ErbB2 Trafficking and Degradation Associated with K48 and K63 Polyubiquitination

Abstract

The overexpressed ErbB2/HER2 receptor is a clinically validated cancer target whose surface localization and internalization mechanisms remain poorly understood. Downregulation of the overexpressed 185 kDa ErbB2 receptor is rapidly (2–6 h) induced by the HSP90 chaperone inhibitor, geldanamycin (GA), while its downregulation and lysosomal degradation are more slowly (24 h) induced by the proteasome inhibitor, bortezomib/PS341. In PS341 treated SK-BR-3 cells, overexpressed ErbB2 co-precipitates with the E3 ubiquitin ligase, c-Cbl, and also with the deubiquitinating enzyme, USP9x; moreover, siRNA downregulation of USP9x enhances PS341 induced ErbB2 down-regulation. Since polyubiquitin linkages via lysine 48 (K48) or 63 (K63) can differentially address proteins for 26S proteasomal degradation or endosome trafficking to the lysosome, multiple reaction monitoring (MRM) mass spectrometry (MS) and polyubiquitin linkage-specific antibodies were used to quantitatively track K48 and K63 linked ErbB2 polyubiquitination following either GA or PS341 treatment of SK-BR-3 cells. MRM/MS revealed that unlike the rapid, modest (4- to 8-fold) and synchronous GA induction of K48 and K63 polyubiquitinated ErbB2, PS341 produces a dramatic (20- to 40-fold) sequential rise in polyubiquitinated ErbB2 consistent with K48 polyubiquitination followed by K63 editing. Fluorescence microscopic imaging confirmed that PS341, but not GA, induces co-localization of K48 and K63 linked polyubiquitin with perinuclear lysosome-sequestered ErbB2. Thus, ErbB2 surface overexpression and recycling appear to depend on its polyubiquitination and deubiquitination; as well, the contrasting effects of PS341 and GA on ErbB2 receptor localization, polyubiquitination and degradation point to alternate cytoplasmic trafficking likely regulated by different K48 and K63 polyubiquitin editing mechanisms.

Introduction

Overexpression of the ErbB2/HER2 receptor tyrosine kinase (RTK) occurs in up to 25% of human breast cancers where it is predictive of aggressive disease and poor clinical outcome, and serves as a validated clinical target for a growing class of anti-ErbB2 therapeutics (1, 2). In addition to therapeutic antibodies and small molecule kinase inhibitors that specifically interfere with ErbB2 receptor function, some targeted agents in clinical development actually depend upon the endocytic recycling and intracellular trafficking of membrane overexpressed ErbB2 (3, 4), calling attention to this poorly understood but critical feature of ErbB2 receptor biology. Yet another class of therapeutics being developed to treat ErbB2-positive cancers include derivatives of the benzoquinoid ansamycin antibiotic, geldanamycin (GA), which bind to and inactivate an essential chaperone of membrane-bound ErbB2, heat shock protein 90 (HSP90), inducing endocytosis and receptor down-regulation by proteasomal and lysosomal mechanisms (5, 6). Given its unique mechanism of action and the particular sensitivity of overexpressed ErbB2 to HSP90 inhibitors, GA has become a favorite tool for studying ErbB2 receptor endocytosis and trafficking (7–10). Less well appreciated is the recent observation that proteasome inhibition by the clinically approved dipeptide boronate, bortezomib (PS341), can also induce ErbB2 internalization and lysosomal decay (11).

Unlike the epidermal growth factor receptor (EGFR) whose ligand-activated endocytosis and intracellular trafficking has become a model for all receptor tyrosine kinases (12, 13), the ligand-less ErbB2 receptor is considered to be endocytosis impaired, although there is poor understanding of how this is achieved (8, 12–14). Instead of being internalized and endosomally routed into vesicles and the multivesicular body (MVB) pathway for subsequent lysosomal degradation as with ligand-activated EGFR, ErbB2 dimers are largely recycled back to the plasma membrane for reactivation (8, 12, 13). ErbB2 can even transfer its endocytosis impairment to EGFR and other heterodimerizing partners, although this impairment can be fully abrogated by HSP90 inhibition which induces rapid ErbB2 ubiquitination followed by receptor down-regulation (15, 16). The importance of ubiquitination in regulating ErbB2 endocytosis and plasma membrane overexpression remains obscure, although it has been suggested that the endocytosis impairment of ErbB2 is due to its intrinsic resistance to ubiquitination (12). Thus, new insight is needed into the mechanisms and ubiquitination codes associated with ErbB2 endocytosis and down-regulation (13, 17).

