Title: RACK1 and CIS Mediate the Degradation of BimEL in Cancer Cells
Abstract: RACK1 is a 7-WD motif-containing protein with numerous downstream effectors regulating various cellular functions. Using a yeast two-hybrid screen, we identified dynein light chain 1 as a novel interacting partner of RACK1. Additionally, we demonstrated that RACK1 formed a complex with DLC1 and Bim, specifically BimEL, in the presence of apoptotic agents. Upon paclitaxel treatment, RACK1, DLC1, and CIS mediated the degradation of BimEL through the ElonginB/C-Cullin2-CIS ubiquitin-protein isopeptide ligase complex. We further showed that RACK1 conferred paclitaxel resistance to breast cancer cells in vitro and in vivo. Finally, we observed an inverse correlation between CIS and BimEL levels in both ovarian and breast cancer cell lines and specimens. Our study suggests a role of RACK1 in protecting cancer cells from apoptosis by regulating the degradation of BimEL, which together with CIS could play an important role of drug resistance in chemotherapy. RACK1 is a 7-WD motif-containing protein with numerous downstream effectors regulating various cellular functions. Using a yeast two-hybrid screen, we identified dynein light chain 1 as a novel interacting partner of RACK1. Additionally, we demonstrated that RACK1 formed a complex with DLC1 and Bim, specifically BimEL, in the presence of apoptotic agents. Upon paclitaxel treatment, RACK1, DLC1, and CIS mediated the degradation of BimEL through the ElonginB/C-Cullin2-CIS ubiquitin-protein isopeptide ligase complex. We further showed that RACK1 conferred paclitaxel resistance to breast cancer cells in vitro and in vivo. Finally, we observed an inverse correlation between CIS and BimEL levels in both ovarian and breast cancer cell lines and specimens. Our study suggests a role of RACK1 in protecting cancer cells from apoptosis by regulating the degradation of BimEL, which together with CIS could play an important role of drug resistance in chemotherapy. Bim (Bcl-2-interacting mediator of cell death), a BH3 domain-containing protein, is expressed predominantly in hematopoietic, neuronal, and epithelial cells (1O'Reilly L.A. Cullen L. Visvader J. Lindeman G.J. Print C. Bath M.L. Huang D.C. Strasser A. Am. J. Pathol. 2000; 157: 449-461Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar). Bim has at least 18 different splicing variants, among which BimEL (extra long), BimL (long), and BimS (short) are the three major isoforms (2Adachi M. Zhao X. Imai K. Cell Death Differ. 2005; 12: 192-193Crossref PubMed Scopus (28) Google Scholar). BimL and BimEL expression are tightly regulated at both the transcriptional and posttranslational levels. Transcriptionally, growth factors can down-regulate Bim mRNA expression through the Akt-FOXO3 pathway (3Dijkers P.F. Medema R.H. Lammers J.W. Koenderman L. Coffer P.J. Curr. Biol. 2000; 10: 1201-1204Abstract Full Text Full Text PDF PubMed Scopus (831) Google Scholar). Posttranslationally, under normal conditions, Bim is regulated either by binding to microtubules via DLC1 (dynein light chain 1) (4Puthalakath H. Huang D.C. O'Reilly L.A. King S.M. Strasser A. Mol. Cell. 1999; 3: 287-296Abstract Full Text Full Text PDF PubMed Scopus (905) Google Scholar) or by directly binding to 14-3-3 (5Qi X.J. Wildey G.M. Howe P.H. J. Biol. Chem. 2006; 281: 813-823Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar). Upon apoptotic stimulation, Bim is released from the microtubules and translocates to the mitochondrial outer membrane. There, Bim promotes apoptosis (4Puthalakath H. Huang D.C. O'Reilly L.A. King S.M. Strasser A. Mol. Cell. 1999; 3: 287-296Abstract Full Text Full Text PDF PubMed Scopus (905) Google Scholar) either through inhibition of anti-apoptotic Bcl-2 proteins, such as Bcl-2, Bcl-xL, and Mcl1 (6Gomez-Bougie P. Bataille R. Amiot M. Eur. J. Immunol. 