Observation of unphosphorylated STAT3 core protein binding to target dsDNA by PEMSA and X-ray crystallography

Area of Science:

• Molecular Biology
• Cancer Research
• Protein-DNA Interactions

Background:

• Signal Transducer and Activator of Transcription 3 (STAT3) is frequently over-expressed in various cancers.
• Previously, STAT3 phosphorylation was considered essential for its direct DNA binding activity.

Purpose of the Study:

• To investigate the DNA binding capabilities of unphosphorylated STAT3 (uSTAT3).
• To elucidate the mechanism of STAT3 core binding to its high-affinity target DNA sequence (M67 DNA).

Main Methods:

• Protein electrophoretic mobility shift assay (PEMSA) was used to assess STAT3-DNA binding.
• X-ray crystallography was employed to determine the structural basis of STAT3-DNA interaction.
• Competition assays with phosphotyrosyl peptides were performed to evaluate the role of phosphorylation.

Main Results:

• Purified unphosphorylated STAT3 core demonstrated complete binding to M67 DNA.
• Binding of uSTAT3 to M67 DNA was inhibited by increasing concentrations of a phosphotyrosyl peptide.
• X-ray crystallography revealed a binding mode for uSTAT3 similar to phosphorylated STAT3.

Conclusions:

• Direct DNA binding of STAT3 is not strictly dependent on its phosphorylation.
• Unphosphorylated STAT3 can bind to its high-affinity DNA target, suggesting alternative regulatory mechanisms.
• These findings offer new insights into STAT3 function in cancer and potential therapeutic strategies.