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Stat91 (a 91 kd protein that acts as a signal transducer and activator of transcription) is inactive in the cytoplasm of untreated cells but is activated by phosphorylation on tyrosine in response to a number of polypeptide ligands, including interferon alpha (IFN-alpha) and IFN-gamma. We report here that the inactive Stat91 in the cytoplasm of untreated cells is a monomer and that upon IFN-gamma-induced phosphorylation it forms a stable homodimer. Only the dimer is capable of binding to a specific DNA sequence directing transcription. Through dissociation and reassociation assays, we show that dimerization of Stat91 is mediated through SH2-phosphotyrosyl peptide interactions. Dimerization involving SH2 recognition of specific phosphotyrosyl peptides may well provide a prototype for interactions among family members of STAT proteins to form different transcription complexes.

The nuclear envelope in eukaryotic cells has important roles in chromatin organization. The inner nuclear membrane contains over 60 transmembrane proteins. LEM [LAP2 (lamina-associated polypeptide 2)/emerin/MAN1] domain-containing proteins of the inner nuclear membrane are involved in tethering chromatin to the nuclear envelope and affect gene expression. They contain a common structural, bihelical motif, the so-called LEM domain, which mediates binding to a conserved chromatin protein, BAF (barrier to autointegration factor). Interestingly, this domain is highly related to other bihelical motifs, termed HeH (helix–extension–helix) and SAP {SAF (scaffold attachment factor)/acinus/PIAS [protein inhibitor of activated STAT (signal transducer and activator of transcription)]} motifs, which are directly linked to DNA. In the present paper, we summarize evidence that the LEM motif evolved from the HeH and SAP domains concomitantly with BAF. In addition, we discuss the potential evolution of HeH/SAP and LEM domain-containing proteins and their role in chromatin tethering and gene regulation from unicellular eukaryotes to mammals.

Review on PIAS3 (protein inhibitor of activated STAT, 3), with data on DNA, on the protein encoded, and where the gene is implicated.

The acute-phase response is an inflammatory process triggered mainly by the cytokine IL-6. Signaling of IL-6 is transduced by activation of STAT3 (signal transducer and activator of transcription 3), which rapidly induces the production of acute-phase proteins such as haptoglobin and fibrinogen. Another target of the IL-6/STAT3 signal transduction pathway is the microRNA cluster miR-17/92. Here, we investigated the interplay of miR-17/92 and STAT3 signaling and its impact on the acute-phase response in primary human hepatocytes and hepatoma (HepG2) cells. Employing a reporter gene system consisting of STAT3-sensitive promoter sequences, we show that the miR-17/92 cluster member miR-18a enhanced the transcriptional activity of STAT3. IL-6 stimulation experiments in miR-18a-overexpressing hepatocytes and HepG2 cells revealed an augmented acute-phase response indicated by increased expression and secretion of haptoglobin and fibrinogen. This effect was due, at least in part, to repression of PIAS3 (protein inhibitor of activated STAT, 3), a repressor of STAT3 activity, which we identified as a novel direct target of miR-18a. Finally, we demonstrate that the expression of miR-17/92 in primary hepatocytes and HepG2 cells is modulated by IL-6. Our data reveal, for the first time, a microRNA-mediated positive feedback loop of IL-6 signal transduction leading to an enhanced acute-phase response in human hepatocytes.

Rainer Poltz1, Raimo Franke1,#, Katrin Schweitzer1, Steffen Klamt2, Ernst-Dieter Gilles2, Michael Naumann11Institute of Experimental Internal Medicine, Otto von Guericke University, Magdeburg, Germany; 2Max Planck Institute for Dynamics of Complex Technical Systems, Magdeburg, Germany; #Present address: Department of Chemical Biology, Helmholtz Centre for Infection Research, Braunschweig, GermanyAbstract: Genotoxic stress is induced by a broad range of DNA-damaging agents and could lead to a variety of human diseases including cancer. DNA damage is also therapeutically induced for cancer treatment with the aim to eliminate tumor cells. However, the effectiveness of radio- and chemotherapy is strongly hampered by tumor cell resistance. A major reason for radio- and chemotherapeutic resistances is the simultaneous activation of cell survival pathways resulting in the activation of the transcription factor nuclear factor-kappa B (NF-κB). Here, we present a Boolean network model of the NF-κB signal transduction induced by genotoxic stress in epithelial cells. For the representation and analysis of the model, we used the formalism of logical interaction hypergraphs. Model reconstruction was based on a careful meta-analysis of published data. By calculating minimal intervention sets, we identified p53-induced protein with a death domain (PIDD), receptor-interacting protein 1 (RIP1), and protein inhibitor of activated STAT y (PIASy) as putative therapeutic targets to abrogate NF-κB activation resulting in apoptosis. Targeting these structures therapeutically may potentiate the effectiveness of radio- and chemotherapy. Thus, the presented model allows a better understanding of the signal transduction in tumor cells and provides candidates as new therapeutic target structures.Keywords: apoptosis, Boolean network, cancer therapy, DNA-damage response, NF-κB

The protein inhibitor of activated signal transducer and activator of transcription 3 (PIAS3) regulates the transcriptional activity of signal transducer and activator of transcription 3 (STAT3) which regulates transcription of genes involved in cell growth, proliferation and apoptosis. The conserved proline, isoleucine, asparagine, isoleucine, threonine (PINIT) domain of PIAS3 is thought to promote STAT3-PIAS3 interaction. The (His)(7) -PINIT domain (PIAS3(85-272) ) was heterologously expressed and purified to homogeneity by nickel affinity and size exclusion chromatography, and shown to be a folded monomer in solution. Using surface plasmon resonance spectroscopy (SPR) the PINIT domain (PIAS3(85-272) ) alone was shown to specifically bind to STAT3 in a concentration dependent manner. L97A, R99N and R99Q mutations of the PINIT domain were found to abrogate binding to STAT3, suggesting that these residues were part of a potential binding surface. An homology model for the PINIT domain was calculated to analyse the potential locations of L97 and R99 in the structure, and to evaluate the potential role of these residues in interactions with STAT3.

The Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway directly links ligand-binding to a membrane-bound receptor with the activation of a transcription factor. This signalling module enables the cell to rapidly initiate a transcriptional response to external stimulation. The main components of this evolutionary conserved module are cytokines that specifically bind to cytokine receptors leading to the activation of receptor-associated Janus tyrosine kinases (JAKs). The receptor-bound JAKs activate STAT transcription factors through phosphorylation of a single tyrosine residue. Activated STAT dimers translocate into the nucleus to induce target gene expression. In this article we will review current opinions on the molecular mechanism and on intracellular dynamics of JAK/STAT signalling with a special focus on the cytokine receptor glycoprotein 130 (gp130) and STAT3. In particular we will concentrate on non-canonical aspects of Jak/STAT signalling including preassembled receptor complexes, preformed STAT dimers, STAT trafficking and non-canonical functions of STATs.

Introduction Dysfunction of the endothelium contributes to pathological conditions of the arterial wall including atherosclerosis as a result of immunological and/or inflammatory responses. Salvianolic acid B (Sal B), a pure and active compound extracted from the Chinese herb Salvia miltiorrhizae (SM) was characterized for its anti-inflammatory and anti-oxidant properties on vascular system. Methods and Results Sal B pretreatment significantly inhibited the IFN-γ-induced phosphorylations of JAK2 (Tyr 1007/1008) and STAT1 (Tyr701 and Ser727). Consistently, IFN-γ-induced STAT1 downstream targets CXC chemokines’ IP-10, Mig, and I-TAC were suppressed by Sal B pretreatment. Sal B inhibited promoter activities of IP-10 and the secretion of IP-10 protein. The monocyte adhesion to IFN-γ-treated ECs was observed to be reduced after Sal B pretreatment. ECs treated with Sal B alone also increased the expression of PIAS1 and SOCS1 which may also contribute to its inhibitory effect on JAK-STAT1 signaling pathways. Conclusions The anti-inflammatory properties of Sal B on IFN-γ-induced JAK-STAT1 activation were demonstrated in the present study which provides a molecular basis for possible therapeutic usage on vascular disorders.

The signal transducers and activators of transcription (STATs) include a class of cytoplasmic signaling proteins whose role in the regulation of cell growth and survival is mediated by phosphorylation of a critical tyrosine residue within the STAT protein. This occurs in response to cytokines and growth factors modulating the expression of specific target genes. In particular, phosphorylation induces STAT:STAT dimer formation between two monomers, via reciprocal phosphoTyr (pTyr)-SH2 domain interactions. To date, seven members of the STAT family, all with different roles, have been identified in mammals. After dimerization, phosphorylated STATs enter the nucleus and, working co-ordinately with other transcriptional co-activators and transcription factors, induce increased transcriptional initiation. In healthy human and animal cells, ligand-dependent activation of STATs is a transient process, lasting for several minutes to several hours. In contrast, in many cancerous cell lines and tumors, where growth factor dysregulation is frequently at the heart of cellular transformation, the STAT proteins (in particular STAT1, 3 and 5) are persistently tyrosine-phosphorylated or activated; abnormal levels of STAT3 activation have been observed in breast, ovarian, prostate, hematological and head and neck cancer cell lines. Thus, in this review, we examine the most important classes of agents designed to disrupt STAT3 signaling, with particular regard to STAT3 dimerization inhibitors, which could play a significant role in the future of cancer and adjuvant cancer therapies.

