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• W1980002411 abstract "The chemokine receptor CXCR4 plays important roles in the immune and nervous systems. Abnormal expression of CXCR4 contributes to cancer and inflammatory and neurodegenerative disorders. Although ligand-dependent CXCR4 ubiquitination is known to accelerate CXCR4 degradation, little is known about counter mechanisms for receptor deubiquitination. CXCL12, a CXCR4 agonist, induces a time-dependent association of USP14 with CXCR4, or its C terminus, that is not mimicked by USP2A, USP4, or USP7, other members of the deubiquitination catalytic family. Co-localization of CXCR4 and USP14 also is time-dependent following CXCL12 stimulation. The physical interaction of CXCR4 and USP14 is paralleled by USP14-catalyzed deubiquitination of the receptor; knockdown of endogenous USP14 by RNA interference (RNAi) blocks CXCR4 deubiquitination, whereas overexpression of USP14 promotes CXCR4 deubiquitination. We also observed that ubiquitination of CXCR4 facilitated receptor degradation, whereas overexpression of USP14 or RNAi-induced knockdown of USP14 blocked CXCL12-mediated CXCR4 degradation. Most interestingly, CXCR4-mediated chemotactic cell migration was blocked by either overexpression or RNAi-mediated knockdown of USP14, implying that a CXCR4-ubiquitin cycle on the receptor, rather than a particular ubiquitinated state of the receptor, is critical for the ligand gradient sensing and directed motility required for chemokine-mediated chemotaxis. Our observation that a mutant of CXCR4, HA-3K/R CXCR4, which cannot be ubiquitinated and does not mediate a chemotactic response to CXCL12, indicates the importance of this covalent modification not only in marking receptors for degradation but also for permitting CXCR4-mediated signaling. Finally, the indistinguishable activation of ERK by wild typeor 3K/R-CXCR4 suggests that chemotaxis in response to CXCL12 may be independent of the ERK cascade. The chemokine receptor CXCR4 plays important roles in the immune and nervous systems. Abnormal expression of CXCR4 contributes to cancer and inflammatory and neurodegenerative disorders. Although ligand-dependent CXCR4 ubiquitination is known to accelerate CXCR4 degradation, little is known about counter mechanisms for receptor deubiquitination. CXCL12, a CXCR4 agonist, induces a time-dependent association of USP14 with CXCR4, or its C terminus, that is not mimicked by USP2A, USP4, or USP7, other members of the deubiquitination catalytic family. Co-localization of CXCR4 and USP14 also is time-dependent following CXCL12 stimulation. The physical interaction of CXCR4 and USP14 is paralleled by USP14-catalyzed deubiquitination of the receptor; knockdown of endogenous USP14 by RNA interference (RNAi) blocks CXCR4 deubiquitination, whereas overexpression of USP14 promotes CXCR4 deubiquitination. We also observed that ubiquitination of CXCR4 facilitated receptor degradation, whereas overexpression of USP14 or RNAi-induced knockdown of USP14 blocked CXCL12-mediated CXCR4 degradation. Most interestingly, CXCR4-mediated chemotactic cell migration was blocked by either overexpression or RNAi-mediated knockdown of USP14, implying that a CXCR4-ubiquitin cycle on the receptor, rather than a particular ubiquitinated state of the receptor, is critical for the ligand gradient sensing and directed motility required for chemokine-mediated chemotaxis. Our observation that a mutant of CXCR4, HA-3K/R CXCR4, which cannot be ubiquitinated and does not mediate a chemotactic response to CXCL12, indicates the importance of this covalent modification not only in marking receptors for degradation but also for permitting CXCR4-mediated signaling. Finally, the indistinguishable activation of ERK by wild typeor 3K/R-CXCR4 suggests