Alix Facilitates the Interaction between c-Cbl and
Platelet-derived Growth Factor

-Receptor and Thereby
Modulates Receptor Down-regulation
*
□SReceived for publication, September 5, 2006, and in revised form, November 2, 2006 Published, JBC Papers in Press, November 2, 2006, DOI 10.1074/jbc.M608489200 Johan Lennartsson1, Piotr Wardega, Ulla Engstro¨m, Ulf Hellman, and Carl-Henrik Heldin
From the Ludwig Institute for Cancer Research, Uppsala University, Biomedical Center, SE-751 24 Uppsala, Sweden
Alix (ALG-2-interacting protein X) is an adaptor protein involved in down-regulation and sorting of cell surface recep-tors through the endosomal compartments toward the lyso-some. In this study, we show that Alix interacts with the C-ter-minal region of the platelet-derived growth factor (PDGF)
-receptor (PDGFR) and becomes transiently tyrosine-phos-phorylated in response to PDGF-BB stimulation. Increased expression levels of Alix resulted in a reduced rate of PDGFR removal from the cell surface following receptor activation, and this was associated with decreased receptor degradation. Fur-thermore, Alix was found to co-immunoprecipitate with the ubiquitin ligase c-Cbl, and elevated Alix levels increased the interaction between c-Cbl and PDGFR. Interestingly, Alix interacted constitutively with both c-Cbl and PDGFR. More-over, c-Cbl was found to be hyperphosphorylated in cells engi-neered to overexpress Alix compared with control cells. The increased c-Cbl phosphorylation correlated with enhanced pro-teasomal degradation of c-Cbl, which in turn correlated with a decreased ubiquitination of PDGFR. Our data suggest that Alix inhibits down-regulation of PDGFRby modulating the interaction between c-Cbl and the receptor, thereby affecting the ubiquitination of the receptor.
Platelet-derived growth factor (PDGF)2is a family of signal-ing proteins that regulates the proliferation, survival, and migration of cells of mesenchymal origin. The PDGF family consists of four protein chains that form five biologically active dimers (PDGF-AA, -AB, -BB, -CC, and -DD) (1). PDGF iso-forms exert their cellular effects via binding to two tyrosine kinase receptors: PDGFR␣, which binds PDGF-A, -B, and -C chains; and PDGFR, which binds PDGF-B and -D chains.
Ligand binding induces dimerization and autophosphorylation of the receptors. The phosphorylated tyrosine residues have key roles in coupling to downstream signaling pathways as they mediate interaction with Src homology 2 domain (SH2)-con-taining molecules.
Alix (ALG-2-interacting protein X; also referred to as AIP1 or HP95) is a protein that was identified as a binding partner for ALG-2 (apoptosis-linked gene 2) (2). Apoptosis induced by var-ious stimuli requires ALG-2, and several studies have indicated a role for Alix in regulation of cell death (3– 6). Alix possesses an N-terminal Bro1 domain and a C-terminal region rich in pro-line and tyrosine residues, suggesting an adaptor/scaffold func-tion. Recently, Alix has been implicated in receptor endocytosis and trafficking (7–9). Work by Schmidtet al.(8) demonstrates that Alix antagonizes epidermal growth factor receptor (EGFR) down-regulation by the c-Cbl䡠CIN85 complex, resulting in reduced ubiquitination and internalization (8). The same group also showed that Alix interacts with and is phosphorylated by Src family kinases and that this phosphorylation reduces the interaction between Alix and EGFR, CIN85, or Pyk2, thereby releasing the inhibitory function of Alix on endocytosis (9). Another role that has emerged for Alix is in the organization of endosomes and formation of multivesicular bodies required for transportation of proteins from the early endosome to the lyso-some (10, 11). Furthermore, it has been demonstrated that Alix interacts with several cytoskeletal proteins,e.g.actin and tubu-lin, and with tyrosine kinases such as FAK and Pyk2, thereby negatively regulating cell adhesion (12). Overexpression of Alix in NIH3T3 cells promotes flat cell morphology and reduced proliferation (13), and Alix overexpression in HeLa cells leads to cell cycle arrest in G1phase (14). In summary, Alix has been shown to be involved in numerous cellular events, such as modulation of cellular architecture, apoptosis, growth, and endocytosis; however, the precise function of Alix is not well understood.
