ErbB2 trafficking and degradation associated with K48 and K63 polyubiquitination.
ErbB2 trafficking and degradation associated with K48 and K63 polyubiquitination.
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DOI：
10.1158/0008-5472.can-09-3768
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发表时间：
2010-05-01
期刊：
影响因子：
11.2
通讯作者：
Benz CC
中科院分区：
文献类型：
--
作者：
Marx C;Held JM;Gibson BW;Benz CC
过表达的ErbB 2/HER 2受体是临床验证的癌症靶点，其表面定位和内化机制仍知之甚少。过度表达的185 kDa ErbB 2受体的下调由HSP 90伴侣抑制剂格尔德霉素（GA）快速（2-6 h）诱导，而其下调和溶酶体降解由蛋白酶体抑制剂硼替佐米/PS341诱导更缓慢（24 h）。在PS341处理的SK-BR-3细胞中，过表达的ErbB 2与E3泛素连接酶c-Cbl以及去泛素化酶USP 9 x共沉淀;此外，USP 9 x的siRNA下调增强了PS341诱导的ErbB 2下调。由于通过赖氨酸48（K48）或63（K63）的多聚泛素连接可以差异地寻址蛋白质以进行26 S蛋白酶体降解或内体运输至溶酶体，因此使用多反应监测（MRM）质谱（MS）和多聚泛素连接特异性抗体来定量追踪GA或PS341处理SK-BR-3细胞后K48和K63连接的ErbB 2多聚泛素化。MRM/MS显示，与K48和K63多聚泛素化ErbB 2的快速，适度（4至8倍）和同步GA诱导不同，PS341产生了与K48多聚泛素化随后K63编辑一致的多聚泛素化ErbB 2的戏剧性（20至40倍）连续上升。荧光显微成像证实，PS341，而不是GA，诱导共定位的K48和K63连接的多聚泛素与核周溶酶体隔离的ErbB 2。因此，ErbB 2表面过表达和再循环似乎取决于其多聚泛素化和去泛素化;此外，PS341和GA对ErbB 2受体定位、多聚泛素化和降解的对比效应指向可能由不同的K48和K63多聚泛素编辑机制调节的交替细胞质运输。
The overexpressed ErbB2/HER2 receptor is a clinically validated cancer target whose surface localization and internalization mechanisms remain poorly understood. Downregulation of the overexpressed 185 kDa ErbB2 receptor is rapidly (2–6 h) induced by the HSP90 chaperone inhibitor, geldanamycin (GA), while its downregulation and lysosomal degradation are more slowly (24 h) induced by the proteasome inhibitor, bortezomib/PS341. In PS341 treated SK-BR-3 cells, overexpressed ErbB2 co-precipitates with the E3 ubiquitin ligase, c-Cbl, and also with the deubiquitinating enzyme, USP9x; moreover, siRNA downregulation of USP9x enhances PS341 induced ErbB2 down-regulation. Since polyubiquitin linkages via lysine 48 (K48) or 63 (K63) can differentially address proteins for 26S proteasomal degradation or endosome trafficking to the lysosome, multiple reaction monitoring (MRM) mass spectrometry (MS) and polyubiquitin linkage-specific antibodies were used to quantitatively track K48 and K63 linked ErbB2 polyubiquitination following either GA or PS341 treatment of SK-BR-3 cells. MRM/MS revealed that unlike the rapid, modest (4- to 8-fold) and synchronous GA induction of K48 and K63 polyubiquitinated ErbB2, PS341 produces a dramatic (20- to 40-fold) sequential rise in polyubiquitinated ErbB2 consistent with K48 polyubiquitination followed by K63 editing. Fluorescence microscopic imaging confirmed that PS341, but not GA, induces co-localization of K48 and K63 linked polyubiquitin with perinuclear lysosome-sequestered ErbB2. Thus, ErbB2 surface overexpression and recycling appear to depend on its polyubiquitination and deubiquitination; as well, the contrasting effects of PS341 and GA on ErbB2 receptor localization, polyubiquitination and degradation point to alternate cytoplasmic trafficking likely regulated by different K48 and K63 polyubiquitin editing mechanisms.