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Preassociation of STAT1 with STAT2 and STAT3 in separate signalling complexes prior to cytokine stimulation
L F Stancato1, M David, C Carter-Su
1Department of Pharmacology, University of Michigan Medical School, Ann Arbor, 48109, USA.
Abstract:
A variety of cytokines and growth factors act through an induction of gene expression mediated by a family of latent transcription factors called STAT (signal transducers and activators of transcription) proteins. Ligand-induced tyrosine phosphorylation of the STATs promotes their homodimer and heterodimer formation and subsequent nuclear translocation. We demonstrate here that STAT protein heterocomplexes exist prior to cytokine treatment. When unstimulated HeLa cells are ruptured in hypotonic buffer without salt or detergent, immunoadsorption of either STAT1 or STAT2 from the resulting cytosol yields coimmunoadsorption of the other STAT protein. Similarly, STAT1-STAT3 heterocomplexes are coimmunoadsorbed from hypotonic cytosol. STAT1 and STAT2 or STAT1 and STAT3 translated in reticulocyte lysate spontaneously form heterocomplexes when the translation lysates are mixed at 0 degrees C. Our data suggest that interferon-alpha /beta-induced tyrosine phosphorylation increases the stability of a preexisting, latent, STAT1-STAT2 signaling complex. Newly translated STAT1 binds in equilibrium fashion to STAT2 and STAT3, but we show that STAT2 and STAT3 exist in separate heterocomplexes with STAT1, consistent with a model in which STAT1 contains a common binding site for other STAT proteins.
Insights
Signal transducers and activators of transcription (STAT) proteins form complexes before cytokine stimulation. This study reveals that STAT1, STAT2, and STAT3 heterocomplexes pre-exist, with phosphorylation stabilizing these interactions.
Area of Science:
- Molecular Biology
- Cell Signaling
- Biochemistry
Background:
- Cytokines and growth factors regulate gene expression via STAT proteins.
- STAT protein activation involves tyrosine phosphorylation, dimerization, and nuclear translocation.
Purpose of the Study:
- To investigate the pre-cytokine treatment existence of STAT protein heterocomplexes.
- To elucidate the role of STAT protein interactions in cellular signaling pathways.
Main Methods:
- Immunoadsorption of STAT proteins from unstimulated HeLa cell cytosol.
- Co-immunoadsorption assays to detect STAT protein interactions.
- In vitro complex formation studies using reticulocyte lysate.
Main Results:
- STAT1 and STAT2, as well as STAT1 and STAT3, form heterocomplexes in unstimulated cells.
- These heterocomplexes are detectable in cytosol and form spontaneously in vitro.
- Interferon-alpha/beta treatment enhances the stability of pre-existing STAT1-STAT2 complexes.
Conclusions:
- STAT protein heterocomplexes are pre-formed and exist in a latent state prior to cytokine signaling.
- STAT1 appears to possess a common binding site for other STAT proteins, facilitating heterocomplex formation.
- Cytokine-induced phosphorylation stabilizes these pre-existing complexes, rather than solely inducing their formation.