Small ubiquitin-like modifier (SUMO) proteins 1, 2 and 3 can be covalently conjugated to specific lysine residues on target proteins in a process dubbed SUMOylation. This conserved posttranslational modification that was reported for the first time in 1996 has emerged as an important regulatory mechanism in cell physiology, especially in nuclear signaling, transport, transcription and DNA replication/repair 1, 2. SUMOylation is thought to provide docking sites for interacting proteins, which leads to altered cellular localization, activity and/or stability of the target proteins. The SUMOylation process is similar to ubiquitylation, but requires a distinct enzymatic E1→E2→E3 cascade. The SUMOs activated by the SAE1/2 heterodimer (E1) are transferred to the UBC9 (E2) that conjugates them to target lysines. The conjugation process is enhanced by SUMO E3 ligases. Protein Inhibitor of Activated STAT (PIAS) 1 and 3 proteins were originally identified as repressors of STAT signaling. However, the four PIAS family members 1, 2, 3 and 4 play a wide role in cell regulation also by promoting target-specific SUMOylation and hence by functioning as SUMO E3 ligases 3. SUMOylation is a reversible modification reversed by a family of SUMO-specific proteases, SENPs. Although, the SUMOylation machinery, its targets as well as PIAS-interacting proteins are enriched in the nucleus, there are rare examples of cytoplasmic SUMOylated proteins that interact with PIAS proteins, such as septins and PIAS ortholog Siz1 in yeast bud neck and PIAS3 and metabotropic glutamate receptor-8 in mammalian cells 4, 5. Recent isolation of Rho-like GTPase Rac1 as a PIAS3-interacting protein by Castillo-Lluva and co-workers adds a prominent and intriguing new entity to the list of cytoplasmic proteins functionally regulated by SUMOylation 6. The group used a cell culture and proteomics-based approach to identify proteins that interact with Rac1 in response to cell migration that was stimulated by hepatocyte growth factor (HGF). Rac1 is a member of the evolutionary conserved Rho/Rac subfamily of the Ras superfamily of GTPases. It is an intracellular transducer that plays a key role in the regulation of cytoskeleton dynamics and organization and cell migration 7, 8. Rac1 can also influence cell cycle and transcriptional dynamics. Cell migration is a central mechanism in the development and maintenance of healthy multicellular organisms 9. The migration that is often initiated by an extracellular signal is a multistep process that consists of alterations in the cytoskeleton, cell adhesions and extracellular matrix. Mesenchymal-type of migration is characterized by an elongated cell shape. It requires extracellular proteolysis and engagement of integrin and it additionally relies on Rac1-mediated cell polarization and lamellipodia formation 9. Therefore, it is hardly surprising that deregulated Rho/Rac signaling can contribute to a wide range of diseases, including cancer. Rac1 was previously known to be regulated through several mechanisms, including control of nucleotide exchange (guanine nucleotide exchange factors, GEFs, activating Rac1) and hydrolysis (GTPase-activating proteins, GAPs, inactivating Rac1), and regulation of subcellular localization by posttranslational prenylation 10. Interestingly, PIAS3 was found to interact primarily with the activated Rac1-GTP complex 6. Although the PIAS3 is mainly found in the nucleus, small amounts of PIAS3 are also localized in the cytoplasm. Activation of cell migration after HGF exposure resulted in relocalization of PIAS3 from the cytoplasm to the cell membrane, where it colocalized with the Rac1-GTP (Figure 1). Rac1 was conjugated to SUMO-1 in response to the HGF treatment and the SUMO-1 modification was enhanced by PIAS3. The importance of PIAS3 in the Rac1 activation and function was shown by RNA interference. Depletion of cellular PIAS3 markedly reduced Rac1 activation, cell migration and impaired lamellipodia-membrane ruffle formation. The induction of Rac1 SUMOylation was not restricted to HGF signaling, but is a more general mechanism in the activation of Rac1, as it was also seen upon calcium-switched cell adhesion. One enigma in protein SUMOylation is the common finding of a very low stoichiometry, i.e. only a small proportion of most of the target proteins are modified at a given time in the cells. In the case of Rac1, interestingly a high proportion of the Rac1-GTP pool was modified 5 min after the stimulus, but the modification was also highly dynamic; rapidly removed from the protein within 30 min after the stimulus. Figure 1 A summary of the role of PIAS3 and SUMOylation in the regulation of Rac1-dependent cell migration. (A) Situation in a cell in the absence of migration stimulus. (B) HGF signaling through transmembrane receptors induces the GEFs, leading to the activation ... As it seems to be the case of an increasing number of SUMO targets, the SUMOylation sites of Rac1 do not conform to the “classic” consensus sequence ψXKE (hydrophobic amino acid-any amino acid-lysine-glutamic acid). Four lysines (188, 183, 184 and 186) in the C-terminal polybasic (PBR) region of Rac1 were identified as potential targets for SUMOylation 6. Their concomitant mutation abolished the SUMOylation of Rac1 without influencing the ubiquitylation of Rac1. However, the mutation did not affect the interaction with PIAS3 or other Rac1-interacting partners, such as IQGAP, GEFs, β-Pix or Tiam1. As a consequence of the SUMOylation deficiency, the mutant Rac1 bound less GTP than the wild-type, but the subcellular localization of Rac1 was not altered. The authors performed elegant rescue experiments in Rac1-null mouse embryonic fibroblasts (MEFs) and showed that compared to the wild-type Rac1, the SUMOylation-deficient Rac1 is severely compromised in the lamellipodia-ruffle formation. Moreover, the Rac1 SUMOylation mutant was unable to restore the migration defect or the reduced invasiveness of the Rac1-null MEFs. Importantly, even in the case of the wild-type Rac1, PIAS3 was also required for an efficient rescue of the migration or lamellipodia-ruffle defect. Consequently, the SUMOylation seems to have an important role in the maintenance of the activated Rac1-GTP. How this is achieved remains to be investigated. One possibility is that the interaction of Rac1 with the GEFs and the GAPs is altered due to the SUMO modification of Rac1. These fascinating data demonstrate for the first time a role for SUMOylation and a PIAS E3 SUMO ligase function in the regulation of Ras superfamily member and cell migration and invasion. The results have ramifications in several diseases, including cancer, and they may in a long run provide translational opportunities for novel therapeutic interventions. As with all first-class research, the current data also provoke several new and important questions. Since the C-terminal PBRs are well-conserved among the Rho/Rac family proteins, it seems likely that a similar SUMOylation- and PIAS-based regulation may take place at least with some other Rho/Rac proteins. However, the PIAS protein in charge may be other than the PIAS3, as Castello-Lluva and co-workers did not see any effect in another Rho/Rac family member Cdc42-GTP levels upon depletion of PIAS3. Interestingly, in the immediate vicinity of the SUMOylation sites of Rac1 also lies the consensus prenylation site 10, 11. The prenylation, covalent addition of geranylgeranyl group to the C-terminus, is known to target the Rho/Rac proteins to the cell membrane and promote their activation, perhaps by facilitating their interaction with GEFs. Therefore, an obvious question is whether there is a certain type of crosstalk between the prenylation and the SUMOylation in the regulation of Rac1 function. Even more intriguingly, the PBR of Rac1 has also been reported to harbor a functional nuclear localization signal, which may be regulated through the prenylation 11, 12. The PBR may thus function as a switchboard, which coordinates the Rac1 functions, possibly also in the case of other Rho/Rac proteins.

