAS proteins, also includes a PHD domain. We found that the PHD domain binds AtSCE1 and contributes to the SUMO ligase function, being partially and absolutely required for AtSCE1 and GTE3 sumoylation, respectively. Based on the capacity of AtSCE1 and GTE3 to associate with both the PHD and SP-RING domains, we propose a model of interactions to explain AtSIZ1-mediated sumoylation of GTE3 and ligase function of the PHD domain.

Although the JAK/STAT signaling pathway is usually involved in antiviral defense, a recent study suggested that STAT might be annexed by WSSV (white spot syndrome virus) to enhance the expression of a viral immediate early gene in infected shrimps. In the present study, we clone and report the first full-length cDNA sequence for a crustacean STAT from Penaeus monodon. Alignment and comparison with the deduced amino acid sequences of other STATs identified several important conserved residues and functional domains, including the DNA binding domain, SH2 domain and C-terminal transactivation domain. Based on these conserved sequences, a phylogenetic analysis suggested that shrimp STAT belongs to the ancient STAT family, while the presence of the functional domains suggested that shrimp STAT might share similar functions and regulating mechanisms with the well-known STATs isolated from model organisms. Real-time PCR showed a decreased transcription level of shrimp STAT after WSSV infection, but a Western blot analysis using anti-phosphorylated STAT antibody showed an increased level of phosphorylated (activated) STAT in the lymphoid organ of shrimp after WSSV infection. We further show that a primary culture of lymphoid organ cells from WSSV-infected shrimp resulted in activated STAT being translocated from the cytoplasm to the nucleus. This report provides experimental evidence that shrimp STAT is activated in response to WSSV infection. Our results support an earlier finding that WSSV does not disrupt JAK/STAT pathway, but on the contrary benefits from STAT activation in the shrimp host.

SummarySignal Transducer and Activator of Transcription (STAT) proteins are a family of latent cytoplasmic transcription factors that are activated by tyrosine phosphorylation after cytokine stimulation. One mechanism by which STAT signaling is regulated is by dephosphorylation through the action of protein tyrosine phosphatases (PTP). We have identified PTP-Basophil like (PTP-BL) as a STAT PTP. PTP-BL dephosphorylates STAT proteins in vitro and in vivo, resulting in attenuation of STAT-mediated gene activation. In CD4+ T cells, PTP-BL deficiency leads to increased and prolonged activation of STAT4 and STAT6, and consequently enhanced T helper 1 (Th1) and Th2 cell differentiation. Taken together, our findings demonstrate that PTP-BL is a physiologically important negative regulator of the STAT signaling pathway.

Cytokine receptors act through a complex signaling network, involving Janus kinases (JAKs) and the signal transducers and activators of transcription (STATs), to regulate diverse biological processes which control growth, development, homeostasis and immune function, among others. The JAK/STAT signaling pathway is attenuated via three mechanisms controlling the initiation, magnitude, and duration of the signal: the PIAS proteins, which prevent STAT dimerization or DNA interaction, the SHP phosphatases, which dephosphorylate activating tyrosine phosphorylations, and the suppressors of cytokine signaling (SOCS), which are transcribed in response to cytokine stimulation and use several interconnected mechanisms to downregulate the signal. Specific studies targeting the SOCS genes in vivo have unveiled SOCS2 as the main regulator of somatic growth through regulation of GH/IGF-1 signaling. In addition, several studies indicate that SOCS2 also has important actions in the central nervous system, the regulation of metabolism, the immune response, the mammary gland development, cancer, and other cytokine-dependent signaling pathways. Consistent with the role of cytokines in human physiology, any SOCS2 imbalance could result in a broad range of pathologies such as cardiovascular diseases, insulin resistance, cancer, and severe infections, among others. Thus, determining the importance of SOCS2 in health and disease will no doubt aid in the development of novel therapeutic strategies. In this review, we attempt to summarize the available information, including our results, regarding the role of SOCS2 in several biological processes.

