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Full Text (PDF) PIASy-Deficient Mice Display Modest Defects in IFN and Wnt Signaling Wera Roth, Claudio Sustmann, Matthias Kieslinger, Andrea Gilmozzi, Denis Irmer, Elisabeth Kremmer, Chris Turck and This information is current as Rudolf Grosschedl of October 2, 2021. J Immunol 2004; 173:6189-6199; ; doi: 10.4049/jimmunol.173.10.6189 http://www.jimmunol.org/content/173/10/6189 Downloaded from References This article cites 74 articles, 44 of which you can access for free at: http://www.jimmunol.org/content/173/10/6189.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average by guest on October 2, 2021 Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2004 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology PIASy-Deficient Mice Display Modest Defects in IFN and Wnt Signaling1 Wera Roth,* Claudio Sustmann,* Matthias Kieslinger,* Andrea Gilmozzi,* Denis Irmer,* Elisabeth Kremmer,† Chris Turck,‡ and Rudolf Grosschedl2* Protein inhibitors of activated STATs (PIAS) represent a small family of nuclear proteins that modulate the activity of many transcription factors and act as E3 ligases for covalent modification of proteins with the small ubiquitin-like modifier (SUMO). In particular, PIASy has been shown to inhibit the activation of gene expression by the IFN-responsive transcription factor STAT1 and the Wnt-responsive transcription factor LEF1. To assess the function of PIASy in vivo, we generated and analyzed mice carrying a targeted mutation of the Piasy gene. We find that homozygous mutant mice have no obvious morphological defects and have a normal distribution of lymphocyte populations. Molecular analysis of signaling in response to IFN-␥ and Wnt agonists revealed a modest reduction in the activation of endogenous and transfected target genes. Two-dimensional analysis of total Downloaded from proteins and SUMO-modified proteins in transformed pre-B cells showed no significant differences between wild-type mice and homozygous mutant mice. Taken together, our data indicate that PIASy has a modest effect on cytokine and Wnt signaling, suggesting a redundancy with other members of the family of PIAS proteins. The Journal of Immunology, 2004, 173: 6189–6199. rotein inhibitors of activated STATs (PIAS)3 have been with other proteins. PIAS1 and PIAS3 inhibit DNA binding of identified as interaction partners for multiple nuclear pro- STAT1 and STAT3, respectively (1, 2). In contrast, PIASy represses P teins, including the STATs and lymphoid enhancer bind- STAT1, LEF1, Smad4 and the AR without interfering with DNA http://www.jimmunol.org/ ing factor (LEF)1/T cell-specific factor (TCF) proteins that medi- binding by these proteins (3, 24, 26, 27). The Piasx gene encodes two ate nuclear responses to cytokines and Wnt signals, respectively splice variants, x␣ (ARIP3) and x␤ (Miz1), which interact with the (1–5). The cytokines IFN-␣ and IFN-␥ are the main cytokines for AR and with the homeodomain protein Msx2, respectively (28, 29). innate immune responses against viral infections. IFN-␣ is pro- In addition, PIASx␤ interacts with STAT4, following IL-12 stimula- duced by many cell types, whereas IFN-␥ is produced predomi- tion of T cells (5). In most cases, the association of PIAS proteins with nately by hemopoietic cells (reviewed in Refs. 6 and 7). The sig- transcription factors results in repression (reviewed in Ref. 30); how- naling pathways for both IFNs are similar and involve the Jak ever, PIAS proteins can also augment gene expression by synergizing tyrosine kinase-mediated activation of STAT proteins that stimu- with other transcriptional coactivators (4, 18). by guest on October 2, 2021 late transcription of target genes alone or in concert with IFN reg- PIAS proteins share a similar domain structure, including an ulatory factors (reviewed in Refs. 8 and 9). The response of cells N-terminal domain, termed SAP domain, which mediates, in part, to Wnt signals involves the stabilization and nuclear translocation the interaction with partner proteins (3, 31) and a central RING ␤ of -catenin, which associates with LEF/TCF proteins and acti- domain that resembles the catalytic domain of ubiquitin ligases vates Wnt-responsive genes (reviewed in Ref. 10). (32, 33). Recent experiments aimed at the mechanisms by which In addition to STATs and LEF1/TCF proteins, many other tran- PIAS proteins modulate the activity of their interaction partners, scription factors interact with PIAS proteins. These include nuclear have shown that PIAS proteins can augment the covalent modifi- hormone receptors, such as the androgen receptor (AR), p53, cation of proteins with the small ubiquitin-related modifier Smad4, Sp3, HMGI-C, Gfi-1, IRF-1, TFII-I