Malaria affects 300 million people worldwide every year and 450,000 in Brazil. In coastal areas of Brazil, the main malaria vector is Anopheles aquasalis, and Plasmodium vivax is responsible for the majority of malaria cases in the Americas. Insects possess a powerful immune system to combat infections. Three pathways control the insect immune response: Toll, IMD, and JAK-STAT. Here we analyze the immune role of the A. aquasalis JAK-STAT pathway after P. vivax infection. Three genes, the transcription factor Signal Transducers and Activators of Transcription (STAT), the regulatory Protein Inhibitors of Activated STAT (PIAS) and the Nitric Oxide Synthase enzyme (NOS) were characterized. Expression of STAT and PIAS was higher in males than females and in eggs and first instar larvae when compared to larvae and pupae. RNA levels for STAT and PIAS increased 24 and 36 hours (h) after P. vivax challenge. NOS transcription increased 36 h post infection (hpi) while this protein was already detected in some midgut epithelial cells 24 hpi. Imunocytochemistry experiments using specific antibodies showed that in non-infected insects STAT and PIAS were found mostly in the fat body, while in infected mosquitoes the proteins were found in other body tissues. The knockdown of STAT by RNAi increased the number of oocysts in the midgut of A. aquasalis. This is the first clear evidence for the involvement of a specific immune pathway in the interaction of the Brazilian malaria vector A. aquasalis with P. vivax, delineating a potential target for the future development of disease controlling strategies., Author Summary Malaria is endemic in 22 countries in the Americas where the Anopheles aquasalis mosquito is an important malaria vector and the Plasmodium vivax parasite is responsible for most malaria cases. This natural vector-parasite pair is difficult to study due to the lack of cultivating system for P. vivax, and to the lack of genome data for A. aquasalis. Moreover, almost all previous studies are based on African and Asian anopheline species. Understanding the interaction mechanisms between mosquito vectors and plasmodia is important for the development of malaria control strategies. Our results showed that the JAK-STAT immune pathway is activated in A. aquasalis after P. vivax challenge and is important to maintain the low levels of P. vivax load observed in this vector. Our results add to the understanding of the A. aquasalis interaction with P. vivax and lead to possible explanations for this vector competence in P. vivax transmission. All information generated here may be used to direct the development of new or specific strategies to block malaria transmission by A. aquasalis in some parts of the Americas.

Protein modification by conjugation of small ubiquitin-related modifier (SUMO) is involved in diverse biological functions, such as transcription regulation, subcellular partitioning, stress response, DNA damage repair, and chromatin remodeling. Here, we show that the serine/arginine-rich protein SF2/ASF, a factor involved in splicing regulation and other RNA metabolism-related processes, is a regulator of the sumoylation pathway. The overexpression of this protein stimulates, but its knockdown inhibits SUMO conjugation. SF2/ASF interacts with Ubc9 and enhances sumoylation of specific substrates, sharing characteristics with already described SUMO E3 ligases. In addition, SF2/ASF interacts with the SUMO E3 ligase PIAS1 (protein inhibitor of activated STAT-1), regulating PIAS1-induced overall protein sumoylation. The RNA recognition motif 2 of SF2/ASF is necessary and sufficient for sumoylation enhancement. Moreover, SF2/ASF has a role in heat shock-induced sumoylation and promotes SUMO conjugation to RNA processing factors. These results add a component to the sumoylation pathway and a previously unexplored role for the multifunctional SR protein SF2/ASF. Fil:Pelisch, F. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:Gerez, J. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:Druker, J. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:Schor, I.E. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:Muñoz, M.J. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:Risso, G. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:Petrillo, E. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:Srebrow, A. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.

Background Signal transducer and activator of transcription (STAT) activation of gene expression is both rapid and transient, and when properly executed it affects growth, differentiation, homeostasis and the immune response, but when dysregulated it contributes to human disease. Transcriptional activation is regulated by alterations to the chromatin template. However, the role of histone modification at gene loci that are activated for transcription in response to STAT signaling is poorly defined. Results Using chromatin immunoprecipitation, we profiled several histone modifications during STAT1 activation of the interferon regulatory factor 1 gene (IRF1). Methylated lysine histone proteins H3K4me2, H3K4me3, H3K79me3, H3K36me3 and monoubiquitinated histone ubH2B are dynamic and correlate with interferon (IFN)γ induction of STAT1 activity. Chemical inhibition of H3K4 methylation downregulates IRF1 transcription and decreases RNA polymerase II (Pol II) occupancy at the IRF1 promoter. MEN1, a component of a complex proteins associated with Set1 (COMPASS)-like complex and the hBRE1 component, RNF20, are localized to IRF1 in the uninduced state and are further recruited when IRF1 is activated. RNAi-mediated depletion of RNF20 lowers both ubH2B and H3K4me3, but surprisingly, upregulates IFNγ induced IRF1 transcription. The dynamics of phosphorylation in the C-terminal domain (CTD) of Pol II are disrupted during gene activation as well. Conclusions H2B monoubiquitination promotes H3K4 methylation, but the E3 ubiquitin ligase, RNF20, is repressive of inducible transcription at the IRF1 gene locus, suggesting that ubH2B can, directly or indirectly, affect Pol II CTD phosphorylation cycling to exert control on ongoing transcription.

In response to a variety of extracellular ligands, the STAT (signal transducer and activator of transcription) transcription factors are rapidly recruited from their latent state in the cytoplasm to cell surface receptors where they are activated by phosphorylation at a single tyrosine residue(1). They then dimerize and translocate to the nucleus to drive the transcription of target genes, affecting growth, differentiation, homeostasis and the immune response. Not surprisingly, given their widespread involvement in normal cell processes, dysregulation of STAT function contributes to human disease, particularly to cancers(2) and autoimmune diseases(3). It is well established that transcription is regulated by alterations to the chromatin template(4,5). These alterations include the activities of ATP-dependent complexes, as well as covalent histone modifications and DNA methylation(6). Because STAT activation of gene expression is both rapid and transient, it requires specific mechanisms for modulating the chromatin template at STAT-dependent gene loci. To define these mechanisms, we characterize the histone modifications and the enzymatic activities that generate them at gene loci that respond to STAT signaling. This protocol describes chromatin immunoprecipitation, a method that is valuable for the study of STAT signaling to chromatin in activated gene expression.

The UPR (unfolded protein response), a cellular defence mechanism against misfolded protein accumulation in the ER (endoplasmic reticulum), is associated with many human diseases such as aging, cancer and diabetes. XBP1 (X-box-binding protein 1), a key transcription factor of the UPR, is critical in maintaining ER homoeostasis. Nevertheless, the mechanism by which XBP1 transcriptional activity is regulated remains unexplored. In the present study we show that XBP1s, the active spliced form of XBP1 protein, is SUMOylated, mainly by PIAS2 [protein inhibitor of activated STAT (signal transducer and activator of transcription) 2] at two lysine residues located in the C-terminal transactivation domain. Ablation of these SUMOylation events significantly enhances the transcriptional activity of XBP1s towards UPR target genes. Thus our results reveal an unexpected role for SUMO (small ubiquitin-related modifier) in the regulation of UPR activation and ER homoeostasis.

Enzymes from the PDE (phosphodiesterase) 4 cAMP-specific PDE family are crucial for the maintenance of compartmentalized cAMP responses in many cell types. Regulation of PDE activity can be achieved via post-translational modification such as phosphorylation by ERK (extracellular-signal-regulated kinase) MAPKs (mitogen-activated protein kinases) and PKA (protein kinase A). In the present paper, we report for the first time that PDE4 isoforms from the PDE4A and PDE4D subfamilies can be selectively modified by SUMO (small ubiquitin-related modifier). We have identified a single SUMO site within a consensus tetrapeptide motif, ΨKXE (where Ψ represents a hydrophobic residue), which lies in the catalytic unit of these enzymes. SUMO modification of PDE4 at this site was observed upon overexpression of the SUMO E3 ligase PIASy [protein inhibitor of activated STAT (signal transducer and activator of transcription) Y] in HEK (human embryonic kidney)-293 cells and we identify PIASy as a novel binding partner for long PDE4 isoforms. Site-directed mutagenesis of the acceptor lysine residue ablated conjugation of PDE4 with SUMO, suggesting the presence of a single SUMO site in the first subdomain of the conserved PDE4 catalytic unit. This observation was supported by both cell-free in vitro SUMOylation assays and analysis of SUMOylated spot-immobilized peptide arrays. SUMO modification of long PDE4 isoforms serves to augment their activation by PKA phosphorylation and repress their inhibition by ERK phosphorylation. Following ligation of β-adrenergic receptors, SUMOylation of PDE4 isoforms sufficiently amplified PKA-stimulated PDE4 activity to reduce markedly the PKA phosphorylation status of the β2-adrenergic receptor. These results highlight a new means whereby cells might achieve the selective regulation of the activity of cAMP-specific PDE4 enyzmes.

A skewed ratio of pro-inflammatory to anti-inflammatory cytokines, elevated growth factor synthesis and T- and B-lymphocyte activation are 3 hallmarks of rheumatoid arthritis (RA) pathology. Interleukin-6 (IL-6), IL-7, IL-17, IL-12/IL-23 and growth factors, granulocyte macrophage-colony stimulating factor, IL-3, and erythropoietin activate the Janus Kinase/Signal Transducers and Activators of Transcription (JAK/STAT) pathway. Evidence showed that STAT protein phosphorylation (p-STAT) by activated JAKs is permissive for p-STAT to act as transcription factors by binding to STAT-responsive gene promoter sequences. This event is critical for perpetuating RA, in part, by up-regulating pro-inflammatory cytokine gene transcription. Activation of JAK/STAT by cytokines and growth factors can induce 'cross-talk' with other signaling pathways by which Stress-Activated Protein/Mitogen-Activated Protein Kinase (SAP/MAPK) and Phosphatidylinositide-3-Kinase (PI3K)-mediated signaling are also activated. JAK-specific small molecule inhibitors (SMIs) were developed to test whether JAK/STAT pathway blockade would regulate autoimmune-mediated inflammation. JAK-specific SMI blockade inhibited p-STAT induced by pro-inflammatory cytokines in vitro. Systemically administered JAK-specific SMI blockade also ameliorated biomarkers of inflammation in well-validated arthritis animal models. A few JAK-specific SMIs have made their way into RA clinical trials. In fact, the JAK3-specific SMI, CP-690,500 is the first JAK/STAT SMI to be assessed for clinical efficacy in a Phase III RA trial.

