Live Cell Imaging Reveals Continuous STAT6 Nuclear Trafficking Hui-Chen Chen and Nancy C. Reich This information is current as J Immunol 2010; 185:64-70; Prepublished online 24 May of September 24, 2021. 2010; doi: 10.4049/jimmunol.0903323 http://www.jimmunol.org/content/185/1/64 Downloaded from Supplementary http://www.jimmunol.org/content/suppl/2010/05/21/jimmunol.090332 Material 3.DC1 References This article cites 42 articles, 25 of which you can access for free at: http://www.jimmunol.org/content/185/1/64.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 by guest on September 24, 2021 • Fast Publication! 4 weeks from acceptance to publication *average 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 © 2010 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Live Cell Imaging Reveals Continuous STAT6 Nuclear Trafficking Hui-Chen Chen and Nancy C. Reich The STAT6 transcription factor is essential for the development of protective immunity; however, the consequences of its activity can also contribute to the pathogenesis of autoimmune disease. Tyrosine phosphorylation is known to activate STAT6 in response to cytokine stimulation, but there is a gap in our understanding of the mechanisms by which it enters the nucleus. In this study, live cell imaging was used in conjunction with photobleaching techniques to demonstrate the continual nuclear import of STAT6, independent of tyrosine phosphorylation. The protein domain required for nuclear entry includes the coiled coil region of STAT6 and functions similarly before or after cytokine stimulation. The dynamic nuclear shuttling of STAT6 seems to be mediated by the classical importin-a–importin-b1 system. Although STAT6 is imported to the nucleus continually, it accumulates in the nucleus following tyrosine phosphorylation as a result of its ability to bind DNA. These findings will Downloaded from impact diagnostic approaches and strategies to block the deleterious effects of STAT6 in autoimmunity. The Journal of Immunology, 2010, 185: 64–70. eciphering the signaling events initiated by specific effects of STAT6 (12–14). Accurate cellular localization is key to cytokines is critical to understanding their biological the function of a transcription factor, but how the STAT6 protein effects. The STAT6 transcription factor was identified as gains access to the nucleus is not well understood. D http://www.jimmunol.org/ a DNA-binding factor activated in response to IL-4 (1–3). It is now Movement of proteins in and out of the nucleus occurs by known to be required for the generation of Th2 lymphocytes, the passage through nuclear pore complexes that span the nuclear normal function of B lymphocytes, and protection against parasitic membrane (15). Typically, nuclear import of a large protein nematodes (4–7); however, collateral damage accompanies its depends on the presence of a nuclear localization signal (NLS). positive effects in the immune response. Hyperactivity of STAT6 The NLS is recognized by a karyopherin transport receptor that predisposes lymphoproliferative disease and is responsible for facilitates import through the nuclear pore complex (16, 17). The diseases associated with Th2 cell pathologies, like asthma (8–10). classical import receptor consists of a dimer with two distinct Given the considerable evidence that STAT6 contributes to an subunits: an importin-a adapter that binds the NLS and effective immune response and plays a dominant role in asthmatic importin-b that binds importin-a and interacts with the nuclear by guest on September 24, 2021 lung pathology, understanding the mechanisms that regulate its pore complex. In the nucleus, importin-b binds Ran-GTP, lead- nuclear trafficking is essential for therapeutic intervention. ing to release of the NLS cargo. Current knowledge of the nu- STAT6 is a member of the family of signal transducers and clear trafficking of STAT factors has shown that their nuclear activators of transcription and is activated by tyrosine phosphor- import is regulated distinctly (18). For example, nuclear import ylation stimulated in response to Th2 cytokines IL-4 and -13 (11). of the STAT1 factor is conditional and dependent on its dimer- Following cytokine binding to cell-surface receptors, associated ization mediated by tyrosine phosphorylation (19). However, the Janus kinases phosphorylate STAT6 specifically on tyrosine 641. STAT3 transcription factor is imported continually