geldanamycin stimulates internalization of erbb2 in...

85Research Article

IntroductionIt is well-established that the receptor tyrosine kinase ErbB2 isoverexpressed in some cancers and has a long half-life at theplasma membrane, where it appears to be the preferredheterodimerization partner for the other ErbB family members(Baulida et al., 1996; Graus-Porta et al., 1997; Wang et al.,1999; Yarden, 2001; Yarden and Sliwkowski, 2001). ErbB2 isassociated with a more aggressive disease, and it is a validateddrug target in breast cancer (Hynes and Stern, 1994; Klapperet al., 2000; McCann et al., 1991; Yarden, 2001). Thus, it isimportant to improve our understanding of the mechanism(s)that underlie possible downregulation of ErbB2.

Inhibition of the chaperone HSP-90 with geldanamycinleads to downregulation of many HSP-90 client proteins, ofwhich ErbB2 is among the most affected (Neckers andIvy, 2003). HSP-90 inhibition causes ErbB2 to becomeubiquitinylated by the co-chaperone CHIP (Xu et al., 2002;Zhou et al., 2003), however the subsequent events haveremained elusive and several different cellular mechanismshave been suggested to take part in the downregulation ofErbB2. Assuming that ErbB2 is a recycling receptor (Austin etal., 2004; Yarden, 2001), geldanamycin could cause a shift inthe traffic of ErbB2 from a recycling pathway to a degradative,lysosomal pathway as suggested by Austin and co-workers(Austin et al., 2004). However, if ErbB2 is an internalization-resistant receptor (Hommelgaard et al., 2004; Longva et al.,

2005; Wang et al., 1999), then it is more plausible thatgeldanamycin stimulates the internalization of ErbB2 fromthe plasma membrane, resulting in delivery to lysosomes.Additionally, Tikhomirov and Carpenter observed that ErbB2becomes fragmented after geldanamycin stimulation and theyproposed a model involving cleavage of the ErbB2 kinasedomain by cytoplasmic proteases, followed by lysosomal andproteasomal degradation of the N-terminal and C-terminalparts of ErbB2, respectively (Tikhomirov and Carpenter,2000). Additionally, proteasomal inhibitors can efficientlyrescue ErbB2 from geldanamycin-induced downregulation(Mimnaugh et al., 1996), and this has led to a wide acceptanceof proteasomal proteolysis as the mechanism degrading ErbB2(Citri et al., 2002; Hong et al., 1999; Way et al., 2004; Xu etal., 2001; Zheng et al., 2000). The following questions arisefrom this. (1) Does geldanamycin induce lysosomaldegradation of ErbB2 by reducing its recycling or byincreasing its internalization? (2) Is cleavage of the ErbB2kinase domain required for internalization? (3) What is the roleof proteasomal activity in the internalization of ErbB2?

To answer these questions, we have used a non-invasivereceptor internalization assay based on confocal microscopy,allowing us to analyze receptor internalization, recycling,transport to lysosomes, as well as cytoplasmic receptorcleavage in both individual cells and cell populations. We showthat without geldanamycin treatment ErbB2 is not internalized

The potent oncoprotein and receptor tyrosine kinaseErbB2 is remarkable because it resists efficientdownregulation. However, ErbB2 can be downregulated bythe HSP-90 inhibitor geldanamycin, but the underlyingcellular mechanisms are uncertain. Apparently, delivery ofErbB2 to lysosomes, cleavage of the ErbB2 kinase domainand proteasomal activity are all processes that are involved.Using a non-invasive confocal microscopical assay allowingquantitative analysis of ErbB2 internalization in cellpopulations, we show that whereas ErbB2 is resistant tointernalization in untreated SK-BR-3 cells, geldanamycinstimulates internalization and subsequent degradationin lysosomes. This process depends on proteasomalactivity, which is a regulatory upstream event in ErbB2internalization rather than the actual mechanism ofdegradation. ErbB2 can be internalized as a full-lengthprotein, thus cleavage of the ErbB2 kinase domain is not a

requirement for geldanamycin-stimulated internalization.Moreover, as shown by FRAP (fluorescence recovery afterphotobleaching) and electron microscopy, geldanamycininduces an increase in the amount of mobile ErbB2 and aredistribution of ErbB2 in the plasma membrane makingthe receptor accessible to endocytosis. Cells with mostErbB2 endocytosis also have the highest fraction of mobileErbB2. It is concluded that geldanamycin stimulatesinternalization of full-length ErbB2 in a proteasome-dependent manner leading to lysosomal degradation.

Supplementary material available online athttp://jcs.biologists.org/cgi/content/full/119/1/85/DC1

Key words: ErbB2, Endocytosis, Geldanamycin, Proteasome,Lysosome

Summary

Geldanamycin stimulates internalization of ErbB2 in aproteasome-dependent wayMads Lerdrup, Anette M. Hommelgaard, Michael Grandal and Bo van Deurs*Structural Cell Biology Unit, Department of Medical Anatomy, The Panum Institute, Blegdamsvej 3C, University of Copenhagen,2200 Copenhagen, Denmark*Author for correspondence (e-mail: [email protected])

Accepted 21 September 2005Journal of Cell Science 119, 85-95 Published by The Company of Biologists 2006doi:10.1242/jcs.02707

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to any significant level, and that the primary action ofgeldanamycin is to stimulate internalization of ErbB2 ratherthan redirecting internalized ErbB2 from a recycling pathwayto lysosomes. Moreover, proteasomal activity is required forthe internalization of ErbB2 and therefore indirectly forlysosomal degradation of ErbB2. Finally, we show that,although cytoplasmic cleavage of ErbB2 is observed in somecells, this cleavage is not required for ErbB2 internalization.

ResultsEstablishing a non-invasive microscopical ErbB2-internalization assayErbB2 does not bind any ligands with high affinity, and theligands that can activate ErbB2 bind to other ErbB familymembers with higher affinity. Thus, classical receptorinternalization assays based on the binding and uptake ofiodinated or fluorescent ligand are of little use, as they mightmeasure internalization of other ErbB family members insteadof ErbB2. Alternative strategies, based on labeled antibody

binding and internalization followed by acid stripping orquenching of surface-bound antibody, require harsh conditionsfor efficient stripping, and importantly the assays maythemselves induce crosslinking and internalization of thereceptors. We therefore used a noninvasive ErbB2internalization assay with no preparative steps before fixation.First, SK-BR-3 cells are fixed, and surface ErbB2 is labeled(termed ErbB2surface hereafter). Next, cells are permeabilizedand ErbB2 labeled again with the same primary antibody(termed ErbB2total hereafter) (Fig. 1A). Confocal microscopyof cells stained in this manner revealed that ErbB2surface andErbB2total colocalize at exposed surfaces (Fig. 1B, left panel),confirming that internalized ErbB2 does not normallyaccumulate intracellularly to any detectable degree, and thatthe amount of ErbB2 in the biosynthetic pathway is very small.Internalization of ErbB2 induced by extensive crosslinking(Guillemard et al., 2005; Hommelgaard et al., 2004) caused afraction of the receptor population to become internalized andinaccessible to ErbB2surface staining (Fig. 1B, right panel).

