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Classical swine fever (CSF) is a highly contagious viral disease causing severe economic losses to the swine industry. As viroporins of viruses modulate the cellular ion balance and then take over the cellular machinery, blocking the activity of viroporin or developing viroporin-defective attenuated vaccines offers new approaches to treat or prevent viral infection. Non-structural protein p7 of CSF virus (CSFV) is a viroporin, which was highly involved in CSFV virulence. Deciphering the interaction between p7 and host proteins will aid our understanding of the mechanism of p7-cellular protein interaction affecting CSFV replication. In the present study, seven host cellular proteins including microtubule-associated protein RP/EB family member 1 (MAPRE1), voltage-dependent anion channel 1 (VDAC1), proteasome maturation protein (POMP), protein inhibitor of activated STAT 1 (PIAS1), gametogenetin binding protein 2 (GGNBP2), COP9 signalosome subunit 2 (COPS2), and contactin 1 (CNTN1) were identified as the potential interactive cellular proteins of CSFV p7 by using yeast two-hybrid (Y2H) screening. Plus, the interaction of CSFV p7 with MAPRE1 and VDAC1 was further evaluated by co-immunoprecipitation and GST-pulldown assay. Besides, the p7-cellular protein interaction network was constructed based on these seven host cellular proteins and the STRING database. Enrichment analysis of GO and KEGG indicated that many host proteins in the p7-cellular protein interaction network were mainly related to the ubiquitin-proteasome system, cGMP-PKG signaling pathway, calcium signaling pathway, and JAK-STAT pathway. Overall, this study identified potential interactive cellular proteins of CSFV p7, constructed the p7-cellular protein interaction network, and predicted the potential pathways involved in the interaction between CSFV p7 and host cells.

The protein inhibitor of activated STAT (PIAS) proteins are important signal transduction modulator family and regulate the innate immune signaling pathway induced by certain transcription factors, including NF-κB, IRF3, and JAK/STAT. The PIAS protein mechanism that regulates innate immune response in mammals has been well described in the literature; however, whether the PIAS gene exists in ducks as well as the role of PIAS in duck IFN-β expression is still unclear. Here, we cloned duck PIAS (duPIAS), finding PIAS2 could repress IFN-β production. DuPIAS2 contains SAP-PINIT-RLD-S/T characteristic domains, and its overexpression could inhibit virus-induced IFN-β promoter activation. Moreover, duPIAS2 interacts with duck interferon regulatory factor 7 (IRF7) and inhibits IFN-β promoter activation induced by duck IRF7. Additionally, its inhibitory function does not rely on its SUMO E3 ligase activity but rather its C-terminal portion. The above results demonstrate that duPIAS2 is a repressor of IFN-β production induced by duck IRF7.

Proinflammatory cytokine activation of the Janus kinase/signal transducers and activators of transcription (JAK/STAT) signal transduction pathway is a critical event in the pathogenesis and progression of rheumatoid arthritis. Under normal conditions, JAK/STAT signaling reflects the influence of negative regulators of JAK/STAT, exemplified by the suppressor of cytokine signaling and protein inhibitor of activated STAT. However, in rheumatoid arthritis (RA) both of these regulators are dysfunctional. Thus, continuous activation of JAK/STAT signaling in RA synovial joints results in the elevated level of matrix metalloproteinase gene expression, increased frequency of apoptotic chondrocytes and most prominently ‘apoptosis resistance’ in the inflamed synovial tissue. Tofacitinib, a JAK small molecule inhibitor, with selectivity for JAK2/JAK3 was approved by the United States Food and Drug Administration (US FDA) for the therapy of RA. Importantly, tofacitinib has demonstrated significant clinical efficacy for RA in the post-US FDA-approval surveillance period. Of note, the success of tofacitinib has spurred the development of JAK1, JAK2 and other JAK3-selective small molecule inhibitors, some of which have also entered the clinical setting, whereas other JAK inhibitors are currently being evaluated in RA clinical trials.

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.

Protein inhibitor of activated STAT (PIAS) plays a critical role in the feedback modulation of the Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling pathway as a negative regulator in mammals and Drosophila, but the function of PIAS in crustaceans is still unclear. In this study, a PIAS termed LvPIAS was cloned and characterized from Litopenaeus vannamei. The full length of LvPIAS was 3065 bp, including a 2361 bp open reading frame (ORF) coding for a protein of 786 aa. LvPIAS expression was most abundant in muscle and could respond to the challenge of LPS, Vibrio parahaemolyticus, Staphhylococcus aureus, Poly I: C and white spot syndrome virus (WSSV). LvPIAS could be induced by the transcription factor LvSTAT, but LvPIAS could inhibit the transcriptional activity of LvSTAT to the LvPIAS promoter conversely, which indicated that there was a negative feedback loop between LvSTAT and LvPIAS. Furthermore, RNAi-mediated knockdown of LvPIAS shrimps showed higher survival rate to WSSV infection than those in the control group (dsGFP injection), suggesting that LvPIAS may play a negatively role against WSSV infection.