2005; 35: 971-976Crossref PubMed Scopus (59) Google Scholar), or through activation of pro-apoptotic proteins, such as Bax (7Harada H. Quearry B. Ruiz-Vela A. Korsmeyer S.J. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 15313-15317Crossref PubMed Scopus (250) Google Scholar). It has been shown that Bim functions as a tumor suppressor and is also a determinant in paclitaxel sensitivity in both renal carcinoma (8Tan T.T. Degenhardt K. Nelson D.A. Beaudoin B. Nieves-Neira W. Bouillet P. Villunger A. Adams J.M. White E. Cancer Cell. 2005; 7: 227-238Abstract Full Text Full Text PDF PubMed Scopus (254) Google Scholar) and non-small cell lung carcinoma (9Li R. Moudgil T. Ross H.J. Hu H.M. Cell Death Differ. 2005; 12: 292-303Crossref PubMed Scopus (111) Google Scholar). Bim-null mice are generally healthy and fertile, although the number of Bim–/– offspring is only about 50% of the normal counterparts. The major defect of these mice is the dysregulation of leukocyte homeostasis, leading to systematic lupus erythematosus-like autoimmune disease in aged mice (10Bouillet P. Metcalf D. Huang D.C. Tarlinton D.M. Kay T.W. Kontgen F. Adams J.M. Strasser A. Science. 1999; 286: 1735-1738Crossref PubMed Scopus (1304) Google Scholar). RACK1, a 7-WD motif-containing protein, regulates numerous cellular properties through its multiple downstream effectors. RACK1 is an important component of the ribosome and has been shown to regulate protein synthesis (11Sengupta J. Nilsson J. Gursky R. Spahn C.M. Nissen P. Frank J. Nat. Struct. Mol. Biol. 2004; 11: 957-962Crossref PubMed Scopus (200) Google Scholar, 12Ceci M. Gaviraghi C. Gorrini C. Sala L.A. Offenhauser N. Marchisio P.C. Biffo S. Nature. 2003; 426: 579-584Crossref PubMed Scopus (327) Google Scholar). Through different signaling pathways, RACK1 is involved in controlling apoptosis and contributes to tumor growth in vivo. First, RACK1 has been shown to mediate c-Jun N-terminal kinase activation via protein kinase C and promote melanoma growth in nude mice (13Lopez-Bergami P. Habelhah H. Bhoumik A. Zhang W. Wang L.H. Ronai Z. Mol. Cell. 2005; 19: 309-320Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar). Second, RACK1 forms a complex with insulin and insulin-like growth factor 1 receptors to regulate STAT3 activation and mediates insulin receptor/insulin-like growth factor 1 receptor-induced protection against apoptosis of ovarian cancer cells (14Zhang W. Zong C.S. Hermanto U. Lopez-Bergami P. Ronai Z. Wang L.H. Mol. Cell. Biol. 2006; 26: 413-424Crossref PubMed Scopus (90) Google Scholar). Additionally, RACK1 interacts with p73 and prevents p73-mediated cell death (15Ozaki T. Watanabe K. Nakagawa T. Miyazaki K. Takahashi M. Nakagawara A. Oncogene. 2003; 22: 3231-3242Crossref PubMed Scopus (42) Google Scholar). Finally, RACK1 interacts with E1A and rescues E1A-induced yeast growth inhibition and mammalian cell apoptosis (16Sang N. Severino A. Russo P. Baldi A. Giordano A. Mileo A.M. Paggi M.G. De Luca A. J. Biol. Chem. 2001; 276: 27026-27033Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). The expression level of RACK1 is elevated during angiogenesis (17Berns H. Humar R. Hengerer B. Kiefer F.N. Battegay E.J. FASEB J. 2000; 14: 2549-2558Crossref PubMed Scopus (107) Google Scholar) and in colon carcinoma, non-small cell lung carcinoma (17Berns H. Humar R. Hengerer B. Kiefer F.N. Battegay E.J. FASEB J. 2000; 14: 2549-2558Crossref PubMed Scopus (107) Google Scholar), and melanomas (13Lopez-Bergami P. Habelhah H. Bhoumik A. Zhang W. Wang L.H. Ronai