Bcr Interacts with Components of the Endosomal Sorting Complex Required for Transport-I and Is Required for Epidermal Growth Factor Receptor Turnover

Research Article

Oyenike O. Olabisi, Gwendolyn M. Mahon, Elena V. Kostenko, Zhuoming Liu, Harvey L. Ozer, and Ian P. Whitehead

Department of Microbiology and Molecular Genetics and University Hospital Cancer Center, New Jersey Medical School, University of Medicine and Dentistry of New Jersey, Newark, New Jersey

Abstract in-frame fusion protein (p210 Bcr-Abl) that contains NH2-terminal Virtually all patients with chronic myelogenous leukemia sequences of Bcr fused to COOH-terminal sequences of Abl. (CML) express an aberrant protein (p210 Bcr-Abl) that Elevated tyrosine kinase activity residing within Abl is considered to be a critical event in the induction of CML. Therapeutic agents contains NH2-terminal sequences from Bcr fused to COOH- terminal sequences from Abl. In a yeast two-hybrid screen, we that specifically target this activity are effective in the treatment of have identified TSG101 as a binding partner for Bcr. Because CML (3), and loss of this activity is invariably associated with TSG101 is a subunit of the mammalian endosomal sorting diminished transforming activity (4). Sequences that reside within the Bcr component of p210 Bcr-Abl are also required for complex required for transport (ESCRT), which regulates 177 protein sorting during endosomal trafficking, this association transformation. Autophosphorylated Tyr binds to growth factor suggests that Bcr may have a related cellular function. The receptor binding protein 2, which complexes with SOS to activate docking site for TSG101 has been mapped to the COOH Ras (5). Loss of this residue results in a dramatic loss in the terminus of Bcr, indicating that this interaction may be transforming activity. The oligomerizing property of the NH2- disrupted in CML. Overexpression studies with full-length terminal domain of Bcr also plays an essential role in the activation TSG101 and Bcr reveal that this interaction can be reca- of Bcr-Abl tyrosine kinase activity (6). Removal of the domain pitulated in mammalian cells. The association can also be abrogates transforming activity, which can be at least partially observed between natively expressed proteins in a panel of restored by the addition of unrelated sequences that possess hematopoietic and nonhematopoietic cell lines, where a oligomerizing ability (7). Finally, the NH2 terminus of Bcr binds to second subunit of the ESCRT complex, vacuolar sorting the SH2 domain of Abl in a phosphotyrosine-independent manner, protein 28 (Vps28), was also found to interact with Bcr. Both constitutively activating the tyrosine kinase (8). Bcr and TSG101 exhibit a punctate cytoplasmic distribution The major product of the BCR gene is a 160 kDa protein that and seem to colocalize in HeLa cells, which would be exists primarily as a soluble 600 kDa tetramer (1). Oligomerization consistent with an in vivo association. Bacterially purified is mediated by an NH2-terminal coiled-coil domain located within Bcr and TSG101 also bind, suggesting that the interaction is residues 1 to 71. Also contained within Bcr are an NH2-terminal direct and is not dependent on ubiquitination. Disruption of (residues 1-414) serine/threonine kinase domain, a centrally located the endosomal pathway with an ATPase-defective Vps4 mutant Rho-specific guanine nucleotide exchange factor (RhoGEF) domain, results in the cellular redistribution of Bcr, and suppression of and COOH-terminal C2 and Rac-specific GTPase-activating protein Bcr in HeLa cells by small interfering RNAimpairs epidermal (RacGAP) domains. RhoGEF domains catalyze the exchange of growth factor receptor turnover. Taken together, these GDP for GTP on Rho family GTPases, thus converting them into observations suggest that Bcr is a component of the their biologically active state (9). Although the C2 domain of Bcr mammalian ESCRT complexes and plays an important role has not been characterized, they have been shown to bind in cellular trafficking of growth factor receptors. (Cancer Res phospholipids in a calcium-dependent manner in other signaling 2006; 66(12): 6250-7) molecules (10). GAP domains, including the one in Bcr, stimulate the intrinsic rate of hydrolysis of GTPases, thus converting them Introduction to the inactive GDP-bound form (11). The native function of Bcr remains largely unknown. The protein Chronic myelogenous leukemia (CML) is a malignant hemato- is broadly expressed and is predominantly associated with poietic stem cell disorder that occurs at a frequency of 1 to 1.5 per cytoplasmic membranes (12). Sporadic reports have also provided 100,000 people (1). The cytologic hallmarkof CML is a trans- evidence for nuclear localization, which is regulated in a cell cycle– location [t(9;22)(q34;q11)], termed the Philadelphia chromosome, dependent manner (12–14). There are no orthologues of Bcr in that occurs in >90% of all patients (2). In virtually all cases, yeast, Caenorhabditis elegans,orDrosophila, and homozygous null translocation breakpoints fall within introns 13 or 14 of the BCR mice are viable, exhibiting mild neurologic disorders (15). The gene, and the first intron of the ABL gene. This produces a 210 kDa simultaneous disruption of both Bcr and the closely related Abr leads to more severe abnormalities in postnatal cerebellar

Requests for reprints: Ian P. Whitehead, Department of Microbiology and development, which is consistent with the high expression of both Molecular Genetics, New Jersey Medical School, University of Medicine and Dentistry proteins that is observed in neural tissue (16, 17). Interestingly, the of New Jersey, 225 Warren Street, Newark, NJ 07103. Phone: 973-972-4483, ext. 25215; neutrophils of bcrÀ/À mice show unusually high levels of reactive Fax: 973-972-8981; E-mail: [email protected]. I2006 American Association for Cancer Research. oxygen species when exposed to Gram-negative endotoxins, which doi:10.1158/0008-5472.CAN-06-0536 would be consistent with chronic activation of Rac2 (18). This

