Protein Inhibitor Of Activated STAT Proteins Research Articles

SUMO Proteomics Analyses Identify Protein Inhibitor of Activated STAT-Mediated Regulatory Networks Involved in Cell Cycle and Cell Proliferation.

Protein inhibitor of activated STAT (PIAS) proteins are E3 SUMO ligases playing important roles in protein stability and signaling transduction pathways. PIAS proteins are overexpressed in the triple-negative breast cancer cell line MDA-MB-231, and PIAS knockout (KO) results in a reduction in cell proliferation and cell arrest in the S phase. However, the molecular mechanisms underlying PIAS functions in cell proliferation and cell cycle remain largely unknown. Here, we used quantitative SUMO proteomics to explore the regulatory role of PIAS SUMO E3 ligases upon CRISPR/Cas9 KO of individual PIAS. A total of 1422 sites were identified, and around 10% of SUMO sites were regulated following KO of one or more PIAS genes. We identified protein substrates that were either specific to individual PIAS ligase or regulated by several PIAS ligases. Ki-67 and TOP2A, which are involved in cell proliferation and epithelial-to-mesenchymal transition, are SUMOylated at several lysine residues by all PIAS ligases, suggesting a level of redundancy between these proteins. Confocal microscopy and biochemical experiments revealed that SUMOylation regulated TOP2A protein stability, while this modification is involved in the recruitment of Ki-67 nucleolar proteins containing the SUMO interacting motif. These results provide novel insights into both the redundant and specific regulatory mechanisms of cell proliferation and cell cycle mediated by PIAS SUMO E3 ligases.

Super-resolution study of PIAS SUMO E3-ligases in hippocampal and cortical neurons.

The SUMOylation machinery is a regulator of neuronal activity and synaptic plasticity. It is composed of SUMO isoforms and specialized enzymes named E1, E2 and E3 SUMO ligases. Recent studies have highlighted how SUMO isoforms and E2 enzymes localize with synaptic markers to support previous functional studies but less information is available on E3 ligases. PIAS proteins - belonging to the protein inhibitor of activated STAT (PIAS) SUMO E3-ligase family - are the best-characterized SUMO E3-ligases and have been linked to the formation of spatial memory in rodents. Whether however they exert their function co-localizing with synaptic markers is still unclear. In this study, we applied for the first time structured illumination microscopy (SIM) to PIAS ligases to investigate the co-localization of PIAS1 and PIAS3 with synaptic markers in hippocampal and cortical murine neurons. The results indicate partial co-localization of PIAS1 and PIAS3 with synaptic markers in hippocampal neurons and much rarer occurrence in cortical neurons. This is in line with previous super-resolution reports describing the co-localization with synaptic markers of other components of the SUMOylation machinery.

GFI1B Splice Variants Differentially Regulate Transcription and Megakaryocyte/Erythrocyte Fate in Hematopoiesis

