SUMOylation Regulates Nuclear Localization of Krüppel-like Factor 5
2008; Elsevier BV; Volume: 283; Issue: 46 Linguagem: Inglês
10.1074/jbc.m803612200
ISSN1083-351X
AutoresJames X. Du, Agnieszka B. Bialkowska, Beth B. McConnell, Vincent W. Yang,
Tópico(s)Cancer-related gene regulation
ResumoSUMOylation is a form of post-translational modification shown to control nuclear transport. Krüppel-like factor 5 (KLF5) is an important mediator of cell proliferation and is primarily localized to the nucleus. Here we show that mouse KLF5 is SUMOylated at lysine residues 151 and 202. Mutation of these two lysines or two conserved nearby glutamates results in the loss of SUMOylation and increased cytoplasmic distribution of KLF5, suggesting that SUMOylation enhances nuclear localization of KLF5. Lysine 151 is adjacent to a nuclear export signal (NES) that resembles a consensus NES. The NES in KLF5 directs a fused green fluorescence protein to the cytoplasm, binds the nuclear export receptor CRM1, and is inhibited by leptomycin and site-directed mutagenesis. SUMOylation facilitates nuclear localization of KLF5 by inhibiting this NES activity, and enhances the ability of KLF5 to stimulate anchorage-independent growth of HCT116 colon cancer cells. A survey of proteins whose nuclear localization is regulated by SUMOylation reveals that SUMOylation sites are frequently located in close proximity to NESs. A relatively common mechanism for SUMOylation to regulate nucleocytoplasmic transport may lie in the interplay between neighboring NES and SUMOylation motifs. SUMOylation is a form of post-translational modification shown to control nuclear transport. Krüppel-like factor 5 (KLF5) is an important mediator of cell proliferation and is primarily localized to the nucleus. Here we show that mouse KLF5 is SUMOylated at lysine residues 151 and 202. Mutation of these two lysines or two conserved nearby glutamates results in the loss of SUMOylation and increased cytoplasmic distribution of KLF5, suggesting that SUMOylation enhances nuclear localization of KLF5. Lysine 151 is adjacent to a nuclear export signal (NES) that resembles a consensus NES. The NES in KLF5 directs a fused green fluorescence protein to the cytoplasm, binds the nuclear export receptor CRM1, and is inhibited by leptomycin and site-directed mutagenesis. SUMOylation facilitates nuclear localization of KLF5 by inhibiting this NES activity, and enhances the ability of KLF5 to stimulate anchorage-independent growth of HCT116 colon cancer cells. A survey of proteins whose nuclear localization is regulated by SUMOylation reveals that SUMOylation sites are frequently located in close proximity to NESs. A relatively common mechanism for SUMOylation to regulate nucleocytoplasmic transport may lie in the interplay between neighboring NES and SUMOylation motifs. SUMOylation is a recently identified process by which cellular and viral proteins are post-translationally modified by small ubiquitin-related modifier (SUMO) 2The abbreviations used are: SUMO, small ubiquitin-related modifier; BSA, bovine serum albumin; CRM1, chromosome region maintenance 1; GFP, green fluorescence protein; HA, hemagglutinin; IP, immunoprecipitation; KLF5, Krüppel-like factor 5; LRS, leucine-rich sequence; NEM, N-ethylmaleimide; NES, nuclear export signal; siRNA, small interfering RNA; SM, SUMOylation motif; LRS, leucine-rich sequence; NLS, nuclear localization signal; GTPγS, guanosine 5′-3-O-(thio)triphosphate. proteins (1Johnson E.S. Annu. Rev. Biochem. 