Phosphorylation selectively increases triiodothyronine receptor homodimer binding to DNA.
Thyroid hormone receptors (TRs) are ligand-regulated transcription factors that bind to thyroid hormone response elements (TREs) as monomers and homodimers, and as heterodimers with nuclear proteins such as TR auxiliary proteins and retinoid X receptors. Recently, bacterially expressed human TR beta-1 (hTR beta-1) was shown to be phosphorylated in vitro by HeLa cytosolic extract. However, little is known about the consequences of phosphorylation on the nature of TR complexes. Therefore, we studied the effect of phosphorylation on TR binding of TREs. Bacterially expressed hTR beta-1 was phosphorylated in vitro with ATP by HeLa cytosolic extract. The ratio of phosphoserine to phosphothreonine was approximately 5:1. We then analyzed phosphorylated hTR beta-1 binding to several TREs by electrophoretic mobility shift assay. Phosphorylated hTR beta-1 bound better as a homodimer to the TREs than hTR beta-1 incubated with preheated cytosolic extract. Alkaline phosphatase treatment of the phosphorylated hTR beta-1 eliminated the enhanced homodimer binding to DNA. In contrast, phosphorylation did not affect TR/TR auxiliary protein or TR/retinoid X receptor heterodimer binding to DNA. Triiodothyronine decreased both phosphorylated and unphosphorylated hTR beta-1 homodimer binding to several TREs, and the addition of okadaic acid did not alter this triiodothyronine effect. These results indicate that phosphorylation, in addition to ligand binding, modulates TR dimer binding to TREs. As such, it is possible that phosphorylation may also participate in TR-mediated regulation of transcription.
Sugawara
A
A
Department of Medicine, Brigham and Women's Hospital, Boston, Massachusetts.
Yen
P M
PM
Apriletti
J W
JW
Ribeiro
R C
RC
Sacks
D B
DB
Baxter
J D
JD
Chin
W W
WW
D015153:Blotting, Western; D004247:DNA / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D006367:HeLa Cells; D006801:Humans; D010766:Phosphorylation; D011233:Precipitin Tests; D011988:Receptors, Thyroid Hormone / Q000378:metabolism*
D011988:Receptors, Thyroid Hormone; D004247:DNA