Z. Mol. Cell. 2005; 19: 309-320Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar). These observations suggest a positive role of RACK1 in protecting cells from apoptosis and in promoting tumorigenesis. RACK1 has been reported as an E3 4The abbreviations used are: E3, ubiquitin-protein isopeptide ligase; SH, Src homology; HA, hemagglutinin; HIF, hypoxia-inducible factor; GST, glutathione S-transferase; siRNA, small interfering RNA; FACS, fluorescence-activated cell sorter; CMV, cytomegalovirus. ligase component to mediate the degradation of ΔNp63α, a member of the p53 family (18Fomenkov A. Zangen R. Huang Y.P. Osada M. Guo Z. Fomenkov T. Trink B. Sidransky D. Ratovitski E.A. Cell Cycle. 2004; 3: 1285-1295Crossref PubMed Scopus (94) Google Scholar), and to interact with ElonginC and mediate the degradation of HIF-1α (19Liu Y.V. Baek J.H. Zhang H. Diez R. Cole R.N. Semenza G.L. Mol. Cell. 2007; 25: 207-217Abstract Full Text Full Text PDF PubMed Scopus (393) Google Scholar), suggesting a role of RACK1 in proteasome-mediated protein degradation. The specificity of proteasome-mediated protein degradation is determined by distinct E3 ligase complexes. The SOCS (suppressor of cytokine signaling)-box-containing E3 ligase family members are composed of ElonginB/C and Cullin2 or Cullin5 and SOCS-1 or SOCS-3. Together, they form the ECS (ElonginB/C-Cullin-SOCS-box)-Roc complex (20Willems A.R. Schwab M. Tyers M. Biochim. Biophys. Acta. 2004; 1695: 133-170Crossref PubMed Scopus (379) Google Scholar). Besides SOCS-1 and SOCS-3, CIS (cytokine-inducible Src homology 2 domain-containing protein) also has the SOCS-box domain and could potentially be part of the E3 complex (21Kamizono S. Hanada T. Yasukawa H. Minoguchi S. Kato R. Minoguchi M. Hattori K. Hatakeyama S. Yada M. Morita S. Kitamura T. Kato H. Nakayama K. Yoshimura A. J. Biol. Chem. 2001; 276: 12530-12538Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar). Overexpression of CIS has been shown to suppress BCR/ABL-mediated cell transformation, whereas proteasome inhibitors relieved the suppressive phenotype, hinting at the involvement of CIS in proteasome-mediated protein degradation (22Tauchi T. Yoshimura A. Ohyashiki K. Exp. Hematol. 2001; 29: 356-361Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar). CIS has been shown to be overexpressed in breast cancer lines and in breast carcinomas (23Raccurt M. Tam S.P. Lau P. Mertani H.C. Lambert A. Garcia-Caballero T. Li H. Brown R.J. McGuckin M.A. Morel G. Waters M.J. Br. J. Cancer. 2003; 89: 524-532Crossref PubMed Scopus (78) Google Scholar). In the CIS transgenic model, the mice displayed aberrantly increased CD4+ T cell population, prolonged survival of T cells after T cell receptor activation (24Li S. Chen S. Xu X. Sundstedt A. Paulsson K.M. Anderson P. Karlsson S. Sjogren H.O. Wang P. J. Exp. Med. 2000; 191: 985-994Crossref PubMed Scopus (86) Google Scholar), and shifted Th1 versus Th2 T cell balance (25Matsumoto A. Seki Y. Kubo M. Ohtsuka S. Suzuki A. Hayashi I. Tsuji K. Nakahata T. Okabe M. Yamada S. Yoshimura A. Mol. Cell. Biol. 1999; 19: 6396-6407Crossref PubMed Scopus (222) Google Scholar). This implies an independent role of CIS other than that in inhibiting the well known cytokine signaling, such as that mediated by interleukin-2, interleukin-3, and growth hormone. CIS transgenic mice resemble the phenotype of Bim-null mice in terms of T cell proliferation and survival (10Bouillet P. Metcalf D. Huang D.C. Tarlinton D.M. Kay T.W. Kontgen F. Adams J.M. Strasser A. Science. 