2004; 73: 355-382Crossref PubMed Scopus (1407) Google Scholar, 2Wilson V.G. Rangasamy D. Exp. Cell Res. 2001; 271: 57-65Crossref PubMed Scopus (86) Google Scholar). SUMO is conjugated to proteins by a mechanism that resembles ubiquitination (1Johnson E.S. Annu. Rev. Biochem. 2004; 73: 355-382Crossref PubMed Scopus (1407) Google Scholar, 3Pichler A. Melchior F. Traffic. 2002; 3: 381-387Crossref PubMed Scopus (159) Google Scholar). SUMOylation occurs at the lysine residue and most proteins are mono-SUMOylated in vivo (1Johnson E.S. Annu. Rev. Biochem. 2004; 73: 355-382Crossref PubMed Scopus (1407) Google Scholar, 2Wilson V.G. Rangasamy D. Exp. Cell Res. 2001; 271: 57-65Crossref PubMed Scopus (86) Google Scholar). The target lysine often reside within a consensus motif ψKXE, where Ψ is a large hydrophobic residue; K, lysine; X, any amino acid; and E, an acidic residue that is primarily glutamate (1Johnson E.S. Annu. Rev. Biochem. 2004; 73: 355-382Crossref PubMed Scopus (1407) Google Scholar, 2Wilson V.G. Rangasamy D. Exp. Cell Res. 2001; 271: 57-65Crossref PubMed Scopus (86) Google Scholar). SUMOylation contributes to many protein functions, one of which is the regulation of nuclear localization (1Johnson E.S. Annu. Rev. Biochem. 2004; 73: 355-382Crossref PubMed Scopus (1407) Google Scholar, 2Wilson V.G. Rangasamy D. Exp. Cell Res. 2001; 271: 57-65Crossref PubMed Scopus (86) Google Scholar). Nuclear accumulation of a protein is dependent on the balance between nuclear import and export. For the most part, nuclear import and export require transport receptors (4Terry L.J. Shows E.B. Wente S.R. Science. 2007; 318: 1412-1416Crossref PubMed Scopus (423) Google Scholar). The main nuclear export receptor is CRM1 (chromosome region maintenance 1) (4Terry L.J. Shows E.B. Wente S.R. Science. 2007; 318: 1412-1416Crossref PubMed Scopus (423) Google Scholar, 5Pemberton L.F. Paschal B.M. Traffic. 2005; 6: 187-198Crossref PubMed Scopus (585) Google Scholar). CRM1 binds to and is the receptor for the classic leucine-rich nuclear export signal (NES), which is present in various proteins for their delivery to the cytoplasm (6Kutay U. Guttinger S. Trends Cell Biol. 2005; 15: 121-124Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar). NES conforms to the consensus sequence of φ-X1-3-φ-X1-3-φ-X-φ (φ = Leu, Ile, Val, Met, or Phe, or less frequently, Tyr) (6Kutay U. Guttinger S. Trends Cell Biol. 2005; 15: 121-124Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar). The presence of regularly spaced, large hydrophobic residues, especially leucines, is the most important feature of the NES (6Kutay U. Guttinger S. Trends Cell Biol. 2005; 15: 121-124Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar). Many SUMOylated proteins are targeted to the nucleus and SUMOylation is known to regulate nuclear transport (1Johnson E.S. Annu. Rev. Biochem. 2004; 73: 355-382Crossref PubMed Scopus (1407) Google Scholar, 3Pichler A. Melchior F. Traffic. 2002; 3: 381-387Crossref PubMed Scopus (159) Google Scholar). However, the mechanism by which SUMOylation modulates this important biological event has not been clearly defined. Reports have suggested that SUMOylation facilitates nuclear localization through nuclear import (1Johnson E.S. Annu. Rev. Biochem. 2004; 73: 355-382Crossref PubMed Scopus (1407) Google Scholar, 3Pichler A. Melchior F. Traffic. 