Ubiquitination is a reversible post-translational modification regulating a wide variety of protein signaling mechanisms including endocytic down-regulation of ErbB family receptors (13, 17). The 76 amino acid ubiquitin (Ub) polypeptide can be covalently attached via its C-terminal glycine (G76) to either a target protein’s lysine (K) ε-amino group or to another target-bound Ub molecule via one of its seven internal K residues, forming topologically distinct polyubiquitin (polyUb) chains. Ub (E3) ligases interact with specific Ub conjugating (E2) enzymes associated with different intracellular sites and linkage specific reaction products. Two different E3 ligases, CHIP (COOH-terminus of HSP70-interacting protein) and c-Cbl, have been reported to associate with ErbB2, each capable of forming different types of polyUb chains (13–16, 18, 19). The most abundant polyUb chains in living cells are K48 linkages, which adopt a closed conformation and serve as a signal for target protein degradation by the 26S proteasome. Next most common, K63 linkages adopt an extended linear conformation as non-proteasome addressing dock sites for such diverse cellular functions as DNA repair, signal transduction, transcription, endosomal trafficking and MVB sorting for lysosomal decay (12, 13, 17, 20). In particular, K63 linked polyubiquitination has been clearly associated with clathrin-dependent endocytosis and lysosomal down-regulation of ligand activated EGFR (13, 21, 22). As well, ubiquitination can be edited via developmentally and subcellularly restricted deubiquitinating isopeptidases (deubiquitylating enzymes, DUBs), regulating the intracellular fate of a target protein (23, 24). Two DUBs potentially relevant to the endocytosis and trafficking of breast epithelial membrane proteins are USP8/UBPy, known to facilitate the down-regulation of EGFR and ErbB3 (25), and USP9x/FAM, an endosomally localized regulator of epithelial stem/progenitor cell function (26, 27).

The present study employed SK-BR-3 cells to explore the dependence of ErbB2 overexpression on both polyubiquitination and deubiquitination mechanisms. The contrasting effects of PS341 and GA on ErbB2 polyubiquitination were assessed using multiple reaction monitoring (MRM) mass spectrometry (MS) and linkage-specific monoclonal antibodies (28), demonstrating that proteasome and HSP90 inhibitors down-regulate overexpressed ErbB2 by alternative cytoplasmic trafficking and degradative pathways linked to different K48 and K63 polyubiquitin chain editing mechanisms.

Materials and Methods

Cells, reagents and antibodies

SK-BR-3 cells were obtained from American Type Culture Collection (Manassas, VA) and grown under ATCC-recommended culture conditions. Bortezomib (PS341) was kindly provided by Millennium Pharmaceuticals (Cambridge, MA); Geldanamycin (GA) and chloroquine was purchased from Sigma-Aldrich (St. Louis, MO). The mouse IgG₁ monoclonal anti-c-ErbB2/c-Neu (Ab-3), developed against the C-terminal 1242–1255 amino acids of human ErbB2, was purchased from Calbiochem (San Diego, CA). Mouse monoclonals to HSP90 and HSP70 were purchased from Stressgen (Ann Arbor, MI). Antibodies to actin, LAMP2b, clathrin heavy chain (CHC), USP9x, and vimentin were purchased from Abcam (Cambridge, MA); those to ERalpha (F10), c-Cbl, CHIP, and Epsin-1 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA); antibody to Eps 15 was purchased from Novus Biologicals (Littleton, CO); and antibody to total ubiquitin was purchased from Dako (UK). Polyubiquitin linkage-specific recombinant IgG antibodies against K48 (Apu2.07) and K63 (apu2.16) chains (28) were a kind gift from Genentech (South San Francisco, CA).

Expression constructs, siRNA and transfection reagents

Sequence confirmed CMV promoter-driven expression plasmids #17608 (pRK5-HA-wtUb) and #17606 (pRK5-HA-Ub-K63), encoding wildtype Ub and mutated Ub capable of only forming K63 polyUb linkages (all other lysines mutated to arginines) respectively (29), were obtained from Addgene (Cambridge, MA). Two additional expression constructs were generated from these using the Stratagene Quickchange kit forming pRK-HA-monoUb from #17606, with all lysines mutated to arginines (unable to form polyUb), and pRK-HA-Ub-K63R from #17608, with lysine 63 mutated to arginine 63 (only incapable of forming K63 linked polyUb). SK-BR-3 transfections were performed in 10mm plates over 4–6h using serum-free media and Lipofectamine 2000 (Invitrogen). siRNA (and control) reagents targeting Epsin, Eps15 and clathrin heavy chain (CHC) were commercially obtained from Dharmacon (Lafayette, CO) and used at final concentrations of 100nmol/L.

