Phosphorylation selectively increases triiodothyronine receptor homodimer binding to DNA

Area of Science:

• Molecular endocrinology
• Nuclear receptor signaling
• Post-translational modifications

Background:

• Thyroid hormone receptors (TRs) are transcription factors regulating gene expression.
• TRs bind DNA as monomers, homodimers, or heterodimers with other nuclear proteins.
• The impact of TR phosphorylation on DNA binding remains largely uncharacterized.

Purpose of the Study:

• To investigate the effect of phosphorylation on human TR beta-1 (hTR beta-1) DNA binding.
• To determine if phosphorylation influences TR homodimer and heterodimer formation and binding.
• To understand the interplay between phosphorylation, ligand binding, and TR DNA interactions.

Main Methods:

• In vitro phosphorylation of bacterially expressed hTR beta-1 using HeLa cytosolic extract and ATP.
• Electrophoretic mobility shift assays (EMSA) to analyze DNA binding of phosphorylated and unphosphorylated hTR beta-1.
• Treatment with alkaline phosphatase to assess the role of phosphorylation in DNA binding.
• Investigation of ligand (triiodothyronine) and okadaic acid effects on binding.

Main Results:

• Phosphorylated hTR beta-1 exhibited enhanced homodimer binding to thyroid hormone response elements (TREs) compared to unphosphorylated TR.
• Alkaline phosphatase treatment reversed the enhanced homodimer binding, confirming the role of phosphorylation.
• Phosphorylation did not alter TR/TR auxiliary protein or TR/retinoid X receptor heterodimer binding.
• Triiodothyronine reduced homodimer binding for both phosphorylated and unphosphorylated TR, an effect not modulated by okadaic acid.

Conclusions:

• Phosphorylation is a significant post-translational modification that modulates TR homodimer binding to TREs.
• This phosphorylation-dependent modulation of DNA binding is distinct from ligand-induced effects.
• Phosphorylation may play a crucial role in the overall regulation of TR-mediated transcription.