2002; 3: 381-387Crossref PubMed Scopus (159) Google Scholar). In contrast, several recent reports suggested that SUMOylation may also regulate nuclear export (7Sobko A. Ma H. Firtel R.A. Dev. Cell. 2002; 2: 745-756Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, 8Wood L.D. Irvin B.J. Nucifora G. Luce K.S. Hiebert S.W. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 3257-3262Crossref PubMed Scopus (101) Google Scholar, 9Carter S. Bischof O. Dejean A. Vousden K.H. Nat. Cell Biol. 2007; 9: 428-435Crossref PubMed Scopus (193) Google Scholar, 10Rosas-Acosta G. Wilson V.G. Virology. 2008; 373: 149-162Crossref PubMed Scopus (14) Google Scholar). Although it has been suspected that SUMOylation inhibits nuclear export by masking NESs (2Wilson V.G. Rangasamy D. Exp. Cell Res. 2001; 271: 57-65Crossref PubMed Scopus (86) Google Scholar), direct evidence in support of this hypothesis exists only for a viral protein, the adenovirus early region 1B (11Endter C. Kzhyshkowska J. Stauber R. Dobner T. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 11312-11317Crossref PubMed Scopus (92) Google Scholar)-55K (12Kindsmuller K. Groitl P. Hartl B. Blanchette P. Hauber J. Dobner T. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 6684-6689Crossref PubMed Scopus (76) Google Scholar). To date, no unified model for how SUMOylation regulates nuclear transport has emerged (13Hay R.T. Mol. Cell. 2005; 18: 1-12Abstract Full Text Full Text PDF PubMed Scopus (1360) Google Scholar). Krüppel-like factor 5 (14McConnell B.B. Ghaleb A.M. Nandan M.O. Yang V.W. 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Kadowaki T. Kurabayashi M. Nagai R. Nat. Med. 2002; 8: 856-863Crossref PubMed Scopus (333) Google Scholar). It is also part of a KLF core circuitry essential to embryonic stem cell renewal (26Jiang J. Chan Y.S. Loh Y.H. Cai J. Tong G.Q. Lim C.A. Robson P. Zhong S. Ng H.H. Nat. Cell Biol. 2008; 10: 353-360Crossref PubMed Scopus (613) Google Scholar, 27Okita K. Ichisaka T. Yamanaka S. Nature. 2007; 448: 313-317Crossref PubMed Scopus (3632) Google Scholar, 28Takahashi K. Tanabe K. Ohnuki M. Narita M. Ichisaka T. Tomoda K. Yamanaka S. Cell. 2007; 131: 861-872Abstract Full Text Full Text PDF PubMed Scopus (15750) Google Scholar, 29Takahashi K. Yamanaka S. Cell. 2006; 126: 663-676Abstract Full Text Full Text PDF PubMed Scopus (19944) Google Scholar). However, how KLF5 is post-translationally regulated is relatively unclear. Moreover, the mechanism that controls the nuclear localization of KLF5 is completely undefined. Although normally nuclear, recent reports have hinted that KLF5 may be localized to the cytoplasm. For example, in a subset of colorectal tumors with wild type KRAS, KLF5 is heavily localized to the cytoplasm, whereas in tumors carrying mutated KRAS, KLF5 is nuclear (23Nandan M.O. McConnell B.B. Ghaleb A.M. Bialkowska A.B. Sheng H. Shao J. Babbin B.A. Robine S. Yang V.W. Gastroenterology. 2008; 134: 120-130Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). In addition, a yeast two-hybrid screen with KLF5 as bait captured several preys that function as transport vesicle proteins, including sorting nexin 3 (30Haft C.R. de la Luz Sierra M. Barr V.A. Haft D.H. Taylor S.I. Mol. Cell. Biol. 1998; 18: 7278-7287Crossref PubMed Scopus (218) Google Scholar), a traffic protein associated with early endosomes (21Du J.X. Yun C.C. Bialkowska A. Yang V.W. J. Biol. Chem. 2007; 282: 4782-4793Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). Thus, KLF5 may shuttle between the nucleus and cytoplasm, which could be associated with distinct functions. In this report, we determine that KLF5 is