Journal of Cell Science 119 (1)

Fig. 1. Measuring ErbB2 internalization insingle cells and cell populations in a non-invasive manner. (A) The method used tostudy ErbB2 internalization and cleavagemicroscopically. The upper image showsfixed, non-permeabilized cells that arestained with an antibody (Sc08)recognizing an extracellular ErbB2epitope followed by Alexa Fluor 488-conjugated goat anti-mouse secondaryantibody (G�M-488) (green, this stainingis termed ErbB2surface). The lower imageshows cells that subsequently arepermeabilized and stained with Sc08 againplus Alexa Fluor 568-conjugated G�Msecondary antibody (G�M-568) (red, thisstaining is termed ErbB2total) and anantibody recognizing a cytoplasmic C-terminal ErbB2 epitope (2242) followedby Alexa Fluor 633-conjugated goat-anti-rabbit secondary antibody (G�R-633)(blue, this staining is termedErbB2Cterminal). (B,C) Confocal images ofSK-BR-3 cells stained for ErbB2 asdescribed in A. ErbB2 is internalized afterantibody crosslinking of ErbB2 (B). Thiswas done by incubating the cells withSc08 for 5 minutes followed by G�M for30 minutes at 37°C. Similarly, ErbB2 isinternalized after 3 �M geldanamycintreatment as indicated (C). Arrows in theenlarged image indicate that surfaceexposed ErbB2 tends to aggregate beforeinternalization. (D) Scatter diagrams ofpixel intensities from an untreated cell anda cell treated with 3 �M geldanamycin for2 hours and stained as in A. The x-axis depicts the fluorescence intensity of ErbB2surface (green), whereas the y-axis depicts the fluorescenceintensity from ErbB2total (red). The analyzed cells are shown in the upper right corners of the scatter diagrams, and the region of interest isencircled in a green oval. The blue overlay represents pixel intensities that are below the threshold and therefore excluded from the analysis.The Pearson’s correlation coefficients, r2

i, are calculated from all pixels above the threshold in the selected area and shown in the top of thediagram. Thus, reduced r2

i reflects internalization because of less correlation between ErbB2surface and ErbB2total. (E) Histograms of thecolocalization values for ErbB2surface and ErbB2total (r2

i) from SK-BR-3 cells treated with 3 �M geldanamycin as indicated and stained asdescribed in A. Note that increased amounts of internalized ErbB2 are seen with time after geldanamycin treatment (decreasing r2

i values).(F) Histogram of the r2

i values from SK-BR-3 cells where ErbB2 is internalized after antibody crosslinking as in B. Cells were stained asdescribed in A. Bars, 20 �m.

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87ErbB2 uptake requires proteasomal activity

Following geldanamycin stimulation of SK-BR-3, T47D orMCF7 cells, a part of ErbB2 relocated from the surface tointracellular vesicles (Fig. 1C, supplementary material Fig. S2,not shown). It is noteworthy that the surface stainingoften appeared punctuated after geldanamycin stimulation,suggesting that the receptor aggregated prior to internalization(Fig. 1C).

For a non-biased study of a larger cell population, we useda semiquantitative approach based on 2D scatter diagrams ofthe pixel intensities of individual cells. First, we analyzedErbB2surface compared with ErbB2total staining (exemplified inFig. 1D) in untreated cells, which because of the high degreeof colocalization yielded a linear correlation with a highPearson’s correlation coefficient (r2) in the 2D pixel scatterdiagram (Fig. 1D). In cells in which a part of ErbB2 wasinternalized due to geldanamycin treatment, the staining ofintracellular structures with only ErbB2total resulted in reducedcorrelation and a lower r2 (Fig. 1D, right panel). Thus, reducedr2, when comparing ErbB2surface and ErbB2total staining, is aquantitative measure of ErbB2 internalization and termed r2

i.(For validation, see Fig. S1 in supplementary material.)

When measuring the r2i values after geldanamycin

stimulation over a 4 hour time course, we observed a gradualdecrease in average r2

i (Fig. 1E). However, at later time points

the amount of intracellular ErbB2 varied widely from cell tocell resulting in a broad range of r2

i values. The variation inthe r2

i values did reflect actual differences in the cells ratherthan uncertainties from the quantification, and could also beobserved in cells where internalization had been induced bycrosslinking antibodies (Fig. 1F).

ErbB2 is degraded in lysosomes after geldanamycintreatmentTo confirm that geldanamycin induces delivery of ErbB2 tolysosomes as previously reported (Austin et al., 2004; Longvaet al., 2005; Tikhomirov and Carpenter, 2000), we showed thatErbB2 colocalizes both with transferrin in endosomes as wellas cathepsin D in late endosomes/lysosomes (Fig. 2A,B). Tofurther establish the nature of the ErbB2-labeled intracellularstructures seen after geldanamycin stimulation, immunogoldlabeling of ultracryosections was used. After 2 hours ofgeldanamycin treatment, typical late endosomes/lysosomes ofthe multivesicular body (MVB) type were ErbB2-labeled, andthe gold particles were confined to the inner vesicles of thestructures rather than to the limiting membrane, as expected forproteins traveling along the degradative pathway (Fig. 2C).Double-labeling experiments confirmed that these ErbB2-labeled late endosomes/lysosomes also contained cathepsin D

Fig. 2. ErbB2 is internalizedand degraded in lysosomes inresponse to geldanamycintreatment. (A,B) Confocalimages of SK-BR-3 controlcells and cells treated with 3�M geldanamycin for 2 hoursat 37°C. (A) Thirty minutesbefore fixation Alexa Fluor633-conjugated transferrin wasadded to the cells. Afterfixation and permeabilizationthe cells were stained withprimary antibody againstErbB2 (Sc08) and G�M-488.Note that ErbB2 is internalizedafter geldanamycin (GA)treatment and colocalizes withtransferrin (arrows). (B) Afterfixation and permeabilizationthe cells were stained withSc08 and A561 againstcathepsin D followed byG�M-568 and GaR-633.Colocalization of internalizedErbB2 and cathepsin D isindicated with arrows. (C)Ultracryosections of SK-BR-3cells treated withgeldanamycin for 2 hoursbefore fixation. Labeling withSc08 followed by G�M-10shows ErbB2 in MVBs. (D)Ultracryosection of ageldanamycin-treated SK-BR-3 cell treated as in C. The section was first immunogold-labeled for cathepsin D (A561, PAG-5) and then forErbB2 (Sc08, G�M-10) as in C. Three MVBs are seen (M1-M3, M2 cut tangentially). In M1 and M3 double-labeling for cathepsin D andErbB2 is seen. (E) Histogram of the r2

i values from SK-BR-3 cells either pre-treated with 200 nM bafilomycin or left untreated for 1 hourbefore both samples were incubated with 3 �M geldanamycin added for 4 hours. Cells were stained as described in Fig. 1A, and the statisticalanalysis done using a two-sided Mann-Whitney U-test. Bars, 20 �m (A,B); 200 nm (C,D).

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(M1 & M3 in Fig. 2D). In contrast, very dense (terminal)lysosomes that were heavily labeled for cathepsin D onlycontained little ErbB2, presumably because internalized ErbB2had been degraded (not shown). In control cells not treated withgeldanamycin, the intracellular labeling for ErbB2 was verylow and labeled MVBs very rarely seen (not shown).

To confirm that degradation of ErbB2 actually took place inlysosomes we co-treated the cells with 200 nM bafilomycin,an inhibitor of lysosomal degradation (Van Deurs et al.,1996). SK-BR-3 or T47D cells that were both treated withbafilomycin and geldanamycin did indeed accumulate moreinternalized ErbB2 than cells treated with geldanamycin alone(Fig. 2E, supplementary material Fig. S2).

Geldanamycin influences ErbB2 internalization ratherthan recyclingTo examine whether the lack of intracellular ErbB2 revealedby our non-invasive internalization assay was due to efficientrecycling from endosomes (Austin et al., 2004) or to impairedinternalization (Hommelgaard et al., 2004; Longva et al., 2005;Wang et al., 1999), we treated the cells with 10 �M monensin,a well-established inhibitor of recycling (Basu et al., 1981;Stein et al., 1984). In SK-BR-3 or T47D cells incubated with

monensin for 2 hours to block receptor recycling, only verylittle ErbB2 was seen intracellularly (Fig. 3A, left panel,supplementary material Fig. S2) as reported by others (Longvaet al., 2005). Although a few cells in the population hadincreased amounts of intracellular ErbB2 and decreased r2

ivalues, the two populations did not differ significantly (P=0.24,two-sided Mann-Whitney U-test) (Fig. 3B, compare black barsin the upper and lower graph). This makes the possibility ofefficient recycling less likely as a general mechanism.Importantly, when geldanamycin was added to SK-BR-3 orT47D cells where recycling was blocked with monensin theamount of intracellular ErbB2 increased a lot (Fig. 3A, rightpanel, supplementary material Fig. S2). This was reflected inthe r2

i values of the population (Fig. 3B, lower graph), showingthat the main effect of geldanamycin is to increaseinternalization of ErbB2.

