Geldanamycin Induces ErbB-2 Degradation by Proteolytic Fragmentation

Abstract

Exposure of carcinoma cell lines to the antibiotic geldanamycin induces the degradation of ErbB-2, a coreceptor tyrosine kinase that is frequently overexpressed in certain tumors. Using ErbB-2 mutants expressed as chimeric receptors or green fluorescent protein fusion proteins, we report that the kinase domain of ErbB-2 is essential for geldanamycin-induced degradation. The kinase domain of the related epidermal growth factor receptor was not sensitive to this drug. The data further indicate mechanistic aspects of ErbB-2 degradation by geldanamycin. The data show that exposure to the drug induces at least one cleavage within the cytoplasmic domain of ErbB-2 producing a 135-kDa fragment and a 23-kDa fragment. The latter represents the carboxyl-terminal domain of ErbB-2, whereas the former represents the ectodomain and part of the cytoplasmic domain. Degradation of the carboxylterminal fragment is prevented by proteasome inhibitors, whereas degradation of the membrane-anchored 135-kDa ErbB-2 fragment is blocked by inhibitors of the endocytosis-dependent degradation pathway. Confocal microscopy studies confirm a geldanamycin-induced localization of ErbB-2 on intracellular vesicles.ErbB-2 is a Type 1 transmembrane tyrosine kinase that functions as a co-receptor by forming dimers with other members of the ErbB receptor family (ErbB-1 (EGF 1 receptor), ErbB-3, and ErbB-4; Refs. 1 and 2). Although ErbB-2 has a potential ligand-binding ectodomain, no direct ligand has yet been identified. In its role as a co-receptor, ErbB-2 enhances the signaling capacity of its dimerization partners. The association of ErbB-2 with these various receptors is, however, entirely ligand-dependent. In the absence of growth factor ErbB-2 is reported to interact with CD44, an adhesion receptor, in ovarian carcinoma cell lines (3) and with a large plasma membrane glycoprotein complex in microvilli of a mammary adenocarcinoma cell line (4). ErbB-2 has also been demonstrated to form ligand-dependent complexes with the IL-6 receptor component gp130 (5) and Trk A (6), the nerve growth factor receptor.ErbB-2 was originally identified as the transforming oncogene neu in which a point mutation in the transmembrane domain is responsible for its oncogenic potential (7,8). ErbB-2 also functions as an oncogene when overexpressed (9, 10) and in humans is frequently overexpressed in breast and ovarian tumors (11). ErbB-2 overexpression in breast cancer is associated with a poor prognosis (12), and hence it is a target for therapeutic reagents, including monoclonal antibodies and drugs (13). Frequently, antibodies that decrease the growth of ErbB-2-expressing tumors also reduce the level of ErbB-2 by a mechanism that is unclear. Hence, the transforming activity of ErbB-2 is related to structural changes or changes in its level of expression.The benzoquinoid ansamycin antibiotics geldanamycin and herbimycin were first isolated from the culture broths of several actinomycete species (14, 15) and described as inhibitors of tyrosine kinase-dependent growth (16...

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

“…The GA-induced cleavage of ErbB2, which has previously been observed in Western blots (Tikhomirov and Carpenter, 2000, 2001, could essentially be a result of proteolysis of internalized ErbB2 in the endosomal/lysosomal degrada- …”

“…Several studies have shown that endocytic downregulation of ErbB2 is impaired in cancer cells (Sorkin et al, 1993; Baulida et al, 1996;Wang et al, 1999;Hommelgaard et al, 2004;Austin et al, 2004;Longva et al, 2005), and ErbB2 can even transmit this property to the related epidermal growth factor receptor (EGFR; Muthuswamy et al, 1999;Wang et al, 1999;Worthylake et al, 1999;Haslekas et al, 2005). Inhibition of HSP90 (e.g., with geldanamycin [GA]) leads to increased internalization and lysosomal degradation of ErbB2 in a manner depending on proteasomal activity (Tikhomirov and Carpenter, 2000;Austin et al, 2004;Lerdrup et al, 2006). HSP90 inhibition also induces cleavage of the cytoplasmic part of ErbB2, resulting in a transmembrane 135-kDa ErbB2 and short-lived cytoplasmic fragments (Tikhomirov and Carpenter, 2000;Lerdrup et al, 2006).…”

“…Inhibition of HSP90 (e.g., with geldanamycin [GA]) leads to increased internalization and lysosomal degradation of ErbB2 in a manner depending on proteasomal activity (Tikhomirov and Carpenter, 2000;Austin et al, 2004;Lerdrup et al, 2006). HSP90 inhibition also induces cleavage of the cytoplasmic part of ErbB2, resulting in a transmembrane 135-kDa ErbB2 and short-lived cytoplasmic fragments (Tikhomirov and Carpenter, 2000;Lerdrup et al, 2006). The cleavage occurs in the kinase domain and a similar cleavage is seen after stimulation with curcumin or staurosporine (Tikhomirov and Carpenter, 2001) or in cells with high levels of ␣6␤1 integrin (Shimizu et al, 2003).…”

The epidermal growth factor receptor family: Biology driving targeted therapeutics

“…HSP90 binds to the N-lobe of the catalytic tyrosine kinase domain of ErbB2, apparently independent of cellular stress. HSP90 has been shown to stabilize ErbB2, likely by preventing ubiquitinylation and proteosomal degradation [108,109]. HSP90 also limits both tyrosine kinase activity and heterodimer formation thus restraining excessive EGF signaling [110].…”

“…The molecular chaperone HSP90 stabilizes ErbB2 at the cell membrane and increased expression of HSP90 in breast cancer is associated with poor prognosis [108,112]. Inhibition of HSP90 with molecules such as geldanamycin leads to ErbB2 degradation and ultimately attenuation of signaling [109]. The in vivo utility of such an approach remains to be determined given the diverse functions of HSP90.…”