Alternative splicing is a powerful way to expand functional genetic diversity without increasing the number of genes. Recent deep sequencing efforts reveal that 100% of human genes are alternatively spliced. Alternative splicing is enriched in genes that control cell cycle dynamics, transcription and organelle organization during erythrocyte differentiation.Growth factor independence (GFI) 1B is comprised of an N-terminal SNAG domain, a C-terminal concatemer of six C2H2 zinc fingers (ZnFs), and a linker that joins them. GFI1B is required for both definitive erythropoiesis and megakaryopoiesis. Elevated GFI1B expression is seen in AML, while GFI1B mutations cause inherited platelet disorders. Understanding GFI1B function is critical to addressing its contributions to diseases of hematopoiesis. GFI1B has two splice variants, GFI1B-full and GFI1B-short . GFI1B-short lacks ZnFs 1 and 2, but is otherwise structurally identical to GFI1B-full. Both splice variants are expressed during erythroid differentiation from megakaryocyte-erythrocyte progenitors (MEPs), but their distinctive functions and relative contibutions to this binary fate decision remain largely unexplored.We used the bipotent, GFI1B-dependent erythroleukemia cell line, K562, to explore fate specifying and mechanistic differences between GFI1B-full and GFI1B-short. Inducible expression of GFI1B-full in K562 cells promotes spontaneous erythroid differentiation and antagonizes 12-O-Tetradecanoylphorbol-13-acetate (TPA)-induced megakaryocyte differentiation. In contrast, GFI1B-short fails to support spontaneous erythroid differentiation and instead enables megakaryocyte fate in response to TPA. These findings suggest ZnFs 1 and 2 control fate-specifying differences between GFI1B splice variants. We have previously shown the GFI1B paralog, GFI1, is SUMOylated and that a portion of the linker region and ZnFs 1-3 provide binding surfaces for the E3 SUMO ligase, Protein Inhibitor of Activated STAT (PIAS)3. To gain additional insights regarding binding relationships between GFI1B and PIAS family proteins, we mapped the binding interface between GFI1B-full and PIAS3 and investigated binding differences between GFI1B-full vs. GFI1B-short for PIAS family members. We find ZnFs 1-3 of GFI1B are necessary and sufficient for PIAS3 binding and predicted impaired PIAS protein binding by GFI1B-short. While modest impairment was observed between GFI1B-short and PIAS proteins, PIASXa binding was completely abolished. We also find that GFI1B-full is SUMOylated on lysine (K)-61 within a yKXD/E SUMOylation consensus element. Yet, GFI1B-short is not SUMOylated, despite this consensus element being present. This suggests that differential recruitment of the SUMOylation machinery by GFI1 splice variants and assembly of distinct, SUMO-dependent transcriptional regulatory complexes may direct binary cell fate decisions. Using proximity-based in situ biotinylation and mass spectral analysis, we recorded the composition of transcriptional regulatory complexes assembled by GFI1B-full and GFI1B-short. We identifed candidate factors whose recruitment by GFI1B may be SUMOylation-dependent, suggesting that GFI1B splicing variation and differential SUMOylation may direct lineage allocation in hematopoiesis. DisclosuresNo relevant conflicts of interest to declare.

SUMOylation regulates nuclear accumulation and signaling activity of the soluble intracellular domain of the ErbB4 receptor tyrosine kinase

Erb-B2 receptor tyrosine kinase 4 (ErbB4) is a kinase that can signal via a proteolytically released intracellular domain (ICD) in addition to classical receptor tyrosine kinase-activated signaling cascades. Previously, we have demonstrated that ErbB4 ICD is posttranslationally modified by the small ubiquitin-like modifier (SUMO) and functionally interacts with the PIAS3 SUMO E3 ligase. However, direct evidence of SUMO modification in ErbB4 signaling has remained elusive. Here, we report that the conserved lysine residue 714 in the ErbB4 ICD undergoes SUMO modification, which was reversed by sentrin-specific proteases (SENPs) 1, 2, and 5. Although ErbB4 kinase activity was not necessary for the SUMOylation, the SUMOylated ErbB4 ICD was tyrosine phosphorylated to a higher extent than unmodified ErbB4 ICD. Mutation of the SUMOylation site compromised neither ErbB4-induced phosphorylation of the canonical signaling pathway effectors Erk1/2, Akt, or STAT5 nor ErbB4 stability. In contrast, SUMOylation was required for nuclear accumulation of the ErbB4 ICD. We also found that Lys-714 was located within a leucine-rich stretch, which resembles a nuclear export signal, and could be inactivated by site-directed mutagenesis. Furthermore, SUMOylation modulated the interaction of ErbB4 with chromosomal region maintenance 1 (CRM1), the major nuclear export receptor for proteins. Finally, the SUMO acceptor lysine was functionally required for ErbB4 ICD-mediated inhibition of mammary epithelial cell differentiation in a three-dimensional cell culture model. Our findings indicate that a SUMOylation-mediated mechanism regulates nuclear localization and function of the ICD of ErbB4 receptor tyrosine kinase.