Whole cell and lysosomal extracts, immunoprecipitation and immunoblotting

Whole cell lysates were prepared in a modified RIPA buffer (50 mM HEPES, pH 7.5, 100 mM NaCl, 2 mM Na3VO4, 1% NP-40, 1% deoxycholate and 0.01% SDS) containing a protease inhibitor cocktail (Mini Complete) and 5 μM ubiquitin aldehyde (Calbiochem). SDS was omitted from lysates used for immunoprecipitation and mass spectrometry. Whole cell lysates were homogenized by sonication (550 Sonic Dismembrator) twice for 10 s each and cleared by centrifugation for 10 min at 4°C. Protein content of supernatants was determined by Bradford assay (Bio-Rad Laboratories). Lysosomal fractions were isolated from cultured cells by density gradient separation (34200 rpm, 2h) using the Lysosome Enrichment Kit for Tissue and Cultured Cells and protocol from Pierce (Rockford, IL). Prior to immunoprecipitation, 0.5 to 1 μg of lysate protein was precleared with protein A Sepharose beads (Santa Cruz Biotechnology) and then incubated with 5 μg of anti-ErbB2 for 3 h at 4°C under continuous agitation. Immune complexes were recovered using 50 μl of Protein A Sepharose, washing three times in lysis buffer and twice with TBE, and resuspension in 75 μl of Laemmli buffer before gel electrophoresis and immunoblotting. Electrophoresis was performed using 4 to 12% Nu-Page Bis-Tris gradient gels (Invitrogen, Carlsbad, CA) with MOPS running buffer (Invitrogen); and proteins were transferred onto polyvinylidene difluoride membranes (Millipore, Billerica, MA) blocked with 5% nonfat milk in PBS with 0.05% Tween 20. For sequential antibody probing, blots were stripping using Restore Western Blot Stripping Buffer (Pierce Biotechnology, Rockford, IL).

Immunofluorescence Imaging

Cells seeded in eight-chamber slides (Lab-Tek II; Nalge Nunc International, Rochester, NY) were cultured overnight, washed with PBS and fixed with 4% PFA for 10 min at room temperature. After cell permeabilization in 0.5% Triton X for 10 min, cell mounted slides were treated for 30 min (room temperature) with 5% normal donkey serum blocking solution and then overnight in primary antibody (2.5% serum dilution). Secondary antibodies were donkey anti-mouse Alexa Fluor 488 and anti-rabbit Alexa Fluor 555 (Invitrogen), serum diluted and incubated for 30 min. Slides were mounted in Prolong Gold (Invitrogen), stained with DAPI and left overnight before fluorescence microscopic analysis.

Multiple Reaction Monitoring Mass Spectrometry (MRM/MS)

ErbB2 immunoprecipitates were resolved on SDS-PAGE gels, and the gel region extending from the full-length ErbB2 band (185 kDa) to just below the visible myosin band (260 kDa) was excised for trypsin digestion. Typically, 30 ErbB2 peptides were identified in each sample; in addition to ErbB2 only ubiquitin could be detected from this gel region. Eluted gel samples were analyzed by nanoLC-MRM/MS on a 4000 QTRAP hybrid triple quadrupole/linear ion trap mass spectrometer (Applied Biosystems, Framingham, MA). Chromatography was performed using an Eksigent (Eksigent, Dublin, CA) NanoLC-2D LC system with buffer A (0.1% formic acid) and buffer B (90% acetonitrile in 0.1% formic acid). Digested samples were loaded at 20 μL/min (0.1% formic acid) onto a 5 mm × 300 μm Dionex (Sunnyvale, CA) reversed phase C18 column (5 μm, 100Ǻ) and eluted at 300 nL/min with a 75 μm inner diameter Integrafrit column (New Objective, Woburn, MA), packed in house with 10–12 cm of ReproSil-Pur C18-AQ 3 μm reversed phase resin (Dr. Maisch GmbH, Germany), using a gradient of 2–70% buffer B over 32 min. Peptides were ionized using a PicoTip emitter (75 μm, 15 μm tip, New Objective, Woburn, MA). Data acquisition was performed with an ion spray voltage of 2450 V, curtain gas of 10 psi, nebulizer gas of 20 psi, an interface heater temperature of 150°C, and unit resolution. Collision energy, declustering potential, and collision cell exit potential were optimized to achieve maximum sensitivity. Ub linked peptides were analyzed as previously reported (30, 31); with the 7 possible (K6, K11, K27, K29, K33, K48, K63) Ub linkages and their respective tryptic peptides interrogated by their corresponding m/z MRM ion transitions as shown in Supplement Table 1.

