Partington G A, Patient R K
Developmental Biology Research Centre, School of Biomedical Sciences, The Randall Institute, King's College London, 26-29 Drury Lane, London WC2B 5RL, UK.
Nucleic Acids Res. 1999 Feb 15;27(4):1168-75. doi: 10.1093/nar/27.4.1168.
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 NAC, 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 phosphoryl-ation and DNA binding of GATA-1 are already optimalin 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.
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在经氯化血红素、丁酸钠（NaB）或曲古抑菌素A诱导红系分化后，或用N - 乙酰半胱氨酸（NAC）处理后，细胞核提取物中GATA - 1 DNA结合活性的水平。相对于未处理细胞的提取物，在所有情况下GATA - 1结合活性均显著增加。然而，免疫印迹分析显示诱导后GATA - 1蛋白水平未发生变化。在进行EMSA之前，用磷酸酶孵育诱导后的但未诱导的K562提取物会减弱结合活性，这表明K562细胞诱导后GATA - 1结合的增加是磷酸化的结果。当用二甲基亚砜（DMSO）、NaB或NAC诱导小鼠红白血病细胞系MEL时，GATA - 1结合活性在DMSO诱导下降低，在NaB诱导下显著升高，而在NAC诱导的细胞中保持在大致相同水平。在这种情况下，免疫印迹显示GATA - 1蛋白水平与EMSA数据一致。用磷酸酶孵育诱导和未诱导的MEL细胞核提取物后，其DNA结合活性均降低，表明GATA - 1的磷酸化和DNA结合在这些细胞中已经处于最佳状态。磷酸酶处理也降低了从MEL细胞中亲和纯化的GATA - 1的DNA结合活性，表明磷酸化/去磷酸化直接影响该因子。此外，当通过EMSA比较未处理或用磷酸酶抑制剂冈田酸孵育的K562和MEL细胞制备的细胞核提取物时，发现K562细胞的GATA - 1结合增加，而MEL细胞的GATA - 1结合没有变化。总体而言，结果表明K562细胞诱导后GATA - 1磷酸化水平增加，而MEL细胞中GATA - 1已经高度磷酸化，磷酸化增加了GATA - 1对典型结合位点的结合亲和力。