In the present report, we show that Alix interacts constitu-tively with PDGFR. It modulates the stability of the E3 ubiq-uitin ligase c-Cbl, thus affecting receptor ubiqubiq-uitination and down-regulation.
EXPERIMENTAL PROCEDURES
Reagents—Recombinant human PDGF-BB was generously provided by Amgen (Thousand Oaks, CA). The following anti-bodies were used for immunoprecipitation and immunoblot-ting: phosphotyrosine (sc-7020) and PDGFR(sc-339) antisera were from Santa Cruz Biotechnology (Santa Cruz, CA); the *This work was supported by grants from the Swedish Research Council,
Åke Wibergs stiftelse, Magnus Bergvalls stiftelse, and the ENDOTRACK integrated European Union project. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
□S The on-line version of this article (available at http://www.jbc.org) contains
supplemental Fig. 1.
1To whom correspondence should be addressed: Ludwig Inst. for Cancer Research, Box 595, Biomedical Center, SE-751 24, Uppsala, Sweden. Tel.: 46-18-160406; Fax: 46-18-160420; E-mail: [email protected]. 2The abbreviations used are: PDGF, platelet-derived growth factor; Alix,
ALG-2-interacting protein X; Cbl, casitas B-lineage lymphoma; EGFR, epidermal growth factor receptor; HA, hemagglutinin; MALDI-TOF, matrix-assisted laser desorption/ionization time-of-flight; PAE cells, porcine aortic endo-thelial cells; PDGFR, PDGF receptor; PLC␥, phospholipase C␥; pTyr, phos-photyrosine; WGA, wheat germ agglutinin.
monoclonal PDGFR antibody recognizing the extracellular domain (GR14L) was from Oncogene Research Products (Cambridge, MA); monoclonal -actin antibody was from Sigma (A5441); the monoclonal HA antibody (12CA5) was from Roche Applied Science; the monoclonal c-Cbl antibody (610442) was from BD Transduction Laboratories; and Ras-GAP antibodies were from Upstate Biotechnology (05-178). An antiserum against Alix was raised by immunizing a rabbit with the synthetic peptide CSYPFPQPPQQSYYPQQ conjugated to keyhole limpet hemocyanin. The antibodies were purified by chromatography over a column of the corresponding peptide immobilized on Sepharose beads. Purified antibodies were stored in 0.15MNaCl, 20 mMHepes, pH 7.4, and 50% glycerol at ⫺70 °C. Rabbit antiserum against PLC␥ was generated by immunizing rabbits with a peptide corresponding to the C ter-minus of bovine PLC␥(15).
Cell Culture and Transfection—Porcine aortic endothelial (PAE) and 293T cells were cultured in Ham’s F-12 and Dulbec-co’s modified Eagle’s medium, respectively, supplemented with 10% fetal bovine serum and 100 units/ml penicillin, 100g/ml streptomycin, and 2 mM L-glutamine. Cells were transfected by
Lipofectamine according to the protocol provided by the man-ufacturer (Invitrogen). For transient transfections, cells were assayed 48 h after transfection. To establish stable transfec-tants, cells were selected 48 h after transfection by the presence of 400g/ml zeocin until only resistant cells were left.
Affinity Purification of Tyrosine-phosphorylated Proteins— Subconfluent PAE cells (total 109 cells) expressing either PDGFR␣or PDGFRwere serum-starved overnight and then stimulated for 5 min with 100 ng/ml PDGF-BB and lysed in 0.5% Triton, 50 mMNaCl, 10 mMNaF, 30 mM tetrasodium
pyrophosphate, 10% glycerol, 1 mMEDTA, 20 mMTris, 1 mM
Pefabloc, 1% Trasylol, and 1 mMsodium orthovanadate, pH 7.4.