that chemotaxis in response to CXCL12 may be independent of the ERK cascade. The CXCR4 (CXC chemokine receptor 4) is a member of the chemokine receptor family, which belongs to the superfamily of G protein-coupled receptors (GPCRs) 2The abbreviations used are: GPCR, G protein-coupled receptor; HEK293 cells, human embryonic kidney 293 cells; RIPA, radioimmunoprecipitation assay; siRNA, short interference RNA; USP, ubiquitin-specific protease; EGFP, enhanced green fluorescence protein; Ub, ubiquitin; ERK, extracellular signal-regulated kinase; GST, glutathione S-transferase; PBS, phosphate-buffered saline; WT, wild type; HA, hemagglutinin. (1Haribabu B. Richardson R.M. Fisher I. Sozzani S. Peiper S.C. Horuk R. Ali H. Snyderman R. J. Biol. Chem. 1997; 272: 28726-28731Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar). Its ligand, CXCL12, also known as SDF-1α, also binds to RDC1, another chemokine receptor that is being proposed to be renamed as CXCR7 (2Balabanian K. Lagane B. Infantino S. Chow K.Y. Harriague J. Moepps B. Arenzana-Seisdedos F. Thelen M. Bachelerie F. J. Biol. Chem. 2005; 280: 35760-35766Abstract Full Text Full Text PDF PubMed Scopus (847) Google Scholar). CXCR4 mediates CXCL12-induced migration of peripheral blood lymphocytes (3Nagasawa T. Hirota S. Tachibana K. Takakura N. Nishikawa S. Kitamura Y. Yoshida N. Kikutani H. Kishimoto T. Nature. 1996; 382: 635-638Crossref PubMed Scopus (2020) Google Scholar), CD34+ progenitor cells (4Aiuti A. Webb I.J. Bleul C. Springer T. Gutierrez-Ramos J.C. J. Exp. Med. 1997; 185: 111-120Crossref PubMed Scopus (1206) Google Scholar), and pre- and pro-B cell lines (5D'Apuzzo M. Rolink A. Loetscher M. Hoxie J.A. Clark-Lewis I. Melchers F. Baggiolini M. Moser B. Eur. J. Immunol. 1997; 27: 1788-1793Crossref PubMed Scopus (287) Google Scholar). CXCR4 also plays an important role in the development of the immune system, because mouse embryos lacking either expression of the CXCR4 receptor or of its CXCL12 ligand are embryonic lethal and also manifest abnormalities in B cell lymphopoiesis and bone marrow myelopoiesis (3Nagasawa T. Hirota S. Tachibana K. Takakura N. Nishikawa S. Kitamura Y. Yoshida N. Kikutani H. Kishimoto T. Nature. 1996; 382: 635-638Crossref PubMed Scopus (2020) Google Scholar, 6Ma Q. Jones D. Borghesani P.R. Segal R.A. Nagasawa T. Kishimoto T. Bronson R.T. Springer T.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9448-9453Crossref PubMed Scopus (1434) Google Scholar, 7Zou Y.R. Kottmann A.H. Kuroda M. Taniuchi I. Littman D.R. Nature. 1998; 393: 595-599Crossref PubMed Scopus (2134) Google Scholar). The altered cerebellar neuron migration in mice null for the CXCR4 receptor also suggests a role for this receptor in central nervous system development. Abnormal expression and/or function of CXCR4 have been implicated in a number of diseases, including human immunodeficiency virus infection (8Feng Y. Broder C.C. Kennedy P.E. Berger E.A. Science. 1996; 272: 872-877Crossref PubMed Scopus (3643) Google Scholar), cardiovascular disease (9Walter D.H. Haendeler J. Reinhold J. Rochwalsky U. Seeger F. Honold J. Hoffmann J. Urbich C. Lehmann R. Arenzana-Seisdesdos F. Aicher A. Heeschen C. Fichtlscherer S. Zeiher A.M. Dimmeler S. Circ. Res. 2005; 97: 1142-1151Crossref PubMed Scopus (287) Google Scholar), allergic inflammatory disease (10Abu El-Asrar A.M. Struyf S. Al-Mosallam A.A. Missotten L. Van Damme J. Geboes K. Br. J. Ophthalmol. 2001; 85: 1357-1361Crossref PubMed Scopus (44) Google Scholar), neuroinflammation (11Rauer M. Pagenstecher A. Schulte-Mönting J. Sauder C. J. Neurovirol. 2002; 8: 168-179Crossref PubMed Scopus (16) Google Scholar), neurodegenerative diseases (12Mines M. Ding Y. Fan G.H. Curr. Med. Chem. 2007; 14: 2456-2470Crossref PubMed Scopus (53) Google Scholar, 13Xia M.Q. Hyman B.T. J. Neurovirol. 