The signal transducers and activators of transcription (Stat) are effector molecules downstream of cytokine receptors. Ligand occupancy of these receptors results in the tyrosine phosphorylation, dimerization and nuclear translocation of the Stat family of transcription factors and by these means regulate gene expression. Prolactin receptors as members of the cytokine-hematopoietin receptor superfamily, are linked to Stat activation. Sexual stimulation leads to an increase in prolactin secretion that might be involved in long-term changes in the protein repertoire associated to prostate hyperplasia. In order to gain insight into this phenomenon, we analyzed the tyrosine phosphorylation and DNA binding activity of two members of the Stat family in the prostate of sexual experienced rats after different number of ejaculations. A significant increase in Stat-1 and Stat-3 tyrosine phosphorylation was found after three ejaculations. Concomitantly an increase in Stat-1 and Stat-3 DNA-binding activity is detected after two and three ejaculation series. These results, favor the notion that ejaculation-induced prolactin secretion activates its prostate receptors resulting in Stat-1 and Stat-3 nuclear translocation, event likely to be associated to the so-called benign prostate hyperplasia.

In the canonical model of JAK/STAT signalling STAT transcription factors are activated by JAK mediated tyrosine phosphorylation following pathway stimulation by external cytokines. Activated STAT molecules then homo- or heterodimerise before translocating to the nucleus where they bind to DNA sequences within the promoters of pathway target genes. DNA-bound STAT dimers then activate transcription of their targets via interaction with components of the basal transcription machinery. Here we describe a missense mutation in the SH2 domain of the single Drosophila STAT92E homologue which results in an amino-acid substitution conserved in both the canonical SH2 domain and STAT-like molecules previously identified in C. elegans and the mosquito Anopheles gambiae. This mutation leads to nuclear accumulation and constitutive DNA binding of Drosophila STAT92E even in the absence of JAK stimulation. Strikingly, this mutant shows only limited transcriptional activity in tissue culture based assays and functions as a dominant-negative at both the phenotypic and molecular levels in vivo. These features represent aspects of both dominant gain-of-function and dominant-negative activities and imply that the functions of DNA binding can be functionally separated from the role of STAT92E as a transcriptional activator. It is thus possible that an alternative post-translational modification, in addition to tyrosine phosphorylation, may be required to allow STAT to act as a transcriptional activator and suggests the existence of an alternative mechanism by which STAT transcriptional activity may be regulated in vivo.

Transcription activators often recruit promoter-targeted assembly of a pre-initiation complex; many repressors antagonize recruitment. These activities can involve direct interactions with proteins in the pre-initiation complex. We used an optimized yeast two-hybrid system to screen mouse pregnancy-associated libraries for proteins that interact with TATA-binding protein (TBP). Screens revealed an interaction between TBP and a single member of the zinc finger family of transcription factors, ZFP523. Two members of the protein inhibitor of activated STAT (PIAS) family, PIAS1 and PIAS3, also interacted with TBP in screens. Endogenous PIAS1 and TBP co-immunoprecipitated from nuclear extracts, suggesting the interaction occurred in vivo. In vitro-translated PIAS1 and TBP co-immunoprecipitated, which indicated that other nuclear proteins were not required for the interaction. Deletion analysis mapped the PIAS-interacting domain of TBP to the conserved TBP(CORE) and the TBP-interacting domain on PIAS1 to a 39-amino acid C-terminal region. Mammals issue seven known PIAS proteins from four pias genes, pias1, pias3, piasx, and piasy, each with different cell type-specific expression patterns; the TBP-interacting domain reported here is the only part of the PIAS C-terminal region shared by all seven PIAS proteins. Direct analyses indicated that PIASx and PIASy also interacted with TBP. Our results suggest that all PIAS proteins might mediate situation-specific regulatory signaling at the TBP interface and that previously unknown levels of complexity could exist in the gene regulatory interplay between TBP, PIAS proteins, ZFP523, and other transcription factors.