The Protein Inhibitor of Activated STAT 1 (PIAS1) is an E3 SUMO ligase that plays important roles in various cellular pathways, including STAT signaling, p53 pathway, and the steroid hormone signaling pathway. PIAS1 can SUMOylate PML (at Lys-65 and Lys-160) and PML-RARα promoting their ubiquitin-mediated degradation. Increasing evidence shows that PIAS1 is overexpressed in various human malignancies, including prostate and lung cancers. To understand the mechanism of action of PIAS1, we developed a quantitative SUMO proteomic approach to identify potential substrates of PIAS1 in a system-wide manner. Our analyses enabled the profiling of 983 SUMO sites on 544 proteins, of which 204 SUMO sites on 123 proteins were identified as putative PIAS1 substrates. These substrates are involved in different cellular processes, such as transcriptional regulation, DNA binding and cytoskeleton dynamics. Further functional studies on Vimentin (VIM), a type III intermediate filament protein involved in cytoskeleton organization and cell motility, revealed that PIAS1 exerts its effects on cell migration and cell invasion through the SUMOylation of VIM at Lys-439 and Lys-445 residues. VIM SUMOylation was necessary for its dynamic disassembly, and cells expressing a non-SUMOylatable VIM mutant showed reduced levels of proliferation and migration. Our approach not only provides a novel strategy for the identification of E3 SUMO ligase substrates, but also yields valuable biological insights into the unsuspected role of PIAS1 and VIM SUMOylation on cell motility.

Background Bovine viral diarrhea virus (BVDV) belongs to the Flaviviridae family and the pestivius virus group. BVDV is responsible for significant economic loss in cattle industry worldwide because of reducing reproductive performance, increasing incidence of other diseases and mortality among young stock. The core (C) protein of the Flaviviridae family member is involved in host antiviral immune response through activation of related signaling pathways that affect the viral replication. However, the influence of C protein-interaction partners in BVDV infections is poorly defined. Methods To explore C-protein-interacting partners, yeast two-hybrid was used to screen the interaction protein of C protein using bovine peripheral blood mononuclear cell (PBMC) cDNA library. The co-immunoprecipitation and confocal assays were manipulated to determine the interaction between potential partners and C protein. Knockdown and overexpression of the partner were used to examine whether the C-protein-interacting partner plays a role in BVDV proliferation and virulence. Meanwhile, qRT-PCR and western blot assays were used to investigate the effect of C protein and C-protein-interacting partner on the immune response of host cells. Results We identified protein inhibitor of activated STAT 4 (PIAS4) as a novel interacting partner of the BVDV C protein. Co-immunoprecipitation and confocal assays demonstrated a strong interaction between C protein and PIAS4. Silencing of PIAS4 with small interfering RNA suppressed C protein expression and BVDV growth, while overexpression of PISA4 increased C protein expression and BVDV growth. The overexpression of PIAS4 increased the cell apoptosis. Meanwhile, the expressions of STAT4, SOCS3, IFITM, IFN-α were negatively regulated by the expression of PIAS4. The expression of C protein suppressed the antiviral proteins expression, and the inhibition effect was enhanced by interaction of PIAS4 and C protein. These results highlighted the beneficial properties of cellular PIAS4 for BVDV protein expression and growth. Conclusions This study provides reliable clues for understanding the roles of PIAS4 in the regulation of BVDV growth.

The aggressive phenotype displayed by fibroblast-like synoviocytes (FLSs) is a critical factor of cartilage destruction in rheumatoid arthritis (RA). Increased FLSs migration and subsequent degradation of the extracellular matrix are essential to the pathology of RA. Protein inhibitor of activated STAT (PIAS), whose family members include PIAS1, PIAS2 (PIASx), PIAS3, and PIAS4 (PIASy), play important roles in regulating various cellular events, such as cell survival, migration, and signal transduction in many cell types. However, whether PIAS proteins have a role in the pathogenesis of RA is unclear. In this study, we evaluated the role of PIAS proteins in FLSs migration, invasion, and matrix metalloproteinases (MMPs) expression in RA. We observed increased expression of PIAS3, but not PIAS1, PIAS2, or PIAS4, in FLSs and synovial tissues from patients with RA. We found that PIAS3 knockdown by short hairpin RNA reduced migration, invasion, and MMP-3, MMP-9, and MMP-13 expression in FLSs. In addition, we demonstrated that PIAS3 regulated lamellipodium formation during cell migration. To gain insight into molecular mechanisms, we evaluated the effect of PIAS3 knockdown on Rac1/PAK1 and JNK activation. Our results indicated that PIAS3-mediated SUMOylation of Rac1 controlled its activation and modulated the Rac1 downstream activity of PAK1 and JNK. Furthermore, inhibition of Rac1, PAK1, or JNK decreased migration and invasion of RA FLSs. Thus, our observations suggest that PIAS3 suppression may be protective against joint destruction in RA by regulating synoviocyte migration, invasion, and activation.

