Buck Institute for Age Research, Novato, California 94945, USA.
Cancer Res. 2010 May 1;70(9):3709-17. doi: 10.1158/0008-5472.CAN-09-3768. Epub 2010 Apr 20.
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 hours) induced by the HSP90 chaperone inhibitor geldanamycin (GA), whereas its downregulation and lysosomal degradation are more slowly (24 hours) induced by the proteasome inhibitor bortezomib/PS341. In PS341-treated SK-BR-3 cells, overexpressed ErbB2 coprecipitates with the E3 ubiquitin ligase c-Cbl and also with the deubiquitinating enzyme USP9x; moreover, siRNA downregulation of USP9x enhances PS341-induced ErbB2 downregulation. Because 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-linked 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-fold to 8-fold), and synchronous GA induction of K48 and K63 polyubiquitinated ErbB2, PS341 produces a dramatic (20-fold to 40-fold) sequential increase in polyubiquitinated ErbB2 consistent with K48 polyubiquitination followed by K63 editing. Fluorescence microscopic imaging confirmed that PS341, but not GA, induces colocalization of K48-linked and K63-linked polyubiquitin with perinuclear lysosome-sequestered ErbB2. Thus, ErbB2 surface overexpression and recycling seem 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.
过度表达的 ErbB2/HER2 受体是一种经过临床验证的癌症靶点，但其表面定位和内化机制仍知之甚少。热休克蛋白 90（HSP90）伴侣抑制剂格尔德霉素（geldanamycin，GA）可迅速（2-6 小时）下调过表达的 185kDa ErbB2 受体，而蛋白酶体抑制剂硼替佐米/PS341 则更缓慢（24 小时）下调 ErbB2 受体并使其溶酶体降解。在 PS341 处理的 SK-BR-3 细胞中，过表达的 ErbB2 与 E3 泛素连接酶 c-Cbl 以及去泛素化酶 USP9x 共沉淀；此外，USP9x 的 siRNA 下调增强了 PS341 诱导的 ErbB2 下调。由于赖氨酸 48（K48）或 63（K63）上的多泛素连接可以通过 26S 蛋白酶体降解或内体转运到溶酶体来区分不同的蛋白质，因此使用多重反应监测（MRM）/质谱（MS）和多泛素连接特异性抗体来定量跟踪 SK-BR-3 细胞用 GA 或 PS341 处理后 K48 连接和 K63 连接的 ErbB2 多泛素化。MRM/MS 显示，与 GA 快速、适度（4 倍至 8 倍）和同步诱导 K48 和 K63 连接的 ErbB2 多泛素化不同，PS341 可显著（20 倍至 40 倍）增加多泛素化 ErbB2 的顺序增加，与 K48 多泛素化后 K63 编辑一致。荧光显微镜成像证实 PS341，但不是 GA，诱导 K48 连接和 K63 连接的多泛素与核周溶酶体隔离的 ErbB2 共定位。因此，ErbB2 表面过表达和再循环似乎依赖于其多泛素化和去泛素化；同样，PS341 和 GA 对 ErbB2 受体定位、多泛素化和降解的影响相反，表明可能受不同 K48 和 K63 多泛素编辑机制调控的替代细胞质运输。