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Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 2006 American Association for Cancer Research. Bcr Interacts with the ESCRT-I Complex suggests that the COOH-terminal RacGAP domain of Bcr may play versions of human Vps4 fused to green fluorescent protein (GFP; ref. 30: an important role in the regulation of the oxidative burst in kindly provided by P. Woodman, Faculty of Life Sciences, University of professional phagocytes. The function of Bcr in other cell types is Manchester, Manchester, United Kingdom). pGEX5X-1-bcr(871-1271) con- unknown. tains a cDNA that encodes the indicated amino acids of Bcr fused at the NH -terminus to glutathione S-transferase (GST; ref. 14). pET21b- TSG101 was originally identified as a mammalian tumor 2 His6-tsg101(223-390) contains a cDNA that encodes the indicated amino suppressor gene through its insertional inactivation in NIH 3T3 acids of TSG101 fused at the COOH terminus to a polyhistidine tag. All PCR mouse fibroblasts (19). An analysis of the protein structure of products that were used in the assembly of these constructs were TSG101 revealed several identifiable domains. These include an sequenced in their entirety. Detailed descriptions of the cloning strategies NH2-terminal noncanonical E2 ubiquitin–conjugating enzyme that were used to generate each construct are available upon request. domain (UEV; residues 23-160); centrally located proline-rich Yeast two-hybrid screening. A Bcr cDNA fragment encoding the full- (residues 139-216) and coiled-coil (residues 242-312) domains; and length protein (residues 1-1,271) was subcloned in frame into pGBT9 a unique domain in the COOH-terminal region (residues 346-391) [pGBT9-bcr(1-1,271)] containing the GAL4 DNA-binding domain. A human that has been implicated in protein stability (20). Like Bcr, TSG101 is HeLa MATCHMAKER cDNA library (S3; Clontech) was screened with the a ubiquitously expressed protein whose subcellular localization is PJ69 yeast strain according to the instructions of the manufacturer (Clontech). Double selection by HIS3 and lacZ followed by retransformation regulated in a cell cycle–dependent manner (21). TSG101 was of yeast with recovered cDNAs identified two independent TSG101 clones initially identified by its ability to transform mouse fibroblasts when (residues 1-390 and 223-390) as binding partners for Bcr. its expression is suppressed (19). Somewhat paradoxically, an Cell culture and transfection. 293T, HeLa, and ts20 3T3 cells (31) were analysis of tsg101À/À mice revealed that the protein is essential for maintained in DMEM (high glucose) supplemented with 10% fetal bovine cell growth and embryonic development (22, 23). Subsequent to its serum (FBS; Sigma, St. Louis, MO). ts20 cells were maintained and passaged identification, TSG101 was implicated in a variety of cellular at the permissive temperature (35jC). K-562 and HL-60 cells (American processes, including transcriptional activation (24), modulation of Type Tissue Culture Collection, Manassas, VA) were maintained in Iscove’s the MDM2/p53 feedbackloop (25), and cell cycle progression (26). modified medium supplemented with 4 mmol/L L-glutamine, 1.5 g/L More recently, it has been firmly established that TSG101 plays a sodium bicarbonate, and 20% FBS (Sigma, St. Louis, MO). Transient central role in the sorting of endosomal cargo. In both yeast and transfection of HeLa and 293T cells was by LipofectAMINE 2000 according to the instructions of the manufacturer (Invitrogen, Carlsbad, CA). mammalian cells, TSG101 is a component of a 350 kDa soluble Protein expression. Protein expression in transiently transfected 293T protein complex [designated endosomal sorting complex required cells, or untransfected HeLa, 293T, K-562, ts20, and HL-60 cells was for transport-1 (ESCRT-I)], which recognizes ubiquitinated cargo on determined by Western blot analysis as described previously (32). Protein the endosomal membrane and mediates their internalization during was visualized with enhanced chemiluminescence reagents (Amersham formation of the multivesicular body (27, 28). TSG101 is thought Pharmacia Biotech, Piscataway, NJ) or by Odyssey IR imaging. to bind to ubiquitinated proteins through its UEV domain and Coimmunoprecipitations. Cells were harvested in GST fluorescence facilitate their distribution into the multivesicular body. Mutations in situ hybridization (FISH) buffer [50 mmol/L Tris-HCl (pH 7.4), 2 mmol/L in Vps23p, the yeast homologue of TSG101, lead to defective MgCl2, 100 mmol/L NaCl, 10% glycerol, 1% NP40] supplemented with transport of membrane proteins into yeast vacuoles, which are the protease inhibitor cocktails (Calbiochem, San Diego, CA). Lysates were A yeast counterparts of the mammalian lysosome (27, 28). precleared with 0.25 g agarose-conjugated normal IgG (Santa Cruz Bio- technology, Santa Cruz, CA) by rotation for 1 hour at 4jC and precipitated In this current study, we have identified TSG101 as a binding with 10 Ag agarose-conjugated monoclonal antibody by rotation at 4jC partner for Bcr. This interaction occurs in mammalian cells, is overnight. The immunoprecipitate was washed thrice with cold lysis buffer direct, and is not mediated by ubiquitination. An association and then resuspended in 2Â SDS loading dye. between natively expressed Bcr and Vps28 has also been detected, In vitro affinity precipitations. GST-Bcr(871-1,271) was expressed in which strongly suggest that Bcr is a bona fide component of the BL21(DE3) cells by induction with 0.1 mmol/L isopropyl-L-thio-h-D- mammalian ESCRT-I complex. Bcr and TSG101 show a similar galactopyranoside (IPTG) and was purified with glutathione Sepharose 4B subcellular distribution in HeLa cells, and targeted suppression of beads according to the instructions of the manufacturer (Amersham both is associated with defective trafficking, suggesting a similar Pharmacia Biotech). His6-TSG101(223-390) was expressed in BL21(DE3) role in the endosomal pathway. Because TSG101 binds to a region cells by induction with 0.4 mmol/L IPTG and was purified with Talon Metal of Bcr that is lost in p210 Bcr-Abl, this raises the possibility that a Affinity Resin according to the instructions of the manufacturer (Clontech). Bcr-mediated defect in trafficking may occur in the context of CML. Expression was confirmed by Coomassie staining and by Western blot using an anti-GST and anti-His antibody, respectively. Approximately 20 Ag glutathione Sepharose–linked GST or GST-Bcr(871-1,271) was incubated