Protein inhibitor of activated STAT (PIAS) proteins are activation-suppressing proteins for signal transducer and activator of transcription (STAT), which involves gene transcriptional regulation. The inhibitory mechanism of PIAS proteins in the Janus kinase (JAK)/STAT signaling pathway has been well studied in mammals and Drosophila. However, the roles of PIAS in crustaceans are unclear. In the present study, we identified PIAS in kuruma shrimp Marsupenaeus japonicus and found that its relative expression could be induced by Vibrio anguillarum stimulation. To explore the function of PIAS in shrimp infected with V. anguillarum, we performed an RNA interference assay. After knockdown of PIAS expression in shrimp subjected to V. anguillarum infection, bacterial clearance was enhanced and the survival rate increased compared with those in the control shrimp (dsGFP injection). Simultaneously, the expression levels of antimicrobial peptides (AMPs), including anti-lipopolysaccharide factor (ALF) A1, C1, C2, and CruI-1, increased. Further study revealed that knockdown of PIAS also enhanced STAT phosphorylation and translocation. Pulldown assay indicated that PIAS interacts with activated STAT in shrimp. In conclusion, PIAS negatively regulates JAK/STAT signaling by inhibiting the phosphorylation and translocation of STAT through the interaction between PIAS and STAT, which leads to the reduction of AMP expression in shrimp. Our results revealed a new mechanism of PIAS-mediated gene regulation of the STAT signal pathway.

Cobalt protoporphyrin (CoPP) is a potent HO-1 inducer and generally known to be an antioxidant in various cell types. Little is known about the CoPP-induced cyclooxygenase-2 (COX-2) expression and its downstream signaling in microglial cells. In current study, CoPP caused concentration- and time-dependent increases in COX-2 expression in microglial cells. Furthermore, activation of apoptosis signal-regulating kinase (ASK) 1/MAP kinase involved in CoPP-induced COX-2 expression in microglia. CoPP also induced P2X7 receptor activation, and treatment of P2X7 inhibitors effectively reduced CoPP-induced COX-2 expression. Protein inhibitor of activated STAT (PIAS) 1 is reported to be involved in modulating anti-inflammatory response through negative regulation of transcription factors. Interestingly, treatment with CoPP markedly induced PIAS1 degradation which is regulated by PI3K, Akt, and glycogen synthase kinase 3α/β (GSK3α/β) signaling pathways. These results suggest that CoPP induces COX-2 expression through activating P2X7 receptors and ASK1/MAP kinases as well as PIAS1 degradation signaling pathways. Our study provides a new insight into the regulatory effect of CoPP on neuroinflammation in microglial cells.

Several nuclear receptor (NR) superfamily members are known to be the molecular target of either the small ubiquitin-related modifier (SUMO) or ubiquitin-signaling pathways. However, little is currently known regarding how these two post-translational modifications interact to control NR biology. We show that SUMO and ubiquitin circuitry coordinately modifies the pregnane X receptor (PXR, NR1I2) to play a key role in regulating PXR protein stability, transactivation capacity, and transcriptional repression. The SUMOylation and ubiquitylation of PXR is increased in a ligand- and tumor necrosis factor alpha -: dependent manner in hepatocytes. The SUMO-E3 ligase enzymes protein inhibitor of activated signal transducer and activator of transcription-1 (STAT1) STAT-1 (PIAS1) and protein inhibitor of activated STAT Y (PIASy) drive high levels of PXR SUMOylation. Expression of protein inhibitor of activated stat 1 selectively increases SUMO(3)ylation as well as PXR-mediated induction of cytochrome P450, family 3, subfamily A and the xenobiotic response. The PIASy-mediated SUMO(1)ylation imparts a transcriptionally repressive function by ameliorating interaction of PXR with coactivator protein peroxisome proliferator-activated receptor gamma coactivator-1-alpha. The SUMO modification of PXR is effectively antagonized by the SUMO protease sentrin protease (SENP) 2, whereas SENP3 and SENP6 proteases are highly active in the removal of SUMO2/3 chains. The PIASy-mediated SUMO(1)ylation of PXR inhibits ubiquitin-mediated degradation of this important liver-enriched NR by the 26S proteasome. Our data reveal a working model that delineates the interactive role that these two post-translational modifications play in reconciling PXR-mediated gene activation of the xenobiotic response versus transcriptional repression of the proinflammatory response in hepatocytes. Taken together, our data reveal that the SUMOylation and ubiquitylation of the PXR interface in a fundamental manner directs its biologic function in the liver in response to xenobiotic or inflammatory stress.