Signal transducer and activator of transcription-3 (STAT-3) is constitutively activated in ovarian and endometrial cancers and is implicated in uncontrolled cell growth. Thus, its disruption could be an effective approach to control tumorigenesis. Curcumin is a dihydroxyphenolic compound, with proven anti-cancer efficacy in various cancer models. We examined the anti-tumor mechanism of curcumin on STAT-3 and on the negative regulators of STAT-3, including suppressors of cytokine signaling proteins (SOCS-1 and SOCS-3), protein inhibitors of activated STAT (PIAS-1 and PIAS-3), and SH2 domain-containing phosphatases (SHP-1 and SHP-2) in ovarian and endometrial cancer cell lines. Treatment of cancer cells with curcumin induced a dose- and time-dependent decrease of constitutive IL-6 expression and of constitutive and IL-6-induced STAT-3 phosphorylation, which is associated with decreased cell viability and increased cleavage of caspase-3. The inhibition of STAT-3 activation by curcumin was reversible, and phosphorylated STAT-3 levels returned to control levels 24 h after curcumin removal. Compared to normal cells baseline expression of SOCS-3 was high in cancer cells and a marked decrease in SOCS-3 expression was seen following curcumin treatment. Overexpression of SOCS-3 in curcumin-treated cells increased expression of phosphorylated STAT-3 and resulted in increased cell viability. Normal ovarian and endometrial cells exhibited high expression of PIAS-3 protein, whereas in cancer cells the expression was greatly reduced. Curcumin increased PIAS-3 expression in cancer cells. Of significance, siRNA-mediated knockdown of PIAS-3 overcomes the inhibitory effect of curcumin on STAT-3 phosphorylation and cell viability. In conclusion, curcumin suppresses JAK-STAT signaling via activation of PIAS-3, thus attenuating STAT-3 phosphorylation and tumor cell growth.

STAT92E is an essential transcription factor in Drosophila melanogaster for the development of several organs and the immune system. The JAK/STAT pathway employs different evolutionary conserved regulatory mechanisms to control biological processes. Numerous transcription factors in both mammals and invertebrates have been shown to be either activated or inhibited by a covalent modification with a small ubiquitin-like modifier (Sumo). Here, we show that Drosophila STAT92E is modified by Sumo at a single lysine residue 187 in S2 cells. Mutation of Lys187 increases the transcriptional activity of STAT92E, thus suggesting that sumoylation of STAT92E has a repressive role in the regulation of the JAK/STAT pathway in Drosophila melanogaster.

Double-stranded DNA breaks, such as those caused by radiation, activate DNA damage responses. One such response is gene transcription by nuclear factor κB (NF-κB); activation of this transcription factor requires several upstream signaling events, including sumoylation of IκB kinase γ [IKKγ; also known as NF-κB essential modulator (NEMO)] by protein inhibitor of activated STAT y (PIASy) and phosphorylation of IKKγ by the kinase ataxia telangiectasia mutated (ATM) (see also McCool and Miyamoto). To investigate how PIASy and ATM are recruited to IKKγ, Stilmann et al. searched for binding partners of IKKγ and identified poly(ADP-ribose)-polymerase 1 (PARP-1). In contrast to cells from wild-type mice, cells from Parp1 –/– mice did not show NF-κB activation in response to radiation, as assessed by nuclear accumulation of the p65 subunit of NF-κB in mouse embryonic fibroblasts (MEFs) and electrophoretic mobility shift assays (EMSAs) in macrophages and small intestinal epithelial cells. NF-κB activation in Parp1 –/– MEFs was rescued by expressing wild-type PARP-1, but not a catalytically inactive mutant or a mutant unable to bind DNA. The association of endogenous PARP-1 with IKKγ was induced by irradiation, transient in nature, and required poly(ADP-ribosyl)ation. Furthermore, PARP-1 enhanced the interaction between IKKγ and PIASy [an effect that required poly(ADP-ribose) binding motifs in PIASy], was required for sumoylation of IKKγ by PIASy, and promoted the association of ATM with IKKγ and PIASy. Parp1 –/– MEFs were more sensitive to irradiation-induced apoptosis compared with wild-type MEFs; in addition, induction of NF-κB target genes that counter apoptosis was impaired in Parp1 –/– MEFs. The authors propose that DNA damage stimulates PARP-1, which, through poly(ADP-ribosyl)ation, mediates the formation of a complex that enables the phosphorylation of IKKγ by ATM, the sumoylation of IKKγ by PIASy, and ultimately the activation of NF-κB. M. Stilmann, M. Hinz, S. C. Arslan, A. Zimmer, V. Schreiber, C. Scheidereit, A nuclear poly(ADP-ribose)-dependent signalosome confers DNA damage-induced IκB kinase activation. Mol. Cell 36 , 365–378 (2009). [PubMed] K. McCool, S. Miyamoto, A PAR-SUMOnious mechanism of NEMO activation. Mol. Cell 36 , 349–350 (2009). [PubMed]

Protein inhibitor of activated STAT3 (PIAS3), the main cellular inhibitor of signal transducers and activator of transcription 3 (STAT3), has been described as a modulator of DNA binding transcription factors. The exploration of the emerging roles of PIAS3 in immune regulation is a growing and fascinating field. Recent discoveries have shed new light on the key role of PIAS3 in the regulation of transcriptional activity, and on the molecular mechanism involved. These findings suggest that the known functions of this signalling molecule are merely the "tip of the iceberg". This article reviews the challenging questions regarding the link between PIAS3 and the intracellular signalling in immune cells. Some of the known functions of PIAS3 that potentially modulate key proteins in the immune system will also be discussed.

The interactions and functions of protein inhibitors of activated STAT (PIAS) proteins are not restricted to the signal transducers and activators of transcription (STATs), but PIAS1, -2, -3 and -4 interact with and regulate a variety of distinct proteins, especially transcription factors. Although the majority of PIAS-interacting proteins are prone to modification by small ubiquitin-related modifier (SUMO) proteins and the PIAS proteins have the capacity to promote the modification as RING-type SUMO ligases, they do not function solely as SUMO E3 ligases. Instead, their effects are often independent of their Siz/PIAS (SP)-RING finger, but dependent on their capability to noncovalently interact with SUMOs or DNA through their SUMO-interacting motif and scaffold attachment factor-A/B, acinus and PIAS domain, respectively. Here, we present an overview of the cellular regulation by PIAS proteins and propose that many of their functions are due to their capability to mediate and facilitate SUMO-linked protein assemblies.

Haploid a and alpha cells of yeast respond to the pheromones alpha- and a-factor, respectively, by increasing the transcription of many genes whose products are essential for mating. The STE12 protein acts in this process by binding to the DNA sequence that mediates the increased transcription of pheromone-responsive genes. We show here that a hybrid protein containing STE12 fused to the DNA-binding domain of GAL4 can activate transcription of a reporter gene containing GAL4-binding sites but only after treatment of cells with pheromone. Thus, STE12 alone, when bound to DNA, is sufficient to mediate pheromone-induced transcription. By constructing hybrids of different STE12 regions with the GAL4 domain, we map the domain of STE12 necessary for this activation to the central third of the protein. Upon alpha-factor treatment, the hybrid of GAL4 with the complete STE12 sequence is rapidly phosphorylated, with kinetics consistent with the observed transcriptional induction of pheromone-responsive genes. The domain of STE12 necessary for this phosphorylation correlates with that involved in transcriptional activation. We propose that induction of pheromone-responsive genes is mediated by phosphorylation of STE12 to alter its activation function but not its DNA-binding ability.

