Caspase-dependent Cleavage of ErbB-2 by Geldanamycin and Staurosporin

Abstract

The geldanamycin-induced degradation of ErbB-2 produces a 23-kDa carboxyl-terminal fragment, which has been isolated and subjected to amino-terminal microsequencing. The obtained sequence indicates that the amino terminus of this fragment corresponds to Gly-1126 of ErbB-2. Analysis of the residues immediately before Gly-1126 suggests that cleavage may involve caspase activity. Site-directed mutagenesis of Asp-1125 in ErbB-2 prevents geldanamycin-provoked formation of the 23-kDa fragment, consistent with the requirement of this residue for caspase-dependent cleavage in known substrates. Also, the addition of the pan-caspase inhibitor Z-VAD-FMK blocks formation of the 23-kDa ErbB-2 fragment in cells exposed to geldanamycin. Interestingly, staurosporin and curcumin are also shown to provoke the degradation of ErbB-2 with formation of the 23-kDa carboxyl-terminal fragment. The generation of this fragment by staurosporin or curcumin is likewise blocked by caspase inhibition. Caspase inhibition does not prevent accelerated degradation of the 185-kDa native ErbB-2 in geldanamycin-treated cells but does significantly prevent staurosporin-stimulated metabolic loss of ErbB-2.ErbB-2, a Type I transmembrane receptor tyrosine kinase, functions as a co-receptor by dimerizing with ligand-occupied members of the ErbB family, the EGF receptor, ErbB-3, or ErbB-4 (1-3). This heterodimerization event is considered to alter the signaling capacity and cellular responses provoked by homodimers of occupied ErbB receptors. No ligand has been identified that directly interacts with the ectodomain of ErbB-2.ErbB-2 was originally identified as the transforming oncogene neu, which contains a point mutation in the transmembrane domain that is responsible for its oncogenic potential (4). Overexpression of ErbB-2 also produces a transformed phenotype in experimental systems (5-7). Importantly, ErbB-2 is frequently overexpressed in carcinomas, particularly mammary and ovarian carcinomas, and is associated with a poor prognosis (8 -10). ErbB-2 antibodies (11, 12) and agents such as interferon (13) or tyrosine kinase inhibitors (14) decrease the growth of ErbB-2-expressing tumor cells and also reduce the cellular level of ErbB-2 (15). In many of these instances the decreased growth provoked by the loss of ErbB-2 is due to increased apoptosis. In contrast, the overexpression of ErbB-2 can prevent the induction of apoptosis (15). Hence, the growth-controlling activity of ErbB-2 is related to structural changes or alterations in its level of expression.The benzoquinoid anasamycin antibiotic geldanamycin was first isolated and described as an inhibitor of tyrosine kinase activity (16). Subsequently, geldanamycin was shown to possess tumoricidal activity toward cell lines that overexpress ErbB-2 (17). Currently geldanamycin derivatives designed to reduce toxicity are in clinical trials for certain cancer patients (18). The addition of geldanamycin to cells results in an increased rate of degradation of several protein kinases, including ErbB-2 (17), S...

Caspase-dependent regulation of the ubiquitin–proteasome system through direct substrate targeting

“…For certain substrates removal of a regulatory domain results in their constitutive activation, as in the case of various kinases (11,12). For others, cleavage results in a loss of function, and the resulting fragments are frequently degraded by the 26S proteasome (5)(6)(7)(8)(9)(10). In this study, we found an interaction between caspases and the ubiquitin-proteasome system.…”

“…Caspase cleavage can inactivate proteins or generate dominant-negative inhibitors, as in the case of gelsolin, RIP1, and eIF4E-BP1 (4). Moreover, caspase cleavage of numerous substrates, including IRF-3, ErbB2, cyclin E, claspin, SSRP1, and Twist, can enhance their turnover by the proteasome (5)(6)(7)(8)(9)(10). Conversely, caspases can also constitutively activate proteins, particularly kinases such as PKC and Mst1 (11,12), or change the function of a protein altogether, as seen in the conversion of antiapoptotic BCL-2 proteins into proapoptotic BAX-like proteins (13).…”

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

“…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).…”

“…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).It has remained elusive whether endocytosis and C-terminal cleavage of ErbB2 are independent or correlated events. If positively correlated, C-terminal cleavage of ErbB2 could promote endocytosis by releasing ErbB2 from a retention mechanism that normally sequesters the receptor at the plasma membrane (Sorkin et al, 1993;Hommelgaard et al, 2004;Lerdrup et al, 2006) or by exposure of an unknown cryptic motif stimulating endocytosis.…”

Receptor Tyrosine Kinases in the Nucleus

“…In the case of several RTKs (ErbB-2 [Tikhomirov and Carpenter 2001;Benoit et al 2004;Strohecker et al 2008], Ret [Bordeaux et al 2000;Cabrera et al 2011], ALK [Mourali et al 2006;Racaud-Sultan et al 2006], TrkC [Tauszig- Delamasure et al 2007], and Met [Tulasne et al 2004;Pozner-Moulis et al 2006;Foveau et al 2007;Deheuninck et al 2009]) there is evidence that caspase activity cleaves the cytoplasmic domain to produce an ICD fragment. Because the fragment is often produced by two cleavage events within the cytoplasmic domain, the fragment is often considerably smaller than that produced by intramembrane proteolysis.…”