1999; 286: 1735-1738Crossref PubMed Scopus (1304) Google Scholar), pointing to a functional relationship between CIS and Bim. To identify novel interacting partners of RACK1, yeast two-hybrid screening was performed. We found that DLC1 interacted with RACK1. Subsequently, we observed that DLC1 interacted with RACK1 upon apoptotic stimulation in mammalian cells. Furthermore, we demonstrated that RACK1 formed a complex with DLC1 and BimEL in the presence of paclitaxel. Moreover, RACK1 promoted proteasome-mediated BimEL degradation via a Cullin/CIS E3 ligase complex. Our study suggests that RACK1 plays an important role in protecting stress or apoptotic agent-treated cancer cells from apoptosis through degradation of BimEL, which may contribute to tumor formation in vivo and drug resistance during cancer therapy. Cells and DNA Transfection—Human embryonic kidney (HEK) 293T cells, ovarian cancer line SKOV3, colon carcinoma-derived ovarian metastasis line SW626, and breast cancer stable cell lines MDA-MB-468, MCF7, and MCF7-I4 were maintained in Dulbecco's modified Eagle's medium with 10% fetal bovine serum (Sigma). Briefly, MCF7-I4 cells were derived from MCF7 by consecutive cycles of selection of invasive MCF7 cells through a Matrigel-coated Boyden chamber (26Cheng G.Z. Chan J. Wang Q. Zhang W. Sun C.D. Wang L.H. Cancer Res. 2007; 67: 1979-1987Crossref PubMed Scopus (460) Google Scholar). SKOV3, SW626, and MCF7 were transfected with Lipofectamine 2000 according to the manufacturer's (Invitrogen) instructions, HEK293T cells were transfected by calcium phosphate coprecipitation methods, and MB-MDA-468 cells were transfected by FuGENE reagent (Roche). Plasmids and Their Construction—phEF-RACK1-HA has been described previously (27Hermanto U. Zong C.S. Li W. Wang L.H. Mol. Cell. Biol. 2002; 22: 2345-2365Crossref PubMed Scopus (174) Google Scholar). pEF-BimEL (mouse) was kindly provided by Dr. David C. Huang (University of California, Berkeley). Expression plasmids containing CIS were kindly provided by Drs. Tracy Willson and Douglas Hilton. cDNA of human DLC1 was purchased from the American Type Culture Collection (ATCC 927281) and cloned into the phEF-Neo vector. pcDNA-HA-ubiquitin and HA-Cullin2 constructs were generous gifts from Dr. Zhen-Qiang Pan at Mount Sinai School of Medicine. Antibodies—Anti-RACK1, goat anti-rabbit horseradish peroxidase, and goat anti-mouse horseradish peroxidase were purchased from BD Biosciences. Anti-GST antibody was from Amersham Biosciences. Anti-CIS antibody was from Santa Cruz Biotechnology. Anti-Bim antibody was purchased from Chemicon. Anti-FLAG, anti-tubulin, rabbit anti-mouse secondary antibody, and anti-rat horseradish peroxidase antibodies were purchased from Sigma. Anti-glyceraldehyde-3-phosphate dehydrogenase and anti-lamin B1 antibodies were purchased from Biodesign Co. Anti-HA antibody was purchased from the Hybridoma Center at Mount Sinai School of Medicine. Preparation of Cell Lysates, Immunoprecipitation, SDS-PAGE, and Immunoblotting—For direct immunoblot analysis, cell lysates were prepared with ice-cold radioimmune precipitation assay buffer (50 mm Tris-Cl, pH 7.4, 150 mm NaCl, 1% Triton X-100, 1% deoxycholate, 5 mm EDTA, 1% aprotinin, 1 mm phenylmethylsulfonyl fluoride, 0.4 mm phenylarsine oxide, and 25 mm NaF). For coimmunoprecipitation, cell lysates were prepared with Nonidet P-40 lysis buffer (20 mm Hepes, pH 7.4, 150 mm NaCl, 1% Nonidet P-40, 10% glycerol, 1 mm EDTA, pH 8.0, 1 mm phenylmethylsulfonyl fluoride, and 1% aprotinin). Cell lysates or immunoprecipitates were separated by SDS-PAGE and immunoblotted with appropriate antibodies as described (14Zhang W. Zong C.S. Hermanto U. Lopez-Bergami P. Ronai Z. Wang L.H. Mol. Cell. Biol. 2006; 26: 413-424Crossref PubMed Scopus (90) Google Scholar, 27Hermanto U. Zong C.S. Li W. Wang L.H. Mol. Cell. Biol. 2002; 22: 2345-2365Crossref