Sensitivity of Mature ErbB2 to Geldanamycin Is Conferred by Its Kinase Domain and Is Mediated by the Chaperone Protein Hsp90

Abstract

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.The ErbB2 gene (also known as Her2/neu), a homolog of the rat neu gene, encodes a 185-kDa receptor-like glycoprotein that is a member of the ErbB family of receptor tyrosine kinases that also include the epidermal growth factor (EGF) 1 receptor (ErbB1) (1), ErbB3 (2), and ErbB4 (3). ErbB receptors are single transmembrane proteins with an extracellular domain (ECD) that bears two cysteine-rich clusters and is responsible for interaction with polypeptide ligands and an intracellular domain (ICD) that contains a tyrosine kinase motif and a long hydrophilic segment at the C-terminal end (4). Binding of the ECD with ligands causes hetero-and/or homodimerization of ErbB proteins, followed by stimulation of their intrinsic kinase activity, leading in turn to the phosphory...

Transactivation of the epidermal growth factor receptor in responses to myocardial stress and cardioprotection

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Endocytic Down-Regulation of ErbB2 Is Stimulated by Cleavage of Its C-Terminus

“…GA-induced degradation of ErbB2 can be inhibited by proteasomal inhibitors (Mimnaugh et al, 1996), and a straightforward interpretation of this is that ErbB2 is degraded by the proteasome itself (Hong et al, 1999;Zheng et al, 2000;Xu et al, 2001;Citri et al, 2002;Way et al, 2004). We recently demonstrated that proteasomal activity is needed for GAinduced internalization and lysosomal degradation of ErbB2 (Lerdrup et al, 2006).…”

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