with f20 Ag bacterially purified His6-TSG101(223-390) with rotation for Materials and Methods 1 hour at 4jC. Beads were then washed briefly thrice in GST FISH buffer, Molecular constructs. The pGBT9 and pGAD(GH) yeast expression pelleted at 500 rpm for 5 minutes, separated by SDS-PAGE, and transferred vectors are as described by the manufacturer (Clontech, Mountain View, to polyvinylidene difluoride membrane (Amersham). Bound TSG101 was CA). pGBT9-bcr(1-1,271), pGBT9-bcr(871-1,271), pGBT9-bcr(911-1,271), detected by Western blot using an anti-TSG101 monoclonal antibody pGBT9-bcr(1,037-1,271), and pGBT9-bcr(1,165-1,271) contain cDNAs that (C2, Santa Cruz Biotechnology) and then confirmed using an anti-histidine- encode the indicated amino acids of the human Bcr protein fused in frame tag monoclonal antibody (H3, Santa Cruz Biotechnology). to the GAL4 DNA-binding domain. pGAD-tsg101(1-390), pGAD-tsg101(223- Immunostaining. Immunostaining of HeLa cells was done as described 390), pGAD-tsg101(1-345), and pGAD-tsg101(223-345) contain cDNAs that previously (33). Briefly, at 24 hours posttransfection, cells were trypsinized encode the indicated amino acids of the human TSG101 protein fused in and plated on 0.02% poly-L-lysine–coated glass coverslips. At 48 hours frame to the GAL4 DNA activation domain. The pAX142 mammalian posttransfection, cells were fixed with 3.7% formaldehyde in PBS for expression vector has been described previously (29). pAX142-bcr(1-1,271) 20 minutes, and then permeabilized and blocked for 1 hour in 0.1% encodes the full-length human Bcr protein fused at the NH2-terminus to a Triton X-100, 3% bovine serum albumin (BSA) in PBS. Coverslips were then hemagglutinin (HA) epitope tag (14). pAX142-tsg101(1-390) encodes the incubated in a humidity chamber with the appropriate monoclonal full-length TSG101 protein fused at the NH2 terminus to a myc-epitope tag. antibody for 1 hour in 0.1% Triton X-100, 0.1% BSA in PBS, washed GFP-hVps4 and GFP-hVps4(KQ) contain wild-type and dominant inhibitory in PBS, and then incubated with either Alexa Fluor 568 (red) or 488 www.aacrjournals.org 6251 Cancer Res 2006; 66: (12). June 15, 2006

(green)–conjugated secondary antibody (Molecular Probes, Carlsbad, CA) in the same buffer for 1 hour in the dark. Cells were viewed with a Zeiss Axiovert 200M microscope fitted with an ApoTome Imaging system. Image stacks in the axial direction were acquired and all images shown are from a representative axial plane. Epidermal growth factor receptor degradation assay. HeLa cells were transiently transfected by SilentFect reagent (Bio-Rad, Hercules, CA) with 20 nmol/L mixed small interfering RNA (siRNA) oligonucleotides targeted at either TSG101 or Bcr (Santa Cruz Biotechnology). Scrambled siRNAs were also included as negative controls. After 24 hours, cells were serum starved for an additional 12 hours and then 150 ng/mL epidermal growth factor (EGF; E9644, Sigma) was added to the medium for 90 minutes. Lysates were then collected and examined by Western blot for expression of the EGF receptor (EGFR; 1005, Santa Cruz Biotechnology), Bcr (N20, Santa Cruz Biotechnology), and TSG101 (C2, Santa Cruz Biotechnology).

Results p160 Bcr interacts with TSG101 in yeast. To identify new binding partners for p160 Bcr, a yeast two-hybrid screen was done.

Figure 2. p160 Bcr interacts with TSG101 and Vps28 in mammalian cells. IP, antibody used in immunoprecipitations; IB, antibody used in a Western blot to detect an interaction. A, 293T cells were cotransfected with the indicated combinations of plasmids [vector, pAX142; Bcr, pAX142-bcr(1-1271); TSG101, pAX142-tsg101(1-390)]. Lysates were collected at 48 hours and examined by Western blot for expression of TSG101 (middle) or Bcr (bottom). Immunoprecipitations were then done with an anti-Myc antibody to detect an interaction (top). B and C, endogenous Bcr and TSG101 interact in hematopoietic (HL60, K562) and nonhematopoietic (HeLa, 293T) cells. B, lysates were collected from K-562, HL-60, HeLa, and 293T cells and then immunoprecipitations were done by using either an anti-TSG101 antibody or an irrelevant anti-HA antibody. Interactions were shown by Western blot using an anti-Bcr antibody that recognizes the amino terminus of p160 Bcr (top). C, specificity of the interaction between endogenous Bcr and TSG101 in HeLa and 293T cells was confirmed using a blocking peptide for the Bcr antibody (N20-P, Santa Cruz Biotechnology). D, p160 Bcr interacts with endogenous Vps28 in HeLa and 293T cells. Lysates were collected and then immunoprecipitations were done with an anti-Vps28 antibody. An interaction was shown by Western blot using an anti-Bcr antibody (N20, Santa Cruz Biotechnology). The specificity of the binding was again confirmed with the Bcr N20 blocking peptide.