SUMO (small ubiquitin-like modifier)/Smt3 (suppressor of mif two) is a member of the ubiquitin-related protein family and is known to conjugate with many proteins. In the sumoylation pathway, SUMO/Smt3 is transferred to substrate lysine residues through the thioester cascade of E1 (activating enzyme) and E2 (conjugating enzyme), and E3 (SUMO ligase) functions as an adaptor between E2 and each substrate. Yeast Ull1 (ubiquitin-like protein ligase 1)/Siz1, a PIAS (protein inhibitor of activated STAT)-type SUMO ligase, modifies both cytoplasmic and nuclear proteins. In this paper, we performed a domain analysis of Ull1/Siz1 by constructing various deletion mutants. A novel conserved N-terminal domain, called PINIT, as well as the RING-like domain (SP-RING) were required for the SUMO ligase activity in the in vitro conjugation system and for interaction with Smt3 in an in vitro binding assay. The most distal N-terminal region, which contains a putative DNA-binding SAF-A/B, Acinus, and PIAS (SAP) motif, was not required for the ligase activity but was involved in nuclear localization. A strong SUMO-binding motif was identified, which interacted with Smt3 in the two-hybrid system but was not necessary for the ligase activity. The most distal C-terminal domain was important for stable localization at the bud neck region and thereby for the substrate recognition of septins. Furthermore, the C-terminal half conferred protein instability on Ull1/Siz1. Taken together, we conclude that the SP-RING and PINIT of Ull1/Siz1 are core domains of the SUMO ligase, and the other domains are regulatory for protein stability and subcellular localization.

Summary STAT proteins are a family of latent cytoplasmic transcription factors that are activated by tyrosine phosphorylation in response to a variety of cytokines, growth factors, and hormones. Once activated, STAT proteins translocate into the nucleus and help coordinate gene transcription. One striking feature of STAT signaling is its rapid and transient activation and deactivation cycle, although the molecular mechanisms responsible for this remain poorly understood. Here, we report on a nuclear protein that contains both PDZ and LIM domains and that interacts with activated STAT4 molecules. We show that SLIM is an ubiquitin E3 ligase that acts on STAT proteins to cause their proteosome-mediated degradation and enhance their dephosphorylation. Overexpression of SLIM leads to impaired STAT1 and STAT4 activity due to reduced STAT protein levels, while SLIM-deficiency results in increased STAT expression and thus enhanced IFNγ production by Th1 cells. These studies suggest that SLIM is a novel ubiquitin E3 ligase whose targets include STAT proteins.

Aldosterone synthase (CYP11B2) is involved in the final steps of aldosterone biosynthesis and expressed exclusively in the adrenal zona glomerulosa cells. Using an electrophoretic mobility shift assay, we demonstrate that COUP-TFI binds to the -129/-114 element (Ad5) of human CYP11B2 promoter. Transient transfection in H295R adrenal cells demonstrated that COUP-TFI enhanced CYP11B2 reporter activity. However, the reporter construct with mutated Ad5 sequences showed reduced basal and COUP-TFI-enhanced activity, suggesting that binding of COUP-TFI to Ad5 is important for CYP11B2 transactivation. To elucidate molecular mechanisms of COUP-TFI-mediated activity, we subsequently screened for COUP-TFI-interacting proteins from a human adrenal cDNA library using a yeast two-hybrid system and identified Ubc9 and PIAS1, which have small ubiquitin-related modifier-1 (SUMO-1) conjugase and ligase activities, respectively. The coimmunoprecipitation assays confirmed that COUP-TFI forms a complex with Ubc9 and PIAS1 in mammalian cells. Immunohistochemistry showed that Ubc9 and PIAS1 are markedly expressed in rat adrenal glomerulosa cells. Coexpression of Ubc9 and PIAS1 synergistically enhanced the COUP-TFI-mediated CYP11B2 reporter activity, indicating that both proteins function as coactivators of COUP-TFI. However, sumoylation-defective mutants, Ubc9 (C93S) and PIAS1 (C351S), continued to function as coactivators of COUP-TFI, indicating that sumoylation activity are separable from coactivator ability. In addition, chromatin immunoprecipitation assays demonstrated that ectopically expressed COUP-TFI, Ubc9, and PIAS1 were recruited to an endogenous CYP11B2 promoter. Moreover, reduction of Ubc9 or PIAS1 protein levels by small interfering RNA inhibited the CYP11B2 transactivation by COUP-TFI. Our data support a physiological role of Ubc9 and PIAS1 as transcriptional coactivators in COUP-TFI-mediated CYP11B2 transcription.

