Abstract
The benzoquinone ansamycin geldanamycin (GA) stimulates proteasome-mediated degradation of plasma membrane–associated ErbB2, a receptor tyrosine kinase. Drug sensitivity is mediated by ErbB2's kinase domain and occurs subsequent to the disruption of Hsp90 interaction with this domain. Full-length ErbB2 is efficiently processed via the endoplasmic reticulum (ER) and Golgi network, so that at steady state most of the detectable protein is plasma membrane associated. However, previous studies have also demonstrated the GA sensitivity of newly synthesized ErbB2, normally a minor component of the total cellular pool of the kinase. Drug sensitivity of nascent ErbB2 is distinguished by 2 characteristics—protein instability and inability to traverse the ER. As nascent ErbB2 can associate with both cytoplasmic Hsp90 and its ER luminal homolog Grp 94, also a GA-binding protein, the purpose of this study was to examine the relative contributions of the cytoplasmic and ER luminal domains of ErbB2 to the GA sensitivity of the nascent kinase. By studying the drug sensitivity of ErbB2/DK, a construct lacking ErbB2's cytoplasmic kinase domain, and by examining the activity of a GA derivative that preferentially binds Hsp90, we conclude that both the stability and the maturation of nascent ErbB2 are regulated by its cytoplasmic, Hsp90-interacting domain.
INTRODUCTION
The ErbB2 gene (also known as Her2/neu) encodes a 185-kDa receptor-like glycoprotein, which is a member of the ErbB family of receptor tyrosine kinases that also include the epidermal growth factor receptor (EGFR/ErbB1) (Ullrich et al 1984), ErbB3 (Kraus et al 1989), and ErbB4 (O'Rourke et al 1997). ErbB receptors are type I transmembrane proteins, and overexpression of ErbB2 in particular causes cell transformation and tumorigenesis in preclinical models (Hudziak et al 1987). ErbB2 is often amplified in various solid tumors, and the clinical implications of its overexpression in breast and ovarian cancers have been described well (Klapper et al 2000).
ErbB2 is unique among the members of the ErbB family in that it is highly sensitive to the Hsp90-binding antibiotic geldanamycin (GA) (Chavany et al 1996; Mimnaugh et al 1996). Drug treatment results in rapid, proteasome-mediated ErbB2 instability, secondary to disruption of Hsp90 association with ErbB2's kinase domain (Xu et al 2001). Although this is most easily seen with mature plasma-membrane ErbB2, the nascent immature protein is also sensitive to GA while in the endoplasmic reticulum (ER) (Chavany et al 1996).
Although GA-induced instability of plasma membrane ErbB2 is mediated solely by drug binding to Hsp90 (Xu et al 2001), the situation with nascent, immature ErbB2 may be more complex. During its stay in the ER, ErbB2 can associate with Hsp90 via its cytoplasmic kinase domain, but also with the Hsp90 homolog Grp94 via its luminal domain (Chavany et al 1996). As GA binds to both Hsp90 and Grp94 with similar affinities (Schulte et al 1999; Xu et al 2001), drug effects on the nascent kinase could be mediated by its interaction with either chaperone. To investigate these possibilities, we have studied the effects of GA on the maturation, trafficking, and stability of ErbB2/DK, a construct that lacks the Hsp90-interacting cytoplasmic domain but retains the complete transmembrane and ER luminal domains (Xu et al 2001). Additionally, we have examined the sensitivity of newly synthesized, full-length ErbB2 to a GA derivative, WX514, that interacts preferentially with Hsp90 and not with Grp94 (Xu et al 2001).
MATERIALS AND METHODS
Antibodies and plasmid
Mouse anti-ErbB2 monoclonal antibody (Ab-5) was purchased from Oncogene Research, Boston, MA, USA, and was used for both immunoprecipitation and immunofluorescent assay. Cy3™-conjugated goat anti-mouse immunoglobulin was obtained from Jackson ImmunoResearch Laboratories Inc., West Grove, PA, USA. Plasmid constructs of full-length and kinase-domain deleted ErbB2 were described previously (Xu et al 2001). pEYFP-Golgi was purchased from ClonTech Inc (Palo Alto, CA, USA).