Multiple independent copies of four structurally unrelated clones were identified that interacted with the full-length p160 Bcr bait. One of these clones encodes TSG101, the mammalian orthologue of yeast Vps23 (Fig. 1A). Using an extended panel of Bcr baits, we were able to map the TSG101 docking site to a COOH-terminal region of Bcr (residues 1,037-1,165) that is lost in p210 Bcr-Abl. Yeast two- hybrid analysis was also used to map the Bcr binding site in Figure 1. Mammalian TSG101 and p160 Bcr interact in yeast. A, mapping of TSG101 to the COOH-terminal 45 amino acids (Fig. 1B; residues the TSG101 binding site within p160 Bcr. Top (top line), domain structure of the 345-390). full-length Bcr protein (O, oligomerization domain; kinase, serine/threonine kinase domain; DH/PH, RhoGEF domain; C2, calcium-binding domain); p160 Bcr interacts with TSG101 in mammalian cells. Next, bottom lines, regions of the protein included in predicted translation products of we wished to determine whether the association between TSG101 the various cDNA derivatives. Bottom, yeast colonies that grew on and Bcr can be recapitulated in mammalian cells. To address this plates lacking leucine and tryptophan (Leu/Trp) were examined for growth on histidine-deficient plates (Leu/Trp/His). Growth in the absence of leucine possibility, we coexpressed Myc epitope–tagged full-length TSG101 indicates the presence of pGAD containing residues 1 to 390 of TSG101, and HA-tagged full-length Bcr in 293T cells and then immunopre- whereas growth in the absence of tryptophan indicates the presence of pGBT9 containing the indicated derivatives of Bcr. Interactions between proteins are cipitated TSG101 (Fig. 2A) with an anti-Myc monoclonal antibody shown by the ability to activate the his3 reporter gene. B, full-length Bcr binds to (9E10, Santa Cruz Biotechnology). Immunoprecipitates were then the carboxyl-terminal region of TSG101. Top (top line), domain structure of the separated by Western blot and probed for the presence of Bcr using full-length TSG101 protein (PP, proline-rich domain; CC, coiled-coil domain; SB, steadiness box). Bottom, interactions between full-length p160 Bcr and the an anti-HA antibody (HAY11, Santa Cruz Biotechnology). Using this indicated derivatives of TSG101, determined as described above. approach, we were readily able to detect an association between

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Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 2006 American Association for Cancer Research. Bcr Interacts with the ESCRT-I Complex the two full-length proteins. To confirm that binding can be the ubiquitination of either protein. To confirm that ubiquitination detected in a more physiologically relevant context, the association does not mediate the association between full-length Bcr and between natively expressed proteins was also examined. Initially, TSG101 in mammalian cells, we tookadvantage of a cell line that we examined the interaction in a panel of hematopoietic (K562 and contains a temperature-sensitive allele of the mammalian E1 (ts20 HL60) and nonhematopoietic (293T and HeLa) cell lines (Fig. 2B). 3T3; ref. 31). We have shown in previous studies that ubiquitination K-562 is a human myeloid cell line that was isolated from a patient does not occur in these cells when they are cultured at the with CML and, thus, is also positive for p210 Bcr-Abl. HL-60 cells restrictive temperature (31). For this analysis, cells were either are a leukemic cell line of myeloid origin that does not contain left at the permissive temperature or shifted to the restrictive p210 Bcr-Abl. In all cell lines, we were able to coimmunoprecipitate temperature for 18 hours and then lysates were collected. The Bcr with an antibody that detects endogenous TSG101 (Fig. 2B, top) defect in the ubiquitination pathway was shown by the accumu- but not with an irrelevant antibody (Fig. 2B, bottom). The specificity lation of cyclin D2 at the restrictive temperature (Fig. 3B; ref. 31). of the interaction was then confirmed in 293T and HeLa cells using Because we were able to coimmunoprecipitate endogenous Bcr a blocking peptide for the Bcr antibody (N20-P, Fig. 2C). As and TSG101 at both the permissive and restrictive temperatures, predicted from our mapping studies, the TSG101 antibody did we conclude that the interaction does not require ubiquitination of not coimmunoprecipitate p210 Bcr-Abl from the K562 cells (not either protein in mammalian cells. shown). TSG101 and Bcr have similar patterns of subcellular TSG101 interacts with Vps28. TSG101 is a component of distribution. Although binding partners for Bcr have been mammalian ESCRT-I, a protein complex that is found on the described previously, they generally do not exhibit a pattern of limiting membrane of the multivesicular body (34). In addition to subcellular distribution that is consistent with the association. To TSG101, this complex also contains Vps28 and Vps37. To determine further confirm the association between TSG101 and Bcr, we did whether the TSG101-Bcr interaction is occurring in the context of dual staining in HeLa cells that express high endogenous levels of ESCRT-I, we determined whether we can also coimmunoprecipi- both proteins. In accordance with previous reports, TSG101 tate endogenous Bcr with a Vps28 antibody (N12, Santa Cruz exhibited a granular cytoplasmic staining that would be consistent Biotechnology). Using an antibody specific for Bcr (N20, Santa Cruz with an association with endosomal vesicles (36). Bcr exhibited an Biotechnology), we were readily able to detect the association almost identical staining pattern, although an additional pool of between endogenous Vps28 and Bcr in HeLa and 293T cells immunoreactivity was identified in the nucleus. The specificity of (Fig. 2D). The specificity of the interaction was also confirmed the Bcr staining was confirmed using two independent antibodies using the blocking peptide for the Bcr antibody (N20-P, Santa Cruz (not shown). The nuclear localization was observed with both Biotechnology). antibodies and is consistent with several previous reports, The interaction between TSG101 and Bcr is direct and including one from our own laboratory (12, 14). When we merged ubiquitin independent. Because TSG101 is thought to bind to its the images of TSG101 and Bcr, we observed that much, but not all, targets in a ubiquitin-dependent manner, we wondered whether of the cytoplasmic immunoreactivity was colocalized. When we did the association between Bcr and TSG101 is mediated by ubiqui- a similar colocalization experiment with Bcr and a marker for the tination (28, 35). To test this possibility, we used two approaches. late endosome (LAMP-I, H5G11, Santa Cruz Biotechnology), First, we purified His6-tagged TSG101 and GST-tagged Bcr in convergence of signal was not obvious. TSG101 was included in bacteria and did an in vitro binding assay. An interaction was this analysis for comparison and also did not colocalize with observed between His6-TSG101(223-390) and GST-Bcr(871-1,271) LAMP-I. This suggests that the association between TSG101 and but not with GST alone, indicating that the interaction is direct Bcr may be occurring at a site other than the surface of the late (Fig. 3A). Because ubiquitination does not occur in prokaryotes, endosomal membrane (Fig. 4). this result would also suggest that the interaction does not require AVps4 mutant that blocks endosomal trafficking interferes with the subcellular distribution of Bcr. To determine whether Bcr can cycle onto the endosomal membrane, we used a dominant- inhibitory mutant of Vps4 [Vps4(KQ), ref. 31]. Vps4 is a member of a family of ATPases that have been implicated in multiple cellular processes, including endosomal/vacuolar sorting (37). In both yeast and mammalian cells, Vps4 is a class E protein that is required for the disassembly of the ESCRT complexes at the completion of the sorting reaction (38–40). The dominant inhibitory mutant lacks ATPase activity and is thought to accumulate on the limiting membranes of the late endosome (30, 41). Importantly, other class E molecules, such as TSG101, have been shown to accumulate on the same membrane compartments in the presence of the mutant Figure 3. The interaction between TSG101 and Bcr is direct and ubiquitin (36). For our analysis, HeLa cells were transiently transfected with independent. A, His6-TSG101(223-390) interacts with GST-Bcr(871-1,271) in an Bcr or TSG101, and either GFP-Vps4(KQ) or GFP-Vps4(WT) (see in vitro affinity precipitation assay. Bacterially purified His6-TSG101(223-390) was incubated with bacterially expressed, Sepharose-linked GST or Fig. 5). GFP alone was also included in the analysis for comparison. GST-Bcr(871-1,271). The beads were then washed and the interaction was There seemed to be no obvious changes in overall cellular shown by Western blot using an anti-TSG101 antibody. B, Bcr and TSG101 interact in mammalian cells in the absence of ubiquitination. ts20 3T3cells were morphology in response to expression of either GFP-Vps4(KQ) or cultured at the permissive (35jC) and restrictive (39jC) temperatures for GFP-Vps4(WT) (Fig. 5). As described previously, GFP-Vps4(WT) 18 hours and then lysates were collected and examined by coimmunoprecipitation exhibited a diffuse staining throughout the cell, whereas GFP- for an interaction between endogenous TSG101 and Bcr. The defect in ubiquitination was shown by the accumulation of cyclin D2 at the restrictive Vps4(KQ) accumulated in clustered cytoplasmic foci (30). In the temperature. RhoB expression was also monitored as a negative control. presence of the mutant, both TSG101 and Bcr also accumulated in www.aacrjournals.org 6253 Cancer Res 2006; 66: (12). June 15, 2006

