Eradication of pathogenic ␤-catenin by Skp1͞Cullin͞ F box ubiquitination machinery Yunyun Su, Shinji Ishikawa, Masayuki Kojima, and Bo Liu* Pittsburgh Cancer Institute and Department of Pathology, University of Pittsburgh, 5117 Centre Avenue, Pittsburgh, PA 15213 Edited by Peter K. Vogt, The Scripps Research Institute, La Jolla, CA, and approved August 28, 2003 (received for review May 29, 2003) The use of Skp1͞Cull 1͞F box (SCF) ubiquitin-conjugation machinery Materials and Methods as a potential knockout tool offers a means of eradicating disease- Tissue Culture and Transient Transfection. 293T, HFF, DLD1-tet-off, causing proteins. Here a chimeric F box protein (CFP) was engineered HCT116-tet-off, LS174-TR, HA␤18, and HA␤85 cells were kindly to achieve selective eradication of pathogenic ␤-catenin in colorectal provided by L. Falo, M. Stinski, B. Vogelstein, Q. Zhan, H. Clevers, cancer. We show that CFP specifically searches for and subsequently and T. Waldman, respectively. BJ fibroblast and colorectal tumor links the abnormal ␤-catenin to the cellular SCF ubiquitination com- cell lines DLD1 and HCT116 were purchased from American Type plex. Introduction of the CFP to colorectal cancer cells induced tar- Culture Collection. Doxycycline-inducible expression cell lines geted ubiquitination and proteolytic degradation of nuclear and were constructed as described (22). Transient transfections were cytoplasmic free ␤-catenin while preserving its normal cellular adhe- performed by using MIRUS TransIT-LT1 reagent (Mirus, Madi- sion counterpart. Elimination of pathogenic ␤-catenin suppressed son, WI). Transfection efficiency was monitored by using an constitutive Wingless͞Wnt signaling and inhibited in vitro and in vivo enhanced GFP (EGFP) expression plasmid pTrackEGFP. Cells tumor cell growth. This study demonstrates a practical utility of a used for the ubiquitination analysis were cultured in the growth SCF-based knockout system as a tool in targeting an abnormal protein medium containing 12.5 ␮M proteasome inhibitor N-acetyl-leucyl- that affects growth and transformation. leucyl-norleucinal (ALLN; Calbiochem). Plasmid Clones. pTrackEGFP, pShuttleCMV, pCMV-APC, pTOP- bnormal accumulation of protein has catastrophic conse- ␤ quences and is the primary cause of several neuronal and Flash, pFOPFalsh, and pcDNA3F- -TrCP-linker were kindly pro- A vided by B. Vogelstein, H. Clevers, and P. Zhou. pShuttleCMV was cancer diseases (1–4). ␤-Catenin, for example, is an essential slightly modified by inserting a hemagglutinin (HA)-tag and re- component of the cell–cell adhesion complex by binding with named as pShuttleHA. E-cadherin 751–878 and TCF4 1–86 were E-cadherin (5). The protein is also required to activate a variety of obtained by PCR amplification of fetal brain cDNA (Clontech). TCF4-regulated genes in response to Wnt signaling during embry- APC 1242-2060, APC 1014-1177, and APCbc, were obtained by ogenesis (6). In the absence of Wnt signaling, non-E-cadherin- ␤ PCR amplification of pCMV-APC. F box-containing constructs associated free -catenin is tightly regulated by a multiprotein were obtained by PCR amplification of fetal brain cDNA. The complex consisting of adenomatous polyposis coli (APC) protein, ͞ ␤ F box–EGFP fusion was constructed by cloning a PCR-amplified Axin, and the serine threonine kinase GSK3 (7–12). The regu- Xho Xba ␤ EGFP into the I– I-digested F box constructs. F2APCbc4 lation process involves the recognition of -catenin by APC and and F3APCbc4 were constructed by replacing EGFP with APCbc4. ␤ subsequent recruitment of Axin and GSK3 . Interaction between PCR-amplified DNA fragments were confirmed by sequencing and ␤ these proteins facilitates phosphorylation of -catenin at its amino- cloned into the pShuttleHA (cloning details and primer sequences ␤ ␤ terminal serine residues by GSK3 . After phosphorylation, -cate- for each of the PCR amplification can be obtained on request). nin becomes an immediate target for cellular Skp1͞Cull 1͞F box (SCF) ubiquitination machinery. In human colorectal cancer Protein–Protein Interaction, Immunoprecipitation, and Western Blot (CRC), mutations in APC are the earliest detectable genetic events. Analyses. Protein–protein interaction, immunoprecipitation (IP), Loss of APC function causes stabilization and, consequently, high and Western blot analyses were performed essentially as described levels of ␤-catenin accumulation in the nucleus of mutant cells (6). (23). For detection of ubiquitinized ␤-catenin, addition of 12.5 ␮M It is believed that nuclear ␤-catenin binds to TCF4 and activates ALLN was supplemented to inhibit proteasome activities. The TCF4 target genes. Constitutive activation of these Wingless͞Wnt following different primary antibodies were used for