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 biological effects of cytokines are mediated by STAT proteins, a family of dimeric transcription factors. In order to elicit transcriptional activity, the STATs require activation by phosphorylation of a single tyrosine residue. Our experiments revealed that fully tyrosine-phosphorylated STAT dimers polymerize via Tyr(P)-Src homology 2 domain interactions and assemble into paracrystalline arrays in the nucleus of cytokine-stimulated cells. Paracrystals are demonstrated to be dynamic reservoirs that protect STATs from dephosphorylation. Activated STAT3 forms such paracrystals in acute phase liver cells. Activated STAT1, in contrast, does not normally form paracrystals. By reversing the abilities of STAT1 and STAT3 to be sumoylated, we show that this is due to the unique ability of STAT1 among the STATs to conjugate to small ubiquitin-like modifier (SUMO). Sumoylation had one direct effect; it obstructed proximal tyrosine phosphorylation, which led to semiphosphorylated STAT dimers. These competed with their fully phosphorylated counterparts and interfered with their polymerization into paracrystals. Consequently, sumoylation, by preventing paracrystal formation, profoundly curtailed signal duration and reporter gene activation in response to cytokine stimulation of cells. The study thus identifies polymerization of activated STAT transcription factors as a positive regulatory mechanism in cytokine signaling. It provides a unifying explanation for the different subnuclear distributions of STAT transcription factors and reconciles the conflicting results as to the role of SUMO modification in STAT1 functioning. We present a generally applicable system in which protein solubility is maintained by a disproportionately small SUMO-modified fraction, whereby modification by SUMO partially prevents formation of polymerization interfaces, thus generating competitive polymerization inhibitors.

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.

// Samadhi Aparicio-Siegmund 1 , Jan Sommer 1 , Niloufar Monhasery 1 , Ralf Schwanbeck 2 , Eric Keil 3 , David Finkenstadt 3 , Klaus Pfeffer 3 , Stefan Rose-John 2 , Jurgen Scheller 1 and Christoph Garbers 1,4 1 Institute of Biochemistry and Molecular Biology II, Medical Faculty, Heinrich-Heine University, Dusseldorf, Germany; 2 Institute of Biochemistry, Christian-Albrechts-University, Kiel, Germany; 3 Institute of Medical Microbiology and Hospital Hygiene, Heinrich-Heine University, Dusseldorf, Germany 4 Present address: Institute of Biochemistry, Christian-Albrechts-University, Kiel, Germany Correspondence: Jurgen Scheller, email: // Christoph Garbers, email: // Keywords : STAT3, cytokines, tumor, oncogene, signal transduction Received : February 11, 2014 Accepted : March 22, 2014 Published : March 23, 2014 Abstract The Janus kinase / signal transducer and activator of transcription (Jak/STAT) pathway can be activated by many different cytokines, among them all members of the Interleukin (IL-)6 family. Dysregulation of this pathway, resulting in its constitutive activation, is associated with chronic inflammation and cancer development. In the present study, we show that activity of protein kinase II (CK2), a ubiquitously expressed serine/threonine kinase, is needed for induced activation of STAT1 and STAT3 by IL-6 classic and trans-signaling, IL-11, IL-27, oncostatin M (OSM), leukemia inhibitory factor (LIF) and cardiotrophin-1 (CT-1). Inhibition of CK2 efficiently prevented STAT phosphorylation and inhibited cytokine-dependent cell proliferation in a Jak1-dependent manner. Conversely, forced activation of CK2 alone was not sufficient to induce activation of the Jak/STAT signaling pathway. Inhibition of CK2 in turn inhibited Jak1-dependent STAT activation by oncogenic gp130 mutations. Furthermore, CK2 inhibition diminished the Jak1- and Src kinase-dependent phosphorylation of a constitutively active STAT3 mutant recently described in human large granular lymphocytic leukemia. In conclusion, we characterize CK2 as an essential component of the Jak/STAT pathway. Pharmacologic inhibition of this kinase is therefore a promising strategy to treat human inflammatory diseases and malignancies associated with constitutive activation of the Jak/STAT 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.

JAK/STAT is a conserved signaling pathway in higher eukaryotes that plays a critical role in different processes of ontogenesis. This article reviews the pathway structure at the molecular level, mainly in Drosophila as a model organism. The review is focused on the data concerning the relationship between this signaling pathway and transcription machinery of higher eukaryotes.