Figure 4. TSG101 and Bcr exhibit a similar subcellular distribution. HeLa cells were examined by indirect immunofluorescence for the cellular distribution of endogenous TSG101, Bcr, and LAMP-1. Cells were viewed with a Zeiss Axiovert 200M microscope fitted with an ApoTome Imaging system. Image stacks in the axial direction were acquired and all images shown are from a representative axial plane. Cells shown are typical of >50 cells that were examined for the expression of the indicated combination of proteins.

strongly staining cytoplasmic foci that were not apparent in the Discussion presence of GFP alone or wild-type Vps4. When we merged the Previous studies that describe putative binding partners for Bcr images, a limited, but reproducible convergence of signal between have provided relatively little information about its cytoplasmic hVps4(KQ) and either Bcr (Fig. 5A) or TSG01 (Fig. 5B) was function. The NH2-terminus of Bcr interacts with and phosphor- observed. The level of convergence that is shown is representative ylates at least five isoforms of the 14-3-3 proteins (44). Because and is observed in 75% of all cells examined. Although these different members of the 14-3-3 family have been implicated in a observations suggest that both Bcr and TSG101 may be variety of cytoplasmic activities, including apoptosis, signal accumulating on intracellular membranes in response to Vps4 transduction, trafficking, and secretion, the meaning of these expression, they both seem to have a distribution that is, for the interactions remains unclear. Bcr contains a COOH-terminal PDZ- most part, discrete from the Vps4 mutant. binding domain through which it is reported to interact with Bcr is required for EGFR degradation. The interaction of Bcr several cytoplasmic proteins, including AF-6, PDZK1, and Mint3 with components of the ESCRT-I complex suggests that it may (45, 46). AF-6 has been colocalized with tight junctions and function in the sorting machinery of the endosome. It has been adhesion junctions and is thought to mediate the interaction previously shown that components of ESCRT-I mediate a sorting between the plasma membrane and the actin cytoskeleton (46). pathway that regulates the internalization and lysosome-mediated PDZ-K1 is also a plasma membrane–associated protein that is degradation of growth factor receptors (27, 35, 42, 43). For predominantly found in association with apical membrane example, interference with TSG101 in HeLa cells blocks EGF- proteins in polarized epithelia (45). Although these associations mediated receptor turnover and promotes increased receptor are consistent with the presence of Rho-modulating domains recycling to the plasma membrane (27). To determine whether within Bcr, most Bcr immunoreactivity is found on intracellular receptor turnover also requires Bcr function, we suppressed Bcr membranes and none is readily observed on the plasma expression in HeLa cells and then monitored the degradation of membrane. This suggests that these associations are either the receptor in response to EGF stimulation (Fig. 6). TSG101 was transient or highly cell specific. Interestingly, the binding partner included as a positive control because it has been shown pre- for Bcr that has the most similar cellular distribution is Mint3 (45). viously that siRNA-mediated suppression of TSG101 in these cells Mint3 is found primarily in the Golgi compartment where it has causes the accumulation of the receptor at the plasma membrane been implicated in protein processing and vesicular trafficking in (27). Consistent with previous observations, we observed an the distal secretory pathway. Mint3 has a punctate cytoplasmic accumulation of the receptor in EGF-stimulated cells in which staining that partially overlaps with Bcr and it has been previously TSG101 expression was suppressed by siRNA. A similar accumu- proposed that Bcr may have a role in cellular trafficking (45). lation of the receptor was observed when Bcr expression was In the current study, we have identified two structurally suppressed. No accumulation was noted when cells were treated unrelated subunits of the endosomal sorting machinery as binding with scrambled control siRNAs. This observation implies over- partners for Bcr. Bcr interacts with TSG101 and Vps28 in HeLa and lapping functions for Bcr and TSG101 in HeLa cells, and suggests 293T cells, and Bcr and TSG101 exhibit a similar subcellular that each may be required for endosome-mediated receptor distribution in these cell types. The association of Bcr with both internalization. TSG101 and Vps28 suggests a very specific role for Bcr in the

