3249 Research Article Introduction The transcription factor STAT3 has multiple functions in fertility, embryonal development, the acute-phase response of the liver and the regulation of immune responses (Levy and Lee, 2002). Only recently, the importance of dysregulated STAT3 activation for the progression of cancer has been fully recognized (Darnell, 2005;Yu and Jove, 2004). STAT3 not only acts cell-autonomously by stimulating cell proliferation and preventing apoptosis but also facilitates immune evasion of tumor cells (Wang et al., 2004). STAT3 promotes tumor angiogenesis through the induction of VEGF (Niu et al., 2002) and by acting downstream of the VEGF receptor (Yahata et al., 2003). Furthermore, a function of STAT3 in tumor invasion and metastasis by upregulation of matrix metalloproteinases has been established (Itoh et al., 2005). STAT3 consists of an N- terminal multimerization domain, followed by a coiled-coil domain, the DNA-binding domain, a single SH2 domain and finally the transactivation domain (TAD). The tyrosine residue Y705 of STAT3 phosphorylated upon activation is located between the SH2-domain and the TAD and mediates STAT3 dimerization through reciprocal phosphotyrosine/SH2-domain interactions (Levy and Lee, 2002). The family of IL-6-type cytokines that signal through the cytokine receptor gp130 are the most potent physiological activators of STAT3 (Heinrich et al., 2003). Upon ligand binding, STAT3 is phosphorylated at Y705 by receptor- associated Jak1. Activated STAT3 dimers accumulate in the nucleus, where they induce target genes (Lütticken et al., 1994; Stahl et al., 1994). Among other proteins, the feedback inhibitor SOCS3 is induced by STAT3. SOCS3 inhibits Jak1 at the receptor leading to attenuation of Jak1/STAT3 signalling (Nicholson et al., 2000; Schmitz et al., 2000). Besides cytokines, some growth factors such as EGF and PDGF activate STAT3. Activation of STAT3 by receptor tyrosine kinases has been reported to be mediated by Src-dependent and Src-independent mechanisms (Olayioye et al., 1999; Sachsenmaier et al., 1999; Wang et al., 2000). Although constitutive activation of STAT3 has been observed in an increasing number of cancer types, not a single case of a naturally occuring oncogenic mutation within the STAT3 gene has been reported. Rather, STAT3 is activated by dysregulated upstream signalling pathways including cytokine and growth factor receptor signalling and other oncogenic tyrosine kinases such as Src family kinases or Alk kinase (Yu and Jove, 2004). v-Src has been identified to be a strong activator of STAT3 (Cao et al., 1996; Yu et al., 1995). Most Persistent activation of the transcription factor STAT3 has been detected in many types of cancer and plays an important role in tumor progression, immune evasion and metastasis. To analyze persistent STAT3 activation we coexpressed STAT3 with v-Src. We found that tyrosine phosphorylation of STAT3 by v-Src is independent of Janus kinases (Jaks), the canonical activators of STATs. The STAT3-induced feedback inhibitor, suppressor of cytokine signaling 3 (SOCS3), did not interfere with STAT3 activation by v-Src. However, the protein inhibitor of activated STAT3 (PIAS3) suppressed gene induction by persistently activated STAT3. We measured nucleocytoplasmic shuttling of STAT3 in single cells by bleaching the YFP moiety of double-labelled STAT3-CFP- YFP in the cytoplasm. Analysis of the subcellular distribution of CFP and YFP fluorescence over time by mathematical modeling and computational parameter estimation revealed that activated STAT3 shuttles more rapidly than non-activated STAT3. Inhibition of exportin- 1-mediated nuclear export slowed down nucleocytoplasmic shuttling of v-Src-activated STAT3 resulting in reduced tyrosine phosphorylation, decreased induction of STAT3 target genes and increased apoptosis. We propose passage of persistently activated STAT3 through the nuclear pore complex as a new target for intervention in cancer. Supplementary material available online at http://jcs.biologists.org/cgi/content/full/120/18/3249/DC1 Key words: STAT3, v-Src, iFLAP, Nucleocytoplasmic shuttling, Apoptosis, Cancer Summary Nucleocytoplasmic shuttling of persistently activated STAT3 Andreas Herrmann 1, * ,‡ , Michael Vogt 1, *, Martin Mönnigmann 2,§ , Thomas Clahsen 1 , Ulrike Sommer 1 , Serge Haan 1 , Valeria Poli 3 , Peter C. Heinrich 1 and Gerhard Müller-Newen 1,¶ 1 Institut für Biochemie, Universitätsklinikum RWTH Aachen, Pauwelsstraße 30, 52074 Aachen, Germany 2 Lehrstuhl für Prozesstechnik, RWTH Aachen, Templergraben 55, 52056 Aachen, Germany 3 Department of Genetics, Biology and Biochemistry, Molecular Biotechnology Center, University of Turin, Via Nizza 52, 10126 Torino, Italy *These authors contributed equally to this work ‡ Present address: Division of Cancer Immunotherapeutics and Tumor Immunology, Beckman Research Institute at the City of Hope National Medical Center, Duarte, CA 91010, USA § Present address: Technische Universität Braunschweig, 38023 Braunschweig, Germany ¶ Author for correspondence (e-mail: mueller-newen@rwth-aachen.de) Accepted 5 July 2007 Journal of Cell Science 120, 3249-3261 Published by The Company of Biologists 2007 doi:10.1242/jcs.03482 Journal of Cell Science