Results

Proteasome and HSP90 inhibition induce different rates of ErbB2 chaperone exchange and down-regulation associated with divergent intracellular trafficking

Given the nanomolar sensitivity of SK-BR-3 cells to both GA and PS341 and the need to monitor effects in viable cells over a 24h exposure period, a maximum GA dose of 20nM was chosen and compared to PS341 doses ranging from 10–50nM. Previous PS341 studies had shown the SK-BR-3 72h IC₅₀ dose to be 4nM, with virtually complete inhibition of SK-BR-3 proteasome activity achieved by 24h treatment with 25nM PS341 (11). As shown in Figure 1 immunoblots, 20nM PS341 caused a 50% reduction in total ErbB2 protein expression after 24h, while the same dose of GA caused a 50% loss of total ErbB2 protein by 6h and more profound reduction by 24h. Both GA and PS341 caused dissociation of HSP90 from ErbB2 and replacement by HSP70, with kinetics reflecting their different rates of ErbB2 down-regulation: ErbB2 chaperone exchange was induced within 2h of HSP90 inhibition but required 24h of proteasome inhibition. Immunofluorescence imaging using the same C-terminal specific anti-ErbB2 antibody used for immunoblotting revealed differential cytoplasmic trafficking of intact ErbB2 following GA and PS341 (Figure 1, panels C and D). Untreated SK-BR-3 cells showed predominant plasma membrane ErbB2 overexpression; within 4h of PS341 treatment ErbB2 appeared partially internalized, within 8h it appeared as lower intensity scattered cytoplasmic aggregates, and within 24h it appeared as intense polar and perinuclear aggregates. Previously we showed that PS341 causes this same pattern of ErbB2 internalization and perinuclear aggregation in another ErbB2 overexpressing breast cancer cell line, BT474 (11). A similar time course of GA treatment in SK-BR-3 cells showed more rapid internalization with appearance of small scattered cytoplasmic ErbB2 aggregates by 2h, followed by ever-diminishing ErbB2 cytoplasmic signals between 2–24h without any appearance of perinuclear ErbB2 aggregates.

Figure 1.

Proteasome and HSP90 inhibition induce differential rates of ErbB2 chaperone exchange, internalization and decay in SK-BR-3 cells. A. Immunoblots (IB) of whole cell extracts showing loss of 185 kDa ErbB2 protein expression (above), and ErbB2 immunoprecipitates (IP) showing exchange in associated HSP90 and HSP70 chaperones (below), 24h after SK-BR-3 treatment with a proteasome inhibiting dose of bortezomib/PS341 (20nM). B. Similar assays as performed in panel A following SK-BR-3 treatment with an HSP90 inhibiting dose of geldanamycin/GA (20nM), showing rapid loss of ErbB2 protein expression and chaperone exchange within 2h. C. Immunofluorescence imaging showing loss of plasma membrane ErbB2 expression by 8h and appearance of polarized perinuclear aggregation of c-terminally intact ErbB2 by 24h treatment with PS341. D. Immunofluorescence imaging showing rapid loss of surface ErbB2 expression and scattered, punctate cytoplasmic ErbB2 appearing within 2h of GA treatment, without subsequent perinuclear aggregation of ErbB2. Image scale bars (20 microns) are indicated in white.

Proteasome inhibition induces clathrin-independent internalization and lysosomal trafficking of ErbB2

To exclude the possibility that proteasome inhibitor treatment induces redistribution of ErbB2 into aggresomes, we co-stained PS341 treated and untreated SK-BR-3 cells for the aggresome marker, vimentin; we found no co-localization of vimentin and ErbB2 indicating that PS341 does not induce redistribution of ErbB2 into aggresomes (data not shown). In contrast, immunofluorescence imaging after 24h of PS341 treatment showed co-localization of ErbB2 with lysosome associated membrane protein-2b (LAMP2b), while untreated cells showed no ErbB2 and LAMP2b co-localization (Figure 2, panel A). GA treatment out to 24h failed to induce any co-localization of ErbB2 with LAMP2b (results not shown). To confirm ErbB2 trafficking to lysosomes, whole cell and lysosomal fractions were immunoblotted for ErbB2 and LAMP2 proteins; lysosomal extracts were enriched in LAMP2, but only the 24h PS341 treated lysosomal fraction was enriched in ErbB2 protein (panel B). While chloroquine had been shown to inhibit lysosomal proteolysis of ErbB2 induced by PS341 (11), it did not inhibit GA induced ErbB2 degradation (results not shown). ErbB2 internalization induced by GA had been reported by some to occur via a clathrin-dependent process (9, 10) and by others via a clathrin-independent process (32, 33). Transfection of SK-BR-3 cells with siRNAs targeting clathrin heavy chain (CHC) or the endocytic adaptors Epsin 1 and Eps15 were performed to achieve >80% target knock down prior to GA or PS341 (24h) culture treatment. We observed slight inhibition of GA induced ErbB2 down-regulation following CHC knock down but not following Epsin 1 or Eps15 knock down (Supplement Figure 1). By comparison, PS341 treatment produced the expected 50% reduction in total ErbB2 levels but this was not prevented by knock down of any of the three regulators of clathrin-dependent endocytosis, consistent with a clathrin-independent internalization process (Figure 2, panel C). Of note, inhibition of new protein synthesis by 24h concurrent cycloheximide treatment prevented both PS341-induced perinuclear trafficking and lysosomal degradation of ErbB2 (Figure 2D).