ErbB2 internalization correlates with changes of itsmobility and distribution in the plasma membraneThe increase in ErbB2 internalization suggests that theproperties of ErbB2 residing in the plasma membrane changeafter geldanamycin treatment. We therefore performed FRAPanalysis of ErbB2-CFP before and after geldanamycintreatment. Live-cell imaging revealed that in SK-BR-3 cellsa high amount of ErbB2-CFP resides in microspikes andirregular ruffles of the plasma membrane, but not inintracellular vesicles (Fig. 4A, upper panel). Aftergeldanamycin treatment ErbB2 association with thesemembrane protrusions became much less pronounced (Fig. 4Alower panel), and ErbB2 had a significantly increased mobilefraction (Fig. 4B). The cells with the highest endocytosis ofErbB2 also had the highest mobile ErbB2-CFP fractions (Fig.4C). This supports the notion that ErbB2 is released from aretention mechanism prior to internalization.

To evaluate whether geldanamycin treatment also influencedthe distribution of ErbB2 in the plasma membrane, we used apre-embedding electron microscopy approach (Hommelgaardet al., 2004). Whereas ErbB2 is preferentially associated withmembrane protrusions in control SK-BR-3 cells (Fig. 4D), itfrequently had a more even distribution in the plasmamembrane after geldanamycin treatment (Fig. 4E,F).Importantly, in contrast to the control situation where ErbB2was almost completely excluded from clathrin-coated pits(0.05% in coated pits), ErbB2 was much more frequently foundin clathrin-coated pits and various membrane invaginationsafter geldanamycin treatment (0.8% in coated pits) (a total of4393 and 1596 gold particles were counted from 20 and 42different cell profiles in control or geldanamycin-treated cells,respectively). However, this redistribution of ErbB2 was lesspronounced than after antibody crosslinking of ErbB2 wheremost ErbB2 was found in the bulk membrane between theprotrusions, in clathrin-coated pits and other invaginations(Fig. 4G) (Hommelgaard et al., 2004). Taken together, thestudies with FRAP and electron microscopy show thatgeldanamycin influences the distribution of ErbB2 in theplasma membrane in a way that favors ErbB2 internalization.

Cleavage of ErbB2s kinase domain is not needed forinternalizationTo study if ErbB2 became cleaved before being internalized,we extended the internalization assay described in Fig. 1 with


Fig. 3. Geldanamycin affects ErbB2 internalization rather thanrecycling. (A) Confocal images of control and geldanamycin-treatedSK-BR-3 cells where recycling was inhibited with monensin. Thecells were treated with 3 �M geldanamycin (GA) and/or 10 �Mmonensin (Mon) and incubated for 2 hours at 37°C. (B) Histogramsof the r2

i values from SK-BR-3 cells. The upper graph shows cellsthat were treated with 3 �M geldanamycin for 2 hours or untreated.The lower graph shows cells that were treated for 2 hours with 3 �Mgeldanamycin and/or 10 �M monensin alone. Cells were stained asdescribed in Fig. 1A, and the statistical analysis done using a two-sided Mann-Whitney U-test. Note that intracellular ErbB2 is seenonly after geldanamycin treatment. Bar, 20 �m.

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a simultaneous labeling of the cytoplasmic C-terminusof ErbB2 (termed ErbB2Cterminal hereafter) (Fig. 1A).Untreated SK-BR-3 cells exhibited a high degree ofoverlap between ErbB2total and ErbB2Cterminal stainings atthe cell surface (Fig. 5A). Importantly, the geldanamycin-treated cells exhibited more diffuse blue ErbB2Cterminalstaining of the cytoplasm, showing that some cleavage takesplace (Fig. 5A). This cleavage was confirmed with anotherantibody against the C-terminal part of ErbB2 (Fig. 5B), andin SK-BR-3 cells transfected with ErbB2-CFP (Fig. 5C).

Triple labeling that simultaneously visualized internalizationand cleavage of ErbB2 in unstimulated cells revealed

colocalization of all three stains at the cell surface(represented in white in Fig. 5D). Althoughsome geldanamycin-treated cells had diffusecytoplasmic ErbB2Cterminal staining, most cells hadErbB2-containing intracellular vesicles that alsostained positive for ErbB2Cterminal (Fig. 5D). Thissuggests that a significant portion of the receptorremains uncleaved before and duringinternalization, and that cleavage of the kinasedomain is not a requirement for ErbB2internalization (Fig. 5D). To substantiate thisfinding with a biochemical assay, we affinity-precipitated biotinylated, internalized ErbB2, andused the purified protein for western blotting. Asexpected, this showed that geldanamycin induceda large increase in the amount of internalizedErbB2 while having no effect on internalization ofthe transferrin receptor. Importantly, the majorityof internalized ErbB2 remained full length,supporting the idea that cleavage is not arequirement for ErbB2 internalization (Fig. 5E).To further support this notion, we performedimmunogold double-labeling of ultracryosectionswith antibodies against the extracellular portion(the N-terminus) and intracellular portion (the C-terminus) of ErbB2. The plasma membrane aswell as MVBs contained both the intra- andextracellular parts of ErbB2 (Fig. 5F,G). Moreover,the ratio between N- and C-terminal ErbB2labeling seen at the cell surface and in MVBs waslargely the same.

The amount of ErbB2 cleavage could bequantified from ErbB2total and ErbB2Cterminalcolocalization in a manner similar to r2

i (Fig. 1).The resulting Pearson’s correlation coefficient wastermed r2

c, and a reduced r2c indicated increased

cleavage. After geldanamycin treatment a largepart of the cell population had low r2

c values andthe average r2

c was significantly lower than in thecontrol cell population (Fig. 6A, upper panel).

To study if geldanamycin-induced lack ofcolocalization between the intra- and extracellularparts of ErbB2 could be inhibited by the pan-caspase inhibitor zVAD-fmk as reported forcleavage of the ErbB2 kinase domain (Tikhomirovand Carpenter, 2001; Tikhomirov and Carpenter,2003), SK-BR-3 cells were either treated with50 �M zVAD-fmk and geldanamycin orgeldanamycin alone. Treatment with zVAD-fmk

could suppress the reduced r2c values observed after

geldanamycin treatment to the level of cells only treated withzVAD-fmk, suggesting that zVAD-fmk could inhibitcytoplasmic cleavage of ErbB2 in this assay too (Fig. 6A, lowerpanel). It was noteworthy that zVAD-fmk did not inhibitinternalization, supporting the idea that cleavage is not neededfor internalization (Fig. 6B).

When a 2D dot plot of the r2i and r2

c values from each cellat different time points was made, the dots were markedlyspread out at each time point (Fig. 6C). For instance, after 2hours of geldanamycin treatment, the cells ranged from a highdegree of ErbB2 internalization and cleavage to virtually

Fig. 4. Geldanamycin treatment induces a redistribution of ErbB2 in the plasmamembrane and increases receptor mobility. (A) Confocal images of live SK-BR-3cells transfected with ErbB2-CFP before and after 1 hour of geldanamycintreatment. ErbB2 resides in protrusions of the plasma membrane beforestimulation, but not after stimulation with geldanamycin. (B) Diagram showingthe mobile fraction of ErbB2-CFP measured using FRAP before and aftertreatment with 3 �M geldanamycin for 15-60 minutes. The P-values are derivedusing a two-sided Student’s t-test (n=42) and the error bars represent 95%confidence intervals. (C) Mobile fractions of ErbB2-CFP in SK-BR-3 cellstreated with 3 �M geldanamycin for 1 hour. The cells were divided into twogroups depending on their degree of internalized ErbB2 as described in theMaterials and Methods. The P-value is derived using a two-sided Student’s t-test(n=79) and the error bars represents 95% confidence intervals. Note that the cellswith high internalization also exhibit a significantly higher mobile fraction.(D-G) Electron micrographs of SK-BR-3 cells immunogold labeled for ErbB2 onthe cell surface (pre-embedding immunogold labeling). CP, clathrin-coatedpits/profiles. (D) A control cell where ErbB2 is primarily associated withmembrane protrusions. (E,F) The surface of two geldanamycin-treated cellswhere the ErbB2 distribution is more even on the surface of the cell. (G) Thesurface of a cell where ErbB2 has been crosslinked to antibody. ErbB2 isassociated with the bulk membrane between protrusions including CPs. Bars,20 �m (A); 200 nm (D-G).