Inappropriate activation of JAK/STAT signaling occurs with high frequency in human cancers and is associated with cancer cell survival and proliferation. Therefore, the development of pharmacologic STAT signaling inhibitors has therapeutic potential in the treatment of human cancers. Here, we report 2-[(3,5-bis-trifluoromethyl-phenyl)-hydroxy-methyl]-1-(4-nitro-phenylamino)-6-phenyl-1,2,4a,7a-tetrahydro-pyrrolo[3,4-b]-pyridine-5,7-dione (AUH-6-96) as a novel small-molecule inhibitor of JAK/STAT signaling that we initially identified through a cell-based high-throughput screening using cultured Drosophila cells. Treatment of Drosophila cells with AUH-6-96 resulted in a reduction of Unpaired-induced transcriptional activity and tyrosine phosphorylation of STAT92E, the sole Drosophila STAT homologue. In human cancer cell lines, AUH-6-96 inhibited both constitutive and interleukin-6-induced STAT3 phosphorylation. Specifically, in Hodgkin lymphoma L540 cells, treatment with AUH-6-96 resulted in reduced levels of tyrosine phosphorylated STAT3 and of the STAT3 downstream target gene SOCS3 in a dose- and time-dependent manner. In addition, AUH-6-96-treated L540 cells showed decreased expression of persistently activated JAK3, suggesting that AUH-6-96 inhibits the JAK/STAT pathway signaling in L540 cells by affecting JAK3 activity and subsequently blocking STAT3 signaling. Importantly, AUH-6-96 selectively affected cell viability only of cancer cells harboring aberrant JAK/STAT signaling. In support of the specificity of AUH-6-96 for inhibition of JAK/STAT signaling, treatment with AUH-6-96 decreased cancer cell survival by inducing programmed cell death by down-regulating the expression of STAT3 downstream target antiapoptotic genes, such as Bcl-xL. In summary, this study shows that AUH-6-96 is a novel small-molecule inhibitor of JAK/STAT signaling and may have therapeutic potential in the treatment of human cancers harboring aberrant JAK/STAT signaling. [Mol Cancer Ther 2008;7(9):2672–80]

Oxidative stress contributes to the development of neurodegenerative diseases. DJ-1, a protein genetically linked to Parkinson's disease (PD), has been implicated in oxidative stress defense and transcriptional regulation. However, it is unclear whether these two aspects of the DJ-1 function are connected. Here, we show that the inactivation of DJ-1 causes decreased expression of the human MnSOD. DJ-1 stimulates the activity of a master regulator of mitochondrial biogenesis and stress response, peroxisome proliferator-activated receptor-gamma co-activator 1alpha (PGC-1alpha), in the transcription of the MnSOD. Although DJ-1 does not interact with PGC-1alpha directly, it inhibits the SUMOylation of a transcriptional repressor, pyrimidine tract-binding protein-associated splicing factor (PSF). PSF binds PGC-1alpha and suppresses its transcriptional activity. In contrast, a SUMOylation-deficient PSF mutant exhibits reduced binding to PGC-1alpha and promotes its activity. SUMO-specific isopeptidase SENP-1 further enhances the synergy between DJ-1 and PGC-1alpha, whereas an SUMO E3 ligase protein inhibitor of activated STAT Y completely blocks the synergy. Conversely, oxidative modification renders DJ-1 unable to inhibit SUMOylation, resulting in attenuated transcriptional synergy between DJ-1 and PGC-1alpha. Therefore, our results validate DJ-1 as a transcriptional regulator in mitochondrial oxidative stress response and imply that the oxidation-mediated functional impairment of DJ-1 leads to gradual dysregulation of the SUMO pathway. Consequent abnormal mitochondrial gene expression may contribute to the development of sporadic PD.

Jun-dimerization protein 2 (JDP2) is a member of the activating protein-1 (AP-1) family of transcription factors. JDP2 dimerizes with other AP-1 proteins such as activating transcription factor-2 (ATF2) and Jun to repress transcription from promoters that contain a cyclic AMP-responsive element (CRE). Interferon regulatory factor-2-binding protein-1 (IRF2-BP1), which is reported to be a transcriptional corepressor of IRF2, was isolated as a JDP2-binding protein using an epitope-tagging method. As anticipated from the presence of a RING-finger domain, IRF2-BP1 enhanced the polyubiquitination of JDP2. Moreover, IRF2-BP1 repressed ATF2-mediated transcriptional activation from a CRE-containing promoter.Structured summaryMINT-6699624:JDP2 (uniprotkb:Q8WYK2) physically interacts (MI:0218) with Valyl-tRNA synthetase (uniprotkb:P26640), IRF2-BP1 (uniprotkb:Q8IU81), ATF7 (uniprotkb:P17544), EEF1G (uniprotkb:P26641), EEF1A1 (uniprotkb:P68104), JUND (uniprotkb:P17535), JUNB (uniprotkb:P17275), EF1D2 (uniprotkb:P29692) and RPLP0 (uniprotkb:P05388) by anti tag coimmunoprecipitation (MI:0007).MINT-6699850:JDP2 (uniprotkb:Q8WYK2) binds (MI:0407) to IRF2-BP1 (uniprotkb:Q8IU81) by pull down (MI:0096).MINT-6699684:JDP2 (uniprotkb:Q8WYK2) physically interacts (MI:0218) with JUNB (uniprotkb:P17275), JUND (uniprotkb:P17535), ATF7 (uniprotkb:P17544) and IRF2-BP1 (uniprotkb:Q8IU81) by anti tag coimmunoprecipitation (MI:0007).MINT-6699839:IRF2-BP1 (uniprotkb:Q8IU81) physically interacts (MI:0218) with JDP2 (uniprotkb:Q8WYK2) by anti bait coimmunoprecipitation (MI:0006).MINT-6699748:JDP2 (uniprotkb:Q8WYK2) physically interacts (MI:0218) with ATF2 (uniprotkb:P15336) and c-JUN (uniprotkb:P05412) by anti tag coimmunoprecipitation (MI:0007).MINT-6699865:ATF2 (uniprotkb:P15336) binds (MI:0407) to IRF2-BP1 (uniprotkb:Q8IU81) by pull down (MI:0096).

The Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway is crucial in controlling cellular activities in response to extracellular cytokines. Dysfunctions of the JAK/STAT pathway result in various hematopoietic and immune disorders. The central events in regulating this pathway are tyrosine phosphorylation and dephosphorylation of the signaling components, which are carried out by protein tyrosine kinases and protein tyrosine phosphatases (PTP), respectively. Here, we review recent advances in the regulatory roles of PTPs, in particular, SHP2 phosphatase, in the JAK/STAT signaling pathway.

SATB1 (special AT-rich sequence-binding protein-1) provides a key link between DNA loop organization, chromatin modification/remodeling, and association of transcription factors at matrix attachment regions (MARs). To investigate the role of SATB1 in cellular events, we performed a yeast two-hybrid screen that identified SUMO-1, Ubc9, and protein inhibitor of activated STAT (PIAS) family members as SATB1 interaction partners. These proteins, working in concert, enhanced SUMO conjugation to lysine-744 of SATB1. Overexpression of SUMO or PIAS in Jurkat cells, which express high levels of endogenous SATB1, exhibited enhanced caspase cleavage of this MAR-associating protein. Sumoylation-deficient SATB1 (SATB1(K744R)) failed to display the characteristic caspase cleavage pattern; however, fusion of SUMO in-frame to SATB1(K744R) restored cleavage. A SUMO-independent interaction of inactive caspase-6 and SATB1 was noted. A subset of total cellular SATB1 localized into promyelocytic leukemia nuclear bodies where enhanced SATB1 cleavage was detected subsequent to caspase activation. These results reveal a novel sumoylation-directed caspase cleavage of this key regulatory molecule. The role of regulated proteolysis of SATB1 may be to control transcription in immune cells during normal cell functions or to assist in efficient and rapid clearance of nonfunctional or potentially damaging immune cells.

International audience; The members of the protein inhibitor of activated STAT (PIAS) family of proteins are implicated in fundamental cellular processes, including transcriptional regulation, either through action as E3 SUMO ligases or through SUMO-independent effects. We report here the identification of FIP200 (focal adhesion kinase family-interacting protein of 200 kDa) as a new PIASy-interacting protein. We show that the interaction depends on the integrity of the RING finger of PIASy and the carboxy terminus of FIP200. Both in vitro and in vivo sumoylation assays failed to reveal any sumoylation of FIP200, suggesting that FIP200 is not a bona fide SUMO substrate. Immunofluorescence microscopy and subcellular fractionation, either upon forced PIASy expression or in the absence of PIASy, revealed that interaction with PIASy redistributes FIP200 from the cytoplasm to the nucleus, correlating with abrogation of FIP200 regulation of TSC/S6K signaling. Conversely, FIP200 enhances the transcriptional activation of the p21 promoter by PIASy whereas PIASy transcription activity is severely reduced upon FIP200 depletion by RNA interference. Chromatin immunoprecipitation analysis demonstrates that endogenous PIASy and FIP200 are corecruited to the p21 promoter. Altogether, these results provide the first evidence for the existence of a close—spatially controlled—mode of regulation of FIP200 and PIASy nucleocytoplasmic functions.