Cell culture and transient transfection
COS7 cells were purchased from American Type Culture Collection (Rockville, MD, USA), and maintained in medium containing 90 % Dulbecco modified Eagle medium (DMEM), 10 % fetal calf serum (FCS), 2 mM glutamine, 1 mM N-2-hydroxythylpiperazine-N′-2-ethane-sulfonic acid (HEPES), and 1 mM sodium pyruvate. For transient transfections, plasmid was premixed with FuGene 6 (Roche Diagnostics Corporation) following the manufacturer's protocol, and was added to cells at 50–70 % confluence. Cells were continually cultured in the same medium until further treated.
[35S] labeling of transfected COS7 cells
COS7 cells were first treated with 1 μM GA or 10 μM WX514, plus proteasome inhibitor N-Acetyl-leu-leu-norleucinal (ALLN) (Sigma, St Louis, MO, USA), 100 μM, at 37°C for 2 hours, and then rinsed twice with warmed methionine- and cysteine-deficient DMEM medium, containing 10 % dialyzed FCS, 2 mM glutamine, 1 mM HEPES, and 1 mM sodium pyruvate, and incubated in the same medium in the presence of GA or WX514. After 30 minutes, [35S]-labeled methionine and cysteine (TRAN35S-LABEL, ICN, Irvine, CA, USA) was added to a final concentration of 100 μCi/mL. Cells were pulsed for 1 hour at 37°C, washed 3 times with regular medium, and chased in regular medium with the same concentration of GA or WX514 for different times.
Immunoprecipitation of [35S]-labeled ErbB2 proteins and digestion with endoglycosidase H
[35S]-labeled cells in 100 × 10–mm plates were washed once with cold phosphate-buffered saline (PBS), pH 7.0, and lysed with scraping in 1 mL TMNSV (50 mM Tris-HCl, pH 7.5, 20 mM Na2MoO4, 0.09 % Nonidet P-40, 150 mM NaCl, 1 mM sodium othovanadate) supplemented with Complete™ proteinase inhibitors (Roche). Cell lysate was clarified by centrifugation at 14 000 rpm (4°C) for 15 minutes. For immunoprecipitation, 400 μL cell lysate was mixed with 1.5 μg mouse monoclonal antibodies and incubated at 4°C for 2 hours, followed by the addition of protein G-Sepharose beads (Life Technologies, Rockville, MD, USA), and rotated overnight at 4°C. The beads were washed 4 times with lysis buffer, resuspended in 60 μL 0.5 % sodium dodecyl sulfate (SDS), and boiled for 5 minutes. For digestion by endoglycosidase H (endoH), 40 μL of supernatant was mixed with an equal volume of digestion buffer (0.1 M sodium acetate, pH 5.5, 0.1 % Triton X-100, 1 mM phenylmethane sulfonyl fluoride) and incubated with endoH (10 mU/tube; Boehringer Mannheim, Indianapolis, IN, USA) overnight at 37°C. Reaction was stopped by the addition of 20 μL 5× SDS sample buffer (400 mM Tris-HCl, pH 6.8, 10 % SDS, 50 % glycerol, 500 mM dithiothreitol, 0.0025 % bromphenol blue), and boiled for 5 minutes. Proteins were separated on 4–20 % SDS–polyacrylamide gel electrophoresis (PAGE). After electrophoresis, the gel was fixed in a buffer containing 10 % acetic acid, 25 % methanol, and 5 % glycerol for 30 minutes. Radioactivity was enhanced with Enlightning solution (NEN Life Science, Boston, MA, USA), the gel was dried at 80°C for 2 hours, and it was exposed to Kodak X-Omat AR film at −70°C.
