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See detailDissecting functions of the N-terminal domain and GAS-site recognition in STAT3
Martincuks, Antons; Fahrenkamp, Dirk; Haan, Serge UL et al

in Cellular Signalling (2016), 28(8), 810-25

Signal transducer and activator of transcription 3 (STAT3) is a ubiquitous transcription factor involved in many biological processes, including hematopoiesis, inflammation and cancer progression ... [more ▼]

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. [less ▲]

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See detailDeterminants governing the potency of STAT3 activation via the individual STAT3-recruiting motifs of gp130.
Lehmann, Ute; Sommer, Ulrike; Smyczek, Tanya et al

in Cellular Signalling (2006), 18(1), 40-9

In recent years, the elucidation of the structures of many signalling molecules has allowed new insights into the molecular mechanisms that govern signal transduction events. In the field of cytokine ... [more ▼]

In recent years, the elucidation of the structures of many signalling molecules has allowed new insights into the molecular mechanisms that govern signal transduction events. In the field of cytokine signalling, the solved structures of cytokine/receptor complexes and of key components involved in signal transduction such as STAT factors or the tyrosine phosphatase SHP2 have broadened our understanding of the molecular basis of the signalling events and provided key information for the rational design of therapeutic approaches to modulate or block cytokine signal transduction. Unfortunately, no structural data on the intracellular parts of cytokine receptors are available. The exact molecular mechanism underlying one of the first steps in signal transduction, namely the recruitment of signalling components to the cytoplasmic parts of cytokine receptors, remains elusive. Here we investigated possible mechanisms underlying the different potency of the STAT3-activating motifs of gp130 after IL-6 stimulation. Our data indicate that the extent of STAT3 activation by the different receptor motifs is not influenced by structural features such as contacts between the two gp130 chains. In addition, the proximity of the negatively regulating motif around tyrosine Y759 to the different STAT3-recruiting motifs does not seem to be responsible for their differential capacity to activate STAT3. However, the potency of a specific motif to activate STAT3 directly reflects the affinity for the binding of STAT3 to this motif. [less ▲]

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See detailMultiple reasons for an inefficient STAT1 response upon IL-6-type cytokine stimulation
Haan, Serge UL; Keller, J. F.; Behrmann, Iris UL et al

in Cellular Signalling (2005), 17(12), 1542-50

IL-6-type cytokines play an important role during inflammation and the immune response. In addition, they are involved in haematopoiesis, liver and neuronal regeneration, embryonic development and ... [more ▼]

IL-6-type cytokines play an important role during inflammation and the immune response. In addition, they are involved in haematopoiesis, liver and neuronal regeneration, embryonic development and fertility. We found that IL-6-type cytokine stimulation of cell lines and primary human macrophages results in a different distribution of the DNA-binding competent STAT dimer species in the cytosol and nucleus as demonstrated by electrophoretic mobility shift assays. In the absence of detergent, STAT3/STAT3, STAT1/STAT3 were the predominant species in the cytoplasm while STAT3/STAT3 was predominant in the nucleus. However, in detergent containing total cellular lysates and nuclear fractions prepared with detergent containing buffers, the STAT1/STAT1 homodimer was as prominent or even more prominent than STAT3/STAT3 and STAT1/STAT3. We were interested in the cause of this discrepancy since STAT1-regulated genes have not been described to be expressed upon IL-6-type cytokine stimulation. In addition to the more transient STAT1 activation, IL-6-type cytokines such as IL-6 and OSM lead to a much less efficient STAT1 activation compared to the potent STAT1 activators IFNgamma and IFNalpha. Studies with STAT1-deficient cells revealed that STAT1 activation does not seem to be an important competitive process to STAT3 activation arguing again for a very inefficient STAT1 activation upon IL-6-type cytokine stimulation. We also describe that pY-STAT3 is much more efficiently shuttled into the nucleus than pY-STAT1. [less ▲]

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