Activating mutations in fibroblast growth factor receptor 3 (FGFR3) cause several human skeletal dysplasias as a result of attenuation of cartilage growth. It is believed that FGFR3 inhibits chondrocyte proliferation via activation of signal transducers and activators of transcription (STAT) proteins, although the exact mechanism of both STAT activation and STAT-mediated inhibition of chondrocyte growth is unclear. We show that FGFR3 interacts with STAT1 in cells and is capable of activating phosphorylation of STAT1 in a kinase assay, thus potentially serving as a STAT1 kinase in chondrocytes. However, as demonstrated by western blotting with phosphorylation-specific antibodies, imaging of STAT nuclear translocation, STAT transcription factor assays and STAT luciferase reporter assays, FGF does not activate STAT1 or STAT3 in RCS chondrocytes, which nevertheless respond to a FGF stimulus with potent growth arrest. Moreover, addition of active STAT1 and STAT3 to the FGF signal, by means of cytokine treatment, SRC-mediated STAT activation or expression of constitutively active STAT mutants does not sensitize RCS chondrocytes to FGF-mediated growth arrest. Since FGF-mediated growth arrest is rescued by siRNA-mediated downregulation of the MAP kinase ERK1/2 but not STAT1 or STAT3, our data support a model whereby the ERK arm but not STAT arm of FGF signaling in chondrocytes accounts for the growth arrest phenotype.

The purpose of our studies is to elucidate how Copine-I antagonizes NF-.B transcription. Nuclear factor-.B (NF-.B) is a dynamic transcription factor that regulates important biological processes involved in cancer initiation and progression. Identifying regulators that control the half-life of NF-.B is important to understanding molecular processes that control the duration of transcriptional responses. In this study we identify Copine-I, a calcium phospholipid-binding protein, as a novel repressor that physically interacts with p65 to inhibit NF-.B transcription. Knockdown of Copine-I by siRNA increases tumor necrosis factor a-stimulated NF-.B transcription, while Copine-I expression blocks endogenous transcription. Copine-I abolishes NF-.B transcription by inducing endoprotease processing of the N-terminus of p65, a process antagonized by I.Ba. Copine-I stimulates endoproteolysis of p65 within a conserved region that is required for base-specific contact with DNA. p65 proteins lacking the N-terminus fail to bind to DNA and act as dominant-negative molecules that inhibit NF-.B transcription. Our work provides evidence that Copine-I regulates the half-life of NF-.B transcriptional responses through a novel mechanism that involves endoproteolysis of the p65 protein. Copine has significance as a potential biomarker for therapeutic intervention in prostate cancer.

It is well established that erythropoietin (EPO) is a pleiotropic cytokine, which has a brain-derived neuroprotective effect in the central nervous system (CNS). Immune abnormality has a close relationship with cerebral palsy (CP), and may be even involved in the development of CP. There is evidence that the amount of EPO in CP children is lower than in normal children, but the levels of proinflammatory cytokines, such as interleukin (IL)-6 and tumor necrosis factor (TNF)-alpha, are higher in the CP children. The signal transduction mediated by EPO that has a neuroprotective effect and mediated by proinflammatory cytokines that lead to brain damage shares the common JAK/STAT pathway. Under acute stress, the JAK/STAT pathway is occupied by massive proinflammatory cytokines, and the negative feedback inhibition factors like suppressor of cytokine signaling (SOCS) proteins are simultaneously activated, which exist in reciprocal inhibition to EPO in the JAK/STAT pathway. As a result, the signal transduction mediated by EPO is prevented or reduced, and the neuroprotective effect of EPO is eventually weakened. In this review, a novel approach to CP treatment through neurodevelopmental treatment (NDT) is put forward by analysis of the interrelationship of signal transduction mediated by EPO and proinflammatory cytokines in the JAK/STAT pathway and their roles in the development of CP, and some reasonable ideas for CP treatment are provided.

The signal transducer and activator of transcription (STAT) proteins are extracellular ligand-responsive transcription factors that mediate broadly diverse biological processes, including cell proliferation, transformation, apoptosis, differentiation, fetal development, inflammation and immune response. Stimulation with multiple cytokines or growth factors all result in the tyrosine phosphorylation of STAT proteins and the subsequent gene regulation via their direct binding to the promoters of responsive genes. Cytokine-regulated gene activation is dependent on the continuous nucleocytoplasmic cycling of STAT signal transducers. The STATs use intricately intertwined karyopherin-dependent and -independent translocation mechanisms to coordinate the activation step at the cell membrane and gene expression in the nucleus. In addition, STATs appear to have cytokine-independent gene regulatory functions that may also depend on their regulated nucleocytoplasmic transfer. Numerous studies have implicated aberrant STAT signalling in cancer, immune defects and inflammatory diseases. Given the central role of intracellular trafficking for the proper signal processing by STAT proteins, pharmacological targeting of STAT nucleocytoplasmic translocation appears to be an attractive strategy to interfere with dysregulated cytokine signalling. This review will discuss possible scenarios that would result from the use of novel modulators of STAT shuttling, which may both increase or decrease STAT activation and, hence, transcriptional activity.

STAT1 (signal transducer and activator of transcription 1) is a critical mediator of IFN-γ (interferon-γ)-induced gene responses, and its function is regulated through phosphorylation of Tyr 701 and Ser 727 . MAPK (mitogen-activated protein kinase) pathways mediate phosphorylation of Ser 727 in response to microbial infections, stress stimuli and growth factors. Recently, STAT1 was found to become modified by PIAS (protein inhibitor of activated STAT)-mediated SUMO-1 (small ubiquitin-related modifier-1) conjugation at Lys 703 , but the regulation of this modification is largely unknown. Here, we have investigated the role of MAPK-induced Ser 727 phosphorylation in regulation of STAT1 SUMOylation. Activation of the p38MAPK pathway by upstream activating kinase, MKK6 (MAPK kinase-6) or osmotic stress enhanced the SUMOylation of STAT1, which was counteracted by the p38MAPK inhibitor SB202190 or by dominant-negative p38MAPK. Activation of the ERK1/2 (extracellular-signal-regulated kinase 1/2) pathway by Raf-1 also enhanced Ser 727 phosphorylation and SUMOylation of STAT1, and this induction was counteracted by PD98059 inhibitor. Mutation of Ser 727 to alanine abolished the p38MAPK-induced SUMOylation. Furthermore, S727D and S727E mutations, which mimic the phosphorylation of Ser 727 , enhanced the basal SUMOylation of STAT1 and interaction between PIAS1 and STAT1. Taken together, these results identify Ser 727 phosphorylation as a regulator of STAT1 SUMOylation and highlight the central role of Ser 727 in co-ordination of STAT1 functions in cellular responses.

NF-κB and STATs regulate multiple cellular processes through the transcriptional activation of genes with diversified functions. Although the molecular mechanisms that can turn on/off the overall NF-κB/STAT signaling have been extensively studied, how NF-κB/STAT-target genes can be differentially regulated is poorly understood. Here we report that PIASy, a member of the PIAS (for protein inhibitor of activated STAT) protein family, is a physiologically important transcriptional repressor of NF-κB and STAT1. Piasy deletion in dendritic cells resulted in enhanced expression of a subset of NF-κB and STAT1-dependent genes in response to LPS or IFN-γ treatment, respectively. Consistently, Piasy null mice are hypersensitive to the LPS-induced endotoxic shock. Furthermore, PIASy and PIAS1 display specific as well as redundant effects on the regulation of NF-κB/STAT1 signaling. Pias1 −/− Piasy −/− embryos died before day 11.5. The disruption of one allele of Pias1 in the Piasy −/− background significantly enhanced the effect of Piasy deletion on the transcriptional induction of NF-κB/STAT1-dependent genes, and vice versa. Our results demonstrate that PIASy cooperates with PIAS1 to regulate the specificity and magnitude of NF-κB/STAT1-mediated gene activation.

The ETS transcription factor Ets-1 (E26 transformation-specific-1) plays a critical role in many physiological processes including angiogenesis, haematopoietic development and tumour progression. Its activity can be regulated by post-translational modifications, such as phosphorylation. Recently, we showed that Ets-1 is a target for SUMO (small ubiquitin-like modifier) modification and that PIASy [protein inhibitor of activated STAT (signal transducer and activator of transcription) Y], a specific SUMO-E3 ligase for Ets-1, represses Ets-1-dependent transcription. In the present study, we demonstrated that Ets-1 is degraded by the proteasome and that overexpression of PIASy increased the stability of endogenous and ectopically expressed Ets-1 protein by preventing proteasomal degradation. Moreover, knockdown of the endogenous PIASy expression by RNA interference reduced the protein level of endogenous Ets-1. The proteasome inhibitor MG132 reversed this effect. Deletion analysis showed that the TAD (transcriptional activation domain), which has been identified as the interaction domain with PIASy, was also required for Ets-1 ubiquitination and proteasomal degradation. However, the Ets-1 stabilization by PIASy was not due to reduced ubiquitination of Ets-1. Our results suggested that PIASy controls Ets-1 function, at least in part, by inhibiting Ets-1 protein turnover via the ubiquitin–proteasome system.