and yeast septins (11– (SUMO) (3, 14–16, 22, 34–36). Two families of SUMO proteins, 25). In mouse and man, four Pias genes (Pias1, Pias3, Piasx, and SUMO-1 and SUMO-2/SUMO-3 resemble ubiquitin proteins and Piasy) have been identified, which encode proteins that share a are also attached to the ⑀-amino groups of lysines in target proteins similar domain structure but differ in their specificity of interaction (37). The sumoylation pathway resembles that of ubiquitination, with the activation of SUMO by an E1 protein and its transfer to an E2 enzyme (37–39). Multiple lines of evidence suggest that *Gene Center and Institute of Biochemistry, University of Munich, †GSF National Research Center for Environment and Health, and ‡Max-Planck-Institute of Psychi- PIAS proteins act as E3 ligases for SUMO. First, PIAS proteins atry, Munich, Germany can cooperate with E1 and E2 enzymes in reconstituted systems to Received for publication May 21, 2004. Accepted for publication August 20, 2004. augment SUMO modification of target proteins (3, 14, 22, 35). The costs of publication of this article were defrayed in part by the payment of page Second, PIAS proteins contain a cysteine-rich RING domain, charges. This article must therefore be hereby marked advertisement in accordance which is a hallmark for ubiquitin ligases and is functionally im- with 18 U.S.C. Section 1734 solely to indicate this fact. portant for the sumoylation activity of PIAS proteins (3, 22, 33– 1 This work was supported by a Grant of the German Research Foundation TR-5. 35). Finally, PIAS proteins display some specificity of protein sub- 2 Address correspondence and reprint requests to Dr. Rudolf Grosschedl at the current strates, although the specificity is not as pronounced as for address: Max-Planck-Institute of Immunobiology, Stuebeweg 51, 79108 Freiburg, Germany. E-mail address: [email protected] ubiquitin ligases (5, 22, 40). 3 Abbreviations used in this paper: PIAS, protein inhibitor of activated STAT; LEF, The function of PIAS proteins as SUMO E3 ligases appears lymphoid enhancer binding factor; TCF, T cell-specific factor; SUMO, small ubiq- to involve multiple mechanisms. The repression of transcription uitin-like modifier; ES, embryonic stem; IRF, IFN regulatory factor; AR, androgen factors by PIAS proteins often correlates with the subnuclear se- receptor; ␤-Gal, ␤-galactosidase; X-Gal, 5-bromo-4-chloro-3-indolyl ␤-D-galacto- side; MuLV, murine leukemia virus; MEF, mouse embryonic fibroblast. questration into speckles, such as promyelocytic leukemia nuclear Copyright © 2004 by The American Association of Immunologists, Inc. 0022-1767/04/$02.00 6190 FUNCTION OF THE SUMO-E3 LIGASE PIASy IN VIVO bodies (3, 20, 21, 23, 34, 41). This observation could account for and hybridized overnight at 42°C. Blots were washed to final stringency of the repression of transcription factor activity without changes in 0.2% SDS, 0.1% SDS at 65°C, and exposed to film. ␤ DNA binding. In addition, PIASx has been found to interact with Immunoblot analysis histone deacetylase-3, which is involved in repression of TFII-I family proteins (25). The activation function of PIAS proteins Total protein extracts were separated by 10% SDS-PAGE and transferred to nitrocellulose. Immunoblots were conducted by the ECL method ac- could also be accounted for by the sumoylation-dependent aug- cording to the manufacturer’s instructions (Amersham Pharmacia Biotech, mentation of protein-protein interactions. In nuclear hormone re- Piscataway, NJ), with the Abs rat anti-PIASy (mouse) mAb. ceptors, the SUMO modification sites coincide with the protein motifs that mediate synergy in the activation of reporters contain- Flow cytometry ing multimerized binding sites for the hormone receptors (42–44). Thymus, spleen, and bone marrow were isolated and dispersed in RPMI Genetic experiments in Drosophila indicated that the PIAS or- 1640, containing antibiotics and glutamine with 10% heat-inactivated FCS. ϫ 6 ␮ thologue dPIAS, also known as Zimp, interacts functionally with Cells (1 10 ) were resuspended in 100 l of FACS buffer (PBS, 1% FCS) and incubated with Ab for 20 min. Cells were washed three times the STAT orthologue stat92E to regulate blood cell and eye de- with FACS buffer, and the process was then repeated with secondary Abs velopment (45). An additional role of PIAS in the regulation of as necessary. After staining, cells were resuspended in 500 ␮l of FACS chromosome structure and function was inferred from the identi- buffer containing propidium iodine. Cells were then analyzed using a FACS- fication and characterization of dPIAS as a suppressor of position- Calibur (BD Biosciences, San Jose, CA).
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