Interaction of Hsp90 with the Nascent Form of the Mutant Epidermal Growth Factor Receptor EGFRvIII

Abstract

EGFRvIII is a mutant epidermal growth factor that promotes aggressive growth of glioblastomas. We made a plasmid that directed the expression of an EGFRvIII with three copies of the Flag epitope at its amino terminus. Flag-tagged EGFRvIII was expressed at the same levels as unmodified EGFRvIII, and showed the same subcellular localization. However, the Flag epitope could only be detected on EGFRvIII present in the endoplasmic reticulum; the epitope was covalently modified during trafficking of the receptor through the Golgi so that it was no longer recognized by anti-Flag antibody. This property was exploited to selectively purify nascent EGFRvIII from glioblastoma cells. Nascent EGFRvIII was found to copurify with a set of other proteins, identified by mass spectrometry as the two endoplasmic reticulum chaperones Grp94 and BiP, and the two cytosolic chaperones Hsc70 and Hsp90. The Hsp90-associated chaperone Cdc37 also co-purified with EGFRvIII, suggesting that Hsp90 binds EGFRvIII as a complex with this protein. Geldanamycin and radicicol, two chemically unrelated inhibitors of Hsp90, decreased the expression of EGFRvIII in glioblastoma cells. These studies show that nascent EGFRvIII in the endoplasmic reticulum associates with Hsp90 and Cdc37, and that the Hsp90 association is necessary to maintain expression of EGFRvIII.Glioblastoma multiforme is an incurable disease with a median survival time that is typically less than 1 year. Surgery and radiation have been shown to increase survival times, but the disease responds poorly to chemotherapy. One of the most common genetic abnormalities found in glioblastoma is amplification of the gene for EGFR.1 Along with amplification, the EGFR gene is frequently mutated. The most common mutation is a deletion of exons 2-7. This deletion is in-frame and results in the expression of a truncated EGFR, known as EGFRvIII, in which amino acids 6 -273 of the extracellular domain are replaced by a single glycine residue (1-4). Multiple studies have demonstrated that EGFRvIII is expressed in approximately half of all glioblastomas (5-8), and there is evidence that its expression is associated with a poorer prognosis (9). Consistent with this, EGFRvIII has been shown to greatly enhance the tumorigenicity of human glioblastoma cells grown as xenografts in nude mice (10).Studies in mouse models of glioblastoma show that EGFRvIII cooperates with mutations at the INK4a/ARF locus to promote the formation of glioblastoma (11). Thus, whereas EGFRvIII expression in normal mice does not give rise to tumors, expression of EGFRvIII in INK4a/ARF(Ϫ/Ϫ) mice does give rise to tumors with a high frequency. The INK4a/ARF locus contains genes coding for two different proteins (12). One of these is Ink4a, a repressor of cyclin-dependent kinase 4 (cdk4); the other is Arf, which has a role in regulating p53 levels. EGFRvIII will also form tumors in mice if cdk4 is overexpressed at the same time, indicating that these two pathways cooperate in the formation of glioblastoma. It is likely that these two ...

Regulation of glioblastoma cell invasion by PKCι and RhoB

“…A172 and U118 cells were from the American Type Culture Collection. Cells were cultured as described previously (Lavictoire et al, 2003).…”

“…Retroviral MSCVpac and MSCVpac-RhoB vectors were a generous gift from Dr G Prendergast (Lankenau Institute for Medical Research, Wynnewood, PA, USA). Retrovirus containing PTEN or RhoB cDNA was made as described previously (Lavictoire et al, 2003). Cells were grown in media containing puromycin (1 mg ml À1 ) to select for transductants.…”

Targeting Cdc37 Inhibits Multiple Signaling Pathways and Induces Growth Arrest in Prostate Cancer Cells

“…This attribute is unique among the HSP90 co-chaperones and may be related to the fact that Cdc37 is the only such molecule to play an essential role in cell cycle regulation (7). At the molecular level, Cdc37 binds to a conserved motif located within the catalytic domains of a large number of protein kinases including molecules whose functions are commonly dysregulated in cancer, such as the Raf-1, Akt, Aurora B, epidermal growth factor receptor (EGFR) and platelet-derived growth factor receptor kinases (8)(9)(10)(11)(12). Although the repertoire of Cdc37 clients is largely restricted to protein kinases, one exception exists, which is of considerable relevance to prostate cancer: its activating function in androgen receptor signaling (13).…”

Heat Shock Protein 90α Recruits FLIPS to the Death-Inducing Signaling Complex and Contributes to TRAIL Resistance in Human Glioma

“…As an example, HSP90 stabilizes Akt and oncogenic forms of mutant epidermal growth factor receptor, both of which contribute to the growth of a variety of cancers including gliomas (13)(14)(15). HSP70, in a complex with HSP90 and HSP40, also serves to regulate the function and turnover of the estrogen receptor, increasing its activation and driving estrogen-dependent cellular responses (16,17).…”