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unaffected cells with r2i and r2

c values resembling those ofuntreated cells (Fig. 6C).

Oddly, when cells were co-treated with zVAD-fmk andgeldanamycin ErbB2 tended to accumulate more in internalvesicular structures than when cells were treated withgeldanamycin only (compare panels C and D in Fig. 6).However, 50 �M zVAD-fmk has been reported to inhibitcathepsins as well (Foghsgaard et al., 2001; Rozman-Pungercaret al., 2003), suggesting that this accumulation might be dueto reduced lysosomal proteolysis. In cells treated withinhibitors of lysosomal proteolysis such as bafilomycin (Fig.6E), Ca074-Me (not shown), or zVAD-fmk (Fig. 6D) aninverse correlation existed between r2

i and r2c in those cells

most affected by the geldanamycin treatment. Thus, the cellswith most internalized ErbB2 also tended to have the leastErbB2 cleavage and vice versa, supporting the idea that theseevents are at least in part competing processes. However, suchan inverse correlation was not evident when the lysosomaldegradation was not inhibited (Fig. 6C) possibly because ofdegradation of internalized ErbB2.

Proteasomal activity is required for ErbB2 internalizationWestern blots of SK-BR-3 cells treated with geldanamycin

with or without the proteasome inhibitor lactacystin showedthat lactacystin could abolish ErbB2 degradation (Fig. 7A). Toexamine if proteasomal activity is needed for internalization ofErbB2 rather than for ErbB2 degradation as such, SK-BR-3cells were either pre-treated with 10 �M lactacystin or controlmedium for 1 hour followed by addition of geldanamycin andincubation for 4 hours. Strikingly, the geldanamycin-inducedinternalization of ErbB2 was almost completely inhibited bylactacystin (Fig. 7B,C), suggesting that the proteasomalactivity is involved in events upstream of ErbB2 internalizationand lysosomal degradation. Similar results were found whenT47D cells were used (supplementary material Fig. S2).In addition, no redistribution of ErbB2 was seen by electronmicroscopy when SK-BR-3 cells were incubated withlactacystin in addition to geldanamycin (data not shown). Inorder to study whether lactacystin also reduced thegeldanamycin-induced lysosomal degradation of ErbB2, weco-treated the cells with 200 nM bafilomycin, geldanamycinand lactacystin. In cells co-treated with all three compoundsvery little ErbB2 accumulated in the intracellularcompartments compared to cells co-treated with bafilomycinand geldanamycin only, confirming that lactacystin reduces thegeldanamycin-induced lysosomal degradation of ErbB2 as


Fig. 5. Cleavage of the ErbB2kinase domain is not neededfor internalization. (A-D).Confocal images of SK-BR-3cells stained for ErbB2 asdescribed in Fig. 1A. (A)ErbB2Cterminal stainingdislocates from ErbB2total andis diffusely localized in thecytoplasm after 3 �Mgeldanamycin treatment,suggesting cytoplasmiccleavage of the receptor. (B) Inaddition, detection of the C-terminus of ErbB2 usinganother antibody (Ab-1) alsoshowed similar dislocationfrom ErbB2total after 3 �Mgeldanamycin treatment for 4hours, supporting cleavage ofErbB2. (C) In SK-BR-3 cellstransfected with ErbB2-CFPand exposed to 3 �Mgeldanamycin for 4 hours, theC-terminal CFP-tag (detectedusing an antibody against thefluorophore) is also dislocatedfrom ErbB2total aftergeldanamycin treatment. (D)ErbB2 is both internalized andcleaved (seen as diffuse bluestaining, indicated with *)after 3 �M geldanamycin treatment. The enlarged region and purple structures show that both the C- and N-termini of ErbB2 are internalized.(E) A biotinylation assay detecting internalized ErbB2. Cells were treated with 30 �M geldanamycin for 3 hours at 37°C, Sc08 1:100 for 1 hourfollowed by G�M 1:100 for 1 hour at 37°C, or left untreated for 3 hours at 4 or 37°C. Both geldanamycin and crosslinking cause increasedinternalization of ErbB2 compared to the control situation but had no effect on the transferrin receptor (TfR). Note that in contrast to the otherexperiments we have used a tenfold increased concentration of geldanamycin. (F,G) Ultracryosections of SK-BR-3 cells treated with 3 �Mgeldanamycin for 2 hours before fixation. C-terminal ErbB2 was labeled with Ab-1 followed by PAG-5 (arrows), and then N-terminal ErbB2with Sc08 followed by G�M-10 (arrowheads) on the cell surface (F) as well as in a MVB (G). Bars, 20 �m (A-D); 200 nm (F,G).

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a consequence of reduced internalization (Fig. 7B,C).Furthermore, lactacystin also inhibited the effect ofgeldanamycin in cells where recycling was inhibited withmonensin, suggesting that proteasomal activity is required atthe level of internalization rather than recycling (Fig. 7D).

DiscussionIn the present study we show that geldanamycin stimulates theinternalization of ErbB2 rather than redirecting the receptorfrom a recycling to a lysosomal pathway. Importantly, theinternalization occurs in a proteasome-dependent way andsubsequently leads to degradation in lysosomes, whereascleavage of the ErbB2 kinase domain is not required forinternalization.

It has been reported that ErbB2 becomes internalized inunstimulated cells, but efficiently recycles to the plasmamembrane (Austin et al., 2004). However, this conclusion wasmainly based on the binding of labeled herceptin (a humanizedmonoclonal antibody against ErbB2, also known astrastuzumab) to live cells followed by removal of non-internalized herceptin. Using an identical setup, Longva et al.(Longva et al., 2005) found that herceptin did not becomeinternalized, but that a significant amount of herceptin

remained at the cell surface after washing and that this couldaffect the readout. In concordance with Longva and co-workers, we found that in cells where recycling was inhibitedby monensin, very little ErbB2 accumulated in intracellularcompartments, supporting the fact that ErbB2 is notinternalized and recycled to any significant level. Austin et al.furthermore found that geldanamycin interferes with theproposed recycling of ErbB2 leading to accumulation ofmore intracellular ErbB2 along the degradative late-endosome–lysosome pathway (Austin et al., 2004). Inopposition, we report here that the amount of ErbB2 thataccumulates in intracellular structures was far higher in cellsthat had been co-treated with geldanamycin and monensin thanin cells treated with monensin alone, suggesting that the effectof geldanamycin is mainly to increase ErbB2 internalization.This internalization depends on proteasomal activity, becauselactacystin can inhibit the geldanamycin-induced accumulationin cells with monensin-blocked recycling.

It is well established that divalent antibodies can induceinternalization of membrane proteins (Huet et al., 1980;Schreiber et al., 1983; Schwartz et al., 1986), we thereforerecommend that caution is taken when exposing living cells tomultivalent probes, especially when the valence is increased

Fig. 6. Cleavage of the ErbB2kinase domain is inverselycorrelated to internalization.(A) The upper left histogramshows r2

c values from cellstreated with 3 �Mgeldanamycin for 4 hours anduntreated cells. The lowerhistogram shows similar r2

cvalues for cells that in additionhave been treated with the pan-caspase inhibitor zVAD-fmkfor 4 hours. The r2

c valueswere calculated for individualcells after staining as describedin Fig. 1A. P-values from two-sided Mann-Whitney U-testsare shown. Note that zVAD-fmk inhibited ErbB2 cleavage.(B) The r2

i values from thesame cells as in A, showingthat zVAD-fmk has no effecton ErbB2 internalization. (C-E) Scatter diagrams, whereeach dot in the scatter diagramrepresents r2

i and r2c values

from a single SK-BR-3 celltreated as indicated and stainedas described in Fig. 1A. (C)Untreated cells, cells treatedwith 3 �M geldanamycin for1, 2 or 4 hours. (D) The cellswere pre-treated with 50 �MzVAD-fmk and either added 3�M geldanamycin or left inzVAD-fmk for 4 hours. The two images to the right show two different situations where either the internalization is dominant and no cleavagecan be observed or where cleavage dominates and internalization is limited, which together with the scatter diagram suggest that theseprocesses are competing events. (E) The cells were treated with 200 nM bafilomycin for 1 hour, then 3 �M geldanamycin was added for 4hours. It is seen that cells with much intracellular ErbB2 had little cleavage and vice versa.