Figure 2.

ErbB2 internalization and lysosomal trafficking induced by proteasome inhibition is clathrin-independent and requires new protein synthesis. A. Immunofluorescence imaging of control and PS341 treated (10nM, 24h) SK-BR-3 cells showing redistribution of ErbB2 (green) from plasma membrane to perinuclear aggregates, relocation of cytoplasmic lysosomes as indicated by the lysosome associated membrane protein LAMP2b (red), and the co-localization of these two signals (merge, yellow). Cell nuclei are counterstained with DAPI (blue). B. Immunoblots of whole cell and LAMP2-enriched lysosomal fractions of control and treated SK-BR-3 cells confirming lysosomal accumulation of full-length (185 kDa) ErbB2 after 24h PS341 treatment. C. Immunoblots of control and siRNA down-regulated (Epsin1, Eps 15, or clathrin heavy chain/CHC) SK-BR-3 cells indicating clathrin independence of PS341-induced loss of ErbB2 receptor expression. SiRNA controls for CHC and Epsin1 show effective siRNA knock down. D. Paired immunoblots and immunofluorescence images of treated SK-BR-3 cells (500μg/ml cycloheximide, 50nM PS341 with/without cycloheximide; 16h) showing abrogation of PS341-induced ErbB2 internalization and lysosomal decay by complete inhibition of protein synthesis. Image scale bars (20 microns) are indicated in white.

ErbB2 overexpression is regulated by both polyubiquitinating and deubiquitinating mechanisms

SK-BR-3 cells were transiently transfected with expression constructs encoding wildtype Ub or different Ub chain mutants, including K48RUb (unable to form K48 polyUb chains), K63RUb (unable to form K63 polyUb chains), monoUb (unable to form any polyUb chains), K48Ub (able to form only K48 polyUb chains) and K63Ub (able to form only K63 polyUb chains). Transfectants were analyzed for short term cell growth and by immunoblotting and immunofluorescence imaging for ErbB2 expression. Transfectants overexpressing the K48Ub, K63Ub, and K48RUb chain mutants showed near normal ErbB2 morphology (results not shown). In contrast, cells overexpressing the K63RUb and monoUb chain mutants lost the uniform surface overexpression of ErbB2 seen in wildtype Ub transfectants and expressed large sub-surface cytoplasmic ErbB2 aggregates (Figure 3, panel A). All but the monoUb transfectants showed near normal short-term culture growth and, when treated for 24h with either GA or PS341, showed control levels of ErbB2 down-regulation (result not shown). In contrast, the monoUb transfectants appeared incapable of short-term growth.

Figure 3.

ErbB2 receptor overexpression in SK-BR-3 is regulated by both polyubiquitinating and deubiquitinating mechanisms. A. Immunofluorescence images of ErbB2 expression in SK-BR-3 cells transiently transfected with constructs overexpressing either wildtype ubiquitin (Ub) or two different lysine (K) mutated variants, one incapable of forming any type of polyubiquitin chain (monoUb) and another capable of forming all types except K63 linked polyubiquitin (K63RUb). Only the wildtype Ub cells show plasma membrane overexpression of ErbB2. Image scale bar (20 microns) is indicated in white. B. (Above) Whole cell lysates of SK-BR-3 cells immunoblotted (IB) for ErbB2 and USP9x after culture treatment with/without PS341 (20nM, 24h) and pretreatment with/without siRNA to knock down USP9x. (Below) ErbB2 (185 kDa) and control (ERalpha) immunoprecipitates (IP) of untreated whole cell SK-BR-3 lysates immunoblotted (IB) for ErbB2 and USP9x. C. (Above) Immunoblots (IB) of whole cell SK-BR-3 lysates after culture treatment with/without PS341 (20nM, 6h) probed for CHIP, c-Cbl, and actin. (Below) ErbB2 immunoprecipitates (IP) of above SK-BR-3 lysates probed for ErbB2, CHIP, and c-Cbl.

