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Mutational analysis of acute-phase response factor/Stat3 activation and dimerization
J Sasse1, U Hemmann, C Schwartz
1Institute of Biochemistry, Rheinisch-Westfälische Technische Hochschule Aachen, Germany.
Abstract:
Signal transducer and transcription (STAT) factors are activated by tyrosine phosphorylation in response to a variety of cytokines, growth factors, and hormones. Tyrosine phosphorylation triggers dimerization and nuclear translocation of these transcription factors. In this study, the functional role of carboxy-terminal portions of the STAT family member acute-phase response factor/Stat3 in activation, dimerization, and transactivating potential was analyzed. We demonstrate that truncation of 55 carboxy-terminal amino acids causes constitutive activation of Stat3 in COS-7 cells, as is known for the Stat3 isoform Stat3beta. By the use of deletion and point mutants, it is shown that both carboxy- and amino-terminal portions of Stat3 are involved in this phenomenon. Dimerization of Stat3 was blocked by point mutations affecting residues both in the vicinity of the tyrosine phosphorylation site (Y705) and more distant from this site, suggesting that multiple interactions are involved in dimer formation. Furthermore, by reporter gene assays we demonstrate that carboxy-terminally truncated Stat3 proteins are incapable of transactivating an interleukin-6-responsive promoter in COS-7 cells. In HepG2 hepatoma cells, however, these truncated Stat3 forms transmit signals from the interleukin-6 signal transducer gp130 equally well as does full-length Stat3. We conclude that, dependent on the cell type, different mechanisms allow Stat3 to regulate target gene transcription either with or without involvement of its putative carboxy-terminal transactivation domain.
Insights
Truncating the carboxy-terminal region of signal transducer and transcription (STAT) factors like Stat3 can lead to constitutive activation. However, the ability of these truncated Stat3 proteins to regulate gene transcription varies by cell type.
Area of Science:
- Molecular Biology
- Cell Signaling
- Transcription Factors
Background:
- Signal transducer and transcription (STAT) factors regulate gene expression in response to extracellular signals.
- Activation of STAT factors involves tyrosine phosphorylation, dimerization, and nuclear translocation.
Purpose of the Study:
- To investigate the functional role of the carboxy-terminal region of Stat3 in its activation, dimerization, and transactivation.
- To analyze how truncations in Stat3 affect its signaling capabilities.
Main Methods:
- Utilized deletion and point mutants of Stat3 in COS-7 and HepG2 cells.
- Employed reporter gene assays to assess transactivation potential.
- Analyzed Stat3 dimerization and activation mechanisms.
Main Results:
- Truncation of 55 carboxy-terminal amino acids resulted in constitutive Stat3 activation in COS-7 cells.
- Mutations affecting residues near and far from Y705 blocked Stat3 dimerization.
- Carboxy-terminally truncated Stat3 failed to transactivate an IL-6-responsive promoter in COS-7 cells but functioned similarly to full-length Stat3 in HepG2 cells.
Conclusions:
- Both carboxy- and amino-terminal regions of Stat3 are crucial for its activation and dimerization.
- Cell-type-specific mechanisms dictate whether Stat3 requires its carboxy-terminal transactivation domain for target gene regulation.