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with antibodies combined with gold particles or secondaryantibodies. Even monovalent Fab-fragments could potentiallyinduce conformational changes or affect protein interactions(Agus et al., 2002; Hipfner et al., 1999; Mijares et al., 2000).This emphasizes the need for non-invasive assays such as theone we have described here when studying receptorinternalization.

In concordance with the geldanamycin-induced changes inthe behavior of ErbB2 at the plasma membrane, we found byFRAP that the mobile fraction of ErbB2-CFP is increased inresponse to geldanamycin. The overall increase in the mobilefraction in response to geldanamycin treatment is limited;however this fits well with the fact that internalization of ErbB2is a relatively slow process. Nonetheless, it was possible toobserve a higher mobile ErbB2 fraction in the cells with mostinternalization, altogether suggesting that ErbB2 is releasedfrom retaining interactions. Despite this, immobilizationcannot be the only mechanism retaining ErbB2 fromendocytosis. If the retention from endocytosis relied on a stableinteraction with immobile constituents of the cell, then ErbB2should be more readily endocytosed in unstimulated cells,where roughly half of the ErbB2-CFP population is mobile.Accordingly, either the postulated retention mechanism mightbe mobile itself or its interaction with ErbB2 is dynamic.

Geldanamycin caused a redistribution of ErbB2 on the cellsurface, from protrusions to the bulk membrane, although lesspronounced than after ErbB2 crosslinking. This supports the

notion that the preferential localization to protrusions plays animportant role in keeping ErbB2 away from endocyticmembrane domains (Hommelgaard et al., 2004). Importantly,0.8% of the ErbB2 gold particles were present in coated pitsafter geldanamycin treatment (a 16-fold increase comparedwith the control situation). It is generally assumed that coatedpits occupy 1-2% of the cell surface area. This means that if areceptor does not have an internalization signal mediating itsconcentration in coated pits, about 1-2% of the plasmamembrane receptor population would be expected to be presentin coated pits at any given time point. Thus 0.8% of ErbB2found in coated pits after geldanamycin treatment is likely torepresent a fraction of the total plasma membrane ErbB2population able to diffuse without restraints in the membraneincluding coated pits. Coated pits can be formed andinternalized within a few minutes (Marsh and McMahon,1999), therefore the fraction of coated-pit-localized ErbB2 caneasily account for the amount of internalized receptor we foundafter geldanamycin treatment for 2 hours. However, moredetailed calculations are not possible because the release fromendocytic restraints and the uptake rate are not likely to beconstant during the 2 hours of geldanamycin stimulation.Clathrin-independent endocytic mechanisms could play a rolein ErbB2 uptake as well.

Tikhomirov and Carpenter (Tikhomirov and Carpenter,2000; Tikhomirov and Carpenter, 2001; Tikhomirov andCarpenter, 2003) proposed that geldanamycin induces cleavage


Fig. 7. Geldanamycin-induced ErbB2internalization requires proteasomalactivity. (A) Western blot of ErbB2 fromSK-BR-3 cells treated with 3 �Mgeldanamycin as indicated. In the lowerpanel the cells were pre-treated with theproteasome inhibitor lactacystin (10 �M)for 1 hour and added geldanamycin. Notethat ErbB2 is degraded after geldanamycintreatment, and that this depends onproteasomal activity. (B) Confocal imagesof SK-BR-3 cells treated with theproteasomal inhibitor lactacystin (10 �M)for 1 hour and then 3 �M geldanamycin(upper right image) for 4 hours. Strikingly,the internalization was inhibited comparedto cells that were only treated withgeldanamycin (upper left image).Likewise, if cells were co-treated with bothbafilomycin (200 nM) and lactacystin(lower right image) before addition ofgeldanamycin there was no internalizationof ErbB2, confirming the importance ofproteasome activity for internalization andas a consequence lysosomal degradation.(C) Upper histogram shows r2

i values fromcells treated with 3 �M geldanamycin for 4hours and cells that were treated withlactacystin for 1 hour and thengeldanamycin for 4 hours. Lowerhistogram shows similar r2

i values for cells that furthermore have been pre-treated with bafilomycin. The r2i values were calculated for

individual cells after staining as described in Fig. 1A, and P-values are from two-tailed Mann-Whitney U-tests. (D) Confocal images showingcells treated with monensin and geldanamycin for 2 hours (left image). When cells were pretreated with 10 �M lactacystin before 10 �Mmonensin and 3 �M geldanamycin were added, the geldanamycin-induced accumulation of intracellular ErbB2 in cells with inhibited recycling(left image) was efficiently inhibited (right image), suggesting that proteasomal activity is needed for ErbB2 internalization. Bars, 20 �m.

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of the ErbB2 kinase domain in the cytosol. This couldpotentially release ErbB2 from restraining protein-proteininteractions or expose cryptic motifs that stimulateinternalization. However, 50 �M zVAD-fmk caused anintracellular accumulation of ErbB2 in geldanamycin-stimulated cells resembling that of cathepsin-inhibition orinhibition of lysosomal acidification, thus this concentration ofzVAD-fmk might reduce ErbB2 degradation by inhibitinglysosomal proteolysis. Both extracellular and cytoplasmic partsof ErbB2 can be internalized and colocalized to endosomes.Additionally, purification of internalized biotinylated ErbB2confirmed that the majority is uncleaved, further suggestingthat ErbB2 is endocytosed as full-length protein and thatcleavage is not a prerequirement for internalization. However,we cannot exclude the fact that cleavage of one ErbB2 in aoligomer complex can induce internalization of the entirecomplex, and therefore several uncleaved ErbB2 molecules.Moreover, some geldanamycin-stimulated cells actually hadlittle colocalization between the intracellular and extracellularparts of ErbB2, and this cleavage was inversely related tointernalization in cells co-treated with cathepsin inhibitorsor bafilomycin, suggesting that these events are partiallycompeting.

The results reported here raise two important questions.By what means is ErbB2 retained from endocytosis inunstimulated cells? And how does proteasomal activityfacilitate the geldanamycin-induced internalization of ErbB2?Internalization of the NMDA receptor GluR1 has beensuggested to depend on preceding proteolytic breakdown of thePDZ-protein PSD-95 or an associated factor (Bingol andSchuman, 2004). PDZ proteins are important regulators ofsubcellular localization, and the PDZ proteins PICK1 andERBIN have previously been described to bind ErbB2 andaffect its signaling and localization in polarized cells (Borg etal., 2000; Jaulin-Bastard et al., 2001). However, in the caseof ErbB2 it seems unlikely that the factor retaining ErbB2from endocytosis is a PDZ protein, because ErbB2-CFP isinternalized as reluctantly as wild-type ErbB2, despite itsaltered C-terminus that is incapable of PDZ-domain binding(our unpublished results). We previously found that theretention mechanism depended on neither the actincytoskeleton nor lipid rafts (Hommelgaard et al., 2004). Thus,future studies will have to address the nature of the retentionmechanism, and if proteasomal degradation of a responsiblefactor is needed for geldanamycin-induced internalization tooccur. Alternatively, the effect of lactacystin on ErbB2internalization could be due to the endocytosis machinerycompeting with the proteasome for free ubiquitin, which isthen sequestered when the proteasome is inhibited withlactacystin. Similarly, Leithe and Rivedal have found thatproteasomal activity is needed for monoubiquitinylation ofconnexin-43 and subsequent endocytosis (Leithe and Rivedal,2004). However, Mimnaugh and co-workers have found thatErbB2 is still ubiquitinylated in cells treated with lactacystinand geldanamycin (Mimnaugh et al., 1996), suggesting thatthis is not the case.

In summary, we propose that geldanamycin-stimulateddownregulation of ErbB2 consists of several phases:proteasome-dependent release of the receptor from itsendocytic restraints; increased receptor mobility; redistributionof ErbB2 in the plasma membrane favoring endocytosis by

random entrapment; internalization; and degradation in thelysosomes. In parallel to this, cleavage of the ErbB2 kinasedomain occurs in some cells (Fig. 8).