ErbB2 immunoprecipitates were probed for their association with the deubiquitylating enzymes, USP8/UBPy and USP9x/FAM. In control and PS341 treated SK-BR-3 cells, ErbB2 was not found to be associated with USP8/UBPy (data not shown). In contrast, ErbB2 immunoprecipitates were found to contain significant USP9x relative to control immunoprecipitates prepared using anti-ERalpha IgG (Figure 3, panel B). Since SK-BR-3 cells do not express ERalpha, this approach controlled for nonspecific protein binding to either the IgG or the protein A conjugated sepharose. Neither GA nor PS341 treatments (6h) produced any significant change in total cell USP9x levels; however, a 24h time course study of PS341 treatment showed persistent USP9x co-association with ErbB2 (data not shown). Following ~90% knock down of USP9x, ErbB2 protein levels were unchanged in control SK-BR-3 cells; as well, this knockdown had no effect on ErbB2 localization in the presence or absence of PS341 or GA, and did not alter GA-induced down-regulation of ErbB2 (data not shown). However, USP9x knock down significantly enhanced PS341 (24h) induced lysosomal decay of ErbB2 (Figure 3, panel B).

We also probed for treatment associated changes in total and ErbB2-associated CHIP and c-Cbl proteins. In whole cell lysates of SK-BR-3, CHIP levels were lower and more difficult to detect than c-Cbl levels; but neither GA nor PS341 treatments (6h) significantly altered cell content of these E3 ligases (Figure 3, panel C). While immunoprecipitates from these same lysates showed no ErbB2 association with CHIP (before or after GA and PS341 treatments; data not shown), these same ErbB2 immunoprecipitates showed PS341 (but not GA) induction of co-precipitating c-Cbl (Figure 3, panel C), implicating this E3 ligase in PS341 induced ErbB2 polyubiquitination.

MRM/MS monitoring of ErbB2 K48 and K63 polyubiquitination

As shown in Figure 4, immunoprecipitated ErbB2 from control and treated SK-BR-3 cells was electrophoretically separated and the full-length band at 185 kDa was excised and trypsin digested along with the gel region from 180 kDa to 250 kDa (panel A). MS analysis determined that there was no protein other than ErbB2 in this excised gel region chosen to enable MRM/MS analysis of varying sizes of polyubiquitinated ErbB2, whose chains were discriminated by the respective diglycine peptide signatures for K48 and K63 (panel B). Three different ErbB2 tryptic peptides (Supplement Table 1) representing extracellular, perimembrane, and intracellular domain fragments were used to normalize for receptor-bound Ub levels between treated samples. ErbB2 K48 and K63 chain linkages were the most abundant polyUb forms detected in all samples; low levels of K29 chain linkages were detected in some treated samples, but there were no K6, K11, K27, or K33 chains detected in any of the samples. With K48 and K63 signals from untreated SK-BR-3 samples establishing the baseline (control) ErbB2 polyUb levels, the differential time-dependent changes observed in K48 and K63 polyUb levels following either PS341 (20nM) or GA (20nM) treatments were plotted as fold changes (panels C and D). Proteasome inhibition produced a gradual 5–15 fold increase in ErbB2 K48 levels between 4–10h after PS341 treatment; in contrast, ErbB2 K63 levels showed a delayed rise beginning 6h after PS341 exposure, nearing ErbB2 K48 levels by 8h, and then more than doubling ErbB2 K48 levels and achieving a 40-fold increase over baseline ErbB2 K63 levels by 10h (panel C). After 24h PS341 exposure, ErbB2 polyUb levels achieved more than 150-fold increase over baseline SK-BR-3 levels (results not shown). Unlike this delayed but dramatic and biphasic K48 and K63 ErbB2 polyUb response to proteasome inhibition, HSP90 inhibition produced a more rapid (within 2h), modest (4- to 6-fold), and synchronous induction of K48 and K63 ErbB2 polyUb (panel D).

Figure 4.

SK-BR-3 editing of ErbB2 polyubiquitin K48 and K63 linkages following proteasome or HSP90 inhibition, as monitored by MRM/MS. A. Full-length (185 kDa) ErbB2 immunoprecipitates from untreated and proteasome inhibited (PS341, 20nM) SK-BR-3 cells were resolved by SDS-PAGE, and the gel regions extending from ErbB2 up to 260 kDa were excised and digested with trypsin. B. Schematic of two representative polyubiquitin chains (K48 and K63) bound to ErbB2 and their diglycine signature peptides formed by trypsin digestion (arrows), based on a previous depiction (31). C. Fold induction in ErbB2 K48 and K63 polyubiquitin linkages with time after proteasome inhibition (PS341) of SK-BR-3 cells, relative to control (CTL) treatment. D. Fold induction in ErbB2 K48 and K63 polyubiquitin linkages with time following HSP90 inhibition (GA) of SK-BR-3 cells, relative to control (CTL) treatment. (Note different ordinate scales in C and D panels.)