Signal transducers and activators of transcription (Stats) comprise a family of seven transcription factors that are activated by a variety of cytokines, hormones and growth factors. Stats are activated through tyrosine phosphorylation, mainly by Jak kinases, that lead to their dimerization, nuclear translocation and regulation of target gene expression. Stat5 was originally identified as a transcription factor that regulates the beta-casein gene in response to prolactin (PRL), but Stat5 is activated also by several other cytokines and growth factors. The molecular mechanisms that underlie Stat5-mediated transcription involve interactions and cooperation with sequence specific transcription factors and transcriptional coregulators. Our studies identified p100 protein as a coactivator for Stat5, and suggest the existence of a positive regulatory loop in PRL-induced transcription, where PRL stabilizes p100 protein, which in turn can cooperate with Stat5 in transcriptional activation. Suppressors of cytokine signaling (SOCS) proteins are important negative regulators of Stats. A target gene for Stat5, the serine/threonine kinase Pim-1, was found to cooperate with SOCS-1 and SOCS-3 to inhibit Stat5 activity suggesting that Pim-1 together with SOCS-1 and SOCS-3 are components of a negative feedback mechanism that allows Stat5 to regulate its own activation.

Stats (for signal transducers and activators of transcription) are a family of transcription factors that regulate cell growth and differentiation. Their activity is latent until phosphorylation by receptor-associated kinases. A sizable body of data from cell lines, mouse models, and human tissues now implicates these transcription factors in the oncogenesis of breast cancer. Because Stat activity is modulated by several posttranslational modifications and protein-protein interactions, these transcription factors are capable of integrating inputs from multiple signaling networks. Given this, the future utilization of Stats as prognostic markers and therapeutic targets in human breast cancer appears likely.

Human pleomorphic adenoma gene 1 (PLAG1), a developmentally regulated proto-oncogene, is consistently rearranged and overexpressed in pleomorphic salivary gland adenomas and lipoblastomas with 8q12 translocations. Together with PLAGL1 and PLAGL2, PLAG1 belongs to a subfamily of C(2)H(2) zinc finger transcription factors that activate transcription through binding to the bipartite consensus sequence GRGGC(N)(6-8)GGG. Ectopic expression of PLAG1 deregulates target genes and presumably results in uncontrolled cell proliferation. To gain insight into molecular mechanisms regulating PLAG transcriptional capacity, we searched for interaction partners using the yeast two-hybrid system and confirmed these by glutathione S-transferase pull-down. Ubiquitin-conjugating enzyme 9 (UBC9) and protein inhibitor of activated STAT (PIAS) proteins were first identified as genuine interacting partners of mouse PlagL2. Because UBC9 and PIAS are components of the small ubiquitin-related modifier (SUMO) modification pathway, we hypothesized that PLAG proteins could be SUMOylated. Here, we report results obtained for founding family member PLAG1. Its endogenous SUMOylation was demonstrated, and SUMOylation of PLAG1 was further investigated in cells co-transfected with PLAG1 and SUMO-1 DNA or a SUMO-1 mutant form and similarly examined in the presence or absence of DNA encoding the various PIAS proteins. Using anti-PLAG1 antibodies, we discovered single and double SUMO-1-modified forms of PLAG1. By mutating predicted SUMO consensus sites, we defined two important target lysines for SUMOylation in PLAG1, Lys-244 and Lys-263. Moreover, mutation of both SUMO consensus sequences, resulting in inhibition of SUMOylation, led to a significant increase of the transactivation capacity of PLAG1. Nuclear distribution of PLAG1 was not measurably influenced. Our results suggest a direct repression of the transactivating capacity of the oncoprotein PLAG1 by SUMOylation.