1999; 5: 32-41Crossref PubMed Scopus (233) Google Scholar), and cancers (14Burger M. Glodek A. Hartmann T. Schmitt-Graff A. Silberstein L.E. Fujii N. Kipps T.J. Burger J.A. Oncogene. 2003; 22: 8093-8101Crossref PubMed Scopus (234) Google Scholar, 15Burger J.A. Kipps T.J. Blood. 2006; 107: 1761-1767Crossref PubMed Scopus (1009) Google Scholar, 16Su L. Zhang J. Xu H. Wang Y. Chu Y. Liu R. Xiong S. Clin. Cancer Res. 2005; 11: 8273-8280Crossref PubMed Scopus (137) Google Scholar, 17Saur D. Seidler B. Schneider G. Algül H. Beck R. Senekowitsch-Schmidtke R. Schwaiger M. Schmid R.M. Gastroenterology. 2005; 129: 1237-1250Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar, 18Kang H. Watkins G. Douglas-Jones A. Mansel R.E. Jiang W.G. Breast. 2005; 14: 360-367Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, 19Cabioglu N. Sahin A. Doucet M. Yavuz E. Igci A. O Yildirim E. Aktas E. Bilgic S. Kiran B. Deniz G. Price J.E. Clin. Exp. Metast. 2005; 22: 39-46Crossref PubMed Scopus (74) Google Scholar, 20Rempel S.A. Dudas S. Ge S. Gutiérrez J.A. Clin. Cancer Res. 2000; 6: 102-111PubMed Google Scholar, 21Oh J.W. Drabik K. Kutsch O. Choi C. Tousson A. Benveniste E.N. J. Immunol. 2001; 166: 2695-2704Crossref PubMed Scopus (104) Google Scholar, 22Zhou Y. Larsen P.H. Hao C. Yong V.W. J. Biol. Chem. 2002; 277: 49481-49487Abstract Full Text Full Text PDF PubMed Scopus (328) Google Scholar, 23Woerner B.M. Warrington N.M. Kung A.L. Perry A. Rubin J.B. Cancer Res. 2005; 65: 11392-11399Crossref PubMed Scopus (90) Google Scholar, 24Hong X. Jiang F. Kalkanis S.N. Zhang Z.G. Zhang X.P. DeCarvalho A.C. Katakowski M. Bobbitt K. Mikkelsen T. Chopp M. Cancer Lett. 2006; 236: 39-45Crossref PubMed Scopus (101) Google Scholar). Stimulation of CXCR4 triggers various intracellular signaling cascades (1Haribabu B. Richardson R.M. Fisher I. Sozzani S. Peiper S.C. Horuk R. Ali H. Snyderman R. J. Biol. Chem. 1997; 272: 28726-28731Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar, 14Burger M. Glodek A. Hartmann T. Schmitt-Graff A. Silberstein L.E. Fujii N. Kipps T.J. Burger J.A. Oncogene. 2003; 22: 8093-8101Crossref PubMed Scopus (234) Google Scholar, 25Wysoczynski M. Reca R. Ratajczak J. Kucia M. Shirvaikar N. Honczarenko M. Mills M. Wanzeck J. Janowska-Wieczorek A. Ratajczak M.Z. Blood. 2005; 105: 40-48Crossref PubMed Scopus (200) Google Scholar, 26Daaka Y. Luttrell L.M. Lefkowitz R.J. Nature. 1997; 390: 88-91Crossref PubMed Scopus (1077) Google Scholar, 27Peng S.B. Peek V. Zhai Y. Paul D.C. Lou Q. Xia X. Eessalu T. Kohn W. Tang S. Mol. Cancer Res. 2005; 3: 227-236Crossref PubMed Google Scholar), such as extracellular signal-regulated kinase (ERK), which likely contribute to CXCR4-induced cell proliferation, differentiation, and/or migration. Ligand stimulation of CXCR4 also induces endocytosis of these receptors, which are targeted to lysosomes for degradation through a pathway involving ubiquitination of the C-terminal lysine residues (28Marchese A. Benovic J.L. J. Biol. Chem. 2001; 276: 45509-45512Abstract Full Text Full Text PDF PubMed Scopus (392) Google Scholar). CXCR4 ubiquitination can be catalyzed by a member of the HECT family of E3 ligases, AIP4 (atrophin-interacting protein 4) (29Pickart C.M. Annu. Rev. Biochem. 