Alteration of proteolytic processing of c-Myb as a consequence of its truncation in murine myeloid leukemia.
Alteration of proteolytic processing of c-Myb as a consequence of its truncation in murine myeloid leukemia.
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小鼠髓系白血病中 c-Myb 截短导致蛋白水解过程发生改变。
DOI：
10.1038/sj.leu.2401326
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发表时间：
1999
期刊：
影响因子：
--
通讯作者：
Wolff,L
中科院分区：
文献类型：
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作者：
Bies,J;Nazarov,V;Wolff,L
蛋白质稳定性的变化有时与致癌活性有关。例如，c-jun、c-fos和c-mos的蛋白产物在转导到逆转录病毒过程中发生截断，与内源性产物相比，导致稳定性和转化能力的增加。同样，由于逆转录病毒插入突变而在小鼠髓系白血病中表达的转录调节因子c-Myb的截短版本被发现比正常蛋白更能抵抗蛋白降解(Bies和Wolff，Oncogene 14：203-212,1997)。在MuLV诱导的小鼠髓系白血病MML RI-4-11和NFS-60以及转化的髓系细胞株VFLJ2中，观察到涉及240-248氨基酸的c-Myb蛋白的羧基末端截断。在所有这些病例中，截短都是由于逆转录病毒插入外显子9和终止5‘逆转录病毒LTR的翻译而引起的。有趣的是，免疫沉淀或Western印迹分析表明，RI-4-11和NFS-60细胞中截短蛋白的稳态水平比M1髓母细胞中的内源性全长蛋白高得多。这种稳定状态水平的增加随后被证明部分是由于截短蛋白质的周转率的变化。当在CT缺失248个氨基酸的蛋白在M1细胞中表达时，它的稳定性大约是在相同细胞中表达的全长内源性c-Myb蛋白的三倍。因此，表达的差异是由于降解率的变化，而降解率反过来又取决于蛋白质结构，而不仅仅是白血病细胞系的内在蛋白分解潜力。随后进行了研究，以确定正常蛋白质的蛋白质分解的基本机制，并试图理解截断形式是如何逃脱这种分解的。通过使用涉及溶酶体、钙蛋白酶和26S蛋白酶体的蛋白分解途径的抑制剂，我们能够证明26S蛋白酶体负责造血细胞中蛋白质的分解。此外，我们还能够证明c-Myb在体外可以被泛素化，这表明c-Myb的加工是通过涉及泛素化和蛋白酶体识别泛素化蛋白的共同途径(Bies和Wolff，Oncogene 14：203-212,1997)。由于已经证明了全长蛋白质的有效泛素化，但不是截短蛋白质的有效泛素化，因此截短蛋白质的低效泛素化很可能导致其逃脱降解。我们最近的研究表明，由于逆转录病毒整合到我们的另一个髓系白血病细胞系中，c-Myb的96个氨基酸的CT截断也会导致稳定，尽管程度低于缺失248个氨基酸的RI-4-11蛋白(见图1)。为了定位影响正常蛋白质降解速率的蛋白质CT部分的区域，我们构建了一系列在COS 7细胞中瞬时表达的缺失突变体，并在脉冲追逐实验中进行了分析。制备了两组缺失突变体。这个
Changes in protein stability have sometimes been associated with oncogenic activation. For example, truncations of the protein products of c-jun, c-fos, and c-mos occurred during transduction into retroviruses and resulted in both increased stability and increased transforming capacity compared to endogenous counterparts. Similarly, truncated versions of the transcriptional regulator c-Myb, found to be expressed in murine myeloid leukemias due to retroviral insertional mutagenesis, have been found to be more resistant to proteolysis than the normal protein (Bies and Wolff, Oncogene 14: 203–212, 1997).Carboxy-terminal (CT) truncations of the c-Myb protein involving 240–248 aa had been observed in MuLV-induced murine myeloid leukemias MML RI-4–11 and NFS-60 and a transformed myeloid cell line, VFLJ2. In all of these cases, truncation was caused by retroviral insertion into exon 9 and termination of translation in the 5′ retrovirus LTR. Interestingly the steady state levels of the truncated protein, as demonstrated by immune precipitation or by Western blot analysis, were much higher in RI-4–11 and NFS-60 cells when compared to the endogenous full length protein in M1 myeloblastic cells. This increase in steady state level was subsequently shown to be due in part to a change in turnover rate of truncated protein. When the protein which was missing 248 aa at the CT was expressed in M1 cells, it was approximately three times more stable than the full length endogenous c-Myb protein expressed in the same cells. Differences in expression, therefore, were due to changes in degradation rates that were in turn dependent upon protein structure and not just to the intrinsic proteolytic potentials of the leukemic cell lines. Studies were subsequently carried out to determine the basic mechanism of proteolysis of the normal protein and to try to understand how the truncated form was escaping this breakdown. With the use of inhibitors of proteolytic pathways involving lysosomes, calpains and the 26S proteasome we were able to show that the 26S proteasome was responsible for breakdown of the protein in hematopoietic cells. In addition, we were able to show that c-Myb can be polyubiquitinated in vitro, suggesting that processing of c-Myb is through the common pathway involving ubiquitination and recognition of ubiquitinated protein by the proteasome (Bies and Wolff, Oncogene 14: 203–212, 1997). Since efficient ubiquitination of the full length, but not truncated protein was demonstrated, it is likely that inefficient ubiquitination of truncated protein leads to its escape from degradation. Our recent studies have shown that CT truncation of 96 aa of c-Myb, a consequence of retrovirus integration in another of our myeloid leukemia cell lines, also results in stabilization although to a lesser extent than that observed for the RI-4–11 protein that is missing 248 aa (see Figure 1). In an attempt to localize areas of the CT portion of the protein that affect rates of degradation of the normal protein, we constructed a series of deletion mutants which were transiently expressed in COS 7 cells and analyzed in a pulse chase experiment. Two sets of deletion mutants were prepared. The