Protein expression abnormalities have been implicated in the pathophysiology of schizophrenia, but the underlying cause of these changes is not known. We sought to investigate ubiquitin and ubiquitin-like (UBL) systems (SUMOylation, NEDD8ylation, and Ufmylation) as putative mechanisms underlying protein expression abnormalities seen in schizophrenia. For this, we performed western blot analysis of total ubiquitination, free ubiquitin, K48- and K63-linked ubiquitination, and E1 activases, E2 conjugases, and E3 ligases involved in ubiquitination and UBL post-translational modifications in postmortem brain tissue samples from persons with schizophrenia (n=13) and comparison subjects (n=13). We studied the superior temporal gyrus (STG) of subjects from the Mount Sinai Medical Center brain collection that were matched for age, tissue pH, and sex. We found an overall reduction of protein ubiquitination, free ubiquitin, K48-linked ubiquitination, and increased K63 polyubiquitination in schizophrenia. Ubiquitin E1 activase UBA (ubiquitin activating enzyme)-6 and E3 ligase Nedd (neural precursor cell-expressed developmentally downregulated)-4 were decreased in this illness, as were E3 ligases involved in Ufmylation (UFL1) and SUMOylation (protein inhibitor of activated STAT 3, PIAS3). NEDD8ylation was also dysregulated in schizophrenia, with decreased levels of the E1 activase UBA3 and the E3 ligase Rnf7. This study of ubiquitin and UBL systems in schizophrenia found abnormalities of ubiquitination, Ufmylation, SUMOylation, and NEDD8ylation in the STG in this disorder. These results suggest a novel approach to the understanding of schizophrenia pathophysiology, where a disruption in homeostatic adaptation of the cell underlies discreet changes seen at the protein level in this illness.

Impaired phosphatase activity leads to the persistent activation of signal transducers and activators of transcription (Stat). In mammals, Stat family members are often phosphorylated or dephosphorylated by the same enzymes. To date, only one Stat similar to mammalian Stat5a/b has been found in crustaceans and there have been few studies in Stat signal regulation in crustaceans. Here, we report that β-arrestin1 interacts with TC45 (45-kDa form of T cell protein tyrosine phosphatase) in the nucleus to attenuate Stat signaling by promoting dephosphorylation of Stat. Initially, we showed that Stat translocates into the nucleus to induce antimicrobial peptide (AMP) expression after bacterial infection. βArr1 enters the nucleus of hemocytes and recruits TC45 to form the βarr1-TC45-Stat complex, which dephosphorylates Stat efficiently. The interaction of TC45 with Stat decreased and Stat phosphorylation increased in βarr1-silenced shrimp (Marsupenaeus japonicus) after challenge with Vibrio anguillarum. βArr1 directly interacts with Stat in nucleus and accelerates Stat dephosphorylation by recruiting TC45 after V. anguillarum challenge. Further study showed that βarr1 and TC45 also affect AMP expression, which is regulated by Stat. Therefore, βarr1 and TC45 are involved in the anti-V. anguillarum immune response by regulating Stat activity negatively to decrease AMP expression in shrimp.

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.

Jak/STAT is an important signaling pathway mediating multiple events in development. We describe participation of metazoan co-activator SAYP/PHF10 in this pathway downstream of STAT. The latter, via its activation domain, interacts with the conserved core of SAYP. STAT is associated with the SAYP-containing co-activator complex BTFly and recruits BTFly onto genes. SAYP is necessary for stimulating STAT-driven transcription of numerous genes. Mutation of SAYP leads to maldevelopments similar to those observed in STAT mutants. Thus, SAYP is a novel co-activator mediating the action of STAT.

Intercellular signal transduction pathways regulate the NK-2 family of transcription factors in a conserved gene regulatory network that directs cardiogenesis in both flies and mammals. The Drosophila NK-2 protein Tinman (Tin) was recently shown to regulate Stat92E, the Janus kinase (JAK) and Signal transducer and activator of transcription (Stat) pathway effector, in the developing mesoderm. To understand whether the JAK/Stat pathway also regulates cardiogenesis, we performed a systematic characterization of JAK/Stat signaling during mesoderm development. Drosophila embryos with mutations in the JAK/Stat ligand upd or in Stat92E have non-functional hearts with luminal defects and inappropriate cell aggregations. Using strong Stat92E loss-of-function alleles, we show that the JAK/Stat pathway regulates tin expression prior to heart precursor cell diversification. tin expression can be subdivided into four phases and, in Stat92E mutant embryos, the broad phase 2 expression pattern in the dorsal mesoderm does not restrict to the constrained phase 3 pattern. These embryos also have an expanded pericardial cell domain. We show the E(spl)-C gene HLHm5 is expressed in a pattern complementary to tin during phase 3 and that this expression is JAK/Stat dependent. In addition, E(spl)-C mutant embryos phenocopy the cardiac defects of Stat92E embryos. Mechanistically, JAK/Stat signals activate E(spl)-C genes to restrict Tin expression and the subsequent expression of the T-box transcription factor H15 to direct heart precursor diversification. This study is the first to characterize a role for the JAK/Stat pathway during cardiogenesis and identifies an autoregulatory circuit in which tin limits its own expression domain.