2001; 70: 503-533Crossref PubMed Scopus (2944) Google Scholar, 30Marchese A. Raiborg C. Santini F. Keen J.H. StenMark H. Benovic J.L. Dev. Cell. 2003; 5: 709-722Abstract Full Text Full Text PDF PubMed Scopus (320) Google Scholar). The ubiquitinated CXCR4 is delivered to the endosomal compartments via a regulated pathway involving several adaptor proteins (31Bhandari D. Trejo J. Benovic J.L. Marchese A. J. Biol. Chem. 2007; 282: 36971-36979Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar). It has been noted that deubiquitination also regulates the fate and function of ubiquitin-conjugated proteins. Deubiquitinating enzymes, which catalyze the removal of ubiquitin from ubiquitin-conjugated proteins, represent the largest family of enzymes in the ubiquitin system, implying the possibility that substrate selectivity is even greater for these enzymes than for those that catalyze ubiquitin ligation. Little is known about the mechanisms of CXCR4 deubiquitination and their regulation by receptor ligands. A proteomics study revealed that the steady state level of USP14 was increased upon CXCL12 stimulation of target cells (32Ding Y. Zhang L. Goodwin J.S. Wang Z. Liu B. Zhang J. Fan G.H. Exp. Cell Res. 2008; 314: 590-602Crossref PubMed Scopus (19) Google Scholar), and preliminary studies revealed that ligand stimulation led to enhanced association of USP14 with the CXCR4. The present studies were undertaken to ascertain the functional consequences of this interaction, the selectivity of CXCR4 for USP14, when compared with three other deubiquitinating enzymes, USP2a, USP4, and USP7, and the impact of modifying the ubiquitinated state of the receptor on CXCR4 turnover, CXCL12-evoked chemotaxis, and CXCL12-induced activation of ERK. Plasmids and siRNAs—Plasmids encoding Myc-CXCR4 and glutathione S-transferase (GST)-conjugated CXCR4 C terminus were obtained from Dr. Gang Pei (Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences). The enhanced green fluorescent protein (EGFP)-conjugated CXCR4 was constructed by amplifying the cDNAs of CXCR4 from HA-CXCR4 in pcDNA3 vector using PCR and inserting the cDNA into the XhoI and BamHI sites of the pEGFP/N1 vector (Clontech). The epitope-tagged CXCR4 has been tested by radioligand binding assay and cyclic AMP assay and was confirmed to function similarly as the nontagged receptor (data not shown). The mutant CXCR4 that cannot be recognized for ubiquitination (HA-3K/R CXCR4) was obtained from Dr. Jeffrey L. Benovic (Thomas Jefferson University, Philadelphia) (28Marchese A. Benovic J.L. J. Biol. Chem. 2001; 276: 45509-45512Abstract Full Text Full Text PDF PubMed Scopus (392) Google Scholar). The USP14 plasmid was constructed by PCR amplification of USP14 cDNA using lymphocyte cDNAs as template and inserted into the pcDNA3 vector. Pre-designed USP14-specific siRNA and pre-designed control (scramble) siRNA were purchased from Ambion. The USP2a plasmid was obtained from Dr. Massimo Loda (Dana Farber Cancer Institute, Harvard Medical School, Boston). The USP4 plasmid was obtained from Dr. Michael Freissmuth (Medical University of Vienna, Vienna, Austria). The pCI-USP7 plasmid was a gift from Dr. Roger Everett (Institute of Virology, University of Glasgow, Scotland, UK). The pcDNA3-USP7 was constructed by amplifying the cDNAs of USP7 in pCI vector using PCR and inserting the cDNA into the XhoI and BamHI sites of the pcDNA3 vector. Cell Culture and Transfection—Human embryonic kidney (HEK293) and HeLa cells were grown in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, 100 units/ml penicillin, and 100 μg/ml streptomycin in 5% CO2, 95% air at 37 °C. Cells were cultured in 100-mm dishes. For studies involving fluorescence microscopy, 22-mm square glass coverslips were placed in the culture dishes before transfection. Transient transfection of recipient HEK293 cells was performed using Lipofectamine 2000 (Invitrogen). Cells stably expressing Myc-CXCR4 or EGFP-CXCR4 were selected with 560 μg/ml geneticin (G418). Co-immunoprecipitation and Western Blot for Determining CXCR4 Complexes—HEK293 cells stably expressing Myc-CXCR4 were transiently transfected with HA-USP14 or USP14-specific siRNA, treated with CXCL12 (10 nm; Pepro-Tech, Inc.) for various time intervals, and lysed in 1 ml of RIPA buffer containing PBS (pH.7.0), 0.1% sodium deoxycholate, 