Melanoma cell lines contain a proteasome-sensitive, nuclear cytoskeleton-associated pool of beta-catenin.
Melanoma cell lines contain a proteasome-sensitive, nuclear cytoskeleton-associated pool of beta-catenin.
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黑色素瘤细胞系含有蛋白酶体敏感的、核细胞骨架相关的 β-连环蛋白池。
DOI：
10.1111/j.1749-6632.1999.tb09418.x
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发表时间：
1999
影响因子：
5.2
通讯作者：
Trepel,J
中科院分区：
文献类型：
--
作者：
Bonvini,P;Hwang,SG;el-Gamil,M;Robbins,P;Neckers,L;Trepel,J
最近的研究表明，含有APC缺失或β-连环蛋白激活突变的结肠癌和黑素瘤细胞含有升高水平的胞质和核β-连环蛋白。1-4在含有野生型β-连环蛋白的细胞的蛋白酶体抑制后报道了类似的结果。此外，在含有突变的β-连环蛋白或野生型β-连环蛋白但不含APC的细胞中，β-连环蛋白蛋白具有异常长的半衰期。这些发现导致以下假设：APC功能的缺乏或β-连环蛋白突变导致继发于可溶性蛋白质的蛋白酶体抗性胞质池升高的核β-连环蛋白水平升高。然而，β-连环蛋白存在于几个亚细胞位置，并且很少知道β-连环蛋白突变/APC缺失对这些池对蛋白酶体抑制的响应的影响。为了进一步研究这个问题，我们研究了三种先前描述的黑色素瘤细胞系，3 1241，其表达APC，但在GSK 3 β磷酸化位点携带Ser 37 → Phe 37（S37 F）β-连环蛋白突变; 928，其表达野生型β-连环蛋白，但没有检测到APC;在这项研究中，三种黑素瘤细胞系用蛋白酶体抑制剂ALLnL（100 μM）5、6处理6小时，并将几种亚细胞组分中的β-catenin稳态水平与未处理细胞的β-catenin稳态水平进行比较。正如先前的研究所预测的，1011细胞中的可溶性β-连环蛋白对蛋白酶体抑制敏感，但928和1241细胞中的可溶性β-连环蛋白组分对蛋白酶体介导的降解具有抗性。这项研究的一个新发现是，来自所有三种细胞系的洗涤剂不溶性β-连环蛋白级分保留了蛋白酶体敏感性。为了澄清这种去污剂不溶性组分的性质，我们在不含去污剂的缓冲液中裂解细胞，并制备可溶性、微粒体和沉淀组分，分析其β-连环蛋白含量。正如预期的那样，在蛋白酶体抑制后，1011细胞中的β-连环蛋白稳态水平主要在可溶性部分中增加。相反，在928和1241细胞中，β-catenin蛋白仅显著积聚，
Recent studies have shown that colon carcinoma and melanoma cells that contain either deletions of APC or activating mutations in β-catenin contain elevated levels of both cytoplasmic and nuclear β-catenin. 1–4 Similar results were reported following proteasome inhibition of cells containing wild-type β-catenin. Furthermore, in cells containing mutated β-catenin or wild-type β-catenin but no APC, the β-catenin protein has an exceptionally long half-life. These findings led to the hypothesis that lack of APC function or β-catenin mutation result in elevated levels of nuclear βcatenin secondary to elevation of a soluble, proteasome-resistant cytosolic pool of the protein. However, β-catenin exists in several subcellular locations, and little is known of the effect of β-catenin mutations/APC deletions on the response of these pools to proteasome inhibition. To further examine this question, we studied three previously described melanoma cell lines, 3 1241, which expresses APC but carries a Ser37Æ Phe37 (S37F) β-catenin mutation in the GSK3ß phosphorylation site; 928, which expresses wild-type β-catenin but no detectable APC; and 1011, which contains both wild-type β-catenin and APC.In this study, the three melanoma cell lines were treated with the proteasome inhibitor ALLnL (100 µM) 5, 6 for 6 hours, and the β-catenin steady-state level in several subcellular fractions was compared with that of untreated cells. As predicted from previous studies, soluble β-catenin in 1011 cells was sensitive to proteasome inhibition, but the soluble β-catenin fraction of both 928 and 1241 cells was resistant to proteasome-mediated degradation. A novel finding of this study is that the detergent-insoluble β-catenin fraction from all three cell lines retained proteasome sensitivity. To clarify the nature of this detergent-insoluble fraction, we lysed cells in detergent-free buffer and prepared soluble, microsomal, and pellet fractions, which were analyzed for β-catenin content. As expected, the β-catenin steady-state level in 1011 cells was increased primarily in the soluble fraction after proteasome inhibition. In contrast, in 928 and 1241 cells, β-catenin protein markedly accumulated only