PubMed Scopus (174) Google Scholar). RNA Interference—Double-stranded siRNA construction and synthesis as well as pRS-shRNA of RACK1 for preparation of MCF7-I4 stable clones have been described previously (13Lopez-Bergami P. Habelhah H. Bhoumik A. Zhang W. Wang L.H. Ronai Z. Mol. Cell. 2005; 19: 309-320Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar, 14Zhang W. Zong C.S. Hermanto U. Lopez-Bergami P. Ronai Z. Wang L.H. Mol. Cell. Biol. 2006; 26: 413-424Crossref PubMed Scopus (90) Google Scholar). The construction and synthesis of double-stranded siRNA of CIS and DLC1 were according to the manufacturer's instruction (Ambion). The target sequence for the CIS siRNA template is 5′-AATGTACGCATTGAGTATGCC-3′; the target sequences for DLC1 are 5′-AACATAGAGAAGGACATTGCG-3′ and 5′-AACTTCGGTAGTTATGTGACA-3′. Annexin V Staining Assay—MCF7-I4 cells that stably overexpress RACK1 and control phEF clones were seeded at 1 × 106 cells and cultured at 37 °C overnight. 200 nm paclitaxel was added into the medium to treat the cells for 72 h. Cells were collected and double-stained with propidium iodide and anti-annexin V-fluorescein isothiocyanate according to the manufacturer's instructions (BD Biosciences). FACS analysis was performed to count the apoptotic cells that were stained with annexin V but not with propidium iodide. Colony Formation Assay and Anoikis Assay—MCF7-I4 cells that stably overexpress RACK1 or MCF7 cells transiently transfected with RACK1 siRNA or CIS siRNA were trypsinized, counted, and resuspended. 5 × 104 cells were used for a colony formation assay (27Hermanto U. Zong C.S. Li W. Wang L.H. Mol. Cell. Biol. 2002; 22: 2345-2365Crossref PubMed Scopus (174) Google Scholar) with or without addition of paclitaxel (10 nm), and 106 cells were used for anoikis assay as described (28Uttamsingh S. Zong C.S. Wang L.H. J. Biol. Chem. 2003; 278: 18798-18810Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar). For quantifying the colony formation assay, the total area of colonies was quantified by ImageTool software; for anoikis assay, cells were stained with trypan blue, and dead cells were counted with a hemocytometer. In Vivo Tumor Growth—BALB/c nude mice were injected with 1 × 106 MCF7-I4-phEF or MCF7-I4-RACK1–3 cells (five mice in each group) in the flank subcutaneously. Tumor growth was monitored by measuring three dimensions and calculated as follows: tumor volume (mm3) = length × width × height × 0.52. 18 days after injection, mice were injected intraperitoneally with either blank vehicle solution or 5 mg/kg paclitaxel (stock solution is formulated with 50% ethanol and 50% Cremophor EL (10 mg/ml), diluted with 0.9% sodium chloride to 0.5 mg/ml for injection). At the end point, mice were sacrificed, and tumors were dissected for tumor weight measuring and protein extraction for biochemical analysis. In all experiments, a two-tailed t test assuming equal variances for non-paired samples was used withα = 0.05; the exact p values were indicated in individual figures. The animal experiments were performed under the protocol approved by the Institutional Animal Care and Use Committee of Mount Sinai School of Medicine. The collection of anonymous tumor samples was according to the institutional review board-approved waived human subject protocol at the University of Southern Florida. RACK1 Associated with DLC1 and BimEL upon Apoptotic Stimulation—To identify novel interacting partners for RACK1, we performed a yeast two-hybrid assay and found that DLC1 interacted with RACK1. Coimmunoprecipitation assay was carried out in HEK293T cells transiently transfected with FLAG-DLC1 to confirm the yeast two-hybrid result. The interaction between RACK1 