Hormone-Activated Phosphorylation of Human β 1 Thyroid Hormone Nuclear Receptor

Abstract

To understand the role of phosphorylation in the hormone-dependent transcriptional activation of thyroid hormone receptors (TRs), the present study evaluated the effect of the thyroid hormone, 3,3′,5-triiodo-L-thyronine (T₃) on the phosphorylation of TR, human subtype β1 (h-TRβ₁). The extent of phosphorylation was compared in cells cultured in T₃-depleted (Td) or T₃-supplemented medium (Td + T₃). T₃ was found to activate phosphorylation of h-TRβ₁ approximately threefold. Taking into account the T₃-induced fourfold downregulation in the expression of h-TRβ₁ in the same period, the specific T₃-activated phosphorylation was increased approximately twelvefold. Phosphoamino acid analysis indicates that the phosphorylation of serine and threonine in a ratio of ∼10:1 was increased approximately threefold by T₃. Comparison of the [³²P]-labeled tryptic maps of h-TRβ₁ phosphorylated in cells cultured in Td medium or Td + T3 medium indicates that the latter had fewer fragments and changes of intensities in several common fragments, indicating that the phosphorylation sites activated by T₃-treatment differed from those of basal phosphorylation. Partial V8 and chymotrypic proteolysis indicates that h-TRβ₁ phosphorylated in cells cultured in Td + T₃ medium was more resistant to proteolysis. These results indicate that T₃-activated phosphorylation altered the protease susceptibility of h-TRβ₁ that could reflect structural changes in h-TRβ₁. These results raise the possibility that T₃-activated phosphorylation may play an important role in transcriptional activation of h-TRβ₁.