Analysis of Stat3 (signal transducer and activator of transcription 3) dimerization by fluorescence resonance energy transfer in living cells

Abstract

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.

IL-6 is required for glioma development in a mouse model

“…Yet, in high-grade gliomas of both human and mouse tumours, we observed, in addition to the expected nuclear localization, a novel distinct plasma membrane-associated immunoreactivity for pY-STAT3. This observation is compatible with recent findings, suggesting that STAT proteins do not float solely as free monomers in the cytosol of mammalian cells but constitute high molecular mass protein complexes in lipid rafts (Sehgal et al, 2002) and/or dimerize prior to its activation (Kretzschmar et al, 2003). Raft proteins reside in specialized microdomains at the cell surface and are thought to play an important role in receptor signalling.…”

“…Live cell imaging (FRET) demonstrated that fusion proteins with spectral variants of green fluorescent protein, CFP and YFP redistributed -like wildtype STAT3 -from a preferentially cytoplasmic to a nuclear localization upon IL-6 stimulation. Normalizing FRETc/YFP signals revealed a gradient from the plasma membrane to the cell interior demonstrating a steady state of STAT3 activation at the plasma membrane and inactivation in the nucleus (Kretzschmar et al, 2003). Thus, the plasma membrane-associated immunoreactivity of pY-STAT3 seen in our gliomas in all likelihood resembles an acute signalling state of a stimulated cell.…”

“…Intriguingly, treatment of cells with the raftbreaking compound methyl-b-cyclodextrin markedly inhibited IL-6-induced signalling (Sehgal et al, 2002). Recently, STAT3 dimerization and subcellular localization was studied in living cells (Kretzschmar et al, 2003). Live cell imaging (FRET) demonstrated that fusion proteins with spectral variants of green fluorescent protein, CFP and YFP redistributed -like wildtype STAT3 -from a preferentially cytoplasmic to a nuclear localization upon IL-6 stimulation.…”

Intracellular protein interaction mapping with FRET hybrids

“…Remarkably, the maximum FRET signal for interacting partners increased 14-fold over that obtained with noninteracting partners (Fig. 2B), a dynamic range substantially greater than that observed by using nonoptimized fluorescent protein FRET pairs to detect protein interactions (25)(26)(27). These results demonstrated that the YPetMona/CyPet-P2 interaction could be specifically detected by using FRET hybrids in complex cell lysates when using FREToptimized probes CyPet and YPet.…”

Molecular tools for cell and systems biology

“…Transcription factors have to enter the nucleus to exhibit their function. Accordingly, fusion proteins of transcription factors such as NFB subunits or STATs have been used to monitor nuclear entry (Birbach et al, 2004;Kretzschmar et al, 2004;Pranada et al, 2004). Another obvious group of proteins are those involved in nuclear import and export (Plafker and Macara, 2002).…”