Immunofluorescence assay
Cells in 2-chamber slides or 6-well plates with cover-slide were pretreated with 1 μM GA for 2 hours, and then transfected with 0.5 μg/well pcDNA3-ErbB2/DK plus 1.5 μg/well pEYFP-Golgi. Sixteen hours after transfection, the cells were rinsed once with PBS, fixed with 3.7 % formaldehyde at room temperature for 10 minutes, rinsed again with PBS, and permeabilized with 0.2 % Triton X-100 at room temperature for 10 minutes. Cells were then washed with PBS, blocked with 1 % bovine serum albumin at 4°C for 1 hour, and incubated with ErbB2 antibody (Ab-5, 10 μg/mL) at 4°C for 1 hour. After washing with PBS, slides were overlaid with fluorescent secondary antibodies (1:500) and incubated for an additional hour at 4°C. Nuclei were visualized by 4′,6-diamidino-2-phenylindole staining. After being thoroughly washed in PBS and water, slides were air dried, and mounted with SlowFade mounting medium (Molecular Probes, Eugene, OR, USA). Fluorescence was visualized by use of a Zeiss Axioskop microscope and an Optronics CCD camera. Specificity of the ErbB2 antibody used was confirmed by lack of signal in the untransfected COS7 cells. Lack of crossover of ErbB2 and pEYFP signals was confirmed in cells stained for either marker alone.
Biotinylation and precipitation of cell surface proteins
COS7 cells in 100 × 10–mm dishes were transfected with 5 μg pcDNA3-ErbB2/DK plasmid DNA. Twenty hours after transfection, the cells were rinsed 3 times with cold borate buffer (10 mM, pH 8.5, 150 mM NaCl). Biotinylation was accomplished by incubating cells in borate buffer containing sulfosuccinimidyl -6- (biotinamido) hexanoate (NHS-LC)-biotin (0.5 mg/mL, Pierce) on ice for 30 minutes. Residual biotinylation reagent was quenched by washing cells 3 times with PBS containing 15 mM glycine. Cells were lysed with TMNSV. ErbB2 proteins were immunoprecipitated as described previously. For precipitation of biotinylated proteins, 1 mg of cell lysate was incubated with 20 μL streptavidin-agarose beads at 4°C for 1 hour, and washed 4 times with lysis buffer. The resultant Western blots were probed with ErbB2 antibody.
All experiments were performed at least 3 times, and consistent results were obtained in each case.
RESULTS
We recently reported that WX514 exhibits a 90-fold weaker affinity for Grp94 when compared with Hsp90. In contrast, WX514's affinity for Hsp90 is only one-third that of GA (Xu et al 2001). Thus, WX514 can be used to preferentially disrupt Hsp90-mediated signaling at concentrations well below its Kd for Grp94. In order to determine the relative contributions of Hsp90 and Grp94 to the stability and maturation of nascent, full-length ErbB2, we compared the ability of GA and WX514 to affect these parameters. Transiently transfected COS7 cells were pulsed with radioactive methionine and cysteine and chased in nonradioactive medium. At the times shown in Figure 1, ErbB2 was immunoprecipitated and treated with endoH. Sensitivity to endoH is characteristic of glycosidic linkages formed in the ER (Tarentino et al 1974). Once glycoproteins reach the trans-Golgi, their carbohydrate residues have been remodeled and have become resistant to endoH. Thus, endoH sensitivity is representative of ER localization as well as of the lack of protein maturation. Although the half-life of ErbB2 was greater than 8 hours in untreated cells, both GA and WX514 dramatically destabilized the nascent protein (Fig 1A, top). Additionally, in the presence of both drugs, the entire pool of detectable ErbB2 remained sensitive to endoH, whereas in untreated cells, the protein rapidly acquired resistance to endoH, even prior to chase (Fig 1A, top). In contrast to full-length ErbB2, the stability and maturation of nascent ErbB2/DK were resistant to both GA and WX514 (Fig 1A, bottom).
Fig 1.