IP: anti-HA effectors initiates the development of colorectal neoplasia (14, 15). monoclonal (clone HA-7, Sigma), anti-Flag monoclonal (clone M2, Although genomic knockout and RNA interference provide pow- Sigma), anti-E-cadherin monoclonal (Pharmingen), and anti-␤- erful reverse genetic tools to study ␤-catenin by silencing its catenin monoclonal (Pharmingen). The antibodies used for West- CELL BIOLOGY expression, these technologies lack the ability to discriminate be- ern blot analysis were rabbit anti-Skp1 polyclonal (Zymed), mouse tween the abnormal protein and its normal counterpart expressed anti-HA monoclonal (clone HA-7, Sigma), mouse anti-Flag mono- from the same gene (16, 17). As ␤-catenin is also required for the clonal (Sigma), mouse anti-c-MYC (clone 9E10, Sigma), mouse ␤ ␣ normal growth of the cell, targeted cells can not survive when the anti- -catenin monoclonal (Pharmingen), rabbit anti- -actin poly- ␤-catenin gene is completely silenced with current knockout tech- clonal (Sigma), rabbit anti-lamin B1 (Zymed), and mouse anti- nologies (18). Targeting proteins to SCF ubiquitination machinery ubiquitin monoclonal (Santa Cruz Biotechnology). The resulting promises an alternative gene-silencing tool (19–21). In this study, signals were visualized by exposure to an x-ray film (Sterling, New we demonstrate that SCF provides a powerful means of eradicating York). Films were scanned and analyzed for signal strength by pathogenic ␤-catenin in CRC. This ability is achieved by engineer- IMAGEQUANT software (Molecular Dynamics). ing a chimeric F box protein (CFP) highly specific in recognizing ␤ abnormal -catenin. We show that introduction of CFP to CRC This paper was submitted directly (Track II) to the PNAS office. cells induced targeted ubiquitination and proteolytic degradation of Abbreviations: SCF, Skp1͞Cullin͞F box complex; APC, adenomatous polyposis coli; CRC, nuclear and cytoplasmic free ␤-catenin while preserving its cellular colorectal cancer; CFP, chimeric F box protein; IP, immunoprecipitation; ALLN, N-acetyl- adhesion counterpart. Elimination of pathogenic ␤-catenin sup- leucyl-leucyl-norleucinal; EGFP, enhanced GFP; HA, hemagglutinin. pressed constitutive Wingless͞Wnt signaling and inhibited in vitro *To whom correspondence should be addressed. E-mail: [email protected]. and in vivo tumor cell growth. © 2003 by The National Academy of Sciences of the USA www.pnas.org͞cgi͞doi͞10.1073͞pnas.2133261100 PNAS ͉ October 28, 2003 ͉ vol. 100 ͉ no. 22 ͉ 12729–12734 Downloaded by guest on September 28, 2021 Fig. 1. Generation of CFP-targeting constructs. (a Left) Schematic repre- sentation of ␤-catenin-binding motifs expressed in 293T cells and a summary of their ability to bind free ␤-catenin and the E-cadherin-associated ␤-cate- nin. (a Right) Coimmunoprecipitation of non-E-cadherin and E-cadherin- associated ␤-catenin with pShuttleHA (control vector), APCbc2, APCbc4, and APCbc6. Lysates from transfected 293T cells were first immunoprecipi- tated with anti-HA antibody, and the amount of ␤-catenin recognized by APCbc2, APCbc4, and APCbc6 was re- vealed by Western blot analysis with mouse anti-␤-catenin antibody (sec- ond blot from the top). The anti-HA antibody-cleared protein lysates were subsequently precipitated with anti-E- cadherin antibody, and the amount of ␤-catenin still associated with E-cad- herin was detected by Western blot analysis with mouse anti-␤-catenin an- tibody (third blot from the top). The expression levels of APCbc2, APCbc4, and APCbc6 in the transfected 293T cell lysates are shown on the bottom. Protein lysates with equal amounts of ␤-catenin from transfected cells were used for the IP (displayed in the top row). (b Left) schematic of F box motifs expressed in 293T cells and summaries of their binding abilities to Skp1 pro- tein. (b Right) Coimmunoprecipita- tion of Skp1 protein with F1, F2, and F3. Protein lysates from transfected 293T cells were immunoprecipitated with anti-HA or anti-Flag monoclonal antibody, and the amount of Skp1 protein associated with F1, F2, F3, and ␤-TrCP was revealed by Western blot analysis with rabbit anti-Skp1 anti- body (shown in the top row). The expression levels of F1EGFP, F2EGFP, F3EGFP, and ␤-TrCP in the transfected cells are shown in the second row. The presence of similar levels of ␣-actin protein in each lane indicates that an equal amount of protein lysates was used for the IP and Western blot analyses (bottom blot). (c Left) Schematic of F2APCbc4 and F3APCbc4 constructs used for transient expression in 293T cells. (c Right) Coimmunoprecipitation of Skp1 and ␤-catenin proteins with APCbc4, F2APCbc4, F3APCbc4, or Flag-tagged ␤-TrCP. Protein lysates from transfected 293T cells were immunoprecipitated with anti-HA or anti-Flag antibody, and