Extracts were cleared by centrifugation, and tyrosine-phospho-rylated proteins were immunoprecipitated with 4G10 antibod-ies coupled to agarose beads (Upstate Biotechnology) for 2 h. The immunocomplex was washed three times in lysis buffer and then eluted twice with 100 mMphenylphosphate in 10 mM
Hepes, pH 7.4, at 4 °C. Eluted proteins were precipitated using the Wessel-Flu¨gge procedure (16) and resuspended in 100l of sample buffer containing 10 mMdithiothreitol. The samples
were treated with 25 mMiodoacetamide for 15 min at room
temperature before separation by SDS-PAGE and visualization by silver staining using an in-gel digestion compatible protocol (17).
Protein Identification—Excised protein bands were de-stained (18) and dried with acetonitrile. A solution of porcine sequence grade trypsin (Promega, Madison, WI) in 50 mM
ammonium bicarbonate, 10% acetonitrile was allowed to soak into the gel pieces on ice for 45 min. Approximately 100 ng trypsin/gel band was applied and incubated at 30 °C overnight. The digestion was stopped by making the samples 1% with respect to trifluoroacetic acid. After dilution in 0.1% trifluoro-acetic acid, samples were concentrated and desalted on a Zip-Tip C18 column (Millipore Corp., Bedford, MA), and the digest mixture was eluted directly onto the matrix-assisted laser de-sorption/ionization (MALDI) target with 70% acetonitrile, con-taining about 8 mg/ml matrix ␣-cyano-4-hydroxycinnamic
acid. Peptide mass fingerprinting was done by MALDI time-of-flight mass spectrometry (MALDI-TOF MS) on a Bruker Ultra-flex TOF/TOF (Bruker Daltonics, Bremen, Germany). The instrument was operated in reflector mode and optimized for analytes up to 4000 Da. The spectra were internally calibrated using autolytic trypsin peptides. The peptide mass lists were used to scan protein sequence data banks for protein identities via the ProFound search engine. Significance of matches were judged after taking several parameters into account, such as “z-score,” error distribution, number of miscuts, and migration behavior.
Immunoprecipitation and Western Blotting—Subconfluent cells were starved in 0.1% fetal bovine serum overnight and incubated and with 100 ng/ml PDGF-BB for the indicated peri-ods of time. Cells were washed with ice-cold phosphate-buff-ered saline and lysed (0.5% Triton, 50 mMNaCl, 10 mMNaF, 30
mMtetrasodium pyrophosphate, 10% glycerol, 1 mMEDTA, 20
mMTris, 1 mMPefabloc, 1% Trasylol, 1 mMsodium
orthovana-date, pH 7.4). Extracts were clarified by centrifugation, and pro-tein concentration was determined by the BCA propro-tein assay system (Pierce). Equal amounts of lysates were incubated with 1
g of antibody as indicated. The immunoprecipitates were col-lected on protein A-Sepharose beads, washed three times in lysis buffer, and then boiled in SDS sample buffer containing dithiothreitol. Immunoprecipitates or equal amounts of total cell lysates were analyzed by SDS-PAGE. For Western blotting, samples were electrotransferred to polyvinylidene difluoride membranes (Immobilon P), which were blocked in 5% bovine serum albumin in phosphate-buffered saline solution contain-ing 0.1% Tween 20. Primary antibodies were used with the con-centrations and buffers recommended by the suppliers and incubated overnight in the cold. After washing, the membranes were incubated with horseradish peroxidase-conjugated anti-mouse or anti-rabbit IgG antibodies (both from GE Healthcare/ Amersham Biosciences), and proteins were visualized using ECL Western blotting detection systems from Roche Applied Science on a cooled charge-coupled device camera (CCD; Fuji, Minami-Ashigata, Japan). Before reprobing, the membranes were stripped with 0.4MNaOH for 10 min at room
tempera-ture, blocked, and incubated with the corresponding antibody. Biotinylation of Cell Surface Proteins—Cells were serum-starved overnight and stimulated with 20 ng/ml PDGF-BB for different periods of time at 37 °C. Next, cells were washed twice in ice-cold phosphate-buffered saline, pH 7.4, and incubated for 1 h on ice with 0.2 mg/ml sulfo-NHS-SS-biotin (Pierce) in phos-phate-buffered saline. To inactivate unbound sulfo-NHS-SS-biotin, cells were treated for 5 min with 50 mMTris, pH 8.0.
