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Functional analysis of AML1 gene in normal hematopoietic cells and leukemia cells

正常造血细胞和白血病细胞中AML1基因的功能分析

基本信息

批准号：
07457229
负责人：
HIRAI Hisamaru
金额：
$ 4.48万
依托单位：
University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1995
资助国家：
日本
起止时间：
1995 至 1996
项目状态：
已结题

项目摘要

The AML1 gene on chromosome 21 is disrupted in the (8 ; 21) (q22 ; q22) and (3 ; 21) (q26 ; q22) translocations associated with myelogenous leukemias and encodes a transcription factor. From AML1 gene, two representative forms of proteins, AML1a and AML1b, are produced by an alternative splicing. Overexpressed AML1a totally suppresses granulocytic differentiation and stimulates cell proliferation in 32Dc13 murine myeloid cells treated with granulocyte colony-stimulating factor. These effects by AML1a were canceled by the concomitant overexpression of AML1b. Such biological phenomena could be explained by our observations that AML1a, which solely has no effects as a transcriptional regulator, dominantly suppresses transcriptional activation by AML1b, and that AML1a exhibits the higher affinity for DNA-binding than AML1b. These antagonistic actions could be important for leukemogenesis and/or myeloid cell differentiation.We also investigate the regulatory mechanisms of AML1 functions through signal transduction pathways. AML1 is phosphorylated in vivo on two serine residues within the proline-, serine- and threonine-rich region, with dependence on the activation of extracellular signal-regulated kinase (ERK). These in vivo phosphorylation sites of AML1 were directly phosphorylated in vitro by ERK.Although alterations in the DNA-binding affinity were not observed between wild AML1 and non-phosphorylated mutants, we have shown that ERK-dependent phosphorylation potentiates the transactivation ability of AML1. Furthermore the phosphorylation site-mutations reduced the transforming capacity of AML1 in fibroblast cells. These data suggest that AML1 functions are regulated by ERK which is activated by cytokine or growth facotr-stimuli. This study would give important clues to clarify unidentified facets of regulatory mechanisms of AML1 function.

21 号染色体上的 AML1 基因在与骨髓性白血病相关的 (8 ; 21) (q22 ; q22) 和 (3 ; 21) (q26 ; q22) 易位中被破坏，并编码转录因子。 AML1 基因通过选择性剪接产生两种代表性的蛋白质形式：AML1a 和 AML1b。在用粒细胞集落刺激因子处理的 32Dc13 小鼠骨髓细胞中，过表达的 AML1a 完全抑制粒细胞分化并刺激细胞增殖。 AML1a 的这些影响被伴随的 AML1b 过度表达所抵消。这种生物学现象可以通过我们的观察来解释，即 AML1a 本身不具有转录调节作用，但主要抑制 AML1b 的转录激活，并且 AML1a 比 AML1b 表现出更高的 DNA 结合亲和力。这些拮抗作用对于白血病发生和/或骨髓细胞分化可能很重要。我们还通过信号转导途径研究了 AML1 功能的调节机制。 AML1 在体内在富含脯氨酸、丝氨酸和苏氨酸的区域内的两个丝氨酸残基上被磷酸化，依赖于细胞外信号调节激酶 (ERK) 的激活。 AML1 的这些体内磷酸化位点在体外被 ERK 直接磷酸化。尽管在野生 AML1 和非磷酸化突变体之间没有观察到 DNA 结合亲和力的变化，但我们已经证明 ERK 依赖性磷酸化增强了 AML1 的反式激活能力。此外，磷酸化位点突变降低了成纤维细胞中 AML1 的转化能力。这些数据表明 AML1 功能受细胞因子或生长因子刺激激活的 ERK 调节。这项研究将为澄清 AML1 功能调节机制的未识别方面提供重要线索。