Zhang Youhong, Biggs Joseph R, Kraft Andrew S
Hollings Cancer Center, the Medical University of South Carolina, 86 Jonathan Lucas St., Charleston, SC 29425, USA.
J Biol Chem. 2004 Dec 17;279(51):53116-25. doi: 10.1074/jbc.M405502200. Epub 2004 Oct 8.
We find that phorbol ester (PE) treatment of K562 cells greatly stimulates promoters (T cell receptor beta, myeloperoxidase, macrophage colony-stimulating factor receptor, and granulocyte macrophage colony-stimulating factor receptor) containing AML1 transcription factor binding sites. This stimulation of AML1c transcriptional activity is mediated by direct phosphorylation of the AML1c molecule on multiple phosphorylation sites. Eleven AML1c (S/T)P sites in the transcriptional activating domain are phosphorylated at a basal level in untreated K562 cells; treatment of the K562 cells with PE results in increased phosphorylation at five of these sites (serines 276, 293, 303, 462, and threonine 300). Mutation of these five sites to alanine inhibits PE-induced transcriptional activity; mutation of the sites to an acidic amino acid, aspartic acid, stimulates constitutive activity. Single mutations in four amino acids or double mutations (serines 276 and 293 or threonine 300 and serine 303) have little effect on AML1c transcriptional activity. Inhibitor assays suggest that the ERK family of protein kinases is activated by PEs to phosphorylate the (S/T)P sites within the AML1c molecule and markedly enhance the transcriptional activity of AML1c.
我们发现，佛波酯（PE）处理K562细胞可极大地刺激含有AML1转录因子结合位点的启动子（T细胞受体β、髓过氧化物酶、巨噬细胞集落刺激因子受体和粒细胞巨噬细胞集落刺激因子受体）。AML1c转录活性的这种刺激是由AML1c分子在多个磷酸化位点的直接磷酸化介导的。转录激活域中的11个AML1c（S/T）P位点在未处理的K562细胞中以基础水平被磷酸化；用PE处理K562细胞会导致其中5个位点（丝氨酸276、293、303、462和苏氨酸300）的磷酸化增加。将这5个位点突变为丙氨酸会抑制PE诱导的转录活性；将这些位点突变为酸性氨基酸天冬氨酸会刺激组成型活性。四个氨基酸的单突变或双突变（丝氨酸276和293或苏氨酸300和丝氨酸303）对AML1c转录活性影响很小。抑制剂分析表明，蛋白激酶的ERK家族被PE激活，使AML1c分子内的（S/T）P位点磷酸化，并显著增强AML1c的转录活性。