0.01% SDS, and 1% Nonidet P-40 to which additional SDS was added to bring the final concentration to ∼10%. The cell debris was removed by centrifugation (13,000 × g, 15 min). The supernatant was pre-cleared by incubation with 40 μl of protein A/G-agarose (Pierce) for 1 h at 4 °C to reduce nonspecific binding. After removing the protein A/G-agarose by centrifugation (13,000 × g, 1 min), the cleared supernatant was collected, and 10 μl of mouse monoclonal anti-Myc antibody (Santa Cruz Biotechnology) was added for an overnight incubation at 4 °C. Protein A/G (40 μl) was then added to this mixture, and the incubation was continued for 2 h at 4 °C. The protein A/G-antibody-antigen complex was collected by centrifugation and washing the pelleted resin three times with ice-cold immunoprecipitation buffer. The final pellets were resuspended in 40 μl of SDS sample buffer containing 5% β-mercaptoethanol and heated to 50 °C for 10 min. Forty microliters of this preparation were separated by 10% SDS-PAGE. The PAGE-embedded proteins were transferred to nitrocellulose membranes (Bio-Rad) by electrophoretic transfer at 60 V for 60 min at 4 °C in a buffer (25 mm Tris, 192 mm glycine, 20% methanol). Proteins were identified on the nitrocellulose filter by Western blotting, using the following antibodies. CXCR4 was detected using a mouse monoclonal antibody directed against the Myc epitope in the N-terminal domain of the receptor. USP14 was detected using a rabbit polyclonal antibody (Abgent). USP2a and USP7 were detected using a rabbit polyclonal antibody (Santa Cruz Biotechnology, Inc.) directed against the HA epitope engineered into the N terminus of the protein. USP4 was detected using a rabbit polyclonal antibody (Santa Cruz Biotechnology) directed against the His epitope engineered into the N terminus of the protein. In Vitro Binding Assay for Protein Association with the C Terminus of CXCR4—For isolating proteins that interact with the CXCR4 C terminus, we exploited a GST-CXCR4 C-terminal fusion protein, using GST alone as a control. For the purification of the GST or GST-CXCR4 C-terminal fusion proteins, DH5α bacteria transformed with plasmids encoding GST or GST-CXCR4 C-terminal fusion proteins were cultured overnight at 37 °C. Isopropyl d-thiogalactopyranoside was added to the culture to induce protein expression, and incubation was continued for another 3 h. The bacteria were lysed in PBST buffer (10 mm sodium phosphate, 2 mm potassium phosphate (pH 7.4), 140 mm NaCl, 3 mm KCl, 0.1% (v/v) Tween 20) and then probe-sonicated on ice for 10 s. The supernatant of the bacterial lysate was incubated with glutathione-Sepharose (Pierce/Thermo Scientific, Inc.) for 30 min at 4 °C to isolate GST or the GST-CXCR4 C-terminal fusion protein. After washing three times with PBST buffer, the purified GST- or GST-CXCR4 C-terminal fusion protein-bound beads were resuspended in PBST buffer and stored on ice until use that day. Cell lysates that were to be incubated with Sepharose-GST or Sepharose-GST-CXCR4 C terminus were prepared in the following way. HEK293 cells not expressing CXCR4 (and not stimulated with CXCL12) but transiently expressing HA-USP14, HA-USP2a, HIS-USP4, or HA-USP7 (with parental cells serving as controls) were lysed using PBST buffer (10 mm sodium phosphate, 2 mm potassium phosphate (pH 7.4), 140 mm NaCl, 3 mm KCl, 0.1% (v/v) Tween 20) 48 h after transfection. Cell lysates were pelleted to remove debris (13,000 × g, 10 min at 4 °C in a microcentrifuge), and the supernatants of this centrifugation were used for the GST pulldown assay. For the GST pulldown assay, the supernatant of the cell lysates was incubated with aliquots of the purified GST or GST-CXCR4 C-terminal fusion proteins (to bring the GST fusion protein concentration to 50 μg/ml) for 2 h at 4 °C