Cells were lysed, and biotinylated proteins were precipitated with streptavidin-agarose (Sigma) for 1 h at 4 °C. The beads were washed three times in lysis buffer and boiled with SDS sample buffer containing dithiothreitol. Eluted proteins were separated using SDS-PAGE and transferred to Immobilon P membranes. The membranes were blocked with 5% bovine serum albumin in phosphate-buffered saline, and the amount of PDGFRwas visualized and quantified by immunoblotting with PDGFRantibodies.
RESULTS
Identification of Tyrosine-phosphorylated Proteins after PDGF Stimulation—We were interested in identifying proteins that become tyrosine-phosphorylated after PDGF stimulation and hence are likely to participate in signal transduction down-stream of the PDGFRs. To this end we used monoclonal anti-phosphotyrosine antibodies (4G10) immobilized on Sepharose beads to immunoprecipitate proteins from PDGF-BB-stimu-lated PAE cells transfected with either PDGFR␣or PDGFR. This allowed us to investigate the tyrosine-phosphorylated pro-teins downstream of the two PDGFR isoforms, expressed at comparable levels in the same cellular background. Bound pro-teins were eluted, separated by SDS-PAGE, and visualized by silver staining (supplemental Fig. 1). Proteins tentatively phos-phorylated after PDGF stimulation were excised and identified by mass spectroscopy as indicated in supplemental Fig. 1. Among the proteins identified, Alix was selected for further study in regard to PDGFRsignal transduction. Interestingly, we observed that Alix was not significantly phosphorylated by PDGFR␣, indicating that Alix is a preferred substrate for PDGFR.
Alix Is Phosphorylated in Response to PDGF and Interacts Constitutively with PDGFR—To verify the mass spectroscopy results, we immunoprecipitated Alix from cells stimulated with PDGF-BB and resolved the immunocomplexes by SDS-PAGE followed by transfer to an Immobilon P membrane. A phospho-tyrosine immunoblot revealed bands with sizes corresponding to Alix (⬃95 kDa) and PDGFR(⬃180 kDa) in the Alix immunopre-cipitate (Fig. 1A,left panels). The 95- and 180-kDa phosphopro-teins were confirmed to be Alix and PDGFR, respectively, by reprobing the membrane with Alix- or PDGFR-specific antibod-ies (Fig. 1A,left panels). Interestingly, we found that Alix inter-acted with PDGFRin a constitutive manner, although its tyro-sine phosphorylation was ligand-dependent. The interaction could also be detected in a reciprocal experiment (Fig. 1A, right panels). Next, we performed a kinetic analysis of Alix phosphorylation. We found that Alix was transiently phos-phorylated in response to PDGF-BB (Fig. 1B,top panel) with kinetics similar to the overall receptor activation as deter-mined by anti-phosphotyrosine immunoblotting of WGA-Sepharose-enriched PDGFR (Fig. 1B, bottom panel). Our results suggest that Alix is phosphorylated by PDGFR. It is possible that Alix is also phosphorylated indirectly by Src family kinases activated by PDGFR; however, such a con-tribution is likely to be minor, because Alix phosphorylation was only slightly inhibited by the low molecular weight inhibitor SU6656 (data not shown).
