Visit our Expo - Redox and Inflammation signaling 2012

VIII. Inflammation specific signaling Poster VIII, 14
Real-time analysis of Jak/STAT signal transduction in single cells
Andreas Herrmann 1 , Michael Vogt 1 , Martin Mönnigmann 2 , Bernd Giese 1 , Michael
Sommerauer 1 , Peter C. Heinrich 1 , Gerhard Müller-Newen 1
1 Institut für Biochemie, Universitätsklinikum der RWTH Aachen, Pauwelsstraße 30,
52057 Aachen, Germany. e-mail: mueller-newen@rwth-aachen.de
2 Lehrstuhl für Prozesstechnik, RWTH Aachen, 52056 Aachen, Germany
The Jak/STAT signal transduction pathway plays a central role in acute and chronic
inflammation. The pro-inflammatory cytokine interleukin-6 (IL-6) signals through the
cytokine receptor gp130. Upon activation, the receptor-associated tyrosine kinases of the
Janus kinase (Jak) family phosphorylate STAT transcription factors. STAT3 (signal
transducer and activator of transcription 3) is preferentially activated in response to IL-6.
Activated STAT3 translocates into the nucleus where it induces target genes that exert
multiple functions in inflammation and carcinogenesis.
We have tagged the cytokine IL-6, its receptor gp130 and the transcription factor STAT3 with
different fluorescent proteins (yellow fluorescent protein (YFP) or cyan fluorescent protein
(CFP)). These fusion proteins enabled us to study ligand-binding and receptor dimerization
(Giese et al. (2005) J. Cell. Sci. 118, 5129-40) as well as nuclear translocation of STAT3
(Pranada et al. (2004) J. Biol. Chem. 279, 15114-23) in single cells by the use of confocal
laser-scanning microscopy and advanced photo-bleaching techniques. We demonstrated that
non-phosphorylated STAT3 constitutively shuttles between the cytoplasm and the nucleus.
Persistent activation of STAT3 is observed in chronic inflammation and cancer. To analyze
persistent activated STAT3 we cotransfected double labelled STAT3-CFP-YFP with v-Src
resulting in constitutive tyrosine phosphorylation and nuclear accumulation of the fluorescent
transcription factor. By bleaching selectively the YFP moiety of STAT3-CFP-YFP in one
cellular compartment and by monitoring the distribution of the CFP and YFP fluorescence
over time with high spatial resolution, we show that persistently activated STAT3 shuttles
between cytoplasm and nucleus. Computational evaluation of the data by model-based
parameter estimations revealed that activated STAT3 shuttles more rapidly than non-activated
STAT3. We propose that inhibition of nucleocytoplasmic shuttling of persistently activated
STAT proteins is a new target for the treatment of chronic inflammation and cancer.
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