The Role of PIAS SUMO E3-Ligases in Cancer

SUMOylation modifies the interactome, localization, activity, and lifespan of its target proteins. This process regulates several cellular machineries, including transcription, DNA damage repair, cell-cycle progression, and apoptosis. Accordingly, SUMOylation is critical in maintaining cellular homeostasis, and its deregulation leads to the corruption of a plethora of cellular processes that contribute to disease states. Among the proteins involved in SUMOylation, the protein inhibitor of activated STAT (PIAS) E3-ligases were initially described as transcriptional coregulators. Recent findings also indicate that they have a role in regulating protein stability and signaling transduction pathways. PIAS proteins interact with up to 60 cellular partners affecting several cellular processes, most notably immune regulation and DNA repair, but also cellular proliferation and survival. Here, we summarize the current knowledge about their role in tumorigenesis and cancer-related processes. Cancer Res; 77(7); 1542-7. ©2017 AACR.

Novel Role for Protein Inhibitor of Activated STAT 4 (PIAS4) in the Restriction of Herpes Simplex Virus 1 by the Cellular Intrinsic Antiviral Immune Response.

ABSTRACTSmall ubiquitin-like modifier (SUMO) is used by the intrinsic antiviral immune response to restrict viral pathogens, such as herpes simplex virus 1 (HSV-1). Despite characterization of the host factors that rely on SUMOylation to exert their antiviral effects, the enzymes that mediate these SUMOylation events remain to be defined. We show that unconjugated SUMO levels are largely maintained throughout infection regardless of the presence of ICP0, the HSV-1 SUMO-targeted ubiquitin ligase. Moreover, in the absence of ICP0, high-molecular-weight SUMO-conjugated proteins do not accumulate if HSV-1 DNA does not replicate. These data highlight the continued importance for SUMO signaling throughout infection. We show that the SUMO ligase protein inhibitor of activated STAT 4 (PIAS4) is upregulated during HSV-1 infection and localizes to nuclear domains that contain viral DNA. PIAS4 is recruited to sites associated with HSV-1 genome entry through SUMO interaction motif (SIM)-dependent mechanisms that are destabilized by ICP0. In contrast, PIAS4 accumulates in replication compartments through SIM-independent mechanisms irrespective of ICP0 expression. Depletion of PIAS4 enhances the replication of ICP0-null mutant HSV-1, which is susceptible to restriction by the intrinsic antiviral immune response. The mechanisms of PIAS4-mediated restriction are synergistic with the restriction mechanisms of a characterized intrinsic antiviral factor, promyelocytic leukemia protein, and are antagonized by ICP0. We provide the first evidence that PIAS4 is an intrinsic antiviral factor. This novel role for PIAS4 in intrinsic antiviral immunity contrasts with the known roles of PIAS proteins as suppressors of innate immunity. IMPORTANCE Posttranslational modifications with small ubiquitin-like modifier (SUMO) proteins regulate multiple aspects of host immunity and viral replication. The protein inhibitor of activated STAT (PIAS) family of SUMO ligases is predominantly associated with the suppression of innate immune signaling. We now identify a unique and contrasting role for PIAS proteins as positive regulators of the intrinsic antiviral immune response to herpes simplex virus 1 (HSV-1) infection. We show that PIAS4 relocalizes to nuclear domains that contain viral DNA throughout infection. Depletion of PIAS4, either alone or in combination with the intrinsic antiviral factor promyelocytic leukemia protein, significantly impairs the intrinsic antiviral immune response to HSV-1 infection. Our data reveal a novel and dynamic role for PIAS4 in the cellular-mediated restriction of herpesviruses and establish a new functional role for the PIAS family of SUMO ligases in the intrinsic antiviral immune response to DNA virus infection.

Protein Inhibitor of Activated STAT3 Regulates Migration, Invasion, and Activation of Fibroblast-like Synoviocytes in Rheumatoid Arthritis.