Protein inhibitor of activated STAT3 (PIAS3) has been shown to regulate the activity of various transcription factors. In this study, we show that the overexpression of PIAS3 in bone marrow-derived monocyte/macrophage lineage cells attenuates osteoclast formation and down-regulates the expression of NFATc1 and osteoclast-associated receptor (OSCAR), which are important modulators in osteoclastogenesis. PIAS3 has been shown to associate with histone deacetylase 1 as well as with transcription factors, including the microphthalmia transcription factor, NFATc1, and c-Fos. Moreover, overexpression of PIAS3 inhibits the transactivation of target genes such as NFATc1 and OSCAR. This inhibitory effect of PIAS3 is possibly mediated by histone deacetylase 1 recruitment to the promoter regions of NFATc1 and OSCAR. Furthermore, silencing of PIAS3 by RNA interference in osteoclast precursors enhances osteoclast formation as well as gene expression of NFATc1 and OSCAR. Taken together, our results reveal that PIAS3 acts as a modulator in osteoclastogenesis.

Leukemia inhibitor factor (LIF) has been shown to potently inhibit HIV-1 replication in vitro and in human organ explant cultures. Furthermore, LIF activates the Jak/Stat signaling pathway with which many viruses, including HIV-1, interfere. We used CXCR4 and the LIF signaling receptor (gp130)-expressing cMAGI cells transfected with CD4, CCR5, and HIV-LTR-beta-galactosidase as a model system to investigate the potential involvement of Stat proteins in the anti-HIV-1 effect of LIF. Pretreatment with recombinant human (rh)LIF resulted in a significantly reduced uptake of HIV-1(BaL) , HIV-1(LAI), and SIVmac251 viral particles without affecting uptake of murine leukemia retroviral particles. HIV-1(BaL), HIV-1(LAI), as well as rhLIF selectively induced phosphorylation of Stat 3 but not Stat 1 or Stat 5. However, treatment of cMAGI cells with rhLIF prior to HIV-1 infection downregulated the HIV-1-mediated Stat 3 phosphorylation. In addition, peripheral blood mononuclear cells (PBMCs) transfected with Stat 3 siRNA prior to HIV-1(LAI) or HIV-1(BaL) infection produced significantly less HIV-1 p24 antigen as compared to nontransfected HIV-1(LAI) and HIV-1(BaL)-infected PBMCs. Thus, the Jak/Stat signaling pathway is important for the HIV-1 replication life cycle and rhLIF excerts its anti-HIV-1 activity by disrupting this signaling cascade.

Background Rheumatoid Arthritis (RA) is a common, chronic immune-mediated inflammatory disease, characterised by synovitis and progressive destruction of articular cartilage and bone. Janus Kinase and Signal Transducer and Activator of Transcription (JAK/STAT), a critical signalling pathway involved in inflammatory mechanisms, has been implicated in the pathogenesis of RA. Objectives In this study we examine the effect of Tofacitinib (JAK inhibitor CP-690,550) on pro-inflammatory pathways in RA using in vitro and ex-vivo culture models. Methods Primary RA synovial fibroblasts (RASFC) and whole tissue ex vivo RA synovial explant cultures were established from RA biopsies obtained at arthroscopy. Phospho-STAT3 (p-STAT3), phospho-STAT1 (p-STAT1), Suppressor of Cytokine Signaling 3 (SOCS3) and Protein Inhibitor of Activated Stat 3 (PIAS3) expression were quantified by Western Blot in RASFC following culture in the presence of IL-17, OSM and IL-6. RASFC were cultured with IL-17, OSM and IL-6 in the presence or absence of Tofacitinib (1uM) and cytokine secretion in supernatants were measured by ELISA. Ex-vivo RA synovial explants were cultured with Tofacitinib (1uM) and spontaneous secretion of IL-6, IL-8, MMP3, Tie2, PIGF and bFGF were quantified in supernatants by ELISA or MSD multiplex. Finally conditioned media from control and Tofacitinib treated RA explants were cultured with RASFC and cell invasion assessed by Transwell Matrigel™ invasion chambers. Results IL-17, OSM and IL-6 induced p-STAT3, p-STAT1 expression and inhibited SOCS3 and PIAS3 expression in RASFC. Tofacitinib decreased IL-17, OSM and IL-6 induced cytokine production from RASFC. Tofacitinib inhibited spontaneous release of IL-6, IL-8, MMP3 and soluble Tie2 receptor from RA synovial explant cultures, with no effect observed for PIGF and bFGF. Finally, conditioned media from Tofacitinib treated RA explants inhibited RASFC invasion. Conclusions This study demonstrates inverse expression of pSTATs and their negative regulators SOCS3/PIAS3 in response to pro-inflammatory mediators in RA. In addition Tofacitinib inhibits pro-inflammatory mediators and RASFC invasion, further confirming a role for JAK-STAT inhibition in the treatment of RA. Disclosure of Interest None declared

Background/Objectives Rheumatoid Arthritis (RA) is a common, chronic immune-mediated inflammatory disease, characterised by synovitis and progressive destruction of articular cartilage and bone. Janus Kinase and Signal Transducer and Activator of Transcription (JAK/STAT), a critical signalling pathway involved in inflammatory mechanisms, has been implicated in the pathogenesis of RA. In this study we examine the effect of Tofacitinib (JAK inhibitor CP-690,550) on pro-inflammatory pathways in RA using in vitro and ex-vivo culture models. Methods Primary RA synovial fibroblasts (RASFC) and whole tissue ex vivo RA synovial explant cultures were established from RA biopsies obtained at arthroscopy. Phospho-STAT3 (p-STAT3), phospho-STAT1 (p-STAT1), Suppressor of Cytokine Signalling 3 (SOCS3) and Protein Inhibitor of Activated Stat 3 (PIAS3) expression were quantified by Western Blot in RASFC following culture in the presence of IL-17, OSM and IL-6. RASFC were cultured with IL-17, OSM and IL-6 in the presence or absence of Tofacitinib (1 uM) and cytokine secretion in supernatants were measured by ELISA. Ex-vivo RA synovial explants were cultured with Tofacitinib (1 uM) and spontaneous secretion of IL-6, IL-8, MMP3, Tie2, PIGF and bFGF were quantified in supernatants by ELISA or MSD multiplex. Finally conditioned media from control and Tofacitinib treated RA explants were cultured with RASFC and cell invasion assessed by Transwell Matrigel TM invasion chambers. Results IL-17, OSM and IL-6 induced p-STAT3, p-STAT1 expression and inhibited SOCS3 and PIAS3 expression in RASFC. Tofacitinib decreased IL-17, OSM and IL-6 induced cytokine production from RASFC. Tofacitinib inhibited spontaneous release of IL-6, IL-8, MMP3 and soluble Tie2 receptor from RA synovial explant cultures, with no effect observed for PIGF and bFGF. Finally, conditioned media from Tofacitinib treated RA explants inhibited RASFC invasion. Conclusion This study demonstrates inverse expression of pSTATs and their negative regulators SOCS3/PIAS3 in response to pro-inflammatory mediators in RA. In addition Tofacitinib inhibits pro-inflammatory mediators and RASFC invasion, further confirming a role for JAK-STAT inhibition in the treatment of RA.

Stroke causes neuronal necrosis and generates inflammation. Pro-inflammatory molecules intervene in this process by triggering glial cell activation and leucocyte infiltration to the injured tissue. Cytokines are major mediators of the inflammatory response. Pro-inflammatory and anti-inflammatory cytokines are released in the ischaemic brain. Anti-inflammatory cytokines, such as interleukin-10, promote cell survival, whereas pro-inflammatory cytokines, such as TNFα (tumour necrosis factor α), can induce cell death. However, deleterious effects of certain cytokines can turn to beneficial actions, depending on particular features such as the concentration, time point and the very intricate network of intracellular signals that become activated and interact. A key player in the intracellular response to cytokines is the JAK (Janus kinase)/STAT (signal transducer and activator of transcription) pathway that induces alterations in the pattern of gene transcription. These changes are associated either with cell death or survival depending, among other things, on the specific proteins involved. STAT1 activation is related to cell death, whereas STAT3 activation is often associated with survival. Yet, it is clear that STAT activation must be tightly controlled, and for this reason the function of JAK/STAT modulators, such as SOCS (suppressors of cytokine signalling) and PIAS (protein inhibitor of activated STAT), and phosphatases is most relevant. Besides local effects in the ischaemic brain, cytokines are released to the circulation and affect the immune system. Unbalanced pro-inflammatory and anti-inflammatory plasma cytokine concentrations favouring an ‘anti-inflammatory’ state can decrease the immune response. Robust evidence now supports that stroke can induce an immunodepression syndrome, increasing the risk of infection. The contribution of individual cytokines and their intracellular signalling pathways to this response needs to be further investigated.