Geldanamycin (GA) and WX514 both affect the stability and maturation of newly synthesized ErbB2. (A) Sixteen hours after transfection of ErbB2 constructs, COS7 cells, untreated or pre-treated for 2 hours with 1 μM GA or 10 μM WX514, were pulsed with radioactive methionine or cysteine, chased for various times in nonradioactive medium, and lysed as described in Materials and Methods. Equal amounts of soluble proteins were immunoprecipitated with anti-ErbB2 antibody and subjected to endoglycosidase H (endoH) digestion. After digestion, proteins were separated on 4–20 % SDS-PAGE. The dried gel was exposed to Kodak X-Omat AR film. In drug-treated samples, drugs were present throughout the pulse-chase period. Because of the size difference of the 2 proteins, cells were cotransfected with ErbB2 and ErbB2/DK. EndoH sensitivity was demonstrated by faster migration after enzyme treatment. Single transfection with either construct alone gave identical results. (B) Cells were treated similarly as in (A), but the proteasome inhibitor ALLN was added 2 hours prior to and during pulse-chase. The ratio of the band densities of mature (slower migrating) and immature (faster migrating) ErbB2 (C) and ErbB2/DK (D) were determined by integrating the optical band densities using NIH Image software, and plotted as a fraction of mature protein vs chase time.
Because both drugs made full-length ErbB2 extremely unstable, it was difficult to assess their effect on ErbB2 maturation. Addition of a proteasome inhibitor along with WX514 permitted the observation that maturation of full-length ErbB2 (eg, loss of endoH sensitivity) was only slightly retarded when compared with untreated cells (Fig 1 B [top],C). Thus, the observed inhibition of maturation of nascent ErbB2 obtained with WX514 appears to be secondary to drug-induced instability of the protein. Similar results were obtained for GA (data not shown). As expected, WX514 had very little effect on the maturation of ErbB2/DK protein (Fig 1 B [bottom],D).
Even though WX514 failed to affect the maturation of full-length ErbB2 in the presence of a proteasome inhibitor, we reasoned that this might be because of the poor affinity of WX514 for Grp94. Thus, in order to evaluate better the role of Grp94 in ErbB2 maturation and to eliminate the possibly complicating destabilizing effects of these drugs on ErbB2, we tested the effect of GA, an equipotent inhibitor of both Hsp90 and Grp94, on the maturation of ErbB2/DK. COS7 cells were transfected with ErbB2/DK, pulsed with radioactive methionine and cysteine, and chased for various times in complete medium. In agreement with the data in Figure 1A, GA did not affect the half-life of ErbB2/DK (Fig 2 A [top],B). Additionally, GA had only a slight effect on ErbB2/DK maturation (Fig 2 A [bottom],C). These results are similar to the small effect of WX514 on the maturation of full-length ErbB2 in the presence of a proteasome inhibitor (Fig 1 B,C). Although GA nonspecifically lowered the amount of protein expressed from the transfected plasmids (as determined by similar inhibition in COS7 cells of a green fluorescent protein (GFP)-expressing plasmid, data not shown), the conclusions we have drawn depend on ErbB2 stability and maturation, not on its rate of synthesis.
Fig 2.
Geldanamycin (GA) affects neither the stability nor the maturation of ErbB2/DK. COS7 cells were transfected with ErbB2/DK and, 16 hours later, left untreated or exposed to GA as in Figure 1. Pulse-chase analysis, endoglycosidase H sensitivity, and graphing of the results were carried out as described in the legend to Figure 1.
In order to document the ability of ErbB2/DK to reach the Golgi in the presence of GA, we cotransfected ErbB2/DK with the Golgi-localizing fusion construct pEYFP-Golgi. COS7 cells were pretreated with GA for 2 hours prior to transfection and during the 16-hour posttransfection period. At the end of this time, cells were fixed, the cellular distribution of ErbB2/DK was visualized by immunofluorescence, and the Golgi were identified by the native fluorescence of the pEYFP-Golgi gene product. As can be seen in Figure 3 (top panels), within 16 hours of transfection, ErbB2/DK could be found throughout the cell (red), including in distinct perinuclear structures that were also labeled by the Golgi marker (green). Overlapping the 2 signals revealed colocalization of the Golgi marker with the perinuclear-localized ErbB2/DK (Fig 3, right panel). The presence of GA throughout the experiment had no effect on ErbB2/DK distribution (Fig 3, bottom panels), confirming that GA did not affect the trafficking of ErbB2/DK from ER to Golgi.
Fig 3.