Materials and MethodsCell cultureThe SK-BR-3 and T47D breast cancer cell lines were obtained from the AmericanType Culture Collection (Manassas, VA). The cells were grown in T25, T75 or T150flasks (Nunc, Roskilde, Denmark) and incubated at 37°C, 5% CO2 in DMEM (SK-BR-3) or RPMI-1640 (T47D) both without Phenol Red (Invitrogen, Carlsbad, CA)supplemented with 10% fetal calf serum (FCS) (Biochrom KG, Berlin), 2 mMglutamine, 10 U/ml penicillin and 10 �g/ml streptomycin (all from Invitrogen).

Subcloning of ErbB2-CFPA construct encoding ErbB2 was kindly provided by Daniel Donoghue (Bell et al.,2000). ErbB2 was PCR amplified from this using the primers 5�-GGCGCGGCTAGCATGATCAT CATGGAGCTG-3� and 5�-CCGCGCGGTA CCTCATACAGGTACATCCAG-3� (TAG Copenhagen, Denmark) containing NheI and KpnIrestriction sites. This product was digested with NheI and KpnI restriction enzymes(New England Biolabs) and subcloned into pcDNA3.1. The pcDNA3.1-ErbB2construct was digested with HindIII and NheI restriction enzymes and ligated intothe pECFP-N1 vector (Clontech, Palo Alto, CA), and the stop codon was removedby mutagenesis.

Immunofluorescence microscopyCells were plated on eight-well chamber slices (Lab-Tek, Naperville, IL). Controlcells were incubated with DMEM-HEPES buffer with 0.2% w/v BSA and 2 mMglutamine for the indicated time at 37°C, washed with PBS, and fixed in 2%paraformaldehyde in PBS (PFA) for 20 minutes at room temperature (RT). Whereindicated, cells were incubated with 50 �g/ml Alexa Fluor 633-conjugatedtransferrin (Molecular Probes, Eugene, OR), 3 �M geldanamycin (Sigma-Aldrich,St Louis, MO), primary anti-ErbB2 mouse monoclonal antibody (Sc08, Santa CruzBiotechnology, 1:100) plus secondary goat anti-mouse antibody (M8642, Sigma-Aldrich, 1:100), 10 �M lactacystin (Sigma-Aldrich), 100 nM bafilomycin (Sigma-Aldrich), 250 �M ALLN (Sigma-Aldrich), 50 �M z-Val-Ala-DL-Asp-CH2F(zVAD-fmk, Bachem, Bubendorff, Switzerland), and/or 100 �M CA-074-Me(Peptides International, Louisville, KY) in DMEM-HEPES buffer with 0.2% w/vBSA and 2 mM glutamine for the indicated time at 37°C before fixation in 2% PFA.Where indicated, cells were immunostained before permeabilization the followingway: non-specific binding was blocked with 5% goat serum (DAKO, Glostrup,Denmark) in PBS for 20 minutes, and cells were incubated with primary antibody(Sc08, 1:400) in 5% goat serum for 1 hour at RT, rinsed three times with PBS,incubated with secondary Alexa Fluor 488-conjugated goat anti-mouse antibody(G�M-488) (1:400, Molecular Probes) in 5% goat serum for 30 minutes at RT, andrinsed three times with PBS. All cells were permeabilized and blocked in blockingbuffer (5% goat serum with 0.2% saponin) for 20 minutes at RT, incubated withprimary antibodies in blocking buffer for 1 hour at RT, rinsed three times with PBS,incubated with secondary antibody in blocking buffer for 30 minutes at RT, rinsedthree times with PBS and mounted with Fluoromount G (Southern Biotechnology

Fig. 8. Tentative model of the mechanisms that influence ErbB2downregulation in response to geldanamycin. Geldanamycin releasesErbB2 from restraints that inhibit its mobility, leading tointernalization of the receptor. These processes require proteasomalactivity. Cleavage of ErbB2 is not needed for internalization of thereceptor, but is a competing event. Finally, ErbB2 is degraded inlysosomes rather than in proteasomes.

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Associates, Birmingham, AL). The primary antibodies used were Sc08 (recognizingan extracellular epitope, 1:400), 2242 (rabbit polyclonal anti-ErbB2 raised againsta synthetic peptide corresponding to residues surrounding Tyr1222 of human ErbB2,1:200, Cell Signaling Technology, Beverly, MA), Ab-1 (rabbit polyclonal anti-ErbB2 raised against a synthetic peptide corresponding to positions 1243-1255(Gullick et al., 1987), 1:100, Neomarkers, Freemont, CA), A561 (rabbit polyclonalanti-cathepsin D, used at 1:100, DAKO), or A11122 (rabbit polyclonal anti-GFP,used 1:1000, Molecular Probes); the secondary antibodies used were Alexa Fluor568-conjugated goat anti-mouse (G�M-568) and/or Alexa Fluor 633-conjugatedgoat anti-rabbit (both 1:400, Molecular Probes). The slides were examined with anLSM 510 Meta confocal microscope (Carl Zeiss, Jena, Germany) equipped with40� and 63� objectives, and using the LSM software v. 3.2 (Carl Zeiss) andoccasionally Adobe Photoshop 7.0.

Image analysis and quantification of colocalizationSeveral images of randomly selected regions from each sample were acquired withsimilar scanning parameters (63� plan-apochromat/1.4 oil, triple track, 824�824pixels, Zoom=1.8, 1.52 �second pixel dwell time, 4� averaging, pinholes 250 �m).Using the LSM software v. 3.2, individual cells were marked as regions of interest,and a fluorescence intensity threshold applied including all fluorescence from cellsbut not the space between. For each region of interest the software calculated thePearson’s correlation coefficient (r2) for Sc08/G�M-488 (ErbB2 extracellularepitope labeling before permeabilization) and Sc08/G�M-568 (ErbB2 extracellularepitope labeling after permeabilization), which we termed r2

i, as well as thePearson’s correlation coefficient for 2242/G�M-633 (ErbB2 intracellular/C-terminal epitope labeling after permeabilization) and Sc08/G�M-568 (ErbB2extracellular epitope labeling after permeabilization), which we termed r2

c.

Pre-embedding immunogold labeling for EMSK-BR-3 cells grown in T25 flasks, either control cells, cells treated withgeldanamycin for 2 hours, or with geldanamycin and lactacystin for 2 and 3 hours,respectively, were washed in PBS and fixed in 0.1% glutaraldehyde and 2% PFA in0.1 M phosphate buffer at RT for 30 minutes. After a wash in PBS the cells wereincubated with Sc08 (Santa Cruz Biotechnology) against the extracellular (N-terminal) part of ErbB2 followed by 10-nm-gold-labeled goat anti-mouse antibody(G�M-10) (Amersham Bioscience AB) and further processed for Epon embeddingand electron microscopy as previously described (Hommelgaard et al., 2004).Antibody crosslinking of ErbB2 with Sc08 followed by G�M-10 was done aspreviously described (Hommelgaard et al., 2004).

Post-embedding immunogold labeling for EMFor detection of intracellular ErbB2, control cells and cells treated withgeldanamycin for 2 hours were processed for immunogold labeling ofultracryosections as previously described (Hommelgaard et al., 2004). To detectErbB2, Sc08 was used, followed by G�M-10. This was in some experimentspreceded by incubation with a rabbit polyclonal anti-cathepsin D antibody (DAKO)followed by protein A conjugated to 5 nm gold (PAG-5). To detect the extracellular(N-terminal) and intracellular (C-terminal) parts of ErbB2 simultaneously, the C-terminal-specific rabbit polyclonal antibodies 2242 (Cell Signal Technology) or Ab-1 (Neomarkers, Freemont, CA) were used followed by PAG-5 or PAG-10, and thenthe sections were incubated with Sc08 and G�M-10 or G�M-5, respectively.