with rotation. To terminate the incubation, Sepharose beads were pelleted by centrifugation (13,000 × g, 2 min) and washed four times with PBST buffer. Proteins bound to the C-terminal CXCR4 receptor-GST fusions protein (or control GST) were released by boiling in SDS-PAGE sample buffer containing 5% β-mercaptoethanol for 10 min, resolved by SDS-PAGE, and analyzed by Western blot, as outlined above. Confocal Microscopy—Our initial studies revealed a selectively greater association of CXCR4 with USP14 compared with other family members; thus, most of our studies focused on CXCR4 and its association with USP14. To examine the localization of USP14 compared with CXCR4 in cells expressing both, HEK293 cells stably expressing EGFP-CXCR4 were transiently transfected with HA-USP14 and grown on coverslips for 1 or 2 days. Cells were treated with CXCL12 (10 nm) for various time intervals and fixed with 100% methanol at room temperature before immediate transfer to 4 °C until further processing. Fixed cells were washed with PBS and incubated with a mouse monoclonal HA antibody (Santa Cruz Biotechnology) for 30 min at room temperature. Cells were washed with PBS and incubated with a CY3-conjugated anti-mouse antibody (Molecular Probes, Eugene, OR) for 30 min at room temperature and then washed twice with PBS and then briefly with deionized water to remove buffer salts. Coverslips were mounted on the microscope slide with a mounting solution. Confocal microscopy was performed on an LSM-510 laser scanning microscope (Carl Zeiss, New York) with a 63 × 1.3 numerical aperture oil immersion lens using dual excitation (488 nm for EGFP and 568 nm for Cy3) and emission (515-540 nm for EGFP and 590-610 nm for Cy3) filter sets. All digital images were captured at the same settings to allow direct quantitative comparison of staining patterns. Final images were processed using Adobe Photoshop software. Time Course of CXCR4 Ubiquitination and Deubiquitination—To assess whether ligand activation of CXCR4 altered the ubiquitination of this receptor, HEK293 cells stably expressing Myc-CXCR4 were incubated with 10 nm CXCL12 for the time intervals indicated in the figures and figure legends. The cells were then lysed in 1 ml of ice-cold RIPA buffer. The cell debris was removed by centrifugation (13,000 × g, 15 min). The supernatant was pre-cleared by incubation with 40 μl of protein A/G-agarose (Pierce) for 1 h at 4 °C to reduce nonspecific binding. After removing the protein A/G-agarose by centrifugation (13,000 × g, 1 min), the cleared supernatant was collected, and 10 μl of mouse monoclonal Myc antibody (Santa Cruz Biotechnology) was added for overnight incubation at 4 °C. Protein A/G (40 μl) was then added, and the incubation was continued for 2 h at 4 °C. The protein A/G-antibody-antigen complex was then collected by centrifugation, as above, and was washed three times with ice-cold immunoprecipitation buffer. The final pellets were resuspended in 40 μl of SDS sample buffer containing 5% β-mercaptoethanol and heated to 50 °C for 10 min. Forty microliters of this preparation were separated by 10% SDS-PAGE, and the proteins transferred to nitrocellulose membranes (Bio-Rad), as described in detail above for Western blotting. The state of ubiquitination of the CXCR4 receptor was detected by Western blot analysis using a rabbit polyclonal anti-ubiquitin antibody (Santa Cruz Biotechnology). Two complementary strategies were used to determine the effect of USP14 on CXCR4 modification, i.e. overexpression of USP14 in CXCR4-expressing HEK293 cells and RNA silencing of endogenous USP14 in cells expressing CXCR4. For USP14 overexpression, cells were transiently transfected with vector (control) or HA-USP14 (see under “Cell Culture and Transfection”) and incubated with ligand 48 h