Bruhat A, Jost J P
Friedrich Miescher Institute, Basel, Switzerland.
Proc Natl Acad Sci U S A. 1995 Apr 25;92(9):3678-82. doi: 10.1073/pnas.92.9.3678.
We have previously shown that estradiol treatment of roosters resulted in a rapid loss of binding activity of the repressor MDBP-2-H1 (a member of the histone H1 family) to methylated DNA that was not due to a decrease in MDBP-2-H1 concentration. Here we demonstrate that MDBP-2-H1 from rooster liver nuclear extracts is a phosphoprotein. Phosphoamino acid analysis reveals that the phosphorylation occurs exclusively on serine residues. Two-dimensional gel electrophoresis and tryptic phosphopeptide analysis show that MDBP-2-H1 is phosphorylated at several sites. Treatment of roosters with estradiol triggers a dephosphorylation of at least two sites in the protein. Phosphatase treatment of purified rooster MDBP-2-H1 combined with gel mobility shift assay indicates that phosphorylation of MDBP-2-H1 is essential for the binding to methylated DNA and that the dephosphorylation can occur on the protein bound to methylated DNA causing its release from DNA. Thus, these results suggest that in vivo modification of the phosphorylation status of MDBP-2-H1 caused by estradiol treatment may be a key step for the down regulation of its binding to methylated DNA.
我们之前已经表明，用雌二醇处理公鸡会导致阻遏蛋白MDBP - 2 - H1（组蛋白H1家族的一员）与甲基化DNA的结合活性迅速丧失，这并非由于MDBP - 2 - H1浓度降低所致。在此我们证明，来自公鸡肝核提取物的MDBP - 2 - H1是一种磷蛋白。磷酸氨基酸分析表明，磷酸化仅发生在丝氨酸残基上。二维凝胶电泳和胰蛋白酶磷酸肽分析表明，MDBP - 2 - H1在多个位点被磷酸化。用雌二醇处理公鸡会引发该蛋白中至少两个位点的去磷酸化。对纯化的公鸡MDBP - 2 - H1进行磷酸酶处理并结合凝胶迁移率变动分析表明，MDBP - 2 - H1的磷酸化对于与甲基化DNA的结合至关重要，并且去磷酸化可发生在与甲基化DNA结合的蛋白上，导致其从DNA上释放。因此，这些结果表明，雌二醇处理引起的MDBP - 2 - H1磷酸化状态的体内修饰可能是其与甲基化DNA结合下调的关键步骤。
