Constitutive ubiquitination and degradation of c-myb by the 26S proteasome during proliferation and differentiation of myeloid cells.
Constitutive ubiquitination and degradation of c-myb by the 26S proteasome during proliferation and differentiation of myeloid cells.
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作者：
S. Feiková;L. Wolff;J. Bies
转录因子的稳态水平在造血细胞的增殖和分化中起重要作用。转录因子c-Myb在鼠和禽白血病中经常被逆转录病毒整合激活。它的失调也与人类急性和慢性白血病以及其他一些非造血系统肿瘤有关。它是一种寿命短的蛋白质，可被26 S蛋白酶体迅速降解。在羧基（COOH）末端的截断，这已经发生在一些致癌形式的c-Myb中，导致对蛋白水解的抗性增加。这种稳定化在体外与较低效率的遍在蛋白化相关。在这里，我们报告的第一个证据的翻译后修饰的c-Myb的泛素在体内使用HA标记的泛素。我们还表明，与不稳定的野生型或氨基（NH 2）-末端截短的c-Myb形式相反，稳定的羧基（COOH）-末端截短的c-Myb不靶向降解泛素在体内通过共价连接。此外，分析后的亚细胞分馏的蛋白质与26 S蛋白酶体抑制剂处理的细胞，我们能够本地化的c-Myb只在核隔室，这表明没有出口到细胞质前蛋白水解加工的要求。此外，从间期细胞或同步于细胞周期的G2/M或G1期的细胞中分离的c-Myb蛋白的脉冲追踪实验没有揭示26 S蛋白酶体的蛋白水解加工的实质性细胞周期依赖性差异。此外，在诱导沿着单核细胞途径分化的髓系祖细胞M1细胞中c-Myb的半衰期与未分化细胞中的半衰期相同的证明表明，c-Myb的蛋白水解分解是增殖和分化期间的组成性过程。
Steady state levels of transcription factors play an important role in proliferation and differentiation of hematopoietic cells. The transcription factor c-Myb is frequently activated by retrovirus integration in murine and avian leukemias. Its deregulation has been also implicated in human acute and chronic leukemias and some other nonhematopoietic tumors. It is a short-lived protein, which is rapidly degraded by the 26S proteasome. Truncation at the carboxyl (COOH) terminus, which has occurred in some oncogenic forms ofc-Myb, results in the increased resistance to proteolysis. This stabilization correlates in vitro with less efficient ubiquitination. Here, we report the first evidence of post-translational modification of c-Myb by ubiquitin in vivo using HA-labeled ubiquitin. We also show that, in contrast to the unstable wild type or amino (NH2)-terminally truncated c-Myb form, stable carboxyl (COOH)-terminally truncated c-Myb is not targeted to degradation by covalent attachment of ubiquitin in vivo. In addition, following an analysis of subcellular fractionation of proteins from cells treated with a 26S proteasome inhibitor we were able to localize c-Myb exclusively in the nuclear compartment, suggesting the absence of a requirement for export to cytoplasm prior proteolytic processing. Furthermore, pulse-chase experiments of c-Myb protein isolated from interphase cells or cells synchronized in the G2/M or G1 phases of cell cycle did not reveal substantial cell cycle dependent differences in proteolytic processing by the 26S proteasome. Also, the demonstration that the half-life of c-Myb in myeloid progenitor M1 cells induced to differentiate along the monocytic pathway is the same as in undifferentiated cells suggested that proteolytic breakdown of c-Myb is a constitutive process during proliferation and differentiation.