Constitutively activated signal transducers and activators of transcription (STAT) are reported to cause uncontrolled transmission of growth signals. In this study, we analyzed the roles of an inhibitor of STAT, protein inhibitor of activated STAT (PIAS) 3, in the development of lung cancer. Treatment with an inhibitor of phosphatidylinositol 3-kinase, LY294002, retarded the growth of human lung cancer cells and rendered them more sensitive to chemotherapeutic agents. However, the inhibition of JAK/STAT by AG490 significantly suppressed cell growth but did not increase drug sensitivity at all. Overexpression of PIAS3 not only significantly inhibited cell growth but also rendered cancer cells up to 12.0-fold more sensitive to the above drugs, which was associated with the suppression of Akt phosphorylation. Inhibition of PIAS3 with small interfering RNA, nevertheless, led cancer cells to accelerate cell proliferation, deteriorate chemosensitivity, and augment Akt phosphorylation. Although the overexpression of suppressors of cytokine signaling 3 in cancer cells also inhibited cell growth and STAT3 phosphorylation, it neither increased sensitivity to chemotherapeutic drugs nor affected the phosphorylation of Akt. These results indicate that PIAS3 may be an attractive candidate for targeting the JAK/STAT and PI3-K/Akt signaling pathways in cancer treatment.

Purpose: Excessive activity of the transcription factors known as signal transducers and activators of transcription (STAT) contributes to the development and progression of malignancy in many organs. It is, therefore, important to develop new drugs to control the STATs, particularly their phosphorylation state, which is required for their transcriptional activity. Experimental Design: Myeloma and lung cancer cells were treated with the new synthetic triterpenoid CDDO-Imidazolide, and STAT phosphorylation and apoptosis were evaluated by immunoblotting and fluorescence-activated cell sorting analysis. Results: We now report that CDDO-Imidazolide, previously shown to be a potent agent for control of inflammation, cell proliferation, and apoptosis, rapidly (within 30-60 minutes) and potently (at nanomolar levels) suppresses either constitutive or interleukin-6-induced STAT3 and STAT5 phosphorylation in human myeloma and lung cancer cells. Furthermore, in these cells, CDDO-Imidazolide also up-regulates critical inhibitors of STATs, such as suppressor of cytokine signaling-1 and SH2-containing phosphatase-1 (a tyrosine phosphatase). Moreover, gene array studies reported here show that CDDO-Imidazolide potently regulates the transcription of important genes that are targets of the STATs. Conclusions: Our new data thus show that CDDO-Imidazolide is a potent suppressor of STAT signaling and provide a further mechanistic basis for future clinical use of this agent to control inflammation or cell proliferation.

Transforming growth factor beta (TGF-beta) plays critical roles in the control of cell proliferation, differentiation, and apoptosis. Smad proteins are substrates of the TGF-beta type I receptor and are responsible for transducing receptor signals to target genes in the nucleus. The PIAS (protein inhibitor of activated STAT) proteins were originally identified as transcriptional co-regulators of the JAK-STAT pathway. Subsequently, cross-talk between the PIAS proteins and other signaling pathways has been shown to be involved in various cellular processes. Importantly, PIAS proteins modulate TGF-beta signaling by regulating the transcriptional activity of Smad3. In this study we tested whether hZimp10, a novel PIAS-like protein, acts as other PIAS proteins to regulate Smad3-mediated transcription. We show that expression of exogenous hZimp10 enhances the transcriptional activity of Smad3, which appears to be Smad4-dependent and responsive to TGF-beta induction. Furthermore, knockdown of endogenous hZimp10 reduced the transcriptional activity of Smad3. A protein-protein interaction between Smad3 and Smad4 with hZimp10 was identified in glutathione S-transferase-pulldown and co-immunoprecipitation assays. The Miz domain of hZimp10 and the MH2 domains of Smad3 and Smad4 were mapped as the regions responsible for binding. Results from immunostaining assays further demonstrated that Smad3, Smad4, and hZimp10 co-localize within cell nuclei. Finally, we demonstrated that Smad3/4-mediated transcription is significantly impaired in response to TGF-beta induction in Zimp10 null (zimp10-/-) embryonic fibroblasts. Taken together, these results provide the first line of evidence to demonstrate a role for Zimp10 in regulating the TGF-beta/Smad signaling pathway.

The Janus Kinase (JAK)/signal transducer and achivator of transcription (STAT) pathway is activated not only by various cytokines such as interferon and interleukin, but also by many growth factors. This pathway plays a crucial role in the signal transduction of these factors. Keywords: Cytokine; JAK; STAT; SOCS; PIAS; PTP

We have identified PIAS1 (protein inhibitor of activated STAT-1), -3, -xalpha, and -xbeta as SOX9-associated polypeptides using the Gal4-based yeast two-hybrid system and a cDNA library derived from a chondrocytic cell line. These PIAS proteins were shown to interact directly with SOX9 in two-hybrid, co-immunoprecipitation, and electrophoretic mobility shift assays. SOX9 was sumoylated in cotransfection experiments with COS-7 cells using PIAS and SUMO-1 (small ubiquitin-like modifier-1) expression vectors. SOX9 was also sumoylated in vitro by PIAS proteins in the presence of SUMO-1, the SUMO-activating enzyme, and the SUMO-conjugating enzyme. In COS-7 cells, PIAS proteins stimulated the SOX9-dependent transcriptional activity of a Col2a1 promoter-enhancer reporter. This increase in reporter activity was paralleled by an increase in the cellular levels of SOX9. Cotransfection with a SUMO-expressing vector further enhanced the transcriptional activity of this SOX9-dependent Col2a1 reporter in COS-7 cells, and this additional activation was inhibited in the presence of either SUMO-1 mutants or PIAS RING domain mutants or by coexpression of a desumoylation enzyme. Immunofluorescence microscopy of SOX9-transfected COS-7 cells showed that the subnuclear distribution of SOX9 became more diffuse in the presence of PIAS1 and SUMO-1. Our results suggest that, by controlling the cellular concentrations of SOX9, PIAS proteins and sumoylation may be part of a major regulatory system of SOX9 functions.

Cytokines activate multiple signal transduction pathways to regulate gene expression. STATs and NF-kB are two important families of transcription factors activated by cytokines. Abnormal regulation of STAT and NF-kB activities has been associated with human diseases. The protein inhibitor of activated STAT (PIAS) protein family has been proposed to interact with over 60 proteins, many of which are transcription factors involved in the immune system. PIAS proteins regulate transcription through several mechanisms, including blocking the DNA-binding activity of transcription factors, recruiting transcriptional co-repressors and promoting protein sumoylation. This article is to review the role of PIAS proteins in the regulation of STAT and NF-kB signaling pathways.

3 páginas, 1 figura., Transcriptional responses to the activation of a signalling pathway are cell-specific. New data show that the sequence-specific transcriptional repressors of the KEN/BCL-6 family play an important role in the selection of STAT targets in vertebrates and invertebrates, indicating that all STAT proteins may share this ancestral mechanism.

Jak2 is a nonreceptor tyrosine kinase that is essential for proper animal development and physiology. It is activated by ligand-occupied cell-surface receptors. Once activated, it then tyrosine phosphorylates the latent cytoplasmic transcription factors, termed the signal transducers and activators of transcription (STAT) proteins. Thus Jak2 is viewed as a classic mediator of ligand-dependent signal transduction. Recent studies, however, suggest that Jak2 may mediate cellular gene expression outside of the classically defined, ligand-activated, Jak/STAT-signaling paradigm. Here we review these studies, provide additional data, and discuss whether Jak2 is a mediator of ligand-independent gene transcription, and, in turn, whether our current understanding of the Jak/STAT signaling paradigm should be modified to incorporate these observations.

Cytokine signalling is dependent on the activation of signal transducer and activator of transcription (STAT) proteins. Activation entails the tyrosine phosphorylation of STATs and their formation of dimers that are retained in the nucleus. These dimers are thought to constitute a diffusible pool in the cell nucleus, from which they are either recruited to DNA and function as transcription regulators, or they are dephosphorylated and resume nucleo-cytoplasmic shuttling. Our research has demonstrated that this model of STAT protein functioning in the nucleus needs to be amended. We have discovered that activated STAT dimers can polymerize and assemble higher-order structures in the nucleus of cytokine-stimulated cells. STAT polymerization can take place both on DNA and in the nucleoplasm. STAT polymers are highly dynamic, with components in constant flux with the surrounding nucleoplasm, whereby DNA-independent polymers can align laterally, leading to well-ordered structures and transition to a liquid crystal-like phase. In contrast to probably all other STAT family members, DNA-independent polymerization of STAT1 can be suppressed by SUMO conjugation. In conjunction with the ability of different STATs to co-polymerize, STAT supramolecular structures may thus constitute a structural base for the cross-regulation of cytokine signal pathways. We have begun to characterize STAT polymerization and its consequences for cytokine signalling. Our results show that phase transitions associated with STAT supramolecular assembly affect cytokine signalling in important ways, examples of which will be presented.