to the nu- Tyrosine phosphorylation promotes the formation of STAT6 dimers cleus, independent of tyrosine phosphorylation (20). The STAT via reciprocal Src homology 2 (SH2) domain and phosphotyrosine molecules share a similar arrangement of functional motifs that interactions. The STAT6 dimer gains the ability to bind DNA tar- including an N terminus, coiled coil domain, DNA-binding do- gets, leading to new gene expression responsible for the biological main, SH2 domain, phosphorylated tyrosine, and carboxyl trans- activation domain. Following tyrosine phosphorylation and Department of Molecular Genetics and Microbiology, Stony Brook University, Stony dimerization, STAT1 gains the function of an NLS within its Brook, NY 11794 DNA-binding domain, whereas STAT3 has a constitutive NLS Received for publication October 15, 2009. Accepted for publication April 18, 2010. within the coiled coil domain, independent of tyrosine phosphor- This work was supported by National Institutes of Health Grant RO1CA122910 (to ylation. N.C.R.). To assess the dynamic movement of STAT6 we used live cell Address correspondence and reprint requests to Dr. Nancy C. Reich, Department of imaging with photobleaching techniques. We provide evidence Molecular Genetics and Microbiology, Stony Brook University, Nicolls Road, Life that STAT6 is imported continually into the nucleus, independent Sciences Building, Stony Brook, NY 11794. E-mail address: nancy.reich@stony brook.edu of tyrosine phosphorylation, and it seems to use the importin- The online version of this article contains supplemental material. a2importin-b1 system. In addition, a region required for NLS Abbreviations used in this paper: anti-pSTAT6, anti-STAT6 phosphotyrosine 641 Ab; function was found to map within the coiled coil domain. c, control; C, cytoplasm; dl, deletion; Fl, fluorescent; FLIP, fluorescence loss in Although nuclear import rates of STAT6 are similar before and photobleaching; FRAP, fluorescence recovery after photobleaching; G, GFP; hIL-4, after tyrosine phosphorylation, nuclear accumulation occurs after human IL-4; impb1, importin-b1; IP, immunoprecipitated; MBP, maltose-binding protein; N, nucleus; NLS, nuclear localization signal; PS, Ponceau S; S, STAT6; phosphorylation, and this is dependent on the DNA-binding abil- SH2, Src homology 2; siRNA, short interfering RNA; STAT6-V5, STAT6 tagged ity of STAT6. Live cell imaging has provided critical insight to the with the V5 epitope; WB, Western blot; wtSTAT6, wild type STAT6. spatial distribution of STAT6, which impacts its function as a tran- Copyright Ó 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00 scription factor. www.jimmunol.org/cgi/doi/10.4049/jimmunol.0903323 The Journal of Immunology 65 Materials and Methods Axiovert 200M microscope using a heating insert coupled with the In- Cell cultures and reagents cubator S (Zeiss). During imaging, the cells were maintained at 37˚C and 5% CO2 using the Zeiss Tempcontrol 37-2 Digital and CTI Controller HeLa and Cos1 cells (American Type Culture Collection, Manassas, VA) 3700. The time-series images for photobleaching assays were taken with were cultured in DMEM with 8% FBS. Cells were treated with human the Zeiss LSM 510 META NLO two-photon laser-scanning microscope rIL-4 (R&D Systems, Minneapolis, MN) at 10 ng/ml. DNA transfections system using a 403 oil objective (Plan-Neofluar, numerical aperture 1.3, were carried out using TransIT-LT1 transfection reagent (Mirus, Madison, differential interference contrast microscopy objective). The excitation WI), according to the manufacturer’s instructions. Rabbit anti-STAT6 Ab wavelength used for GFP was 488 nm, and emission was detected with (Santa Cruz Biotechnology, Santa Cruz, CA), anti-STAT6 phosphotyrosine a 505-nm filter. For fluorescence recovery after photobleaching (FRAP) 641 Ab (anti-pSTAT6) (Cell Signaling Technology, Danvers, MA), and mu- analysis, a region in the nucleus was bleached at 100% power of an argon rine anti-GFP Ab (Roche Diagnostic Systems, Indianapolis, IN) were used laser at 488 nm for 70 s. For fluorescence loss in photobleaching (FLIP) for Western blotting at a 1:1000 dilution. HRP-conjugated anti-rabbit and analysis, a region in the nucleus or cytoplasm was bleached every 12 s at anti-mouse Ig were used as secondary Abs for Western blotting (1:5000). maximum laser intensity for 5 or 50 min. Images were acquired using LSM GFP Ab and murine