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Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 2006 American Association for Cancer Research. Bcr Interacts with the ESCRT-I Complex endosomal pathway. In eukaryotic cells, transmembrane proteins that targets proteins into this pathway (28). The formation of the can be removed from the cell surface by endocytosis, followed by multivesicular body and the sorting of ubiquitinated proteins is lysosome-mediated degradation. On the limiting membrane of the controlled by a large collection of proteins that was originally multivesicular body, proteins that are being transported in this identified in yeast as the class E Vps (reviewed in ref. 47). Because pathway are sorted into vesicles that first bud into the lumen of the both TSG101 and Vps28 are class E Vps proteins in yeast and compartment and then are delivered to the lysosome (34). mammalian cells, we propose that Bcr may also be a member of Although the mechanism by which cargo is sorted as it passes this class. through the multivesicular body is poorly understood, there is Vps proteins are organized into three discrete complexes, termed evidence to suggest that monoubiquitination is used as the flag ESCRT-I, ESCRT-II, and ESCRT-III, that are recruited from the

Figure 5. A dominant inhibitory mutant of Vps4 alters the cellular distribution of Bcr and TSG101. HeLa cells were transiently transfected with GFP alone or GFP fused to wild-type or dominant-inhibitory Vps4. Cells were examined at 48 hours by indirect immunofluorescence for the cellular distribution of Vps4 and Bcr (A), or Vps4 and TSG101 (B). Cells were viewed with a Zeiss Axiovert 200M microscope fitted with an ApoTome Imaging system. Image stacks in the axial direction were acquired and all images shown are from a representative axial plane. Cells shown are typical of >50 cells that were examined for the expression of the indicated combination of proteins.

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multivesicular body in response to growth factor stimulation where it identifies and routes the internalized receptors to the lysosome. TSG101 binds directly to ubiquitinated proteins and thus may serve to recognize the targeted receptors (28, 35). Because the interaction between TSG101 and Bcr does not require ubiquitination, it is unlikely that Bcr is simply a preferred endosomal cargo in this pathway that is being recognized by TSG101. Once the receptor internalization is complete, the ESCRT complexes are disassembled from the limiting membrane of the multivesicular body in response to the action of the Vps4 ATPase. Interference of TSG101 through siRNA or Vps28 by neutralizing antibodies causes an accumulation of the EGFR on the cell surface in response to treatment with EGF (35, 49). Similarly, we have observed that siRNAs directed against both TSG101 and Bcr cause equivalent accumulations of EGFR on the cell surface. Thus, the association of Bcr with a complex that includes TSG101 and Vps28 seems to have functional relevance Figure 6. Bcr is required for ligand-mediated turnover of the EGFR. A, siRNAs with regard to the efficient turnover of growth factor receptors. were used to transiently suppress the expression Bcr or TSG101 in HeLa cells. Despite the fact that our binding studies suggest that Bcr Cells were then treated for 90 minutes with 150 ng/mL EGF and then lysates were collected and examined for expression of EGFR (anti-EGFR), Bcr interacts with ESCRT complexes, we do not observe a good (anti-Bcr), and TSG101 (anti-TSG101). B, relative levels of receptor expression convergence of signal between either TSG101 or Bcr, and a marker were determined using an Odyssey IR imager and expressed as integrated for the late endosomal membrane (LAMP-I). Because ESCRT intensity units. Average of three independent experiments. complexes cycle between the cytoplasm and the limiting membrane of the late endosome, we assume that the majority of the cytoplasm to act sequentially at the surface of the multivesicular TSG101-Bcr complexes in HeLa cells are in the soluble, resting body (28, 39, 48). ESCRT-I is thought to recognize ubiquitinated state. Our observation that the dominant-inhibitory Vps4 mutant proteins and trigger the assembly and activation of ESCRT-II. causes both TSG101 and Bcr to accumulate on densely staining ESCRT-II, in turn, is required for the recruitment of ESCRT-III cytoplasmic foci suggests that at least some of these complexes are whose role is to concentrate the multivesicular body cargo for being cycled onto membranes and can be trapped by the action of internalization and remove the ubquitination tag. Once sorting is the mutant before membrane dissociation. completed, Vps4 binds to ESCRT-III and disassembles the complex Our observation that Bcr interacts with subunits of the ESCRT (37, 38). The Vps proteins are highly conserved from yeast to complexes raises the possibility that endosomal trafficking may be mammals, and mutations in the mammalian orthologues exhibit impaired in CML. Although the interaction between Bcr and phenotypes consistent with defects in endosomal trafficking. For TSG101 is still observed in K562 cells that express p210 Bcr-Abl, we most components of the ESCRT complexes, one or more mam- have also observed that incomplete suppression of either Bcr or malian counterparts have now been identified. Because the mam- TSG101 by siRNA is sufficient to impair growth factor receptor malian counterpart of the ESCRT-I complex contains both TSG101 turnover in HeLa cells. Thus, the reduced dosage of Bcr that occurs and Vps28 (reviewed in ref. 34), our binding data suggests that Bcr in Philadelphia chromosome–positive cells may in fact be sufficient is either a component or a regulator of this complex. Whether Bcr to cause impairment in trafficking. Alternatively, p210 Bcr-Abl may also binds directly to Vps28 in this complex is unclear. Because it exhibit a dominant-inhibitory effect with respect to the normal has been shown previously that TSG101 interacts with mammalian endosomal function of Bcr. Although the TSG101 docking site is Vps28 in HeLa cells, our immunoprecipitations with the Vps28 lost in p210 Bcr-Abl, the NH2 terminus of Bcr may interact with antibody may be detecting Bcr indirectly through its association additional components of the sorting complex, such as Vps28. with TSG101 (36). To resolve this issue, in vitro studies are currently Thus, p210 Bcr-Abl may be recruited to the endosome and interfere under way to examine the ability of Bcr to directly interact with the with the normal function of Bcr. Both of these possibilities are various components of the mammalian ESCRT complexes. currently under investigation. However, regardless of whether the interaction with Vps28 is direct, the existence of a complex containing Bcr, TSG101, and Vps28 in mammalian cells strongly suggests that Bcr is a bona fide Acknowledgments component of the mammalian endosomal sorting machinery. Received 2/10/2006; revised 3/29/2006; accepted 4/12/2006. Support for a role for Bcr in endosomal sorting also comes from Grant support: NIH grants R01 CA097066 (I. Whitehead), RO1 AG04821 (H. Ozer), our functional studies. In mammalian cells, the ESCRT-I complex is F31 CA117049 (O. Olabisi), and F32 CA113049 (E. Kostenko). The costs of publication of this article were defrayed in part by the payment of page required for the down-regulation of growth factor receptors (27, 35, charges. This article must therefore be hereby marked advertisement in accordance 42, 43). The complex is recruited from the cytoplasm to the with 18 U.S.C. Section 1734 solely to indicate this fact.