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.

Transcriptional Activity of Erythroid Kruppel-like Factor (EKLF/KLF1) Modulated by PIAS3 (Protein Inhibitor of Activated STAT3)

Erythroid Kruppel-like factor (EKLF or KLF1) is a transcription factor crucial for red cell development that is directly involved in regulation of a large number of erythroid genes. EKLF serves mostly as an activator of expression of these genes; however, it can act also as a repressor. Here, we present evidence that EKLF interacts with proteins from the PIAS (protein inhibitor of activated STAT) family that convey repressive activity to EKLF in the absence of sumoylation. Our studies identify PIAS3 as a transcriptional corepressor of EKLF for at least a subset of its target genes during erythropoiesis (e.g. β-globin, α-hemoglobin stabilizing protein). We demonstrate an interaction between EKLF and PIAS proteins confirmed by in vivo coimmunoprecipitation assays with both exogenous and endogenous proteins. We identified an LXXLL signature motif located near the N terminus of PIAS proteins that, although not involved in the EKLF-PIAS3 interaction, is required for the transrepression activity. Knockdown of endogenous PIAS3 accelerates differentiation of both murine erythroleukemia cells, as well as fetal liver cells, whereas an increase in PIAS3 levels inhibits this increase. Using chromatin immunoprecipitation assays, we show that PIAS3 preferentially occupies the β-globin promoter in undifferentiated murine erythroleukemia cells. Together these results demonstrate that an interaction between EKLF and PIAS3 provides a novel mode of regulation of EKLF activity in the absence of sumolylation and furthermore shows an important involvement of PIAS proteins in erythropoiesis.

The Pias-Like Coactivator ZMIZ1 Drives C-MYC Induction in Collaboration with NOTCH1 in T-Cell Acute Lymphoblastic Leukemia.

Abstract 2380Activating NOTCH1 mutations are found in 50–60% of human T-cell acute lymphoblastic leukemia (T-ALL) samples. In mouse models, these mutations generally fail to induce leukemia. Cooperating oncogenes must be recruited by NOTCH1 to fully induce leukemia. Murine insertional mutagenesis screens previously implicated ZMIZ1 as a possible NOTCH1 collaborator in leukemia (Uren et al., Cell, 2008; Dupuy et al., Nature, 2005; Berquam-Vrieze et al., Blood, 2011). ZMIZ1 is a transcriptional co-activator of the Protein Inhibitor of Activated STAT (PIAS)-like family. It shares a zinc finger domain, the MIZ domain, with PIAS proteins. The MIZ domain mediates interactions with DNA-binding transcription factors and sumoylation. Previously, we showed that ZMIZ1 promotes T-ALL in collaboration with leukemia-associated NOTCH1 alleles in mouse models. ZMIZ1 and activated NOTCH1 were co-expressed in a subset of human patients. Genetic ZMIZ1 inhibition slowed leukemic cell growth and overcame resistance of some T-ALL cell lines to NOTCH inhibitors. ZMIZ1 may be a new clinically relevant oncogene. Here we sought to determine the downstream target genes of ZMIZ1 in leukemia. Validation of gene expression profiling data identified C-MYC and IL7RA as downstream targets of ZMIZ1. Targeting the C-MYC or IL-7 pathways using genetic and pharmacological inhibitors partly phenocopied the growth inhibitory effects we previously saw with ZMIZ1 inhibition. In order to determine whether these genes are direct or indirect targets of ZMIZ1, we generated an estrogen fusion protein, ZMIZ1-ER. ZMIZ1-ER induced C-MYC and IL7RA expression in the presence of tamoxifen, but failed to induce these genes with the addition of cycloheximide. These data suggest that C-MYC and IL-7RA are indirect targets. Like the PIAS proteins, ZMIZ1 appeared to have a broad effect on transcription to exert its functions. We next sought to elucidate the biochemical mechanism of ZMIZ1. Ectopic expression of ZMIZ1 or NOTCH1 had weak effects on endogenous c-Myc expression and failed to rescue a C-MYC-dependent T-ALL cell line after withdrawal of ectopic C-MYC. In contrast, ZMIZ1 in combination with NOTCH1 dramatically induced C-MYC expression by several fold and rescued the C-MYC dependent cell line. ZMIZ1 enhanced the ability of even weak NOTCH1 mutants to induce C-MYC, suggesting a mechanism by which ZMIZ1 may increase resistance to NOTCH inhibitors. ZMIZ1 did not influence C-MYC expression post-transcriptionally. It functioned primarily as a transcriptional activator. Although both C-MYC and IL7RA are both NOTCH1 target genes, ZMIZ1 did not directly interact with NOTCH1 or influence the expression of several other NOTCH1 target genes such Ptcra, Hes1, Dtx1, and Cd25. Thus, ZMIZ1 did not pan-activate NOTCH signaling. Based on bioinformatic analysis, we generated mutants that deleted individual domains of ZMIZ1. All mutants expressed at high levels by Western blot. Deletion of the transcriptional activation domain or the N-terminal domain (NTD) abolished the ability of ZMIZ1 to induce c-Myc and drive proliferation. Surprisingly, deletion of the PAT-like, Proline-rich, and MIZ domains or all three domains simultaneously had no effect on ZMIZ1 function. The 120-amino acid NTD has a predicted helical structure without significant sequence homology to any known domain. It is not found in ZMIZ2 or PIAS proteins. In summary, the mechanism of ZMIZ1 appears to be novel, indirect, transcriptional, and independent of canonical NOTCH and PIAS functions. Our study demonstrates the importance of characterizing genetic collaborations between parallel leukemic pathways that may be therapeutically targeted. They also raise new inquiries into potential NOTCH-ZMIZ1 collaboration in a variety of C-MYC-driven cancers. Disclosures:No relevant conflicts of interest to declare.