Alix Binds to the C-terminal Region of PDGFR—To deter-mine the binding region for Alix in PDGFR, we decided to analyze different C-terminally truncated mutants of the recep-tor for their abilities to interact with Alix. By expressing dele-tion mutants of the receptor in 293T cells, we found a partial loss of interaction when the 75 most C-terminal amino acid residues of PDGFRwere deleted and an almost complete loss of binding when the last 98 amino acid residues were deleted (Fig. 2A). Because Alix, despite its not being significantly phos-phorylated by PDGFR␣, was also found to interact with this
receptor (data not shown), we compared the amino acid sequence of PDGFR␣and -in the region important for bind-ing. We observed that the sequence surrounding Tyr-1021 in PDGFRis similar between both receptor isoforms (Fig. 2B). Furthermore, this region is rich in proline residues, which can interact with SH3 domain or WW domain-containing proteins in a constitutive manner. To clarify whether this region is responsible for the interaction with Alix, we synthesized pep-tides corresponding to the amino acid sequence between resi-dues 1015 and 1030, with the addition of an N-terminal cysteine to facilitate coupling to a solid support (Fig. 2B), with Tyr-1021 either phosphorylated or not phosphorylated. Next we per-formedin vitrointeraction assays using this peptide immobi-lized on beads followed by immunoblotting for target proteins. We found that Alix interacted with both peptides, independent of phosphorylation, in agreement with the constitutive binding observedin vivo(Fig. 2C). As a control, we analyzed the inter-action between these peptides and its known interinter-action part-ner PLC␥1. Indeed, we could detect a phosphorylation-dependent interaction with PLC␥1 (Fig. 2C). As a negative control we used a peptide containing tyrosine 771 in the PDGFR, which is known to bind RasGAP. As seen in Fig. 2C, this peptide, when phosphorylated on tyrosine, could pull down RasGAP but not Alix. Thus, we concluded that Alix can interact with the PDGFRin the region between residues 1015 and 1030 in a receptor phosphorylation-independent manner. However, other regions in the PDGFRmay contribute, because there was a weak residual binding also when the last 98 amino acids of the receptor were deleted.
FIGURE 1.Alix is tyrosine-phosphorylated in response to PDGF-BB and interacts with PDGFR.A, PAE cells expressing PDGFRwere serum-starved overnight and stimulated for 5 min with 100 ng/ml PDGF-BB at 37 °C. Alix was immunoprecipitated (Ip) and the filter subjected to immunoblotting (Ib) with antibodies against phosphotyrosine (pTyr) (top left panel), Alix (middle left panel), or PDGFR(bottom left panel). Theright panelsshow the reciprocal experiment.B, cells were serum-starved and stimulated for the indicated peri-ods of time with 100 ng/ml PDGF-BB at 37 °C. Alix was immunoprecipitated and the filter subjected to immunoblotting with pTyr antibodies (top panel) or Alix (middle panel). PDGFRwas precipitated with WGA-Sepharose and the filter probed with pTyr antibody to determine overall receptor phosphoryla-tion (bottom panel).
Alix Inhibits PDGF-BB-induced Removal of PDGFRfrom the Cell Surface—To investigate whether the expression level of Alix influenced the rate of PDGFRcell surface clearance, we analyzed cells that were engineered to overexpress Alix (Fig. 3A). To measure the amount of receptors on the cell surface, we stimulated cells for different periods of time and biotinylated the cell surface proteins prior to lysis. Biotinylated proteins were purified using streptavidin-agarose, and the levels of PDGFRwere then analyzed by immunoblotting (Fig. 3B). As can be seen, the PDGF-BB-induced clearance of PDGFRfrom the cell surface was reduced in Alix-overexpressing cells as compared with mock-transfected control cells.
