title: Dissecting functions of the N-terminal domain and GAS-site recognition in STAT3 nuclear trafficking.
abstract: Signal transducer and activator of transcription 3 (STAT3) is a ubiquitous transcription factor involved in many biological processes, including hematopoiesis, inflammation and cancer progression. Cytokine-induced gene transcription greatly depends on tyrosine phosphorylation of STAT3 on a single tyrosine residue with subsequent nuclear accumulation and specific DNA sequence (GAS) recognition. In this study, we analyzed the roles of the conserved STAT3 N-terminal domain (NTD) and GAS-element binding ability of STAT3 in nucleocytoplasmic trafficking. Our results demonstrate the nonessential role of GAS-element recognition for both cytokine-induced and basal nuclear import of STAT3. Substitution of five key amino acids within the DNA-binding domain rendered STAT3 unable to bind to GAS-elements while still maintaining the ability for nuclear localization. In turn, deletion of the NTD markedly decreased nuclear accumulation upon IL-6 treatment resulting in a prolonged accumulation of phosphorylated dimers in the cytoplasm, at the same time preserving specific DNA recognition ability of the truncation mutant. Observed defect in nuclear localization could not be explained by flawed importin-α binding, since both wild-type and NTD deletion mutant of STAT3 could precipitate both full-length and autoinhibitory domain (∆IBB) deletion mutants of importin-α5, as well as ∆IBB-α3 and ∆IBB-α7 isoforms independently of IL-6 stimulation. Despite its inability to translocate to the nucleus upon IL-6 stimulation, the NTD lacking mutant still showed nuclear accumulation in resting cells similar to wild-type upon inhibition of nuclear export by leptomycin B. At the same time, blocking the nuclear export pathway could not rescue cytoplasmic trapping of phosphorylated STAT3 molecules without NTD. Moreover, STAT3 mutant with dysfunctional SH2 domain (R609Q) also localized in the nucleus of unstimulated cells after nuclear export blocking, while upon cytokine treatment the subcellular localization of this mutant had not changed. Our findings support the concept that basal nucleocytoplasmic shuttling of STAT3 is different from active cytokine-induced nuclear import and does not require conserved N- or SH2-terminal domains, preformed dimer formation and GAS-element-specific DNA recognition.
authors.affiliation: Institute of Biochemistry and Molecular Biology, Faculty of Medicine, RWTH Aachen University, Pauwelsstraße 30, 52074 Aachen, Germany.
authors.forename: Antons
authors.initials: A
authors.lastname: Martincuks
authors.affiliation: Institute of Biochemistry and Molecular Biology, Faculty of Medicine, RWTH Aachen University, Pauwelsstraße 30, 52074 Aachen, Germany.
authors.forename: Dirk
authors.initials: D
authors.lastname: Fahrenkamp
authors.affiliation: Institute of Biochemistry and Molecular Biology, Faculty of Medicine, RWTH Aachen University, Pauwelsstraße 30, 52074 Aachen, Germany; Molecular Disease Mechanisms Group, Life Sciences Research Unit, University of Luxembourg, 162A Avenue de la Faïencerie, L-1511, Luxembourg, Luxembourg; Signal Transduction Group, Life Sciences Research Unit, University of Luxembourg, 162A Avenue de la Faïencerie, L-1511, Luxembourg, Luxembourg.
authors.forename: Serge
authors.initials: S
authors.lastname: Haan
authors.affiliation: Institute of Biochemistry and Molecular Biology, Faculty of Medicine, RWTH Aachen University, Pauwelsstraße 30, 52074 Aachen, Germany; Beckman Research Institute, City of Hope National Medical Center, Duarte, CA, USA.
authors.forename: Andreas
authors.initials: A
authors.lastname: Herrmann
authors.affiliation: Institute of Biochemistry and Molecular Biology, Faculty of Medicine, RWTH Aachen University, Pauwelsstraße 30, 52074 Aachen, Germany.
authors.forename: Andrea
authors.initials: A
authors.lastname: Küster
authors.affiliation: Institute of Biochemistry and Molecular Biology, Faculty of Medicine, RWTH Aachen University, Pauwelsstraße 30, 52074 Aachen, Germany. Electronic address: mueller-newen@rwth-aachen.de.
authors.forename: Gerhard
authors.initials: G
authors.lastname: Müller-Newen
mesh_terms: D021581:Active Transport, Cell Nucleus / Q000187:drug effects; D000818:Animals; D001483:Base Sequence; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism*; D004247:DNA / Q000378:metabolism; D006801:Humans; D015850:Interleukin-6 / Q000494:pharmacology; D008024:Ligands; D051379:Mice; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D000072417:Protein Domains; D055503:Protein Multimerization / Q000187:drug effects; D050796:STAT3 Transcription Factor / Q000737:chemistry* / Q000378:metabolism*; D017384:Sequence Deletion; D013329:Structure-Activity Relationship; D028901:alpha Karyopherins / Q000378:metabolism
chemical_list: D015850:Interleukin-6; D008024:Ligands; D050505:Mutant Proteins; D050796:STAT3 Transcription Factor; D028901:alpha Karyopherins; D004247:DNA
keywords: DNA-binding; Gene induction; Importin; N-terminal domain; Nuclear translocation; STAT3