SUMO wrestling in cell movement

SUMO wrestling in cell movement

Abstract 1177: Genomic abnormality and genetic alterations of a novel ZMIZ1 gene at human chromosome 10q22.3 in oncogenesis and prognosis

Abstract Chromosomal abnormalities and genomic instability in 10q22.3 region have been associated with lung cancer development, histopathological staging, and poor prognosis. Genetic mapping identified several genes including ZMIZ1, SFTPA1 and SFTPA2 in this critical region. Loss, interruption or gain of function of these genes in association with 10q22.3 deletions, translocations, or amplifications that were frequently detected in human primary lung cancer and cancer cell lines may promote oncogenesis. Human ZMIZ1, also named hZimp10, is a novel PIAS (protein inhibitor of activated STAT)-like protein that shares a zing finger domain, MIZ (MSX-interacting zinc finger), with other PIAS proteins. Fusion of ZMIZ1 to ABL1 with a t(9;10)(q34;q22.3) translocation was identified in a B-cell acute lymphoblastic leukemia patient. In this study, we performed fluorescence in situ hybridization (FISH) using genomic DNA probes covering ZMIZ1 and SFTPA gene loci at 10q22.3 to determine chromosomal aberrations involving the ZMIZ1 gene in primary lung tumor samples and lung cancer cell lines. Chromosomal abnormalities at 10q22.3 loci, including deletions, polysomy, and translocations were frequently detected in tumors and tumor-derived cell lines. We analyzed copy number variations specifically linked to ZMIZ1 gene loci by mining the published genome-wide SNP dataset from 370 NSCLC tumor samples and confirmed a significant copy number gain with average copy numbers (CN) > 5 or loss with CN < 1.5, respectively. A high frequency of loss of heterozygosity (LOH) of ZMIZ1 and ABL1 genes in lung cancer cell lines and high level amplifications of ZMIZI gene (CN > 7) in breast and prostate cancer cell lines were also detected in Sanger SNP and CGP databases, respectively. We performed Spectral karyotyping (SKY) analysis to identify chromosomal abnormalities and potential translocations involved in the 10q region in lung cancer cell lines. We found that the 10q region was translocated and fused with other chromosomes extensively in H1819 cells. This result was consistent with the high frequency 10q chromosomal abnormalities detected in human lung and other cancer cell lines in the NCI/NCBI SKY/M-FISH & CGH database. Furthermore, we developed and performed a high-throughput RACE-PCR-based gene-specific RNA sequencing analysis using next-generation parallel sequencing technology (454 Life Sciences, Inc.) to search for potential transcriptional variants and fusion partners of the ZMIZ1 gene in lung cancer cell lines which has revealed potential fusion mRNA. These results suggest that ZMIZ1 may play an important role in lung cancer development and serve as a useful biomarker for lung cancer molecular staging and prognosis. (Supported by NIH/NCI grants SPORE P50CA70907 and RO1CA116322) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1177.