and DLC1 was detected upon treatment of the cells with UV irradiation, paclitaxel, or staurosporine (Fig. 1A). Based on this observation and a previous report of the Bim-DLC1 complex (4Puthalakath H. Huang D.C. O'Reilly L.A. King S.M. Strasser A. Mol. Cell. 1999; 3: 287-296Abstract Full Text Full Text PDF PubMed Scopus (905) Google Scholar), we hypothesized that RACK1 may be involved in the regulation of Bim-DLC1-related apoptosis. To test our hypothesis, we first examined whether RACK1 also interacted with Bim. In MCF7 breast cancer cells, endogenous RACK1 was able to be coimmunoprecipitated with anti-Bim antibody, which was greatly enhanced upon paclitaxel or staurosporine treatment (Fig. 1B). As previously reported by Puthalakath et al. (4Puthalakath H. Huang D.C. O'Reilly L.A. King S.M. Strasser A. Mol. Cell. 1999; 3: 287-296Abstract Full Text Full Text PDF PubMed Scopus (905) Google Scholar), endogenous DLC1 associated with BimEL constitutively in HEK293T cells (Fig. 1C). Interaction between endogenous DLC1 and RACK1 was significantly enhanced by paclitaxel treatment (Fig. 1C). To examine whether RACK1 can interact with DLC1 and BimEL directly, in vitro binding assays were carried out with GST-DLC1, His-RACK1 or its C-terminal deletion mutant His-WD1–4, the N-terminal deletion mutant His-WD5–7, and His-BimEL. These constructs were expressed and purified from Escherichia coli. An equal amount of GST-DLC1 was incubated with GST (negative protein control), RACK1, or its deletion mutants. His-RACK1 and His-WD1–4 directly interacted with GST-DLC1 (Fig. 1D, left panel). His-WD5–7 failed to pull down DLC1 (Fig. 1D, left panel). Because His-WD1–4 is able to bind to GST-DLC1, we have focused our initial effort within this sequence. According to a predicted model of RACK1 (29McCahill A. Warwicker J. Bolger G.B. Houslay M.D. Yarwood S.J. Mol. Pharmacol. 2002; 62: 1261-1273Crossref PubMed Scopus (333) Google Scholar), we synthesized a peptide, 138CKYTVQDES146, including the entire outskirt β sheet of blade 3 and named pWD4. We found that pWD4 was able to inhibit the RACL1-DLC1 interaction in a dose-dependent manner (Fig. 1D, right panel), suggesting that it may overlap with the RACK1 binding sequence for DLC1. By contrast, an unrelated peptide, pSH2, had no effect on the RACK1-DLC1 interaction. RACK1 also interacted with BimEL directly in vitro, which was further enhanced in the presence of DLC1 (Fig. 1E). The interaction between RACK1 and BimEL was also through the N-terminal portion of RACK1, the WD1–4 domain (supplemental Fig. S1). These in vitro binding data support the intracellular association of these proteins described above and suggest that RACK1 interacted directly with both DLC1 and BimEL. Because Bim is sequestered to microtubules by DLC1 and released to mitochondria upon apoptotic stimulation, we tested whether RACK1 is involved in this process. To this end, velocity sedimentation in a glycerol gradient was performed with cell lysates from MCF7 cells and HEK293T cells overexpressing FLAG-DLC1. Under normal conditions, BimEL distributed along with DLC1 and β-tubulin, confirming that BimEL is sequestered to microtubules through DLC1 (supplemental Fig. S2A); upon paclitaxel treatment, both DLC1 and BimEL shifted from the heavier to the lighter fractions 16–20 (peaked at 18–19 and 18–20 for BimEL and DLC1, respectively) and corresponded to the sedimentation positions of a mitochondrial outer membrane protein, Tom20 (supplemental Fig. S2A). No global distribution change of tubulin, RACK1, BimL, and BimS (data not shown) was observed, suggesting the paclitaxel-induced release of BimEL and DLC1 from microtubules was specific in these two cell lines. To