Biotin labelingCells were plated in T25 flasks. The cells were rinsed twice in ice-cold PBS withCa2+ and Mg2+ (PBS-CM) for 10 minutes at 4°C. Sulfo-NHS-SS-biotin (0.5 mg/ml,Pierce) was dissolved in PBS-CM and added to the cells at 4°C on a shaking table.After 20 minutes new 0.5 mg/ml sulfo-NHS-SS-biotin was added to the cells andfurther incubated at 4°C for 20 minutes. The cells were washed with DMEM-HEPES buffer with 1% w/v BSA and 2 mM glutamine (DMEM-BSA) for 10minutes at 4°C. Control cells were incubated with DMEM-BSA for 2 hours at 4 or37°C. Some cells were incubated with either 30 �M geldanamycin or Sc08 diluted1:100 in DMEM-BSA for 60 minutes at 37°C. The cells incubated with Sc08 werewashed and further incubated for 60 minutes at 37°C with G�M-488 diluted 1:400.The treatment was stopped by transferring the tubes back to ice, and the cells wererinsed twice with ice-cold DMEM-BSA. The biotin on the membrane surface wascleaved by incubating the cells in reducing solution [50 mM 2-sodium 2-mercaptoethanesulfonate (Sigma), 100 mM NaCl, 50 mM Tris-HCl pH 8.7, 2.5 mMCaCl2] for 20 minutes at 4°C, which was repeated twice. The cells were washedthree times with PBS-CM. Then, the cells were scraped off in lysis buffer [1% TritonX-100, 50 mM Tris-HCl pH 7.4, 150 mM NaCl, 2 mM EDTA, 10 mM NaF, 1 mMvanadate, and phosphatase inhibitor cocktail 1:100 (Sigma)], lysed for 20 minutesat 4°C, sonicated twice, and centrifuged for 5 minutes at 6000 g at 4°C. Proteinconcentrations were determined (Bio-Rad) and the samples standardized.Streptavidin-coated beads (20 �l, Sigma) were added to the samples overnight at4°C. The cells were centrifuged for 30 seconds at 4°C at 16,000 g, the pellet waswashed four times in lysis buffer and centrifuged at high speed for 30 seconds at4°C after each wash. The pellet was then dissolved in lysis buffer and Laemmli

buffer with 50 mM DTT added, heated for 5 minutes at 95°C, and finally processedfor western blotting.

Western blottingThe samples were resolved on 8% bis-tris-acrylamide gels, transferred to apolyvinylidene difluoride membrane (Amersham Biosciences AB, Uppsala,Sweden), and non-specific binding was blocked with 5% milk powder (Bio-Rad) inPBS containing 0.1% Tween 20 (blocking buffer). Blots were probed with primaryantibody (mouse monoclonal anti-ErbB-2, 1:3000, Ab-17, Neomarkers; or mousemonoclonal anti-TfR, 1:1000, 13-6800, Zymed) in blocking buffer, followed by ahorseradish peroxidase (HRP)-conjugated goat anti-mouse secondary antibody(1:2000, DAKO, Carpentaria, CA) in blocking buffer. The HRP signal was detectedusing ECL+ (Amersham Biosciences AB) chemiluminescence reagent and Hyper-ECL films (Amersham Biosciences AB).

FRAP, data analysis and endocytosis estimationSK-BR-3 cells were plated on custom-made 60 mm plates with a 0.2 mm coverglass inserted in the centre, allowed to grow for 24 hours, and transfected with 1.0�g ErbB2-CFP using Fugene 6 (Roche, Basel, Switzerland). After 48 hours themedium was substituted with 37°C HEPES buffer (20 mM HEPES pH 7.5, 140 mMNaCl, 2 mM CaCl2, 10 mM KCl, 1 mg/ml Glucose), and the cells were studied at37°C using a heated microscope stage. CFP was excited and photobleached usinga 458 nm Argon laser, and CFP fluorescence was separated from background usingthe Meta detector of a Zeiss LSM510-Meta and linear unmixing. Fluorescencerecovery of bleached regions was recorded for ~40 frames, and compensated for theloss of fluorescence in unbleached regions due to scanning. The data were exportedto Excel, corrected for background fluorescence and bleaching, and exported toOrigon 7.0 for non-linear analysis using the function for simple diffusion:F(t)=(F(0)+F�(t/T1/2))/(1+t/T) (Yguerabide et al., 1982). The obtained F(0), F� andT1/2 were used to calculate the diffusion coefficient D and the mobile fraction Mf.The mobile fraction was calculated as: Mf=(F�–F(0))/(Fbefore–F(0)), where Fbefore isthe fluorescence in the region of interest before bleaching. The diffusion coefficientD was calculated as: D=�r2/(4T1/2), where r is the radius of the bleached region,and the � parameter depends on the percentage of fluorescence bleached and canbe found in Table 1 of Yguerabide et al. (Yguerabide et al., 1982).

The degree of internalization was estimated by three people who independentlyassigned the values 0 (no internalization), 1, 2, 3 (most internalization), or X (notdetermined) to the images without knowing the identity of the cell images, and theaverage value was calculated for each cell disregarding Xs. The approach wasvalidated by comparing the obtained values of control cells to geldanamycin-treatedcells, which resulted in a highly significant increase in the degree of internalization(data not shown). The geldanamycin-treated cells were then divided into twopopulations, depending on whether their assigned value was above or below theaverage degree of internalization.

We appreciate the skilled technical assistance offered by UllaHjortenberg, Mette Ohlsen, and Izabela Rasmussen. We thank SilasBruun, Kirstine Roepstorff and Frederik Vildhardt for valuablediscussions. This study was supported by grants from the DanishCancer Society, the Danish Medical Research Counsil, the NovoNordic Foundation, and the John and Birthe Meyer Foundation.

ReferencesAgus, D. B., Akita, R. W., Fox, W. D., Lewis, G. D., Higgins, B., Pisacane, P. I.,

Lofgren, J. A,, Tindell, C., Evans, D. P., Maiese, K. et al. (2002). Targeting ligand-activated ErbB2 signaling inhibits breast and prostate tumor growth. Cancer Cell 2,127-137.

Austin, C. D., De Maziere, A. M., Pisacane, P. I., van Dijk, S. M., Eigenbrot, C.,Sliwkowski, M. X., Klumperman, J. and Scheller, R. H. (2004). Endocytosis andsorting of ErbB2 and the site of action of cancer therapeutics trastuzumab andgeldanamycin. Mol. Biol. Cell 15, 5268-5282.

Basu, S. K., Goldstein, J. L., Anderson, R. G. and Brown, M. S. (1981). Monensininterrupts the recycling of low density lipoprotein receptors in human fibroblasts. Cell24, 493-502.

Baulida, J., Kraus, M. H., Alimandi, M., Di Fiore, P. P. and Carpenter, G. (1996). AllErbB receptors other than the epidermal growth factor receptor are endocytosisimpaired. J. Biol. Chem. 271, 5251-5257.

Bell, C. A., Tynan, J. A., Hart, K. C., Meyer, A. N., Robertson, S. C. and Donoghue,D. J. (2000). Rotational coupling of the transmembrane and kinase domains of the Neureceptor tyrosine kinase. Mol. Biol. Cell 11, 3589-3599.

Bingol, B. and Schuman, E. M. (2004). A proteasome-sensitive connection betweenPSD-95 and GluR1 endocytosis. Neuropharmacology 47, 755-763.

Borg, J. P., Marchetto, S., Le, B. A., Ollendorff, V., Jaulin-Bastard, F., Saito, H.,Fournier, E., Adelaide, J., Margolis, B. and Birnbaum, D. (2000). ERBIN: abasolateral PDZ protein that interacts with the mammalian ERBB2/HER2 receptor.Nat. Cell Biol. 2, 407-414.

Citri, A., Alroy, I., Lavi, S., Rubin, C., Xu, W., Grammatikakis, N., Patterson, C.,Neckers, L., Fry, D. W. and Yarden, Y. (2002). Drug-induced ubiquitylation and


Jour

nal o

f Cel

l Sci

ence


degradation of ErbB receptor tyrosine kinases: implications for cancer therapy. EMBOJ. 21, 2407-2417.

Foghsgaard, L., Wissing, D., Mauch, D., Lademann, U., Bastholm, L., Boes, M.,Elling, F., Leist, M. and Jaattela, M. (2001). Cathepsin B acts as a dominantexecution protease in tumor cell apoptosis induced by tumor necrosis factor. J. CellBiol. 153, 999-1010.

