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

21号染色体上的AML1基因在与髓系白血病相关的(8；21)(q22；q22)和(3；21)(q26；q22)易位中被破坏，并编码一个转录因子。AML1基因通过选择性剪接产生两种具有代表性的蛋白质AML1a和AML1b。过表达的AML1a完全抑制粒细胞分化，并刺激粒细胞集落刺激因子处理的32Dc13小鼠髓系细胞增殖。AML1a的这些作用被伴随的AML1b的过度表达所抵消。这种生物现象可以用我们的观察来解释，AML1a完全没有转录调节作用，主要是抑制AML1b的转录激活，并且AML1a比AML1b表现出更高的DNA结合亲和力。这些拮抗作用可能在白血病发生和/或髓系细胞分化中起重要作用。我们还通过信号转导途径研究了AML1功能的调节机制。AML1在体内的两个丝氨酸残基上被磷酸化，这些残基位于富含脯氨酸、丝氨酸和苏氨酸的区域，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。尽管野生型AML1与非磷酸化突变体之间的DNA结合亲和力没有变化，但我们发现ERK依赖的磷酸化增强了AML1的反式激活能力。此外，磷酸化位点突变降低了AML1在成纤维细胞中的转化能力。这些数据表明，AML1的功能受ERK的调节，ERK由细胞因子或生长因子刺激激活。这项研究将为阐明AML1功能调节机制的未知方面提供重要线索。

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.