detect the distribution of RACK1-BimEL-DLC1 complexes, a coimmunoprecipitation experiment was done with the glycerol gradient fractions. Little interaction between RACK1 and BimEL (supplemental Fig. S2B) or between RACK1 and DLC1 (supplemental Fig. S2C) was detected in the controlled cells; however, paclitaxel treatment led to the interaction between RACK1 and BimEL in MCF7 cells (supplemental Fig. S2B) and between RACK1 and DLC1 in HEK293T cells (supplemental Fig. S2C). Because of technical restriction, we failed to coimmunoprecipitate all three proteins in the same cells. The observation suggested that the interaction between RACK1 and the BimEL-DLC1 complex might be a regulatory step for BimEL translocation. RACK1 Promoted BimEL Degradation in Paclitaxel-treated Cells—Having established that RACK1 interacts with both DLC1 and BimEL, we proceeded to examine the role of RACK1 in the BimEL level because we found that the proteasome inhibitor MG132 could significantly enhance the interaction between RACK1 and BimEL in MCF7 cells (Fig. 1F). To further examine whether RACK1 is involved in controlling the BimEL level, different cancer cells were transiently transfected with either RACK1 or RACK1 siRNA. In the RACK1-overexpressing breast cancer lines, MDA-MB-468 and MCF7, BimEL expression decreased dramatically at 48 and 24 h after paclitaxel treatment, respectively (Fig. 2A). A similar observation was made in the ovarian cancer lines, SKOV3 and SW626, respectively (supplemental Fig. S3A). The reduction of BimEL by RACK1 overexpression was proteasome-dependent, because the proteasome inhibitor, MG-132, significantly reversed the BimEL level (Fig. 2A). Reverse transcription-PCR was performed to assess whether the RACK1-mediated decrease of BimEL was due to reduced transcription. As shown in supplemental Fig. S3B, the BimEL mRNA level did not change in RACK1-overexpressing paclitaxel-treated cells. These observations suggest that RACK1-mediated regulation was not at the level of mRNA but at the level of protein stability.FIGURE 2RACK1 mediated BimEL ubiquitination and degradation in paclitaxel-treated cells. A, MDA-MB-468 or MCF7 cells were transiently transfected with phEF-Neo or phEF-RACK1. 24 h later, cells were treated with 100 nm paclitaxel in the presence or absence of MG132 for the indicated hours. The relative intensity of the bands was quantified as in Fig. 1 and normalized to the loading control. B, MDA-MB-468 cells were transiently transfected with two independent siRNAs of RACK1 or scrambled control for siRNA1. 72 h later, cells were treated with 100 nm paclitaxel for 24 h. C, MCF7 cells were transiently transfected with 1 μg of HA-ubiquitin (Ub) expression plasmid and either phEF-RACK1 or phEF-Neo. 24 h later, cells were treated with 100 nm paclitaxel, with or without 1 μm MG132 for 6 h. Cell lysates were collected with Nonidet P-40 lysis buffer, and immunoprecipitation was performed with 1 mg of total protein lysates. The result was the representative of three independent experiments. Con, control; Pac., paclitaxel; IP, immunoprecipitation; IB, immunoblotting; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To confirm that RACK1 regulated the BimEL level in paclitaxel-treated cells, RACK1 siRNAs were transiently transfected into MDA-MB-468 cells. Knockdown of RACK1 resulted in a higher level of BimEL compared with the control in paclitaxel-treated MDA-MB-468 cells (Fig. 2B). Analogous to RACK overexpression results, down-regulation of RACK1 by siRNA in MCF7 cells did not affect the BimEL mRNA level (supplemental Fig. S3B). Similar results were observed in MCF7-I4 cells (a subline of MCF7) stably expressing short hairpin RNA of RACK1 (supplemental Fig. S3C). Additionally, we showed that overexpression of RACK1 in HEK293T cells promoted the polyubiquitination of BimEL upon paclitaxel treatment, which was further enhanced by the treatment of MG132 (Fig. 2C). Taken together, RACK1 reduced the BimEL protein level in paclitaxel-treated cells through a proteasome-dependent pathway. CIS Regulated BimEL Ubiquitination and Degradation—RACK1 has been implicated as a component in the E3 ligase complex to enhance the ubiquitination and degradation of ΔNp63α, a member of the p53 family (18Fomenkov A. Zangen R. Huang Y.P. Osada M. Guo Z. Fomenkov T. Trink B. Sidransky D. Ratovitski E.A. Cell Cycle. 2004; 3: 1285-1295Crossref PubMed Scopus (94) Google Scholar). A recent report showed that RACK1 interacted with ElonginC and mediated the degradation of HIF-1α (19Liu Y.V. Baek J.H. Zhang H. Diez R. Cole R.N. Semenza G.L. Mol. Cell. 2007; 25: 207-217Abstract Full Text Full Text PDF PubMed Scopus (393) Google Scholar). We observed previously that paclitaxel enhanced the interaction between RACK1 and CIS in HEK293T cells (data not shown). CIS belongs to SOCS family and is implicated to be a subunit in the RING domain-based E3 ligase complex, containing ElonginB/C-Cullin2-SOCS family members (21Kamizono S. Hanada T. Yasukawa H. Minoguchi S. Kato R. Minoguchi M. Hattori K. Hatakeyama S. Yada M. Morita S. Kitamura T. Kato H. Nakayama K. Yoshimura A. J. Biol. Chem. 2001; 276: 12530-12538Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar). These observations prompted us to investigate whether RACK1 and CIS were involved in E3 ligase complex-mediated BimEL ubiquitination and degradation. To determine whether CIS is involved in the degradation of BimEL, we overexpressed CIS in HEK293T cells. The overexpression of CIS led to a decreased BimEL level in a dose-dependent manner (Fig. 3A). Inversely, transient knockdown of CIS resulted in significant accumulation of BimEL in MCF7 cells (Fig. 3A). Similar results were also observed in SKOV3 and SW626 cells (Fig. 3B), supporting that CIS plays a regulatory role in BimEL degradation. Furthermore, we found that CIS overexpression could significantly promote the ubiquitination of endogenous BimEL, which was further enhanced by paclitaxel and MG132 treatment (Fig. 3C). These data point to the role of CIS as a component in the E3 ligase complex to regulate the degradation of BimEL. CIS, RACK1, and DLC1 Were Mutually Dependent to Mediate the Degradation of BimEL via the E3 Ligase Complex—To examine whether RACK1, BimEL, and CIS form a protein complex, a coimmunoprecipitation assay was performed. The paclitaxel treatment caused enhanced interaction between RACK1 and CIS (Fig. 4A, left panel), which confirmed our previous unpublished observation. 5W. Zhang, G. Z. Cheng, J. Gong, U. Hermanto, C. S. Zong, J. Chan, J. Q. Cheng, and L.-H. Wang, unpublished observations. Reciprocal coimmunoprecipitation revealed that the interaction between BimEL and CIS was enhanced upon paclitaxel treatment (Fig. 4A, right panels). Together with the observations described above, we have shown that 1) RACK1, CIS, and BimEL can interact and potentially form a triple-protein complex and 2) both CIS and RACK1 regulated BimEL protein levels via a proteasome-dependent pathway. CIS, like other SOCS/CIS family members, contains a short N-terminal domain, a central SH2 domain, and a C-terminal SOCS-box. Because of the structural similarity, CIS can potentially associate, via its SOCS-box, with ElonginB and ElonginC (the Elongin B/C complex) and Cullin2, thereby forming the RING domain-based E3 ligase complex. Additionally, Cullin2 has already been shown to form an E3 ligase complex with other SOCS