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

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Bcr Interacts with Components of the Endosomal Sorting Complex Required for Transport-I and Is Required for Epidermal Growth Factor Receptor Turnover Research Article 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, 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 Cancer Res 2006; 66: (12). June 15, 2006 6250 www.aacrjournals.org 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
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