Wang Suiquan, Zhang Youhong, Soosairajah Juliana, Kraft Andrew S
Hollings Cancer Center, Medical University of South Carolina, Charleston, SC 29425, USA.
Leuk Res. 2007 Jun;31(6):839-51. doi: 10.1016/j.leukres.2006.08.016. Epub 2006 Oct 4.
The acute myeloid leukemia 1 (AML1, RUNX1) transcription factor is a key regulator of hematopoietic differentiation both in embryonic stem cells and mature hematopoietic progenitors. The RUNX1 protein is thought to play a role in the control of progression through the cell cycle. We have shown that post-transcriptional regulation of RUNX1 activity occurs, in part, through phosphorylation. To investigate whether transit through the cell cycle is associated with changes in the phosphorylation of RUNX1, we have derived phospho-specific antibodies against three of the five major phosphorylation sites in the transcriptional activation domain of RUNX1, S276, S303 and S462. Using these antibodies we demonstrate that treatment of Jurkat T-cells with nocodazole, a G2/M blocking compound, causes an increase in phosphorylation of these three amino acids. By elutriating the Jurkat cells, we are able to demonstrate that these amino acids are normally phosphorylated at the G2/M phase of the cell cycle. Using in vivo inhibitors and in vitro assays this phosphorylation appears to be dependent on Cdk1. We find that RUNX1 degradation occurs at the G2/M-G1 transition and is regulated by both Cdc20 and phosphoryation, suggesting that the anaphase promoting complex plays a role in modifying the level of this protein. Regulation of the extent of phosphorylation of RUNX1 may play a role in controlling the degradation of the protein, implying that additional E3 ligases may also be involved.
急性髓系白血病1（AML1，RUNX1）转录因子是胚胎干细胞和成熟造血祖细胞中造血分化的关键调节因子。RUNX1蛋白被认为在控制细胞周期进程中发挥作用。我们已经表明，RUNX1活性的转录后调控部分通过磷酸化发生。为了研究细胞周期进程是否与RUNX1磷酸化的变化相关，我们针对RUNX1转录激活域中五个主要磷酸化位点中的三个，即S276、S303和S462，制备了磷酸特异性抗体。使用这些抗体，我们证明用诺考达唑（一种G2/M期阻断化合物）处理Jurkat T细胞会导致这三个氨基酸的磷酸化增加。通过淘洗Jurkat细胞，我们能够证明这些氨基酸在细胞周期的G2/M期正常磷酸化。使用体内抑制剂和体外试验，这种磷酸化似乎依赖于Cdk1。我们发现RUNX1降解发生在G2/M-G1期转换，并且受Cdc20和磷酸化调节，这表明后期促进复合体在调节该蛋白水平中发挥作用。RUNX1磷酸化程度的调节可能在控制该蛋白降解中起作用，这意味着可能还涉及其他E3连接酶。