Alix Overexpression Enhances Interaction between c-Cbl and PDGFR and Phosphorylation of c-Cbl—Ubiquitination of receptor tyrosine kinases is important for their internalization, and c-Cbl is a major ubiquitin ligase connected with PDGFR. Because we noticed a reduced rate of PDGFR down-regula-tion from the cell surface in cells overexpressing Alix (Fig. 3B), we investigated the effect of increased Alix levels on c-Cbl phosphorylation and association with the PDGFRafter ligand
stimulation. We found that elevated Alix levels resulted in increased interaction between c-Cbl and PDGFR (Fig. 4). In addition, we observed that Alix co-immunopre-cipitated with c-Cbl. Interestingly, neither the interaction between c-Cbl and Alix nor that between c-Cbl and PDGFRwas induced by PDGF stimulation, but it existed in a constitutive manner both in con-trol and Alix-overexpressing cells. Moreover, PDGF-induced c-Cbl phosphorylation was augmented in cells overexpressing Alix compared with control cells (Fig. 4).
Elevated Levels of Alix Lead to Reduced PDGFRDown-regulation but Increased c-Cbl Degradation in Response to PDGF-BB—Next, we wanted to determine whether the reduced rate of PDGFR removal from the cell surface in cells overex-pressing Alix was associated with changes in receptor stability. We therefore stimulated cells with PDGF-BB for different periods of time and determined the amount of PDGFR by immunoblotting. Indeed, ligand-induced PDGFR degradation was reduced in Alix-overexpressing cells compared with mock-transfected cells (Fig. 5). In contrast, we could not observe any effects of Alix overexpression on PDGFR phosphorylation. How-ever, c-Cbl was hyperphosphoryla-ted in cells overexpressing Alix (Figs. 4 and 5). We found that the enhanced c-Cbl phosphoryl-ation in response to PDGF-BB stimulphosphoryl-ation in cells with elevated levels of Alix correlated with increased c-Cbl degradation (Fig. 5). The enhanced rate of c-Cbl degradation was completely inhibited by MG132, suggesting that the degradation occurred in proteasomes (Fig. 6).
Elevated Alix Expression Decreases Ligand-induced PDGFR Ubiquitination—The observation that increased Alix expres-sion resulted in increased proteasomal degradation of c-Cbl suggested that the level of PDGFRubiquitination might also be affected. To investigate this possibility, we transfected Alix-overexpressing or mock-transfected cells with ubiquitin fused with an HA epitope and performed immunoprecipitation of PDGFR followed by immunoblotting with HA antibodies. Indeed, we found that the PDGFRwas significantly less ubiq-uitinated in cells overexpressing Alix (Fig. 7). However, after shorter periods (5–15 min) of PDGF-BB stimulation, we could not demonstrate a significant difference in level of PDGFR ubiquitination between control and Alix-overexpressing cells (data not shown). This suggests that the bulk of c-Cbl FIGURE 2.Alix binds to the C-terminal tail of PDGFR.A, 293T cells were transiently transfected with PDGFR
constructs with progressively longer deletions of the C-terminal tail (CT) and stimulated with PDGF-BB (100 ng/ml) for 5 min. Alix was immunoprecipitated (Ip) and the membrane immunoblotted (Ib) with antibodies recognizing the extracellular domain PDGFR. The membrane was then reprobed with Alix antibodies. To verify comparable receptor expression and activation in all transfections, PDGFRs were precipitated with WGA-Sepharose, and the filter was immunoblotted with pTyr and subsequently reprobed for total receptor.WT, wild type.B, the amino acid sequence of PDGFRcorresponding to the region between CT98 and CT75 is shown in bold letters. The PDGFR␣sequence is alignedbelow. On theright, theposition numbersfor the C-terminal residues are indicated. In addition, the amino acid sequence for the peptide used forin vitrointeraction studies is shown, to which an N-terminal cysteine was added to facilitate coupling to Sulfolink beads.C, a synthetic peptide corresponding to the amino acid sequence surrounding tyrosine 1021 (residues 1015–1030, seepanel B) or tyrosine 771 (residue 764 –781), either phosphorylated or not, was used to investigate the interaction among Alix, PLC␥1, and RasGAPin vitroby immunoblotting as indicated.
proteins must first be degraded before the reduction in ubiquitination can be observed. In summary, Alix overex-pression stabilizes the PDGFR, possibly through promot-ing enhanced proteasomal degradation of c-Cbl in response to PDGF-BB and thereby reducing c-Cbl-mediated ubiquiti-nation of the PDGFR.
