Bernardin-Fried Florence, Kummalue Tanawan, Leijen Suzanne, Collector Michael I, Ravid Katya, Friedman Alan D
Division of Pediatric Oncology, Johns Hopkins University, Baltimore, Maryland 21231, USA.
J Biol Chem. 2004 Apr 9;279(15):15678-87. doi: 10.1074/jbc.M310023200. Epub 2004 Jan 27.
AML1/RUNX1, a member of the core binding factor (CBF) family stimulates myelopoiesis and lymphopoiesis by activating lineage-specific genes. In addition, AML1 induces S phase entry in 32Dcl3 myeloid or Ba/F3 lymphoid cells via transactivation. We now found that AML1 levels are regulated during the cell cycle. 32Dcl3 and Ba/F3 cell cycle fractions were prepared using elutriation. Western blotting and a gel shift/supershift assay demonstrated that endogenous CBF DNA binding and AML1 levels were increased 2-4-fold in S and G(2)/M phase cells compared with G(1) cells. In addition, G(1) arrest induced by mimosine reduced AML1 protein levels. In contrast, AML1 RNA did not vary during cell cycle progression relative to actin RNA. Analysis of exogenous Myc-AML1 or AML1-ER demonstrated a significant reduction in G(1) phase cells, whereas levels of exogenous DNA binding domain alone were constant, lending support to the conclusion that regulation of AML1 protein stability contributes to cell cycle variation in endogenous AML1. However, cytokine-dependent AML1 phosphorylation was independent of cell cycle phase, and an AML1 mutant lacking two ERK phosphorylation sites was still cell cycle-regulated. Inhibition of AML1 activity with the CBFbeta-SMMHC or AML1-ETO oncoproteins reduced cyclin D3 RNA expression, and AML1 bound and activated the cyclin D3 promoter. Signals stimulating G(1) to S cell cycle progression or entry into the cell cycle in immature hematopoietic cells might do so in part by inducing AML1 expression, and mutations altering pathways regulating variation in AML1 stability potentially contribute to leukemic transformation.
AML1/RUNX1是核心结合因子（CBF）家族的成员之一，通过激活谱系特异性基因来刺激髓系造血和淋巴系造血。此外，AML1通过反式激活诱导32Dcl3髓系细胞或Ba/F3淋巴系细胞进入S期。我们现在发现AML1水平在细胞周期中受到调控。使用淘析法制备32Dcl3和Ba/F3细胞周期组分。蛋白质印迹法以及凝胶迁移/超迁移分析表明，与G1期细胞相比，S期和G2/M期细胞中内源性CBF DNA结合活性和AML1水平增加了2至4倍。此外，含羞草碱诱导的G1期阻滞降低了AML1蛋白水平。相比之下，在细胞周期进程中，相对于肌动蛋白RNA，AML1 RNA没有变化。对外源Myc-AML1或AML1-ER的分析表明，G1期细胞中其水平显著降低，而单独的外源DNA结合结构域水平保持恒定，这支持了以下结论：AML1蛋白稳定性的调控导致内源性AML1的细胞周期变化。然而，细胞因子依赖性AML1磷酸化与细胞周期阶段无关，并且缺乏两个ERK磷酸化位点的AML1突变体仍受细胞周期调控。用CBFβ-SMMHC或AML1-ETO癌蛋白抑制AML1活性会降低细胞周期蛋白D3 RNA表达，并且AML1结合并激活细胞周期蛋白D3启动子。刺激未成熟造血细胞从G1期进入S期或进入细胞周期的信号可能部分是通过诱导AML1表达来实现的，而改变调控AML1稳定性变化途径的突变可能会导致白血病转化。