Geldanamycin (GA) does not affect endoplasmic reticulum to Golgi trafficking of ErbB2/DK. COS7 cells, untreated or pretreated with 1 μM GA for 2 hours, were transfected with pcDNA3-ErbB2/DK plus pEYFP-Golgi. Sixteen hours after transfection, cells were stained with anti-ErbB2. Panels A–C, untreated; panels D–F, GA treated. Panels A and D, ErbB2/DK; panels B and E, pEYFP-Golgi; panels C and F, overlay. Nuclei are visualized by 4′,6-diamidino-2-phenylindole (blue) staining. Golgi vary in size in the different fields examined in this and similar experiments, and no consistent effect of GA on Golgi size could be documented
Finally, to determine unambiguously whether GA affected the trafficking of ErbB2 from the ER to the plasma membrane, we transfected ErbB2/DK ± GA, as in the previous experiment. Sixteen hours after transfection, we labeled the surface of intact cells with biotin-X-NHS, lysed the cells, and divided the lysates in half. One half of each lysate was immunoprecipitated with antibody to ErbB2, and the other half of each lysate was affinity purified with streptavidin agarose. After SDS-PAGE and electrotransfer, the membrane was immunoblotted with antibody to ErbB2. Whereas immunoprecipitation with ErbB2 antibody recognizes both intracellular and extracellular ErbB2, affinity precipitation with streptavidin agarose identifies only the plasma membrane pool of ErbB2, whose extracellular domain is accessible to biotinylation. As can be seen from the data in Figure 4, although GA somewhat reduced the overall expression of ErbB2/DK, it did not affect the fraction of total ErbB2/DK that could be biotinylated (and thus was at the cell surface) 16 hours after transfection. Thus, a similar proportion of newly synthesized ErbB2/DK attains a mature glycosylation state, traffics to the Golgi, and reaches the plasma membrane in the presence or absence of GA.
Fig 4.
GA does not affect the proportion of newly synthesized ErbB2/DK reaching the cell surface. COS7 cells were transfected with pcDNA3-ErbB2/DK. Twenty hours after transfection, cell surface proteins were biotinylated with NHS-LC-biotin. Cells were lysed, and total cellular ErbB2 proteins were immunoprecipitated as described earlier. For precipitation of biotinylated proteins, cell lysate was incubated with streptavidin-agarose beads. Western blots were probed with ErbB2 antibody (Ab-3). SA, streptavidin
DISCUSSION
GA and WX514 equally affected the stability of nascent, full-length ErbB2 at the level of the ER. These data support the hypothesis that Hsp90 interaction with the cytoplasmic kinase domain of ErbB2 is required for both the stability and the resultant maturation of the newly synthesized protein. WX514 was able to efficiently destabilize nascent, full-length ErbB2 at a concentration comparable to that of GA but far below the drug's affinity for Grp94. Likewise, neither the stability, nor the maturation, nor the trafficking of nascent ErbB2/DK was significantly affected by GA, even though we previously demonstrated that the drug disrupts ErbB2 association with Grp94 (Chavany et al 1996). Taken together, these data support the hypothesis that Hsp90 association with the cytoplasmic domain of nascent, full-length ErbB2 is both necessary and sufficient for the protein's maturation in and transit through the ER, whereas Grp94 appears to have a limited role in these processes. Although the function of Grp94 in protein maturation and processing in the ER remains unclear, its lack of involvement in ErbB2 biogenesis is surprising. Nonetheless, in agreement with our data, a recent study emphasized the importance of Hsp90 in the stability and trafficking of another nascent transmembrane protein, cystic fibrosis transmembrane regulator (Loo et al 1998).
Finally, these data are of potential clinical significance because a GA derivative, 17-allylaminogeldanamycin (17-AAG), is currently in a multi-institution Phase I clinical trial in cancer patients. Like GA, 17-AAG binds to Hsp90 and Grp94 with similar affinities (Neckers, unpublished observations). Some of the dose-limiting toxicities observed with 17-AAG might be caused by disruption of an as yet unknown Grp94 function. As both newly synthesized and mature ErbB2 are destabilized by disruption of their association with Hsp90 only, an Hsp90-specific inhibitor similar to WX514 may ultimately prove to be most effective in vivo.
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