A Novel Role for Protein Inhibitor of Activated STAT (PIAS) Proteins in Modulating the Activity of Zimp7, a Novel PIAS-like Protein, in Androgen Receptor-mediated Transcription

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

Specific Domain Structures Control Abscisic Acid-, Salicylic Acid-, and Stress-Mediated SIZ1 Phenotypes

SIZ1 (for yeast SAP and MIZ1) encodes the sole ortholog of mammalian PIAS (for protein inhibitor of activated STAT) and yeast SIZ SUMO (for small ubiquitin-related modifier) E3 ligases in Arabidopsis (Arabidopsis thaliana). Four conserved motifs in SIZ1 include SAP (for scaffold attachment factor A/B/acinus/PIAS domain), PINIT (for proline-isoleucine-asparagine-isoleucine-threonine), SP-RING (for SIZ/PIAS-RING), and SXS (for serine-X-serine, where X is any amino acid) motifs. SIZ1 contains, in addition, a PHD (for plant homeodomain) typical of plant PIAS proteins. We determined phenotypes of siz1-2 knockout mutants transformed with SIZ1 alleles carrying point mutations in the predicted domains. Domain SP-RING is required for SUMO conjugation activity and nuclear localization of SIZ1. Salicylic acid (SA) accumulation and SA-dependent phenotypes of siz1-2, such as diminished plant size, heightened innate immunity, and abscisic acid inhibition of cotyledon greening, as well as SA-independent basal thermotolerance were not complemented by the altered SP-RING allele of SIZ1. The SXS domain also controlled SA accumulation and was involved in greening and expansion of cotyledons of seedlings germinated in the presence of abscisic acid. Mutations of the PHD zinc finger domain and the PINIT motif affected in vivo SUMOylation. Expression of the PHD and/or PINIT domain mutant alleles of SIZ1 in siz1-2 promoted hypocotyl elongation in response to sugar and light. The various domains of SIZ1 make unique contributions to the plant's ability to cope with its environment.

The PHD Domain of Plant PIAS Proteins Mediates Sumoylation of Bromodomain GTE Proteins

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

The Novel PIAS-like Protein hZimp10 Enhances Smad Transcriptional Activity

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.

Sumoylation of CCAAT/Enhancer-binding Protein α and Its Functional Roles in Hepatocyte Differentiation

