Xu W, Mimnaugh E, Rosser M F, Nicchitta C, Marcu M, Yarden Y, Neckers L
Department of Cell and Cancer Biology, Medicine Branch, NCI, National Institutes of Health, Rockville, Maryland 20850, USA.
J Biol Chem. 2001 Feb 2;276(5):3702-8. doi: 10.1074/jbc.M006864200. Epub 2000 Nov 8.
ErbB receptors are a family of ligand-activated tyrosine kinases that play a central role in proliferation, differentiation, and oncogenesis. ErbB2 is overexpressed in >25% of breast and ovarian cancers and is correlated with poor prognosis. Although ErbB2 and ErbB1 are highly homologous, they respond quite differently to geldanamycin (GA), an antibiotic that is a specific inhibitor of the chaperone protein Hsp90. Thus, although both mature and nascent ErbB2 proteins are down-regulated by GA, only nascent ErbB1 is sensitive to the drug. To reveal the underlying mechanism behind these divergent responses, we made a chimeric receptor (ErbB1/2) composed of the extracellular and transmembrane domains of ErbB1 and the intracellular domain of ErbB2. The ErbB1/2 protein is functional since its kinase activity was stimulated by epidermal growth factor. The sensitivity of ErbB1/2 to GA was similar to that of ErbB2 and unlike that of ErbB1, indicating that the intracellular domain of the chimera confers GA sensitivity. This finding also suggests that the GA sensitivity of mature ErbB2 depends on cytosolic Hsp90, rather than Grp94, a homolog of Hsp90 that is restricted to the lumen of the endoplasmic reticulum, although both chaperones bind to and are inhibited by GA. Lack of Grp94 involvement in mediating ErbB2 sensitivity to GA is further suggested by the fact that a GA derivative with low affinity for Grp94 efficiently depleted ErbB2 protein in treated cells. To localize the specific region of ErbB2 that confers GA sensitivity, we made truncated receptors with progressive deletions of the cytoplasmic domain and tested the GA sensitivity of these molecules. We found that ErbB2 constructs containing an intact kinase domain retained GA sensitivity, whereas those lacking the kinase domain (ErbB2/DK) lost responsiveness to GA completely. Hsp90 co-immunoprecipitated with all ErbB2 constructs that were sensitive to GA, but not with ErbB2/DK or ErbB1. Both tyrosine-phosphorylated and non-phosphorylated ErbB2 proteins were similarly sensitive to GA, as was a kinase-dead ErbB2 mutant. These data suggest that Hsp90 uniquely stabilizes ErbB2 via interaction with its kinase domain and that GA stimulates ErbB2 degradation secondary to disruption of ErbB2/Hsp90 association.
表皮生长因子受体（ErbB）家族是一类配体激活的酪氨酸激酶，在细胞增殖、分化和肿瘤发生过程中发挥核心作用。超过25%的乳腺癌和卵巢癌中存在ErbB2的过表达，且其与预后不良相关。尽管ErbB2和ErbB1高度同源，但它们对格尔德霉素（GA）的反应却截然不同，GA是一种抗生素，是伴侣蛋白Hsp90的特异性抑制剂。因此，尽管成熟和新生的ErbB2蛋白都能被GA下调，但只有新生的ErbB1对该药物敏感。为了揭示这些不同反应背后的潜在机制，我们构建了一种嵌合受体（ErbB1/2），它由ErbB1的细胞外和跨膜结构域以及ErbB2的细胞内结构域组成。ErbB1/2蛋白具有功能，因为其激酶活性可被表皮生长因子激活。ErbB1/2对GA的敏感性与ErbB2相似，与ErbB1不同，这表明嵌合体的细胞内结构域赋予了对GA的敏感性。这一发现还表明，成熟ErbB2对GA的敏感性取决于胞质Hsp90，而非Grp94（Hsp90的同源物，局限于内质网腔），尽管这两种伴侣蛋白都能与GA结合并被其抑制。对Grp94低亲和力的GA衍生物能有效降低处理细胞中ErbB2蛋白的含量，这一事实进一步表明Grp94不参与介导ErbB2对GA的敏感性。为了定位赋予ErbB2对GA敏感性的特定区域，我们构建了一系列胞质结构域逐渐缺失的截短受体，并测试了这些分子对GA的敏感性。我们发现，含有完整激酶结构域的ErbB2构建体保留了对GA的敏感性，而那些缺乏激酶结构域的构建体（ErbB2/DK）则完全丧失了对GA的反应性。Hsp90能与所有对GA敏感的ErbB2构建体进行共免疫沉淀，但不能与ErbB2/DK或ErbB1进行共免疫沉淀。酪氨酸磷酸化和非磷酸化的ErbB2蛋白对GA的敏感性相似，激酶失活的ErbB2突变体也是如此。这些数据表明，Hsp90通过与ErbB2的激酶结构域相互作用独特地稳定了ErbB2，并且GA通过破坏ErbB2/Hsp90的结合刺激了ErbB2的降解。