DISCUSSION
Down-regulation of receptor tyrosine kinases is essential for proper response to ligand, and disturbances in this process may contribute to transformation (19). It has been shown that monoubiquitination of receptor tyrosine kinases is an impor-tant mechanism that regulates receptor internalization and intracellular trafficking (20). c-Cbl is a ubiquitin ligase targeting several receptor tyrosine kinases including the PDGFRs. In this report, we present data on c-Cbl and the multifunctional adap-tor protein Alix and on their involvement in PDGFR down-regulation. In general, our results are consistent with earlier work with the EGFR (8, 9).
We found that Alix forms a constitutive complex with PDGFR and is transiently phosphorylated in response to ligand stimulation. An activation-independent interaction has also been reported between Alix and the EGFR (8). We mapped the binding site of Alix to the C-terminal tail of PDGFR. How-ever, contributions from other regions of the receptor in this interaction cannot be excluded. The C-terminal region con-tains a proline-rich sequence that may mediate the interaction with Alix. However, Alix does not contain any domain that is
known to interact with proline-rich regions. Thus, it is possible the interaction between Alix and PDGFRis indirect, through an adaptor protein. In fact, Alix is believed to bind indirectly to the EGFR because the association cannot be observed using recombinant proteins (8). Moreover, the interaction between Alix and PDGFRwas not affected by mutation of tyrosines 1009 and 1021 in the C-terminal tail of the receptor to pheny-lalanine (data not shown).
It was recently shown that overexpression of Alix results in a reduced EGFR internalization (8). We found that overexpres-sion of Alix also reduced the rate of ligand-induced PDGFR removal from the cell surface compared with mock-transfected cells; this reduction was accompanied by a decreased rate of receptor degradation. Moreover, we observed c-Cbl hyper-phosphorylation in response to PDGF-BB in Alix-overexpress-ing cells that was associated with enhanced proteasomal degra-dation of c-Cbl. These observations are in concurrence with a study from Baoet al.(21) showing that expression of active Src (or overexpression of normal Src) promotes c-Cbl hyperphos-FIGURE 3.Alix overexpression inhibits PDGFRdown-regulation.PAE
cells expressing PDGFRwere stably transfected with a mock vector (PAE/ PDGFR) or an Alix expression vector (PAE/PDGFR-Alix).A, total cell ly-sate (TCL) was separated and immunoblotted with Alix antibodies to deter-mine Alix overexpression.B, cells were serum-starved overnight and treated with PDGF-BB for the indicated periods of time at 37 °C. Cell surface proteins were then biotinylated before lysis. Biotinylated proteins were precipitated with streptavidin-agarose, separated on SDS-PAGE, and transferred onto Immobilon P membranes. The level of receptor was determined by immuno-blotting (Ib) with PDGFRantibodies. The intensity of the PDGFRbands was measured and plotted as percent of initial receptor levels.
FIGURE 4.c-Cbl exists in a constitutive complex with Alix and PDGFR, and overexpression of Alix increases c-Cbl phosphorylation. PAE/ PDGFR-Alix and PAE/PDGFR cells were serum-starved overnight and treated with 100 ng/ml PDGF-BB for 5 min at 37 °C. Lysates were prepared, and c-Cbl was immunoprecipitated (Ip). Proteins were separated by SDS-PAGE, transferred onto Immobilon P membranes, and subjected to immuno-blotting (Ib) with pTyr, PDGFR, Alix, and c-Cbl antibodies as indicated. PDGFRwas precipitated with WGA-Sepharose, and the filter was probed with pTyr antibodies to verify equal receptor stimulation.
phorylation and, as a consequence, its degradation (21). How-ever, the low molecular weight Src inhibitor SU6656 had no significant effect on PDGF-BB-induced c-Cbl phosphorylation in our cell model (data not shown).
