Lin K H, Ashizawa K, Cheng S Y
Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892.
Proc Natl Acad Sci U S A. 1992 Aug 15;89(16):7737-41. doi: 10.1073/pnas.89.16.7737.
The role of phosphorylation on the gene activation activity of the human beta 1 thyroid hormone nuclear receptor (h-TR beta 1) was examined. h-TR beta 1 was found to be a phosphoprotein when expressed in COS-1 cells, with serine, threonine, and tyrosine (85:10:5) as the phosphorylation sites. Okadaic acid (a potent inhibitor of phosphatases 1 and 2A) at 0.1, 0.25, and 0.5 microM increased the phosphorylation of h-TR beta 1 by 3-, 7-, and 11-fold, respectively. The increase in phosphorylation was accompanied by a concomitant increase in phosphorylation was accompanied by a concomitant increase in receptor-mediated transcription in transient transfection assays. h-TR beta 1 purified from Escherichia coli was phosphorylated in vitro by the endogenous kinase from cellular extracts. Serine, threonine, and tyrosine were phosphorylated in a similar ratio to that found in COS-1 cells. The in vitro phosphorylation was stimulated by okadaic acid. Phosphorylation did not affect the binding of h-TR beta 1 to 3,3',5-triiodo-L-thyronine. However, phosphorylation of h-TR beta 1 resulted in an increase of its binding to DNA and conferred on it the ability to bind to nuclear accessory proteins. The results indicate that phosphorylation plays an important role in the transcriptional activity of h-TR beta 1.
研究了磷酸化对人β1甲状腺激素核受体（h-TRβ1）基因激活活性的作用。当在COS-1细胞中表达时，h-TRβ1被发现是一种磷蛋白，其磷酸化位点为丝氨酸、苏氨酸和酪氨酸（比例为85:10:5）。0.1、0.25和0.5微摩尔的冈田酸（蛋白磷酸酶1和2A的有效抑制剂）分别使h-TRβ1的磷酸化增加了3倍、7倍和11倍。磷酸化的增加伴随着瞬时转染实验中受体介导的转录的相应增加。从大肠杆菌中纯化的h-TRβ1在体外被细胞提取物中的内源性激酶磷酸化。丝氨酸、苏氨酸和酪氨酸的磷酸化比例与在COS-1细胞中发现的相似。体外磷酸化受到冈田酸的刺激。磷酸化不影响h-TRβ1与3,3',5-三碘-L-甲状腺原氨酸的结合。然而，h-TRβ1的磷酸化导致其与DNA的结合增加，并赋予其与核辅助蛋白结合的能力。结果表明，磷酸化在h-TRβ1的转录活性中起重要作用。
