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。从AML1基因，两种具有代表性的蛋白质形式，AML1a和AML1b，是通过选择性剪接产生的。在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的这些作用被AML1b的过表达所抵消。这一生物学现象可以通过我们的观察来解释，AML1a完全没有作为转录调节剂的作用，而主要抑制AML1b的转录激活，并且AML1a比AML1b具有更高的dna结合亲和力。这些拮抗作用可能对白血病发生和/或骨髓细胞分化很重要。我们还通过信号转导途径研究了AML1功能的调控机制。在体内，AML1在富含脯氨酸、丝氨酸和苏氨酸区域的两个丝氨酸残基上磷酸化，依赖于细胞外信号调节激酶（ERK）的激活。这些AML1的体内磷酸化位点在体外被ERK直接磷酸化。虽然在野生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的转激活能力。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的功能受细胞因子或生长因子刺激激活的ERK调控。这项研究将为阐明AML1功能调控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.