Our results suggest that that c-Cbl, Alix, and PDGFR inter-act constitutively, although our data could not distinguish whether a ternary complex or separate binary complexes are formed. However, elevated Alix expression enhances the inter-action between c-Cbl and PDGFR. Notably, the constitutive interaction between c-Cbl and PDGFRwas also detected in
mock-transfected cells and hence is not an artifact of overex-pression. The enhanced c-Cbl phosphorylation observed in cells overexpressing Alix may be explained by enhanced inter-action between c-Cbl and the receptor, which may allow a more efficient phosphorylation.
Given that c-Cbl is a major ubiquitin ligase downstream of receptor tyrosine kinases (22), we anticipated that an increase in c-Cbl degradation would be reflected in a decreased level of receptor ubiquitination. Indeed, we observed that Alix overex-pression correlated with a reduced level of PDGFR ubiquiti-nation. It has been suggested that degradation of hyperphos-phorylated c-Cbl allows the EGFR to recycle back to the plasma membrane instead of being sorted for lysosomal degradation (21), potentially because of a lack of sustained ubiquitination (23, 24). This is compatible with our results showing that Alix overexpression leads to increased c-Cbl phosphorylation and degradation, as well as to a reduced rate of PDGFRclearance from the cell surface. It remains to be determined whether this FIGURE 5. Alix overexpression inhibits PDGFR degradation and
enhances Cbl degradation.A, PAE/PDGFR-Alix and PAE/PDGFRcells were serum-starved overnight and treated with 100 ng/ml PDGF-BB for the indicated periods of time at 37 °C. Lysates were prepared, and PDGFRwas immunoprecipitated (Ip) using WGA-Sepharose and c-Cbl. Proteins were sep-arated by SDS-PAGE, transferred onto Immobilon P membranes, and sub-jected to immunoblotting (Ib) as indicated. As a loading control, a fraction of total cell lysate (TCL) was subjected to immunoblotting for-actin levels. B, the average intensities from three experiments were measured and plotted
⫾1 S.D. as indicated. The value for unstimulated cells was used for normaliza-tion.a.u., arbitrary units.
FIGURE 6.Cbl is degraded via the proteasomal pathway.A, PAE/PDGFR -Alix and PAE/PDGFRcells were serum-starved overnight and treated with 25 MMG132 or dimethyl sulfoxide (DMSO) for 4 h followed by stimulation with 100 ng/ml PDGF-BB for the indicated periods of time at 37 °C. Lysates were prepared, and c-Cbl was immunoprecipitated (Ip), separated by SDS-PAGE, transferred onto Immobilon P membranes, and immunoblotted (Ib) as indi-cated. As a loading control a fraction of total cell lysate (TCL) was immuno-blotted for-actin levels.B, the intensities of the bands shown inAwere measured and plotted as indicated. The value for unstimulated mock cells was used for normalization.a.u., arbitrary units.
is because of decreased internalization or increased recycling. In conclusion, Alix may function as an adaptor/scaffold protein regulating PDGFRubiquitination and consequently intracel-lular sorting by modulating the degradation of c-Cbl.
Acknowledgment—The Alix expression construct was a kind gift from Dr. Luciano D’Adamino.
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J. Biol. Chem.279,37153–37162 FIGURE 7.Alix overexpression leads to a reduced PDGFR
ubiquitina-tion.A, HA-tagged ubiquitin was transfected into cells having normal or ele-vated level of Alix expression. These cells were serum-starved overnight and stimulated with 100 ng/ml PDGF-BB for the indicated periods of time at 37 °C. Lysates were prepared, and PDGFRwas immunoprecipitated (Ip), separated by SDS-PAGE, transferred onto Immobilon P membranes, and immuno-blotted (Ib) with antibodies against HA-ubiquitin (Ub) or PDGFR.B, the inten-sities of the bands shown inAwere measured and plotted as indicated.a.u., arbitrary units.
