Adler H T, Nallaseth F S, Walter G, Tkachuk D C
Veterans Administration Puget Sound Health Care System, Seattle Division, Seattle, Washington 98108, USA.
J Biol Chem. 1997 Nov 7;272(45):28407-14. doi: 10.1074/jbc.272.45.28407.
One of the most common chromosomal abnormalities in acute leukemia is a reciprocal translocation involving the HRX gene at chromosome locus 11q23, resulting in HRX fusion proteins. Using the yeast two-hybrid system, in vitro binding studies, and human cell culture coimmunoprecipitation experiments, we show here that a region of the HRX protein that is consistently retained in HRX leukemic fusion proteins interacts directly with SET, another protein implicated in leukemia. We have identified the binding sites on HRX for SET and show that these sequences are clustered near the A.T hooks that have been shown to bind DNA. We also show that carboxyl-terminal SET sequences, possibly the acidic tail of SET, bind to HRX. We have also found serine/threonine-specific protein phosphatase activity in anti-HRX coimmunoprecipitates. Using the phosphatase inhibitor okadaic acid and Western blotting, the phosphatase was identified as protein phosphatase 2A (PP2A). Mutation of a single amino acid in one of the SET binding sites of HRX resulted in lower amounts of both coimmunoprecipitated SET protein and coimmunoprecipitated PP2A. These results suggest that the leukemogenic effects of HRX fusion proteins may be related to interactions with SET and PP2A.
急性白血病中最常见的染色体异常之一是涉及染色体位点11q23上HRX基因的相互易位，从而产生HRX融合蛋白。通过酵母双杂交系统、体外结合研究以及人类细胞培养共免疫沉淀实验，我们在此表明，HRX白血病融合蛋白中始终保留的HRX蛋白区域直接与SET相互作用，SET是另一种与白血病相关的蛋白。我们已经确定了HRX上与SET的结合位点，并表明这些序列聚集在已证明能结合DNA的A.T钩附近。我们还表明，SET的羧基末端序列，可能是SET的酸性尾巴，与HRX结合。我们还在抗HRX共免疫沉淀中发现了丝氨酸/苏氨酸特异性蛋白磷酸酶活性。使用磷酸酶抑制剂冈田酸和蛋白质印迹法，该磷酸酶被鉴定为蛋白磷酸酶2A（PP2A）。HRX的一个SET结合位点中的单个氨基酸突变导致共免疫沉淀的SET蛋白和共免疫沉淀的PP2A数量减少。这些结果表明，HRX融合蛋白的致白血病作用可能与与SET和PP2A的相互作用有关。