References 3. O’Dwyer ME, Druker BJ. Chronic myelogenous leu- ABL-induced oncogenesis is mediated by direct inter- kaemia—new therapeutic principles. J Intern Med 2001; action with the SH2 domain of the GRB-2 adaptor 1. Maru Y. Molecular biology of chronic myeloid 250:3–9. protein. Cell 1993;75:175–85. leukemia. Int J Hematol 2001;73:308–22. 4. Lugo TG, Pendergast AM, Muller AJ, Witte ON. 6. McWhirter JR, Galasso DL, Wang JY. A coiled-coil 2. Nowell PC, Hungerford DA. The etiology of leukemia: Tyrosine kinase activity and transformation potency of oligomerization domain of Bcr is essential for the some comments on current studies. Semin Hematol bcr-abl oncogene products. Science 1990;247:1079–82. transforming function of Bcr-Abl oncoproteins. Mol Cell 1966;3:114–21. 5. Pendergast AM, Quilliam LA, Cripe LD, et al. BCR- Biol 1993;13:7587–95.

Cancer Res 2006; 66: (12). June 15, 2006 6256 www.aacrjournals.org

7. MaruY,AfarDE,WitteON,ShibuyaM.The TSG101 deficiency. Proc Natl Acad Sci U S A 1998;95: recruited to VPS4-induced endosomes. J Biol Chem dimerization property of glutathione S-transferase 1595–600. 2001;276:11735–42. partially reactivates Bcr-Abl lacking the oligomerization 22. Ruland J, Sirard C, Elia A, et al. p53 accumulation, 37. Babst M, Sato TK, Banta LM, Emr SD. Endosomal domain. J Biol Chem 1996;271:15353–7. defective cell proliferation, and early embryonic lethality transport function in yeast requires a novel AAA-type 8. Muller AJ, Young JC, Pendergast AM, et al. BCR first in mice lacking tsg101. Proc Natl Acad Sci U S A 2001;98: ATPase, Vps4p. EMBO J 1997;16:1820–31. exon sequences specifically activate the BCR/ABL 1859–64. 38. Babst M, Wendland B, Estepa EJ, Emr SD. The Vps4p tyrosine kinase oncogene of Philadelphia chromo- 23. Wagner KU, Krempler A, Qi Y, et al. Tsg101 is AAA ATPase regulates membrane association of a Vps some-positive human leukemias. Mol Cell Biol 1991;11: essential for cell growth, proliferation, and cell survival protein complex required for normal endosome func- 1785–92. of embryonic and adult tissues. Mol Cell Biol 2003;23: tion. EMBO J 1998;17:2982–93. 9. Whitehead IP, Campbell S, Rossman KL, Der CJ. Dbl 150–62. 39. Babst M, Katzmann DJ, Snyder WB, Wendland B, family proteins. Biochim Biophys Acta 1997;1332:F1–23. 24. Watanabe M, Yanagi Y, Masuhiro Y, et al. A putative Emr SD. Endosome-associated complex, ESCRT-II, 10. Nalefski EA, Falke JJ. The C2 domain calcium-binding tumor suppressor, TSG101, acts as a transcriptional recruits transport machinery for protein sorting at the motif: structural and functional diversity. Protein Sci suppressor through its coiled-coil domain. Biochem multivesicular body. Dev Cell 2002;3:283–9. 1996;5:2375–90. Biophys Res Commun 1998;245:900–5. 40. Odorizzi G, Katzmann DJ, Babst M, Audhya A, Emr 11. Chuang TH, Xu X, Kaartinen V, Heisterkamp N, 25. Li L, Liao J, Ruland J, MakTW, Cohen SN. A TSG101/ SD. Bro1 is an endosome-associated protein that Groffen J, Bokoch GM. Abr and Bcr are multifunctional MDM2 regulatory loop modulates MDM2 degradation functions in the MVB pathway in Saccharomyces regulators of the Rho GTP-binding protein family. Proc and MDM2/p53 feedbackcontrol. Proc Natl Acad Sci cerevisiae. J Cell Sci 2003;116:1893–903. Natl Acad Sci U S A 1995;92:10282–6. U S A 2001;98:1619–24. 41. Yoshimori T, Yamagata F, Yamamoto A, et al. The 12. Wetzler M, Talpaz M, Van Etten RA, Hirsh-Ginsberg 26. Zhong Q, Chen Y, Jones D, Lee WH. Perturbation of mouse SKD1, a homologue of yeast Vps4p, is required C, Beran M, KurzrockR. Subcellular localization of Bcr, TSG101 protein affects cell cycle progression. Cancer for normal endosomal trafficking and morphology in Abl, and Bcr-Abl proteins in normal and leukemic cells Res 1998;58:2699–702. mammalian cells. Mol Biol Cell 2000;11:747–63. and correlation of expression with myeloid differentia- 27. Babst M, Odorizzi G, Estepa EJ, Emr SD. Mammalian 42. Bache KG, Slagsvold T, Cabezas A, Rosendal KR, tion. J Clin Invest 1993;92:1925–39. tumor susceptibility gene 101 (TSG101) and the yeast Raiborg C, StenmarkH. The growth-regulatory protein 13. Laurent E, Talpaz M, Wetzler M, KurzrockR. homologue, Vps23p, both function in late endosomal HCRP1/hVps37A is a subunit of mammalian ESCRT-I Cytoplasmic and nuclear localization of the 130 and trafficking. Traffic 2000;1:248–58. and mediates receptor down-regulation. Mol Biol Cell 160 kDa Bcr proteins. Leukemia 2000;14:1892–7. 