Phosphorylation of GATA-1 increases its DNA-binding affinity and is correlated with induction of human K562 erythroleukaemia cells
Phosphorylation of GATA-1 increases its DNA-binding affinity and is correlated with induction of human K562 erythroleukaemia cells
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DOI：
10.1093/nar/27.4.1168
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发表时间：
1999-02-15
影响因子：
14.9
通讯作者：
Patient, RK
中科院分区：
文献类型：
--
作者：
Partington, GA;Patient, RK
我们通过电泳迁移率变动分析 (EMSA) 研究了人红白血病细胞系 K562 制备的核提取物中 GATA-1 DNA 结合活性的水平，在氯高铁血红素、丁酸钠 (NaB) 或曲古抑菌素 A 诱导红细胞后，或用 N-乙酰半胱氨酸 (NAC) 处理后，相对于未处理细胞的提取物，GATA-1 结合活性在所有情况下均显着增加，然而，免疫印迹分析显示，GATA-1 结合活性的水平没有变化。诱导后GATA-1蛋白。在 EMSA 之前将诱导的而非未诱导的 K562 提取物与磷酸酶一起温育会减弱结合活性，这表明 K562 细胞诱导后 GATA-1 结合的增加是磷酸化的结果。当小鼠红白血病细胞系 MEL 用二甲基亚砜 (DMSO)、NaB 或 NAG 诱导时，GATA-1 结合活性随 DMSO 下降，随 NaB 显着上升，并在 NAC 诱导的细胞中保持在大致相同的水平。在这种情况下，免疫印迹显示 GATA-1 蛋白水平与 EMSA 数据一致。通过与磷酸酶孵育，诱导和未诱导的 MEL 细胞核提取物的 DNA 结合活性降低，表明磷酸化和GATA-1 的 DNA 结合在这些细胞中已经达到最佳状态。来自 MEL 细胞的亲和纯化的 GATA-1 的 DNA 结合活性也因磷酸酶处理而降低，表明磷酸化/去磷酸化直接影响该因子。此外，当通过 EMSA 对由未经处理或用磷酸酶抑制剂冈田酸孵育的 K562 和 MEL 细胞制备的核提取物进行比较时，发现 GATA-1 结合在 K562 细胞中增加，而在 MEL 细胞中 GATA-1 结合没有变化，总体而言，结果表明，K562 诱导后 GATA-1 磷酸化水平增加，但 MEL 细胞则不然，其中 GATA-1 已经高度磷酸化。此外，磷酸化增加了 GATA-1 对典型结合位点的结合亲和力。
We have investigated by electrophoretic mobility shift assay (EMSA) the level of GATA-1 DNA-binding activity in nuclear extracts prepared from the human erythroleukaemic cell line, K562, after erythroid induction by hemin, sodium butyrate (NaB) or Trichostatin A or treatment with N-acetylcysteine (NAC), Relative to extract from untreated cells, GATA-1 binding activity increased markedly in all cases, However, immunoblot analysis revealed unchanged levels of GATA-1 protein after induction. Incubation of induced but not uninduced K562 extracts with phosphatase prior to EMSA weakened the binding activity, suggesting that the increase in GATA-1 binding following induction of K562 cells was a consequence of phosphorylation. When the mouse erythroleukaemic cell line MEL was induced with dimethylsulphoxide (DMSO), NaB or NAG, GATA-1 binding activity fell with DMSO, rose significantly with NaB and remained at about the same level in NAC-induced cells, In this case immunoblotting revealed that GATA-1 protein levels were in accord with the EMSA data, The DNA-binding activities of induced and uninduced MEL cell nuclear extracts were decreased by incubation with phosphatase, showing that phosphorylation and DNA binding of GATA-1 are already optimal in these cells. The DNA-binding activity of affinity-purified GATA-1 from MEL cells was also reduced by phosphatase treatment, showing that phosphorylation/dephosphorylation is directly affecting the factor, Furthermore, when a comparison was made by EMSA of nuclear extracts prepared from K562 and MEL cells untreated or incubated with okadaic acid, a phosphatase inhibitor, GATA-1 binding was seen to increase with K562 cells, whereas with MEL cells there was no change in GATA-1 binding, Overall the results suggest that the level of GATA-1 phosphorylation increases after the induction of K562, but not MEL cells, where GATA-1 is already highly phosphorylated. Furthermore, phosphorylation increases the binding affinity of GATA-1 for a canonical binding site.