The sumoylation of CCAAT/enhancer-binding proteins (C/EBPs) by small ubiquitin-related modifier-1 (SUMO-1) has been reported recently. In this study, we investigated the functional role of the sumoylation of C/EBPalpha in the differentiation of hepatocytes. The amount of sumoylated C/EBPalpha gradually decreased during the differentiation, which suggests that the sumoylation is important for the control of growth/differentiation especially in the fetal liver. To analyze the function of the sumoylation of C/EBPalpha in liver-specific gene expression, we studied its effects on the expression of the albumin gene. The C/EBPalpha-mediated transactivation of the albumin gene was reduced by sumoylation of C/EBPalpha in primary fetal hepatocytes. The enhancement of C/EBPalpha-mediated transactivation by BRG1, a core subunit of the SWI/SNF chromatin remodeling complex, was hampered by sumoylation in a luciferase reporter assay. In addition, we discovered that sumoylation of C/EBPalpha blocked its inhibitory effect on cell proliferation by leading to the disruption of a proliferation-inhibitory complex because of a failure of the sumoylated C/EBPalpha to interact with BRG1. BRG1 was recruited to the dihydrofolate reductase promoter in nonproliferating C33a cells but was not detected in proliferating cells where C/EBPalpha, BRG1, and SUMO-1 were overexpressed. This result suggests that BRG1 down-regulates the expression of the dihydrofolate reductase gene. These findings provide the insight that SUMO acts as a space regulator, which affects protein-protein interactions.

Interaction of Protein Inhibitor of Activated STAT (PIAS) Proteins with the TATA-binding Protein, TBP

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

Regulation of cytokine signaling pathways by PIAS proteins

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.

PIAS proteins are involved in the SUMO-1 modification, intracellular translocation and transcriptional repressive activity of RET finger protein

Ret finger protein (RFP) is a nuclear protein that is highly expressed in testis and in various tumor cell lines. RFP functions as a transcriptional repressor and associates with Enhancer of Polycomb 1 (EPC1), a member of the Polycomb group proteins, and Mi-2β, a main component of the nucleosome remodeling and deacetylase (NuRD) complex. We show that RFP binds with PIAS (protein inhibitor of activated STAT) proteins, PIAS1, PIAS3, PIASxα and PIASy at their carboxyl-terminal region and is covalently modified by SUMO-1 (sumoylation). PIAS proteins enhance the sumoylation of RFP in a dose-dependent manner and induce the translocation of RFP into nuclear bodies reminiscent of the PML bodies. In addition, co-expression of PIAS proteins or SUMO-1 strengthened the transcriptional repressive activity of RFP. Finally, our immunohistochemical results show that RFP, SUMO-1 and PIASy localize in a characteristic nuclear structure juxtaposed with the inner nuclear membrane (XY body) of primary spermatocytes in mouse testis. These results demonstrate that the intracellular location and the transcriptional activity of RFP are modified by PIAS proteins which possess SUMO E3 ligase activities and suggest that they may play a co-operative role in spermatogenesis.

Repression and Coactivation of CCAAT/Enhancer-binding Protein ϵ by Sumoylation and Protein Inhibitor of Activated STATx Proteins

CCAAT/enhancer-binding protein epsilon (C/EBPepsilon) is a neutrophil-specific transcription factor whose activity is controlled by juxtaposed activating and regulatory domains. We previously determined that the function of the major regulatory domain (RD1) in C/EBPepsilon was dependent on the integrity of a five-amino acid motif that was identical to the recognition site for members of the small ubiquitin-like modifier (SUMO) family of ubiquitin-related proteins. We show here that the SUMO attachment site (the regulatory domain motif) is necessary and sufficient both for the intrinsic inhibitory function of RD1 and for coactivation by PIASxalpha and PIASxbeta, two members of the protein inhibitor of activated STAT (PIAS) family of SUMO E3 ligases. PIASxbeta was a more potent coactivator than PIASxalpha of both full-length C/EBPepsilon and fusion proteins containing the N-terminal portion of C/EBPepsilon, whereas PIASxalpha was more active on fusion proteins containing a heterologous activation domain. Two modes of coactivation were observed, one that was dependent on the integrity of the RING finger (RF) domain and was shared by both PIASxalpha and PIASxbeta and a second mode that was independent of the RF and was only observed with PIASxbeta. Sumoylation of C/EBPepsilon was enhanced by coexpression of PIASxalpha, suggesting that this modification is associated with the enhanced activity of the target protein. These results suggest that a complex interplay of accessory factors, including SUMO and PIAS proteins, modulates the activity of C/EBPepsilon.