Protein Inhibitor of Activated Signal Transducer and Activators of Transcription 3 (PIAS3) is a molecule that regulates STAT3 and has antiproliferative properties. Glioblastoma and squamous cell lung cancer lack PIAS3 expression. To test the hypothesis that PIAS3 transcriptional effects are STAT3-independent, we developed models for STAT3 knockdown and PIAS3 over-expression. PIAS3 expression results in a distinct transcriptional profile that does not occur with STAT3 knockdown. We identify novel transcription factor binding partners for PIAS3 including ETS, EGR1, NR1I2, and GATA1. PIAS3 binds to these factors and regulates their transcriptional effects resulting in alterations in canonical pathways including Wnt/β-catenin signaling and functions such as cell death and proliferation. A model is proposed by which PIAS3 effects EGR1 regulated pathways.

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 Janus kinase (JAK) signal transducer and activator of transcription (STAT) pathway is one of the main signaling pathways in eukaryotic cells. This pathway is used during diverse growth and developmental processes in multiple tissues to control cell proliferation, differentiation, survival, and apoptosis. In addition to its role during development, the JAK/STAT pathway has also been implicated in tumorigenesis. Drosophila melanogaster is a powerful genetic tool, and its eyes have been used extensively as a platform to study signaling pathways. Many reports have demonstrated that the JAK/STAT pathway plays pleiotropic roles in Drosophila eye development. Its functions and activation are decided by its interplay with other signal pathways and the epigenetic status. In this review, we focus on the functions and regulation of the JAK/STAT pathway during eye development and provide some insights into the study of this pathway in tumorigenesis. Developmental Dynamics 239:2522–2533, 2010. © 2010 Wiley-Liss, Inc.

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 Janus kinase/signal transducer and activator of transcription (JAK–STAT) pathway mediates important responses in immune cells. Activation of any of the four JAK family members leads to phosphorylation of one or more of seven STAT family members. Phosphorylation of STAT family members leads to their dimerization and translocation into the nucleus, in which they bind specific DNA sequences to activate gene transcription. Regulation of JAKs and STATs therefore has a significant effect on signal transduction and subsequent cellular responses. Mast cells are important mediators of allergic disease and asthma. These cells have the ability to cause profound inflammation and vasodilation upon the release of preformed mediators, as well as subsequent synthesis of new inflammatory mediators. The regulation of mast cells is therefore of intense interest for the treatment of allergic disease. An important regulator of mast cells, STAT5, is activated downstream of the receptors for immunoglobulin E, interleukin-3 and stem cell factor. STAT5 contributes to mast cell homeostasis, by mediating proliferation, survival, and mediator release. Regulators of the JAK–STAT pathway, such as the suppressors of cytokine signaling (SOCS) and protein inhibitor of activated STAT (PIAS) proteins, are required to fine tune the immune response and maintain homeostasis. A better understanding of the role and regulation of JAKs and STATs in mast cells is vital for the development of new therapeutics.

The family of STAT proteins consists of seven members that mediate highly specific functions in cytokine signaling. STAT6 is a critical regulator of transcription for interleukin-4 (IL-4)-induced genes. Activation of gene expression involves recruitment of coactivator proteins that function as bridging factors connecting sequence-specific transcription factors to the basal transcription machinery, and as chromatin-modifying enzymes. In this report, we show that the coacitivator p100 protein can interact with STAT6 through its SN domain both in vivo and in vitro, resulting in enhancement of STAT6-mediated gene transcriptional acitivation. Consistent with our previous reports, we identified intracellular localization of p100 and STAT-6 by confocal microscopy examined in response to IL-4. Moreover, in consideration of STAT molecules sharing significant homology in structure and function, we detected whether p100 can associate with STAT-1. In conclusion, this study found no evidence that p100 functions as a transcriptional coactivator for STAT1-dependent gene regulation. Anat Rec, 2010. © 2010 Wiley-Liss, Inc.

Protein inhibitors of activated STATs (PIAS) regulate the interferon-gamma (IFN-gamma) signaling pathway, which has numerous effects on tumor development and tumor cell biology. PIAS's also regulate STAT family members not directly involved in IFN-gamma signaling. This project was designed to assess PIAS1 expression in colon cancer.To determine whether PIAS1, one of the PIAS family members, or IFN-gamma signaling pathway components could be used to stratify colon tumors, we stained tissue microarrays for PIAS1, interferon regulatory factor-1 (IRF-1) and STAT1alpha.PIAS1 staining of the colon cancer tissue microarrays indicated a strong correlation of normal colon cells, and adenomas, with high expression of both PIAS1 and IRF-1.The PIAS1 results in particular may represent a basis for new approaches for efficiently distinguishing adenomas from colon cancer.

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.