SUMOylated in vivo, and this modification occurs at two specific lysine residues, Lys151 and Lys202, in the mouse sequence. We further show that a small fraction of KLF5 is localized to the cytoplasm, including structures that resemble intracellular vesicles. In close proximity to the first SUMOylation site of KLF5 is a putative NES. This leucine-rich NES matches the classic NES consensus, directs a fused GFP to the cytoplasm, binds CRM1, and is inhibited by leptomycin. Moreover, this NES activity is abolished by mutating two critical valine/leucine residues within the consensus sequence. SUMOylation facilitates nuclear localization of KLF5 and inhibits nuclear export by specifically inhibiting the NES. In addition, SUMOylation of KLF5 enhances the ability of KLF5 to stimulate anchorage-independent growth of HCT116 colorectal cancer cells. A survey of proteins that nuclear localizations are facilitated by SUMOylation reveals that SUMOylation sites and NESs are often located in close proximity to each other. This report therefore provides direct evidence that SUMOylation facilitates protein nuclear localization by inhibiting the nuclear export signal adjacent to its target site. Plasmid Constructs—KLF5 mutants were constructed with the QuikChange site-directed mutagenesis kit (Stratagene). SUMOylation site lysine residues were replaced with arginines, and SUMOylation motif glutamate residues were replaced with alanines. The two hydrophobic residues predicted to be crucial for the NES of KLF5 were replaced with alanines. Plasmids expressing GFP fusion proteins were prepared by inserting DNA fragments encoding the indicated KLF5 peptides into the EcoRI-SalI site of pEGFPC1 (Clontech). The amounts of plasmids introduced in the transfection were adjusted for equal amounts of protein expression in the experiments. Plasmids GFP-SUMO-1, HIS-SUMO-1, and pMT3/HA-KLF5 have been described previously (15Conkright M.D. Wani M.A. Anderson K.P. Lingrel J.B. Nucleic Acids Res. 1999; 27: 1263-1270Crossref PubMed Scopus (144) Google Scholar, 31Fogal V. Gostissa M. Sandy P. Zacchi P. Sternsdorf T. Jensen K. Pandolfi P.P. Will H. Schneider C. Del Sal G. EMBO J. 2000; 19: 6185-6195Crossref PubMed Scopus (322) Google Scholar, 32Muller S. Berger M. Lehembre F. Seeler J.S. Haupt Y. Dejean A. J. Biol. Chem. 2000; 275: 13321-13329Abstract Full Text Full Text PDF PubMed Scopus (353) Google Scholar, 33Dang D.T. Zhao W. Mahatan C.S. Geiman D.E. Yang V.W. Nucleic Acids Res. 2002; 30: 2736-2741Crossref PubMed Google Scholar). SUMOylation Assays—COS-1 cells were transfected with the indicated plasmids and disrupted in lysis buffer (20 mm Tris-HCl, pH 7.4, 1% Nonidet P-40, 135 mm NaCl, 20 mmN-ethylmaleimide (NEM, Sigma), and complete protease inhibitor mixture (Roche)). The lysates were immunoprecipitated with a rabbit HA antibody (Sigma) for 2 h followed by incubation with EZview Red protein G affinity gel (Sigma) for 1 h. The immune complexes were washed with the lysis buffer four times and subjected to Western blotting with a mouse HA (Covance) or GFP (Roche) antibody. Alternatively, the transfected cells were disrupted by boiling in SDS lysis buffer (62.5 mm Tris-HCl, pH 6.8, 2% SDS, 8.75% glycerol, 5% 2-mecaptoethanol) containing NEM, phenylmethylsulfonyl fluoride, and aprotinin as described previously (21Du J.X. Yun C.C. Bialkowska A. Yang V.W. J. Biol. Chem. 2007; 282: 4782-4793Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). Western blot analysis was performed with mouse HA (Covance), HIS (Qiagen), or β-actin (Sigma) antibodies. Fluorescence Microscopy—COS-1 cells were transfected with the indicated plasmids. On the following day, cells were fixed with methanol, blocked with 2% bovine serum albumin (BSA) in phosphate-buffered saline for 1 h, and incubated overnight with a rabbit HA antibody (Sigma) followed by RRX-conjugated donkey anti-rabbit secondary antibody (Jackson ImmunoResearch). Cells were also stained with Hoechst dye to reveal the nuclei. To inhibit nuclear export, cells were either mock treated or treated for 12 h with 10 ng/ml leptomycin A and B (Sigma) (34Hamamoto T. Gunji S. Tsuji H. Beppu T. J. Antibiot. (Tokyo). 1983; 36: 639-645Crossref PubMed Scopus (137) Google Scholar, 35Hamamoto T. Seto H. Beppu T. J. Antibiot. (Tokyo). 1983; 36: 646-650Crossref PubMed Scopus (131) Google Scholar, 36Buschbeck M. Ullrich A. J. Biol. Chem. 2005; 280: 2659-2667Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). For endogenous KLF5 staining, COS-1 cells were fixed with methanol, blocked with 10% horse serum, 3% BSA, and 3% nonfat dry milk for 1 h, and incubated overnight with a rabbit antibody raised against KLF5 (Santa Cruz) and a RRX-conjugated donkey anti-rabbit antibody (Jackson ImmunoResearch). For endogenous KLF5 staining after small interfering RNA (siRNA) knockdown, KLF5 siRNA (Invitrogen) or the corresponding negative control siRNA-transfected DLD-1 cells were fixed with 3% formaldehyde, blocked with 3% BSA and 0.02% Triton X-100 in phosphate-buffered saline, and stained with a rabbit KLF5 antibody (CeMines) and an Alexa Fluor 488-conjugated goat anti-rabbit secondary antibody (Molecular Probes). For endogenous co-staining of KLF5 and SNX3, COS-1 cells were fixed with methanol, blocked, and incubated overnight with a rabbit KLF5 antibody (23Nandan M.O. McConnell B.B. Ghaleb A.M. Bialkowska A.B. Sheng H. Shao J. Babbin B.A. Robine S. Yang V.W. Gastroenterology. 2008; 134: 120-130Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar) and a goat SNX3 antibody (Santa Cruz), followed by fluorescein isothiocyanate-conjugated donkey anti-rabbit and RRX-conjugated donkey anti-goat secondary antibodies (Jackson ImmunoResearch). Cells were stained with Hoechst dye to reveal the nuclei. Fluorescence was observed under a LSM 510 laser confocal microscope (Zeiss). For the localization of GFP-LRS proteins, COS-1 cells were transfected with the indicated plasmids. On the next day, the cells were either mock treated or treated for 16 h with 10 ng/ml leptomycin A and B (34Hamamoto T. Gunji S. Tsuji H. Beppu T. J. Antibiot. (Tokyo). 1983; 36: 639-645Crossref PubMed Scopus (137) Google Scholar, 35Hamamoto T. Seto H. Beppu T. J. Antibiot. (Tokyo). 1983; 36: 646-650Crossref PubMed Scopus (131) Google Scholar, 36Buschbeck M. Ullrich A. J. Biol. Chem. 2005; 280: 2659-2667Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). The green fluorescence was viewed under an Eclipse TS100 inverted microscope (Nikon). Heterokaryon Assay—Heterokaryon assay was performed as described (37Pinol-Roma S. Dreyfuss G. Nature. 1992; 355: 730-732Crossref PubMed Scopus (753) Google Scholar, 38Roth J. Dobbelstein M. Freedman D.A. Shenk T. Levine A.J. EMBO J. 1998; 17: 554-564Crossref PubMed Scopus (536) Google Scholar, 39Salinas S. Briancon-Marjollet A. Bossis G. Lopez M.A. Piechaczyk M. Jariel-Encontre I. Debant A. Hipskind R.A. J. Cell Biol. 2004; 165: 767-773Crossref PubMed Scopus (85) Google Scholar, 40Tao W. Levine A.J. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 6937-6941Crossref PubMed Scopus (502) Google Scholar). Briefly, COS-1 cells were transfected with HA-KLF5. Eight h after transfection, NIH3T3 cells were plated and co-cultured with the transfected COS-1 cells. Fifteen h later, the co-cultured cells were treated for one-half h with 50 μg/ml cycloheximide (Sigma), which was also present throughout the following steps. Cells were then either treated with 50% (w/v) polyethylene glycol 8000 in the culture medium for 2 min to induce fusion or mock treated as the control. After three washes, the fused cells were cultured for 2 h to allow interkaryon shuttling, and fixed and stained with a chicken anti-HA antibody (Chemicon) and fluorescein isothiocyanate-conjugated donkey anti-chicken secondary antibody. To distinguish between monkey (COS-1) and mouse (NIH3T3) nuclei, the cells were simultaneously stained with Hoechst dye. The murine nuclei were readily identified by their unique punctate pattern of fluorescence as previously described (37Pinol-Roma S. Dreyfuss G. Nature. 1992; 355: 730-732Crossref PubMed Scopus (753) Google Scholar, 38Roth J. Dobbelstein M. Freedman D.A. Shenk T. Levine A.J. EMBO J. 1998; 17: 554-564Crossref PubMed Scopus (536) Google Scholar, 39Salinas S. Briancon-Marjollet A. Bossis G. Lopez M.A. Piechaczyk M. Jariel-Encontre I. Debant A. Hipskind R.A. J. Cell Biol. 2004; 165: 767-773Crossref PubMed Scopus (85) Google Scholar, 40Tao W. Levine A.J. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 6937-6941Crossref PubMed Scopus (502) Google Scholar). Small Interfering RNA—Stealth Select siRNA against human KLF5 and the corresponding negative control siRNA were obtained from Invitrogen and prepared according to the manufacturer's instruction as described (23Nandan M.O. McConnell B.B. Ghaleb A.M. Bialkowska A.B. Sheng H. Shao J. Babbin B.A. Robine S. Yang V.W. Gastroenterology. 2008; 134: 120-130Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). Cells were transfected with the siRNA using Lipofectamine RNAiMAX reagent in Opti-MEM I Reduced Serum Medium (Invitrogen). After transfection, the cells were either collected for subcellular fractionation and Western blotting or immunostained with a rabbit anti-KLF5 antibody. Subcellular Fractionation—COS-1 cells were subjected to subcellular fractionation as described (41Guillemin I. Becker M. Ociepka K. Friauf E. Nothwang H.G. Proteomics. 2005; 5: 35-45Crossref PubMed Scopus (87) Google Scholar). The fractionated proteins were probed with rabbit KLF5 and mouse histone H1 (Upstate), α-tubulin (Sigma), and Na+/K+-ATPase (Millipore) antibodies. DLD-1 cells were subjected to nuclear and cytoplasmic fractionation as described (42Bialkowska A. Zhang X.Y. Reiser J. BMC Genomics. 2005; 6: 113Crossref PubMed Scopus (9) Google Scholar). The fractionated proteins were probed with rabbit KLF5 and mouse histone H1 (Upstate), and β-actin (Sigma) antibodies. Immunoprecipitation—To detect binding to nuclear export receptor, COS-1 cells were transfected with the indicated plasmids. Two days later, cells were lysed in lysis buffer containing phosphatase inhibitors (Active Motif). Immunoprecipitation was performed with a nuclear complex co-IP kit (Active Motif) according to the manufacturer's instruction. Briefly, the lysates were blocked with horse serum and EZview protein G affinity gel (Sigma) or protein A-agarose beads (Upstate) by rocking for 2 h, and immunoprecipitated with either a mouse monoclonal CRM1 antibody (BD Transduction