Immunoblotting and cell imaging of ErbB2 polyUb using K48 and K63 linkage-specific antibodies

Recently developed K63 and K48 linkage-specific antibodies (28) were used to complement the quantitative MRM/MS monitoring of ErbB2 polyUb. Parallel sets of ErbB2 immunoprecipitates from PS341 treated SK-BR-3 cells were immunoblotted to detect ErbB2 associated with total Ub, K48 linked polyUb, and K63 linked polyUb (Supplement Figure 2, panel A). Consistent with the biphasic MRM/MS results, ErbB2 associated K63 polyUb appeared absent until 4–8h after proteasome inhibition followed by a more dramatic increase at 10h, while ErbB2 associated K48 polyUb increased earlier. These antibodies were also used to image the intracellular trafficking of total Ub as well as total K48 and K63 polyUb in relation to total ErbB2 (Supplement Figure 2, panels B and C). Following PS341 treatment (20nM, 24h), total intracellular Ub appeared scattered throughout the cell nucleus and cytoplasm, with perinuclear accumulation appearing in some cells. Only this perinuclear Ub appeared to co-localize with the internalized and perinuclear ErbB2 (panel B). In untreated SK-BR-3 cells, K63 polyUb was predominantly extranuclear while K48 polyUb was both nuclear (excluding the nucleolus) and cytoplasmic; some membrane co-localization of ErbB2 with the two different forms of polyUb was apparent, more pronounced for K63 polyUb (panel C). Scattered cytoplasmic co-localization of ErbB2 with K63 and K48 polyUb was weakly detectable 4h after PS341 treatment. By 24h, two significant differences were notable: some but not all of the total intracellular K48 and K63 polyUb was redistributed and polarized in perinuclear cytoplasm, and internalized ErbB2 co-localized only with the perinuclear K48 and K63 polyUb (panel C). A comparable set of ErbB2, K48 and K63 polyUb immunoblots and immunofluorescent imaging studies were performed on GA (20nM, 0–24h) treated SK-BR-3 cells and showed dissimilar temporal and spatial effects on total intracellular K48 and K63 polyUb relative to PS341 treated cells (Supplement Figure 3).

Discussion

Although there is little understanding and still some controversy behind the conclusion that ErbB2 is an endocytosis impaired receptor system (12, 13), interest in this aspect of ErbB2 biology has increased with the development of novel therapeutics that either modulate or employ the ErbB2 endocytosis mechanism (1–4). While GA inhibition of HSP90 has emerged as an invaluable tool for investigating mechanisms regulating the maintenance of ErbB2 surface expression and its endocytic down-regulation (5–10, 12, 13), controversies arising from these studies as well as comparisons between ErbB2 and EGFR internalization mechanisms now include: clathrin dependence or independence of ErbB2 internalization (9, 10, 12, 13, 32, 33), endocytic trafficking of ErbB2 to either proteasome or lysosome for receptor degradation (5, 9, 10), and the role of ErbB2 ubiquitination in mediating any of these processes (12–17). Given the extensive investigations into ErbB2 internalization activated by HSP90 inhibition, the present study attempted to cast new light on these processes by evaluating ErbB2 internalization and down-regulation activated by proteasome inhibition using the approved therapeutic, bortezomib (PS341).

Unlike the rapid ErbB2 down-regulating effects of HSP90 inhibition by GA (50% by 6h), complete proteasome inhibition in SK-BR-3 by PS341 induced a more delayed exchange in the ErbB2-associated chaperones (HSP70 for HSP90), followed by surface-to-perinuclear redistribution of ErbB2 and a 50% reduction in total ErbB2 protein expression by 24h. This perinuclear sequestration of ErbB2 co-localized with lysosomal proteins and occurred by a clathrin-independent internalization process that required new protein synthesis; and, as previously shown, chloroquine inhibition of lysosomal proteolysis prevented the PS341-induced degradation of ErbB2 but not its lysosomal sequestration (11). While recent studies have been inconclusive about either the clathrin dependence or lysosomal trafficking of GA-induced ErbB2 endocytosis (9, 10, 32, 33), our SK-BR-3 studies indicate that GA-induced down-regulation of ErbB2 may be clathrin-dependent and is unaffected by chloroquine and, therefore, unassociated with lysosomal trafficking. Furthermore, our SK-BR-3 imaging and molecular studies of full-length ErbB2 clearly indicate that proteasome and HSP90 inhibition result in different ErbB2 chaperone exchange and internalization rates, intracellular trafficking mechanisms, and degradative fates. Associated with these clear mechanistic differences in ErbB2 down-regulation, we have also shown that PS341 and GA differentially affect ErbB2 receptor ubiquitination as well as its association with the E3 ligase, c-Cbl, and the deubiquitylating enzyme, USP9x.