A member of the PIAS (protein inhibitor of activated STAT) family of proteins, PIAS1, have been reported to serve as an E3-type SUMO ligase for tumor suppressor p53 and its own. It also was proposed that the N-terminal domain of PIAS1 interacts with DNA as well as p53. Extensive biochemical studies have been devoted recently to understand sumoylations and its biological implications, whereas the structural aspects of the PIAS family and the mechanism of its interactions with various factors are less well known to date. In this study, the three-dimensional structure of the N-terminal domain (residues 1-65) of SUMO ligase PIAS1 was determined by NMR spectroscopy. The structure revealed a unique four-helix bundle with a topology of an up-down-extended loop-down-up, a part of which the helix-extended loop-helix represented the SAP (SAF-A/B, Acinus, and PIAS) motif. Thus, this N-terminal domain may be referred to as a four-helix SAP domain. The glutathione S-transferase pull-down assay demonstrated that this domain possesses a binding ability to tumor suppressor p53, a target protein for sumoylation by PIAS1, whereas gel mobility assays showed that it has a strong affinity toward A/T-rich DNA. An NMR analysis of the four-helix SAP domain complexed with the 16-bp-long DNA demonstrated that one end of the four-helix bundle is the binding site and may fit into the minor groove of DNA. The three-dimensional structure and its binding duality are discussed in conjunction with the biological functions of PIAS1 as a SUMO ligase.

Members of the Stat transcription factor family are specifically activated by cytokines, and each Stat mediates its biological effects through the trans-activation of a unique profile of target genes. This specificity is achieved even when Stat proteins mediating opposite transcriptional effects bind to the same palindromic Stat sites in target genes. We show here that the non-conserved sequences of Stat transcription activation domains (TADs) contribute to specificity in promoter activation. Chimeric proteins in which the Stat6 TAD was replaced by that from Stat1alpha or Stat5 exhibited normal interleukin-4-inducible DNA binding activity, but at best modest trans-activation of reporters containing Stat6 binding sites, and a failure to activate the endogenous CD23 promoter in primary B cells. The p160 coactivator nuclear coactivator-1 (Src-1) was specifically recruited by and coactivated Stat6 but not the chimeric Stat6 molecules. Strikingly, transcriptional responses exhibited distinct requirements for the nuclear coactivator-1 interaction motif of the Stat6 C terminus. Together, these findings indicate that the Stat6 TAD contributes to promoter specificity by the differential recruitment of and requirement for a p160-class coactivator.

The SP-RING or Miz zinc finger domain that is related to the classical RING-finger motif, defines a class of proteins that can act as E3-like factors in the pathway of small ubiquitin-related modifier (SUMO) conjugation. This family includes the mammalian protein inhibitor of activated STAT (PIAS) proteins and related proteins from lower eukaryotes. Here we report the existence of a gene in Leishmania infantum, present as two identical copies placed upstream of each MAT2 gene copy, and transcribed as a single approximately 2.2 kb mRNA both in the logarithmic and stationary phases of the promastigote stage. This gene encodes a 47 kDa protein that has been named LORIEN. LORIEN is circumscribed to the cell periphery and it is antigenic during L. infantum infection of dogs and hamsters. Strikingly, this novel protein contains a highly conserved SP-RING/Miz zinc finger domain, raising the possibility that a SUMO or ubiquitin-like system may exist in this microorganism.

Smads proteins play a key role in the intracellular signaling of the transforming growth factor (TGF)-beta family of growth factors, which exhibits a diverse set of cellular responses, including cell proliferation and differentiation. In particular, Smad7 acts as an antagonist of TGF-beta signaling, which could determine the intensity or duration of its signaling cascade. In this study we identified a protein inhibitor of activated STAT (signal transducers and activators of transcription), PIASy, as a novel interaction partner of Smad7 by yeast two-hybrid screening using the MH2 domain of Smad7 as bait. The association of Smad7 and PIASy was confirmed using co-expressed tagged proteins in 293T cells. Moreover, we found that other Smads including Smad3 also associated with PIASy through its MH2 domain, and PIASy suppressed TGF-beta-mediated activation of Smad3. PIASy also stimulated the sumoylation of Smad3 in vivo. Furthermore, endogenous PIASy expression was induced by TGF-beta in Hep3B cells. These findings provide the first evidence that a PIAS family protein, PIASy, associates with Smads and involves the regulation of TGF-beta signaling using the negative feedback loop.