Serum response has been used as a model for studying signaling transduction for many biological events such as cell proliferation and survival. Although expression of many genes is up- or down-regulated after serum stimulation, the Notch effector Hes1 displays oscillatory response. However, the precise mechanism and biological significance of this oscillation remain to be determined. Here, we identified serum-induced ultradian oscillators, including molecules in Stat and Smad signaling. Stat and Smad oscillations involve activation of Stat3 and Smad1 and delayed negative feedback by their inhibitors Socs3 and Smad6, respectively. Moreover, Stat oscillations induce oscillatory expression of Hes1 by regulating its half-life, and loss of Hes1 oscillations leads to G 1 phase retardation of the cell cycle. These results indicate that coupled Stat and Hes1 oscillations are important for efficient cell proliferation and provide evidence that expression modes of signaling molecules affect downstream cellular events.

The tumor suppressor, p53, plays critical roles in the cell cycle progression, DNA repair and apoptosis. The PIAS proteins (protein inhibitor of activated STAT) were originally identified as inhibitors of the JAK-STAT pathway. Subsequently, crosstalk between the PIAS proteins and other signaling pathways has been shown to be involved in various cellular processes. Particularly, previous studies have demonstrated that PIAS proteins regulate p53-mediated transcription through sumoylation. hZimp10, also named zmiz1, is a novel PIAS-like protein and functions as a transcriptional co-activator. We recently identified p53 to be an hZimp10 interacting protein in the yeast two-hybrid screen. The interaction between p53 and hZimp10 was confirmed by GST pull-down and co-immunoprecipitation assays. Co-localization of p53 and hZimp10 proteins was also observed within cell nuclei by immunostaining. Moreover, we show that expression of exogenous hZimp10 enhances the transcriptional activity of p53 and knockdown of endogenous hZimp10 reduces the transcriptional activity of p53. Furthermore, using chromatin immunoprecipitation assays, we demonstrate that hZimp10 binds to p53 on the p21 promoter. Finally, p53-mediated transcription is significantly impaired in Zimp10 null embryonic fibroblasts. Taken together, these results provide the first line of evidence to demonstrate a role for Zimp10 in regulating p53 function.

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.

The present study investigated human retinoid X receptor alpha (hRXRα) as a substrate for modification with small ubiquitin like modifier (SUMO) and how members of the protein inhibitor of activated STAT (PIAS) family may impact upon this process. In agreement with a previous study, we validate Ubc9 to facilitate SUMOylation of hRXRα at lysine 108 but note this modification to occur for all isoforms rather than specifically with SUMO1 and to preferentially occur with the unliganded form of hRXRα. SUMOylation of hRXRα is significantly enhanced through PIAS4-mediated activity with lysine 245 identified as a specific SUMO2 acceptor site modified in a PIAS4-dependent fashion. While individual mutations at lysine 108 or 245 modestly increase receptor activity, the combined loss of SUMOylation at both sites significantly potentiates the transcriptional responsiveness of hRXRα suggesting both sites may cooperate in a DNA element-dependent context. Our findings highlight combinatorial effects of SUMOylation may regulate RXRα-directed signalling in a gene-specific fashion.

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.

STAT transcription factors have been implicated in many biological processes, particularly host immune defense and development. Here we characterize a STAT orthologue from the nematode, C. elegans. We show that this protein, termed STA-1, is structurally and functionally related to other vertebrate and invertebrate STAT proteins, recognizing a cis DNA element conserved through phylogeny. Unexpectedly, STA-1 lacks the conserved amino-terminal oligomerization domain found in vertebrate and other invertebrate STAT proteins, a feature also lacking in orthologues from a distantly related nematode species and from the slime mold, Dictyostelium discoideum. This absence suggests that a primordial STAT protein lacked this domain, which was accreted later in evolution to provide further regulatory control of STAT signaling. Derivation of null mutants demonstrated that STA-1 is not required for nematode viability, despite its widespread expression in multiple tissues of the worm. However, mutant STA-1 proteins that lack functional coiled-coil and DNA binding domains could still be activated and accumulated in the nucleus, suggesting that DNA binding is not a necessary prerequisite for nuclear retention of activated STAT proteins. Our results shed new light on the evolution and function of the STAT signaling pathway and on the structural requirements for STAT activation.

Cytokine signaling is essential for intercellular communication and affects cell proliferation, differentiation, and survival. In the immune system, cytokines coordinate the activities of many cell types ultimately leading to both innate and adaptive immune responses. Dysregulation of these processes can result in a wide spectrum of diseases ranging from defective host responses to invading pathogens to autoimmunity. Most cytokines signal through the Janus kinase-signal transducer and activator of transcription (Jak-STAT) pathway initiated by the cytokine binding to its cell surface receptor, which leads to the activation of STAT proteins, their binding to response elements near target promoters ultimately changing the transcription of STAT-responsive genes. STAT proteins do not exist in isolation but act in concert with other transcription factors and cofactors which can either stimulate or inhibit their activity. One such factor is a ligand-dependent transcriptional regulator termed the glucocorticoid (GC) receptor (GR), which transduces the information conveyed by GC hormones and their synthetic analogs. GR is known for its anti-inflammatory and immunosuppressive properties; GC-like molecules have been used as drugs for inflammatory, autoimmune and lymphoproliferative diseases since the 1950s. In contrast, cytokines frequently promote activation of the immune system. In last several years, functional interactions have been described between virtually every member of the STAT family and GR or its cofactors. This review focuses on the recent literature on the modes and levels of interactions between these seemingly unrelated regulators and potential biological implications of STAT : GR cross-talk.

