Analysis of Stat3 (signal transducer and activator of transcription 3) dimerization by fluorescence resonance energy transfer in living cells.
Signal transducer and activator of transcription 3 (Stat3) dimerization is commonly thought to be triggered by its tyrosine phosphorylation in response to interleukin-6 (IL-6) or other cytokines. Accumulating evidence from in vitro studies, however, suggests that cytoplasmic Stat3 may be associated with high-molecular-mass protein complexes and/or dimerize prior to its activation. To directly study Stat3 dimerization and subcellular localization upon cytokine stimulation, we used live-cell fluorescence spectroscopy and imaging microscopy combined with fluorescence resonance energy transfer (FRET). Stat3 fusion proteins with spectral variants of green fluorescent protein (GFP), cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) were constructed and expressed in human hepatoma cells (HepG2) and human embryonic kidney cells (HEK-293). Like wild-type Stat3, the fusion proteins redistributed from a preferentially cytoplasmic to nuclear localization upon IL-6 stimulation and supported IL-6-dependent target gene expression. FRET studies in cells co-expressing Stat3-CFP and Stat3-YFP demonstrated that Stat3 dimers exist in the absence of tyrosine phosphorylation. IL-6 induced a 2-fold increase of this basal FRET signal, indicating that tyrosine phosphorylation either increases the dimer/monomer ratio of Stat3 or induces a conformational change of the dimer yielding a higher FRET efficiency. Studies using a mutated Stat3 with a non-functional src-homology 2 (SH2) domain showed that the SH2 domain is essential for dimer formation of phosphorylated as well as non-phosphorylated Stat3. Furthermore, our data show that visualization of normalized FRET signals allow insights into the spatiotemporal dynamics of Stat3 signal transduction.
Kretzschmar
Antje K
AK
Institute of Clinical Immunology and Transfusion Medicine, University Hospital, Leipzig Johannisallee 30, D-04103 Leipzig, Germany.
Dinger
Michaela C
MC
Henze
Christian
C
Brocke-Heidrich
Katja
K
Horn
Friedemann
F
D021581:Active Transport, Cell Nucleus; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019281:Dimerization; D031541:Fluorescence Resonance Energy Transfer; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins / Q000737:chemistry / Q000235:genetics; D008856:Microscopy, Fluorescence; D008958:Models, Molecular; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D050796:STAT3 Transcription Factor; D015534:Trans-Activators / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D050796:STAT3 Transcription Factor; C494086:STAT3 protein, human; D015534:Trans-Activators; C054562:yellow fluorescent protein, Bacteria; D049452:Green Fluorescent Proteins