PIASx and PIAS1, two members of the conserved protein inhibitor of activated STAT (PIAS) family, are able to interact with and modulate activities of several distinct nuclear proteins, including androgen receptor (AR). PIASx and PIAS1 also function as E3-type ligases in small ubiquitin-related modifier 1 modifications. To gain better insight into the physiological roles of PIASx and PIAS1 in vivo, their cell-type specific expression and regulation were analyzed during testicular development and spermatogenesis in the rat. The expression of both PIASx and PIAS1 was low or undetectable in newborn rats. PIASx mRNA started to accumulate after day 20 of postnatal life, whereas expression of PIAS1 mRNA increased around day 30 after birth. In the adult rat testis, both PIASx and PIAS1 mRNA were present in Sertoli cells and in germ cells in the seminiferous epithelium at all stages. However, PIASx mRNA was more abundant in spermatocytes than in other cell types, whereas higher levels of PIAS1 mRNA were detected in late spermatocytes and round spermatids than in early spermatocytes. Since PIASx and PIAS1 accumulate in developing male germ cells, their regulatory functions are not only restricted to AR in Sertoli cells, but they also participate in molecular processes during meiosis.

Signal transducers and activators of transcription (STAT) factors are cytoplasmic proteins that can be activated by Janus kinases (JAK) and that modulate gene expression in response to cytokine receptor stimulation. STAT proteins dimerize, translocate into the nucleus, and activate specific target genes. In the present study, we show for the first time that interleukin-6 (IL), in the presence of its soluble receptor (sIL-6R), induces activation of JAK1, JAK2, and STAT1/STAT3 proteins in bovine articular chondrocytes. Western blotting and mobility shift assays demonstrated that this effect is accompanied by the DNA binding of the STAT proteins. The mitogen-activated protein kinase pathway was also activated in response to IL-6/sIL-6R association, as reflected by phosphorylation of ERK1 and ERK2 proteins. In these conditions, the expression of cartilage-specific matrix genes, type II collagen, aggrecan core, and link proteins was found to be markedly down-regulated. This negative effect was abolished by addition of parthenolide, an inhibitor of the STAT activation, whereas blockade of the MAP kinases with PD098059 was without significant effect. Thus, activation of the STAT signaling pathways, but not ERK-dependent pathways, is essential for down-regulation of the major cartilage-specific matrix genes by IL-6. In addition, a parallel reduction of Sox9 expression, a key factor of chondrocyte phenotype, was found in these experimental conditions. These IL-6 effects might contribute to the phenotype loss of chondrocytes in joint diseases and the alteration of articular cartilage associated with this pathology.

The cytoplasmic region of human epidermal growth factor receptor (EGFR) contains an intrinsic tyrosine kinase (697-955) followed by a 231-residue-long COOH-terminal tail (C-tail), which contains multiple tyrosine residues. To examine the role of the EGFR C-tail in signal transducer and activator of transcription (STAT) activation, a series of EGFR C-tail truncations were constructed. Transient transfection of 293 cells with EGFR lacking the C-tail, i.e. Y974DeltaEGFR or Y992DeltaEGFR, led to EGF-independent or constitutive STAT activation, whereas EGF-dependent STAT activation was restored with truncations made COOH-terminal to the next tyrosine residue, i.e. EGFR-Y1045Delta. Transfection with the-truncated form EGFR-Y954Delta resulted in the loss of STAT activation, suggesting that the sequence between Tyr(974) and Tyr(954) is essential for STAT activation. Phosphopeptide competition analysis revealed multiple tyrosine residues within the C-tail that can act as the docking sites for both Stat1 and Stat3. A region that negatively regulated STAT activation was also identified, extending from Tyr(1114) to Glu(1172), consistent with the ability of this region to recruit a suppressor of cytokine signaling factors SOCS1 and SOCS3. When cotransfected with the full-length EGFR, but not Y992DeltaEGFR, SOCS1 or SOCS3 inhibited STAT activation by EGF in 293 cells. This suggests that both SOCS1 and SOCS3 can negatively regulate EGFR activation, presumably by inducing ubiquitination-dependent EGFR degradation upon ligand binding. These findings may therefore offer clues to how the EGF receptor C-tail regulates STAT activity.