The protein inhibitor of activated STAT (PIAS) family of proteins has been proposed to regulate the activity of many transcription factors, including signal transducer and activator of transcription proteins (STATs), nuclear factor-kappaB, SMA- and MAD-related proteins (SMADs), and the tumour-suppressor protein p53. PIAS proteins regulate transcription through several mechanisms, including blocking the DNA-binding activity of transcription factors, recruiting transcriptional corepressors or co-activators, and promoting protein sumoylation. Recent genetic studies support an in vivo function for PIAS proteins in the regulation of innate immune responses. In this article, we review the current understanding of the molecular basis, specificity and physiological roles of PIAS proteins in the regulation of gene-activation pathways in the immune system.

The cytokine-activated Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway plays an important role in the control of a wide variety of biological processes. When misregulated, JAK/STAT signaling is associated with various human diseases, such as immune disorders and tumorigenesis. To gain insights into the mechanisms by which JAK/STAT signaling participates in these diverse biological responses, we carried out a genome-wide RNA interference (RNAi) screen in cultured Drosophila cells. We identified 121 genes whose double-stranded RNA (dsRNA)-mediated knockdowns affected STAT92E activity. Of the 29 positive regulators, 13 are required for the tyrosine phosphorylation of STAT92E. Furthermore, we found that the Drosophila homologs of RanBP3 and RanBP10 are negative regulators of JAK/STAT signaling through their control of nucleocytoplasmic transport of STAT92E. In addition, we identified a key negative regulator of Drosophila JAK/STAT signaling, protein tyrosine phosphatase PTP61F, and showed that it is a transcriptional target of JAK/STAT signaling, thus revealing a novel negative feedback loop. Our study has uncovered many uncharacterized genes required for different steps of the JAK/STAT signaling pathway.

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.

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.

Human leukemias are frequently associated with the aberrant expression of activated fusion tyrosine kinases or activated protein tyrosine kinases carrying insertional or point mutations. The activated kinase enzymes typically phosphorylate one or more signal transducer and activator of transcription (STAT) factors, which translocate to the cell nucleus and regulate the expression of genes associated with survival and proliferation. The phosphorylation and activation of STAT family members has been described in a wide range of human leukemias. Furthermore, animal models of leukemia have demonstrated the pivotal contribution of STAT activation to leukemic pathogenesis. This review discusses evidence for the functional importance of STAT activation in the biology of leukemia and current opportunities for modulating STAT proteins in the therapy of this group of diseases.

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.

Signal transducer and activator of transcription (STAT) proteins are a 7-member family of cytoplasmic transcription factors that contribute to signal transduction by cytokines, hormones, and growth factors. STAT proteins control fundamental cellular processes, including survival, proliferation, and differentiation. Given the critical roles of STAT proteins, it was hypothesized that inappropriate or aberrant activation of STATs might contribute to cellular transformation and, in particular, leukemogenesis. Constitutive activation of mutated STAT3 has in fact been demonstrated to result in transformation. STAT activation has been extensively studied in leukemias, and mechanisms of STAT activation and the potential role of STAT signaling in leukemogenesis are the focus of this review. A better understanding of mechanisms of dysregulation of STAT signaling pathways may serve as a basis for designing novel therapeutic strategies that target these pathways in leukemia cells.

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.

Extracellular proteins bound to cell-surface receptors can change nuclear gene expression patterns in minutes, with far-reaching consequences for development, cell growth and homeostasis. The signal transducer and activator of transcription (STAT) proteins are among the most well studied of the latent cytoplasmic signal-dependent transcription-factor pathways. In addition to several roles in normal cell decisions, dysregulation of STAT function contributes to human disease, making the study of these proteins an important topic of current research.

PIAS3, a member of the protein inhibitor of activated STAT family, was found to interact in vivo and in vitro with TIF2, a previously described coactivator for nuclear receptors. The interaction is mediated by two distinct non-contiguous regions of TIF2. We found that TIF2–PIAS3 interaction occurs through a unique domain of PIAS3, very rich in acidic residues and conserved throughout the PIAS family. PIAS3 modulates the ability of TIF2 to mediate ligand-enhanced transcription activation positively or negatively, for different steroid receptors. Taken together, our results indicate a potential role of PIAS3 as transcriptional modulator of TIF2-mediated signalling.