Laboratories) or a rabbit GFP antibody (Santa Cruz) overnight followed by incubation with BSA-blocked EZview protein G affinity gel or protein A-agarose beads for 1 h. The immune complexes were washed three times with BSA-containing IP high buffer supplemented with 2 mm NEM, salt, detergent, phosphatase inhibitors, and complete protease inhibitor mixture, and twice with IP high buffer without BSA. The precipitates were immunoblotted with goat anti-GFP (Rockland) and mouse anti-CRM1. In Vitro SUMOylation and Binding Assays—To detect binding to CRM1 in vitro, COS-1 cells were transfected with the indicated plasmids. Two days later, cells were lysed, and the GFP fusion proteins were purified by immunoprecipitation with anti-GFP antibody and protein A beads. The immunoprecipitates were extensively washed with 20 mm Tris-HCl, pH 7.4, and 5 mm MgCl2, and resuspended in SUMOylation buffer (LAE Biotech) containing purified SUMO-1, and E1 (SAEI/SAEII) and E2 (Ubc9) enzymes (LAE Biotech). The reactions were rocked at 37 °C for 1 h, followed by incubation for 3 days with rocking at 4 °C. The samples were centrifuged and washed, and the precipitates were resuspended in lysis buffer containing 50 μm GTPγS (Sigma), purified CRM1 (Abnova) and Ran (Sigma) that have been preincubated with 10 mm GTPγS, and rocked at 4 °C overnight. The mixtures were centrifuged, and the pellets were washed three times with the BSA-containing IP high buffer, and twice with the IP high buffer without BSA. Fifty μm GTPγS was supplemented during all washing steps. The samples were immunoblotted with goat anti-GFP and mouse anti-CRM1. Colony Formation Assay—To determine the transformation potential of KLF5 and its SUMOylation mutants, HCT116 cells were transfected with the indicated plasmids, and colony formation assay in soft agar was performed as described (43Cox A.D. Der C.J. Methods Enzymol. 1994; 238: 277-294Crossref PubMed Scopus (87) Google Scholar). Colonies were counted 3 weeks later. KLF5 Is SUMOylated—KLF5 contains two highly conserved sequences that resemble the consensus SUMOylation motif, ψKXE (1Johnson E.S. Annu. Rev. Biochem. 2004; 73: 355-382Crossref PubMed Scopus (1407) Google Scholar, 2Wilson V.G. Rangasamy D. Exp. Cell Res. 2001; 271: 57-65Crossref PubMed Scopus (86) Google Scholar) (Fig. 1). We first determined whether KLF5 is SUMOylated in cells. COS-1 cells were co-transfected with HA-tagged KLF5 and GFP-tagged SUMO-1. Cell lysates were prepared in the presence of deSUMOylation inhibitor NEM (1Johnson E.S. Annu. Rev. Biochem. 2004; 73: 355-382Crossref PubMed Scopus (1407) Google Scholar) and immunoprecipitated with a rabbit HA antibody. Immunoblots were then performed on the immunoprecipitates with a mouse HA or GFP antibody (Fig. 2A). Three slow-migrating HA-KLF5 species were detected (Fig. 2A, lane 1, *, +, and ++). These three forms were SUMOylated as confirmed by blotting with the GFP antibody (Fig. 2A, lane 5). The slowest migrating HA-KLF5 (*) was fully SUMOylated, whereas the other two slow-migrating species (+ and ++) were partially SUMOylated (see below). Quantitative analysis of chemiluminescence showed that SUMOylated KLF5 represented on average 7.5 ± 3.8% (n = 4) of total KLF5.FIGURE 2SUMOylation of KLF5.A, SUMOylation of KLF5 with GFP-tagged SUMO-1 and identification of the two SUMOylation lysine residues within the ΨKXE motifs. COS-1 cells were co-transfected with GFP-SUMO-1 and one of the following: pMT3/HA-KLF5 (WT), pMT3/HA-KLF5-K151R (K151R), pMT3/HA-KLF5-K202R (K202R), or pMT3/HA-KLF5-K151R/K202R (K151R/K202R). Lysates were immunoprecipitated with a rabbit HA antibody followed by Western blotting with a mouse HA (lanes 1-4) or GFP (lanes 5-8) antibody. B, SUMOylation of KLF5 with His-tagged SUMO-1 and confirmation of the two SUMOylation sites. COS-1 cells were co-transfected with HIS-SUMO-1 and pMT3/HA-KLF5 (WT), pMT3/HA-KLF5-K151R (K151R), pMT3/HA-KLF5-K202R (K202R), or pMT3/HA-KLF5-K151R/K202R (K151R/K202R). The transfected cells were disrupted by boiling in the presence of SDS and NEM and Western blot was performed with a mouse HA, His, or β-actin antibody. C, SUMOylation of endogenous KLF5. Endogenous KLF5 is SUMOylated in the presence (lane 1, HS1) or absence (lane 2) of HIS-SUMO-1 transfection. D, loss in KLF5 SUMOylation by mutation at the two essential glutamate residues within the ΨKXE motifs. COS-1 cells were co-transfected with GFP-SUMO-1 and pMT3/HA-KLF5 (WT), pMT3/HA-KLF5-E153A (E153A), or pMT3/HA-KLF5-E153A/E204A (E153A/E204A). Lysates were immunoprecipitated with rabbit anti-HA followed by Western blot with mouse anti-HA (lanes 1-4) or GFP (lanes 5-8). A short exposure of the panel in lanes 5-8 is also shown. In all panels, * represents the di-SUMOylated form of KLF5; + and ++, mono-SUMOylated forms.View Large Image Figure ViewerDownload Hi-res image Download (PPT) As an alternative method to detect SUMOylation of KLF5, we transfected COS-1 cells with HA-KLF5 and His-tagged SUMO-1 and disrupted the cells under denaturing conditions by boiling in the presence of SDS and NEM to preserve SUMOylation (1Johnson E.S. Annu. Rev. Biochem. 2004; 73: 355-382Crossref PubMed Scopus (1407) Google Scholar). Upon Western blotting against HA, SUMOylated KLF5 was apparent in the whole cell lysates (Fig. 2B, lane 1). Due to the smaller His tag, all three SUMOylated forms of KLF5 migrated faster than the three GFP-SUMO-1-modified counterparts although they exhibited a similar relative pattern of migration. These results confirm that KLF5 is SUMOylated. We next determined whether endogenous KLF5 is SUMOylated. COS-1 cells were either transfected with HIS-SUMO-1 or mock transfected, and the whole cell lysates were blotted with a KLF5 antibody to reveal endogenous KLF5 (Fig. 2C). In cells transfected with His-SUMO-1, SUMOylated endogenous KLF5 was detected (Fig. 2C, lane 1). In mock transfected cells, three slow-migrating forms with slightly faster mobility than those in His-SUMO-1-transfected cells were detected (Fig. 2C, lane 2; *, +, and ++). These represent SUMOylated KLF5 by endogenous SUMO. We estimate that less than 1% of endogenous KLF5 was SUMOylated without SUMO overexpression. This is consistent with the observations that only a small percentage of a given protein, often less than 1%, is SUMOylated at steady state (1Johnson E.S. Annu. Rev. Biochem. 2004; 73: 355-382Crossref PubMed Scopus (1407) Google Scholar). These experiments show that the endogenous KLF5 is also SUMOylated. Identification of the SUMOylation Sites in KLF5—We next sought to identify the sites of SUMOylation in KLF5. SUMOylation typically occurs at lysine residues within a consensus sequence ψKXE (1Johnson E.S. Annu. Rev. Biochem. 2004; 73: 355-382Crossref PubMed Scopus (1407) Google Scholar, 2Wilson V.G. Rangasamy D. Exp. Cell Res. 2001; 271: 57-65Crossref PubMed Scopus (86) Google Scholar). The lysine residues within the consensus SUMOylation motifs of KLF5 are located at amino
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