Members of the protein inhibitor of activated signal transducer and activator of transcription (STAT) family (PIAS family) of proteins act as negative regulators of STATs in cytokine signaling. We report here that PIAS proteins have proapoptotic activity. PIAS1 induced apoptosis in both human 293T cells and human osteosarcoma U2OS cells. PIAS1 is localized in the nucleus as distinct nuclear dots. Ectopic expression of PIAS1 in U2OS cells activated JNK1 (c-Jun NH(2)-terminal kinase). A dominant-negative JNK1, capable of inhibiting PIAS1-induced JNK1 activation, blocked PIAS1-mediated apoptosis. Furthermore, a mutant PIAS1, lacking the first 9 amino acid residues, failed to repress Stat1-mediated gene activation although it retained its ability to activate JNK and to trigger apoptosis. Our results identify a novel function of PIAS1 in the induction of JNK-dependent apoptosis, independent of the previously known inhibitory activity of PIAS1 in STAT-mediated gene activation.

Objective T lymphocytes can be activated by soluble factors such as cytokines or through direct cell-cell interactions. Although cytokine receptors are known to signal through STAT family transcription factors, the mechanisms by which other cell-surface molecules, such as CD2, transduce signals is unclear. The goal of this study was to determine whether stimulation of T cells through CD2 recapitulates aspects of cytokine-induced T-cell activation by use of STAT transcription factors. Materials and methods T cells were treated with anti-CD2 antibodies or cells bearing the natural CD2 ligand CD58, after which signaling through STAT transcription factors was assessed. Results Stimulation of CD2 on primary T lymphocytes leads to the tyrosine phosphorylation, nuclear translocation, and DNA binding of STAT1. In contrast to stimulation by cytokines, the activation of STAT1 in response to CD2 ligation is delayed and does not involve Jak kinases. Furthermore, while STAT phosphorylation induced by cytokines is generally transient, STAT1 phosphorylation following CD2 stimulation persists for a period of days. Transcription of key target genes such as IRF1 and c-fos proceeds with delayed kinetics following CD2 stimulation, suggesting that this unique pattern of STAT activation may lead to a distinct cellular response following CD2 ligation. This pathway appears to be restricted to T cells, as stimulation of CD2 on NK cells does not lead to STAT1 activation. Conclusion Stimulation of T cells through cell-surface molecules such as CD2 involves activation of STAT transcription factors, thus recapitulating elements of cytokine signaling.

Signal transducers and activators of transcription (STAT) factors are cytoplasmic proteins that induce gene activation in response to cytokine receptor stimulation. Following tyrosine phosphorylation, STAT proteins dimerize, translocate into the nucleus, and activate specific target genes. Activation is transient, and down-regulation of STAT signaling occurs within a few hours. In the present study, we show that the cyclin-dependent kinase inhibitor p21(WAF1/CIP1/SDI1) inhibits STAT3 transcriptional activation. Following leukemia inhibitory factor stimulation, p21(WAF1/CIP1/SDI1) was found to associate with STAT3 proteins in coimmunoprecipitation and pull down assays. In vivo, overexpression of p21(WAF1/CIP1/SDI1) reduced transcriptional activation by STAT3 proteins but did not modify DNA binding activity. Interestingly, pull down experiments showed that p21(WAF1/CIP1/SDI1) could interact with the CREB-binding coactivator protein, and inhibition of STAT3 activity by p21(WAF1/CIP1/SDI1) did not occur when CREB-binding protein was overexpressed. These results suggest a model by which p21(WAF1/CIP1/SDI1) functions as an inhibitor of STAT3 signaling and highlight a new activity for this cyclin-dependent kinase inhibitor.