Ubiquitination, primarily in the form of K63 linked polyubquitin chains, is known to play an essential role in the endosomal trafficking and lysosomal degradation of EGFR (25), yet its potential role in mediating plasma membrane overexpression or endocytosis of ErbB2 remains obscure (13, 17). Transient transfection into SK-BR-3 of constructs expressing mutated Ub unable to form any polyUb chains, or specifically unable to form K63 linked polyUb, prevented surface overexpression of ErbB2, and in the former case also prevented SK-BR-3 cell growth. Given the myriad intracellular mechanisms affected by polyubiquitination, it is likely that the observed abnormalities induced by these constructs reflect a general impairment in plasma membrane protein trafficking rather than direct alteration of ErbB2 polyubiquitination. Direct and indirect ErbB2 effects may also explain our observation that siRNA knock down of the deubiquitylating enzyme, USP9x, significantly enhanced PS341-induced lysosomal decay of ErbB2, although the observed physical association between ErbB2 and USP9x/FAM in PS341 treated and untreated cells implicates direct mediation of this DUB in the ErbB2 internalization and ubiquitination induced by proteasome inhibition. Unlike this ErbB2 association with USP9x, neither of the two E3 ligases (CHIP or c-Cbl) were found to co-precipitate with overexpressed ErbB2 in untreated SK-BR-3 cells. However, within 6h of PS341 (but not GA) treatment the intact and internalized ErbB2 receptor became associated with c-Cbl (but not with CHIP) and remained so for at least 24h, during which time ErbB2 polyubiquitination topology was changing from predominantly K48 to K63 chain linkages while the c-terminally intact receptor was trafficking into the lysosome compartment.

Since K48 linked polyUb is known to be required for 26S proteasome recognition and degradation (13, 14, 17), proteasome inhibition might be expected to result in a buildup of K48 linked polyubiquitinated proteins otherwise destined for proteasomal degradation. In contrast, the proteasome system is not inhibited in GA treated cells so an early buildup to a steady state limit of K48 ErbB2 polyUb might also be expected if K48 ErbB2 polyUb was rapidly induced and then proteasomally degraded at a constant rate. It is interesting to note that the modest yet balanced buildup in both K48 and K63 ErbB2 polyUb following HSP90 inhibition was not associated with any detectable lysosomal trafficking and proteolysis, suggesting that GA-induced ErbB2 polyUb contains mixed chain linkages that lack the extended linear topology of K63 ErbB2 polyUb essential for endosomal sorting to the MVB and lysosome. In contrast, the early buildup of K48 ErbB2 polyUb followed by the later (≥8h) buildup of K63 ErbB2 polyUb, during proteasome inhibition and when ErbB2 is associated with c-Cbl, suggests a sequential polyubiquitin editing process rather than unbalanced mixed ubiquitin chain formation, superimposing K63 chain formation on a primary base of K48 linkages to enable lysosomal trafficking. Although CHIP has been shown capable of producing either K48, K63 or even mixed polyUb chain linkages depending on its associated E2 enzyme (34), we were unable to detect any GA-induced ErbB2 association with CHIP in SK-BR-3 cells. In contrast, we found that upon exposure to PS341, ErbB2 associates with c-Cbl; and recent studies indicate that c-Cbl can also produce either K48 or K63 ubiquitin chains (35, 36), suggesting that depending on its subcellular context and E2 partner, c-Cbl may also be able to form mixed polyUb chain linkages and participate in ErbB2 polyubiquitin editing.

Further investigations are needed to understand the various mechanistic controls determining K48 and K63 ErbB2 polyubiquitination, as well as the roles these topologically distinct forms of ErbB2 polyUb play in determining ErbB2 intracellular trafficking and degradation. Nonetheless, the translational importance of these processes is becoming clearer. Recently it was shown that combined treatment with trastuzumab and an HSP90 inhibitor produces greater ErbB2 ubiquitination and degradation than can be achieved by either treatment alone (19). Therefore, ErbB2 intracellular trafficking mechanisms activated by either GA or PS341 treatment must be better delineated for optimal clinical development of the next generation of ErbB2 targeted therapeutics, which will also include drug-encapsulated anti-ErbB2 immunoliposomes (4) and antibody-drug conjugates like trastuzumab-DM1 (3) that depend upon ErbB2 internalization and endocytosis to deliver their cytotoxic cargos.

Abbreviations list

K48

lysine 48

K63

lysine 63

Ub

ubiquitin

polyUb

polyubiquitin

DUB

deubiquitylating enzyme

HSP90

heat shock protein-90

GA

geldanamycin

PS341

bortezomib

MVB

multivesicular body

MRM/MS

multiple reaction monitoring mass spectrometry