Geldanamycin stimulates internalization of ErbB2 in a proteasome-dependent way

Abstract

The potent oncoprotein and receptor tyrosine kinase ErbB2 is remarkable because it resists efficient downregulation. However, ErbB2 can be downregulated by the HSP-90 inhibitor geldanamycin, but the underlying cellular mechanisms are uncertain. Apparently, delivery of ErbB2 to lysosomes, cleavage of the ErbB2 kinase domain and proteasomal activity are all processes that are involved. Using a non-invasive confocal microscopical assay allowing quantitative analysis of ErbB2 internalization in cell populations, we show that whereas ErbB2 is resistant to internalization in untreated SK-BR-3 cells, geldanamycin stimulates internalization and subsequent degradation in lysosomes. This process depends on proteasomal activity, which is a regulatory upstream event in ErbB2 internalization rather than the actual mechanism of degradation. ErbB2 can be internalized as a full-length protein, thus cleavage of the ErbB2 kinase domain is not a requirement for geldanamycin-stimulated internalization. Moreover, as shown by FRAP (fluorescence recovery after photobleaching) and electron microscopy, geldanamycin induces an increase in the amount of mobile ErbB2 and a redistribution of ErbB2 in the plasma membrane making the receptor accessible to endocytosis. Cells with most ErbB2 endocytosis also have the highest fraction of mobile ErbB2. It is concluded that geldanamycin stimulates internalization of full-length ErbB2 in a proteasome-dependent manner leading to lysosomal degradation.

PMCA2 regulates HER2 protein kinase localization and signaling and promotes HER2-mediated breast cancer

“…We found HER2 and HSP-90 colocalized in membrane protrusions in SKBR3 cells (Fig. 5C) (12,26), but their colocalization was abolished in PMCA2KD cells (Fig. 5C).…”

“…Several groups have reported that HER2 resides within microvillus-like membrane protrusions, and Hommelgaard and colleagues hypothesized that these structures support the retention of HER2 on the cell surface (33)(34)(35)(36). Previous experiments also showed that interactions with HSP-90 are important for HER2 stabilization within membrane protrusions and its resistance to internalization (12,26). Thus, it is noteworthy that PMCA2 levels correlate with HSP-90 levels in human tumors and that knockdown of PMCA2 is associated with decreased interactions between HSP-90 and HER2, the internalization of HER2, and the disruption of the membrane protrusions.…”

“…Interactions with HSP-90 stabilize HER2 at the cell membrane, prevent its internalization, and facilitate HER2 signaling (12,(25)(26)(27)(28). Therefore, we examined the previously described tissue microarray (TMA) and microarray databases and found that HSP-90 expression correlates with PMCA2 expression in human breast cancers at both a protein (Fig.…”

Antitumor activity of efrapeptins, alone or in combination with 2-deoxyglucose, in breast cancer in vitro and in vivo

“…It is well known that ErbB2/HER2 is a plasma protein that resists internalization and exhibits a long half-life on the surface of the cells. Because it relies on Hsp90 for stable expression, exposure of ErbB2/HER2-expressing cells to an Hsp90 inhibitor such as geldanamycin leads to rapid internalization, ubiquitination, and degradation of the protein (Mimnaugh et al 1996;Lerdrup et al 2006). Pedersen et al (2008) have argued that ubiquitination of ErbB2/HER2 following inhibition of Hsp90 is essential for presentation of the internalized protein to the lysosomes for degradation.…”

“…Pedersen et al (2008) have argued that ubiquitination of ErbB2/HER2 following inhibition of Hsp90 is essential for presentation of the internalized protein to the lysosomes for degradation. Concurrent treatment of ErbB2/HER2-expressing cells with geldanamycin and lactacystin, a well known proteasomal inhibitor, rescues ErbB2/HER2 from degradation and results in the accumulation of internalized, ubiquitinated protein (Lerdrup et al 2006). We hypothesize that accumulation of internalized and thus inactive, ubiquitinated ErbB2/HER2 protein in MCF-7 cells transfected to express ErbB2/HER2 and treated with EF leads to such an increase in the cellular stress that cells prefer to die than enter a cytostatic phase following treatment with EF.…”