Graus-Porta, D., Beerli, R. R., Daly, J. M. and Hynes, N. E. (1997). ErbB-2, thepreferred heterodimerization partner of all ErbB receptors, is a mediator of lateralsignaling. EMBO J. 16, 1647-1655.

Guillemard, V., Nedev, H. N., Berezov, A., Murali, R. and Saragovi, H. U. (2005).HER2-mediated internalization of a targeted prodrug cytotoxic conjugate is dependenton the valency of the targeting ligand. DNA Cell Biol. 24, 350-358.

Gullick, W. J., Berger, M. S., Bennett, P. L., Rothbard, J. B. and Waterfield, M. D.(1987). Expression of the c-erbB-2 protein in normal and transformed cells. Int. J.Cancer 40, 246-254.

Hipfner, D. R., Mao, Q., Qiu, W., Leslie, E. M., Gao, M., Deeley, R. G. and Cole, S.P. (1999). Monoclonal antibodies that inhibit the transport function of the 190-kDamultidrug resistance protein, MRP. Localization of their epitopes to the nucleotide-binding domains of the protein. J. Biol. Chem. 274, 15420-15426.

Hommelgaard, A. M., Lerdrup, M. and van Deurs, B. (2004). Association withmembrane protrusions makes ErbB2 an internalization-resistant receptor. Mol. Biol.Cell 15, 1557-1567.

Hong, R. L., Spohn, W. H. and Hung, M. C. (1999). Curcumin inhibits tyrosine kinaseactivity of p185neu and also depletes p185neu. Clin. Cancer Res. 5, 1884-1891.

Huet, C., Ash, J. F. and Singer, S. J. (1980). The antibody-induced clustering andendocytosis of HLA antigens on cultured human fibroblasts. Cell 21, 429-438.

Hynes, N. E. and Stern, D. F. (1994). The biology of erbB-2/neu/HER-2 and its role incancer. Biochim. Biophys. Acta 1198, 165-184.

Jaulin-Bastard, F., Saito, H., Le, B. A., Ollendorff, V., Marchetto, S., Birnbaum, D.and Borg, J. P. (2001). The ERBB2/HER2 receptor differentially interacts withERBIN and PICK1 PSD-95/DLG/ZO-1 domain proteins. J. Biol. Chem. 276, 15256-15263.

Klapper, L. N., Kirschbaum, M. H., Sela, M. and Yarden, Y. (2000). Biochemical andclinical implications of the ErbB/HER signaling network of growth factor receptors.Adv. Cancer Res. 77, 25-79.

Leithe, E. and Rivedal, E. (2004). Ubiquitination and down-regulation of gap junctionprotein connexin-43 in response to 12-O-tetradecanoylphorbol 13-acetate treatment. J.Biol. Chem. 279, 50089-50096.

Longva, K. E., Pedersen, N. M., Haslekas, C., Stang, E. and Madshus, I. H. (2005).Herceptin-induced inhibition of ErbB2 signaling involves reduced phosphorylation ofAkt but not endocytic down-regulation of ErbB2. Int. J. Cancer 116, 359-367.

Marsh, M. and McMahon, H. T. (1999). The structural era of endocytosis. Science 285,215-220.

McCann, A. H., Dervan, P. A., O’Regan, M., Codd, M. B., Gullick, W. J., Tobin, B.M. and Carney, D. N. (1991). Prognostic significance of c-erbB-2 and estrogenreceptor status in human breast cancer. Cancer Res. 51, 3296-3303.

Mijares, A., Lebesgue, D., Wallukat, G. and Hoebeke, J. (2000). From agonist toantagonist: Fab fragments of an agonist-like monoclonal anti-beta(2)-adrenoceptorantibody behave as antagonists. Mol. Pharmacol. 58, 373-379.

Mimnaugh, E. G., Chavany, C. and Neckers, L. (1996). Polyubiquitination andproteasomal degradation of the p185c-erbB-2 receptor protein-tyrosine kinase inducedby geldanamycin. J. Biol. Chem. 271, 22796-22801.

Neckers, L. and Ivy, S. P. (2003). Heat shock protein 90. Curr. Opin. Oncol. 15, 419-424.

Rozman-Pungercar, J. et al. (2003). Inhibition of papain-like cysteine proteases andlegumain by caspase-specific inhibitors: when reaction mechanism is more importantthan specificity. Cell Death. Differ. 10, 881-888.

Schreiber, A. B., Libermann, T. A., Lax, I., Yarden, Y. and Schlessinger, J. (1983).Biological role of epidermal growth factor-receptor clustering. Investigation withmonoclonal anti-receptor antibodies. J. Biol. Chem. 258, 846-853.

Schwartz, A. L., Ciechanover, A., Merritt, S. and Turkewitz, A. (1986). Antibody-induced receptor loss. Different fates for asialoglycoproteins and the asialoglycoproteinreceptor in HepG2 cells. J. Biol. Chem. 261, 15225-15232.

Stein, B. S., Bensch, K. G. and Sussman, H. H. (1984). Complete inhibitionof transferrin recycling by monensin in K562 cells. J. Biol. Chem. 259, 14762-14772.

Tikhomirov, O. and Carpenter, G. (2000). Geldanamycin induces ErbB-2 degradationby proteolytic fragmentation. J. Biol. Chem. 275, 26625-26631.

Tikhomirov, O. and Carpenter, G. (2001). Caspase-dependent cleavage of ErbB-2 bygeldanamycin and staurosporin. J. Biol. Chem. 276, 33675-33680.

Tikhomirov, O. and Carpenter, G. (2003). Identification of ErbB-2 kinase domainmotifs required for geldanamycin-induced degradation. Cancer Res. 63, 39-43.

Van Deurs, B., Holm, P. K. and Sandvig, K. (1996). Inhibition of the vacuolar H(+)-ATPase with bafilomycin reduces delivery of internalized molecules from maturemultivesicular endosomes to lysosomes in HEp-2 cells. Eur. J. Cell Biol. 69, 343-350.

Wang, Z., Zhang, L., Yeung, T. K. and Chen, X. (1999). Endocytosis deficiency ofepidermal growth factor (EGF) receptor-ErbB2 heterodimers in response to EGFstimulation. Mol. Biol. Cell 10, 1621-1636.

Way, T. D., Kao, M. C. and Lin, J. K. (2004). Apigenin induces apoptosis throughproteasomal degradation of HER2/neu in HER2/neu-overexpressing breast cancer cellsvia the phosphatidylinositol 3-kinase/Akt-dependent pathway. J. Biol. Chem. 279,4479-4489.

Xu, W., Mimnaugh, E., Rosser, M. F., Nicchitta, C., Marcu, M., Yarden, Y. andNeckers, L. (2001). Sensitivity of mature Erbb2 to geldanamycin is conferred by itskinase domain and is mediated by the chaperone protein Hsp90. J. Biol. Chem. 276,3702-3708.

Xu, W., Marcu, M., Yuan, X., Mimnaugh, E., Patterson, C. and Neckers, L. (2002).Chaperone-dependent E3 ubiquitin ligase CHIP mediates a degradative pathway for c-ErbB2/Neu. Proc. Natl. Acad. Sci. USA 99, 12847-12852.

Yarden, Y. (2001). Biology of HER2 and its importance in breast cancer. Oncology 61,1-13.

Yarden, Y. and Sliwkowski, M. X. (2001). Untangling the ErbB signalling network. Nat.Rev. Mol. Cell Biol. 2, 127-137.

Yguerabide, J., Schmidt, J. A. and Yguerabide, E. E. (1982). Lateral mobility inmembranes as detected by fluorescence recovery after photobleaching. Biophys. J. 40,69-75.

Zheng, F. F., Kuduk, S. D., Chiosis, G., Munster, P. N., Sepp-Lorenzino, L.,Danishefsky, S. J. and Rosen, N. (2000). Identification of a geldanamycin dimer thatinduces the selective degradation of HER-family tyrosine kinases. Cancer Res. 60,2090-2094.

Zhou, P., Fernandes, N., Dodge, I. L., Reddi, A. L., Rao, N., Safran, H., DiPetrillo,T. A., Wazer, D. E., Band, V. and Band, H. (2003). ErbB2 degradation mediated bythe co-chaperone protein CHIP. J. Biol. Chem. 278, 13829-13837.

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