28. Katzmann DJ, Babst M, Emr SD. Ubiquitin-depen- 2004;15:4337–46. 14. Mahon GM, Wang Y, Korus M, et al. The c-Myc dent sorting into the multivesicular body pathway 43. Lu Q, Hope LW, Brasch M, Reinhard C, Cohen SN. oncoprotein interacts with Bcr. Curr Biol 2003;13: requires the function of a conserved endosomal protein TSG101 interaction with HRS mediates endosomal 437–41. sorting complex, ESCRT-I. Cell 2001;106:145–55. trafficking and receptor down-regulation. Proc Natl 15. Voncken JW, Baram TZ, Gonzales-Gomez II, et al. 29. Whitehead I, KirkH, Tognon C, Trigo-Gonzalez G, Acad Sci U S A 2003;100:7626–31. Abnormal stress response and increased fighting Kay R. Expression cloning of lfc, a novel oncogene with 44. Reuther GW, Fu H, Cripe LD, Collier RJ, Pendergast behavior in mice lacking the bcr gene product. Int J structural similarities to guanine nucleotide exchange AM. Association of the protein kinases c-Bcr and Bcr- Mol Med 1998;2:577–83. factors and to the regulatory region of protein kinase C. Abl with proteins of the 14-3-3 family. Science 1994;266: 16. Kaartinen V, Nagy A, Gonzalez-Gomez I, Groffen J, J Biol Chem 1995;270:18388–95. 129–33. Heisterkamp N. Vestibular dysgenesis in mice lacking 30. Bishop N, Woodman P. ATPase-defective mammalian 45. Malmberg EK, Andersson CX, Gentzsch M, et al. Bcr Abr and Bcr Cdc42/RacGAPs. Dev Dyn 2002;223:517–25. VPS4 localizes to aberrant endosomes and impairs (breakpoint cluster region) protein binds to PDZ- 17. Kaartinen V, Gonzalez-Gomez I, Voncken JW, et al. cholesterol trafficking. Mol Biol Cell 2000;11:227–39. domains of scaffold protein PDZK1 and vesicle coat Abnormal function of astroglia lacking Abr and Bcr 31. Chowdary DR, Dermody JJ, Jha KK, Ozer HL. protein Mint3. J Cell Sci 2004;117:5535–41. RacGAPs. Development 2001;128:4217–27. Accumulation of p53 in a mutant cell line defective in 46. Radziwill G, Erdmann RA, Margelisch U, Moelling K. 18. Voncken JW, van Schaick H, Kaartinen V, et al. the ubiquitin pathway. Mol Cell Biol 1994;14:1997–2003. The Bcr kinase downregulates Ras signaling by phos- Increased neutrophil respiratory burst in bcr-null 32. Whitehead IP, Lambert QT, Glaven JA, et al. phorylating AF-6 and binding to its PDZ domain. Mol mutants. Cell 1995;80:719–28. Dependence of Dbl and Dbs transformation on MEK Cell Biol 2003;23:4663–72. 19. Li L, Cohen SN. Tsg101: a novel tumor susceptibility and NF-nB activation. Mol Cell Biol 1999;19:7759–70. 47. Katzmann DJ, Odorizzi G, Emr SD. Receptor down- gene isolated by controlled homozygous functional 33. Rossman KL, Cheng L, Mahon GM, et al. Multifunc- regulation and multivesicular-body sorting. Nat Rev Mol knockout of allelic loci in mammalian cells. Cell 1996; tional roles for the PH domain of Dbs in regulating Rho Cell Biol 2002;3:893–905. 85:319–29. GTPase activation. J Biol Chem 2003;278:18393–400. 48. Babst M, Katzmann DJ, Estepa-Sabal EJ, Meerloo T, 20. Feng GH, Lih CJ, Cohen SN. TSG101 protein steady- 34. Babst M. A protein’s final ESCRT. Traffic 2005;6:2–9. Emr SD. Escrt-III: an endosome-associated heterooligo- state level is regulated posttranslationally by an evolu- 35. Bishop N, Horman A, Woodman P. Mammalian class meric protein complex required for mvb sorting. Dev tionarily conserved COOH-terminal sequence. Cancer E vps proteins recognize ubiquitin and act in the Cell 2002;3:271–82. Res 2000;60:1736–41. removal of endosomal protein-ubiquitin conjugates. 49. Doyotte A, Russell MR, Hopkins CR, Woodman PG. 21. Xie W, Li L, Cohen SN. Cell cycle-dependent J Cell Biol 2002;157:91–101. Depletion of TSG101 forms a mammalian ‘‘Class E’’ subcellular localization of the TSG101 protein and 36. Bishop N, Woodman P. TSG101/mammalian VPS23 compartment: a multicisternal early endosome with mitotic and nuclear abnormalities associated with and mammalian VPS28 interact directly and are multiple sorting defects. J Cell Sci 2005;118:3003–17.

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Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 2006 American Association for Cancer Research. Bcr Interacts with Components of the Endosomal Sorting Complex Required for Transport-I and Is Required for Epidermal Growth Factor Receptor Turnover

Oyenike O. Olabisi, Gwendolyn M. Mahon, Elena V. Kostenko, et al.

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