later. For knockdown of USP14, cells were transiently transfected with scrambled siRNA (control) or USP14-siRNA (Ambion, Inc.). In either case, cells were incubated with CXCL12 for 10 min to assess the impact of ligand on the ubiquitination of CXCR4. To assess the reversibility of the ubiquitination, another set of cells was incubated for 10 min with CXCL12 followed by a 60-min “recovery from stimulation” period (after removal of ligand and replacement of fresh medium after the 10 min of stimulation). Incubations were terminated by cell lysis in 1 ml of ice-cold RIPA buffer. The cells were processed as described above for assessment of CXCR4 ubiquitination. Receptor Degradation Assay—To assess the impact of USP14 and reversal of ubiquitination on CXCR4 turnover and degradation, HEK293 cells expressing CXCR4 and transiently expressing HA-USP14 (see figure legends) were pretreated with cycloheximide (5 μg/ml) for 15 min at 37 °C to prevent new protein synthesis during the course of our experiments. Cells were incubated with CXCL12 (10 nm) for 8 h at 37 °C to maximally induce receptor ubiquitination and degradation. To terminate the incubation, cells were transferred to ice and then lysed in ice-cold RIPA buffer. Lysates containing equal amounts of proteins were subjected to 10% SDS-PAGE. CXCR4 was detected by Western blot analysis using an anti-CXCR4 antibody (Abcam). The blots were stripped and reprobed with anti-tubulin antibody to confirm equal loading (Santa Cruz Biotechnology). To assess the effect of USP14 modulation on endogenous CXCR4 turnover and degradation, HeLa cells, which endogenously express CXCR4, were transiently transfected with HA-USP14 or USP14-specific siRNA. Cells were pretreated with cycloheximide (5 μg/ml) for 15 min at 37 °C, to prevent new protein synthesis during the course of our experiments, and incubated with CXCL12 (10 nm) for varying time points at 37 °C (see figure legends). To terminate the incubation, cells were transferred to ice and then lysed in ice-cold RIPA buffer. Lysates containing equal amounts of proteins were subjected to 10% SDS-PAGE. CXCR4 was detected by Western blot analysis using an anti-CXCR4 antibody (Abcam). The blots were stripped and reprobed with anti-tubulin antibody to confirm equal loading (Santa Cruz Biotechnology). Densitometric Analysis of Western Blot Bands—The relative amount of all Western blot bands was measured using UNSCAN-IT gel version 6.1 (Silk Scientific Corp.). The relative density of the protein bands was calculated in the area encompassing the immunoreactive protein band following subtraction of the density of the background signal detected in an adjacent area without protein signal in the same lane as the protein of interest. Statistical Analysis—Student's t tests were performed to test statistical significance for the paired data comparisons. Analysis of variance tests were used to test the significant differences for the group data comparisons. Chemotaxis Assay—The migration of HEK293 cells stably expressing Myc-CXCR4 and transiently expressing vector alone (control), HA-USP14, USP14-specific siRNA, or scrambled (control) siRNA was evaluated using a chemotaxis assay (36Luo C. Pan H. Mines M. Watson K. Zhang J. Fan G.H. J. Biol. Chem. 2006; 281: 30081-30093Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). Briefly, polycarbonate filters (10-μm pore size) coated with 20 μg/μl human collagen type IV were placed between the upper and lower compartments of the Boyden chambers (Neuroprobe, Gaithersburg, MD). The lower compartment of the chamber was loaded with 400-μl aliquots of 1 mg/ml ovalbumin/Dulbecco's modified Eagle's medium (chemotaxis buffer) or CXCL12 (0.01-100 nm) diluted in chemotaxis buffer. Cells (5 × 105/100 μl) were loaded into the upper compartment and incubated for 4 h at 37 °C in a 5% CO2 atmosphere. Cells that had migrated through the filter into the bottom chamber of medium or medium containing CXCL12 were counted under the microscope (×20 objective) after being stained with a Diff-Quik kit. The migration index was calculated and was defined as number of cells crossing the filter toward CXCL12 (various concentrations)/number of cells migrating toward medium alone (control). Each experiment was performed at least three times in duplicate. To confirm that the modulation of chemotaxis was being mediated via the receptor itself, and not some other USP14-interacting protein, and to confirm the necessity of a ubiquitination/deubiquitination cycle, we assessed the effect of CXCL12 stimulation on mutant CXCR4-mediated, HA-3K/R CXCR4, cell migration. As such, the chemotactic index of HEK293 cells transiently transfected with HA-WT CXCR4 or HA-3K/R CXCR4 was calculated as described above. CXCL12-induced CXCR4-mediated ERK Activation—To exploit a possible mechanism for the inhibition of CXCL12-induced CXCR4-mediated cell chemotaxis, we explored possible changes in the activation of ERK, a signaling molecule located downstream of CXCR4 activation. HEK293 cells transiently transfected with HA-WT CXCR4 or HA-3K/R CXCR4 were stimulated with CXCL12 (10 nm) for varying time points (see figure legends) at 37 °C. Stimulation was terminated by transferring cells to ice and then lysed with ice-cold RIPA buffer. Lysates containing equal amounts of protein were subjected to 10% SDS-PAGE, and activated ERK was detected using an anti-P-ERK antibody (Santa Cruz Biotechnology). The blots were stripped and reprobed with an antibody (anti-ERK2 antibody; Santa Cruz Biotechnology) that recognizes ERK in both its phosphorylated and nonphosphorylated states to confirm equal loading. An anti-HA antibody (Santa Cruz Biotechnology) was also employed to detect the level of expression of HA-CXCR4. CXCL12 Activation Leads to a Time-dependent Association of USP14 with CXCR4—A previous series of experiments revealed the possibility that USP14 was a CXCR4-interacting protein (37Ding Y. Li M. Zhang J. Li N. Xia Z. Hu Y. Wang S. Fan G.H. Mol. Pharmacol. 2006; 69: 1269-1279Crossref PubMed Scopus (15) Google Scholar). This study verified that CXCL12 incubation of HEK293 cells stably expressing the receptor and transiently expressing HA-USP14 leads to a time-dependent association of USP14 with CXCR4 (Fig. 1A). USP14 association with CXCR4 was not readily detected before ligand stimulation, but exposure of CXCR4-expressing cells to CXCL12 led to detection of USP14 association with the receptor at the earliest time point evaluated (2 min). Association continued for ∼5 min, after which time detectable receptor-dependent association declined (Fig. 1B), despite sustained receptor expression in the cells (Fig. 1A). It was not necessary to overexpress USP14 to detect this interaction, because a similar time course for CXCL12-induced association of USP14 with myc-CXCR4 was also detected in HEK cells expressing endogenous USP14 (data not shown). Of further interest was our finding that USP14 interaction with CXCR4 is relatively selective, compared with other members of the USP family, includ" @default.
• W1980002411 created "2016-06-24" @default.
• W1980002411 creator A5050517398 @default.
• W1980002411 creator A5061028143 @default.
• W1980002411 creator A5077963861 @default.
• W1980002411 creator A5082501983 @default.
• W1980002411 creator A5086900416 @default.
• W1980002411 date "2009-02-01" @default.
• W1980002411 modified "2023-10-15" @default.
• W1980002411 title "Deubiquitination of CXCR4 by USP14 Is Critical for Both CXCL12-induced CXCR4 Degradation and Chemotaxis but Not ERK Activation" @default.
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