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• W2018304918 abstract "Homeodomain-interacting protein kinase 2 (HIPK2) is a member of the nuclear protein kinase family, which induces both p53- and CtBP-mediated apoptosis. Levels of HIPK2 were increased by UV irradiation and cisplatin treatment, thereby implying the degradation of HIPK2 in cells under normal conditions. Here, we indicate that HIPK2 is ubiquitinated and degraded by the WD40-repeat/SOCS box protein WSB-1, a process that is blocked under DNA damage conditions. Yeast two-hybrid screening was conducted to identify the proteins that interact with HIPK2. WSB-1, an E3 ubiquitin ligase, was characterized as an HIPK2-interacting protein. The coexpression of WSB-1 resulted in the degradation of HIPK2 via its C-terminal region. Domain analysis of WSB-1 showed that WD40-repeats and the SOCS box were required for its interaction with and degradation of HIPK2, respectively. In support of the degradation of HIPK2 by WSB-1, HIPK2 was polyubiquitinated by WSB-1 in vitro and in vivo. The knockdown of endogenous WSB-1 with the expression of short hairpin RNA against WSB-1 increases the stability of endogenous HIPK2 and resulted in the accumulation of HIPK2. The ubiquitination and degradation of HIPK2 by WSB-1 was inhibited completely via the administration of DNA damage reagents, including Adriamycin and cisplatin. These findings effectively illustrate the regulatory mechanisms by which HIPK2 is maintained at a low level, by WSB-1 in cells under normal conditions, and stabilized by genotoxic stresses. Homeodomain-interacting protein kinase 2 (HIPK2) is a member of the nuclear protein kinase family, which induces both p53- and CtBP-mediated apoptosis. Levels of HIPK2 were increased by UV irradiation and cisplatin treatment, thereby implying the degradation of HIPK2 in cells under normal conditions. Here, we indicate that HIPK2 is ubiquitinated and degraded by the WD40-repeat/SOCS box protein WSB-1, a process that is blocked under DNA damage conditions. Yeast two-hybrid screening was conducted to identify the proteins that interact with HIPK2. WSB-1, an E3 ubiquitin ligase, was characterized as an HIPK2-interacting protein. The coexpression of WSB-1 resulted in the degradation of HIPK2 via its C-terminal region. Domain analysis of WSB-1 showed that WD40-repeats and the SOCS box were required for its interaction with and degradation of HIPK2, respectively. In support of the degradation of HIPK2 by WSB-1, HIPK2 was polyubiquitinated by WSB-1 in vitro and in vivo. The knockdown of endogenous WSB-1 with the expression of short hairpin RNA against WSB-1 increases the stability of endogenous HIPK2 and resulted in the accumulation of HIPK2. The ubiquitination and degradation of HIPK2 by WSB-1 was inhibited completely via the administration of DNA damage reagents, including Adriamycin and cisplatin. These findings effectively illustrate the regulatory mechanisms by which HIPK2 is maintained at a low level, by WSB-1 in cells under normal conditions, and stabilized by genotoxic stresses. Homeodomain-interacting protein kinase 2 (HIPK2) 5The abbreviations used are: HIPK2homeodomain-interacting protein kinase 2SCFSkp1-Cullin-F-boxECSElonginB/C-Cul2/5-SOCSSOCS boxsuppressor of cytokine signaling boxGFPgreen fluorescent proteinMyc tagMyc epitope tagGSTglutathione S-transferaseUbubiquitinE1ubiquitin-activating enzymeE2ubiquitin-conjugating enzymeE3ubiquitin ligaseaaamino acid(s)HAhemagglutininshRNAshort hairpin RNA. 5The abbreviations used are: HIPK2homeodomain-interacting protein kinase 2SCFSkp1-Cullin-F-boxECSElonginB/C-Cul2/5-SOCSSOCS boxsuppressor of cytokine signaling boxGFPgreen fluorescent proteinMyc tagMyc epitope tagGSTglutathione S-transferaseUbubiquitinE1ubiquitin-activating enzymeE2ubiquitin-conjugating enzymeE3ubiquitin ligaseaaamino acid(s)HAhemagglutininshRNAshort hairpin RNA. is a member of a novel family of nuclear protein kinases and is well conserved from Drosophila to humans (1Choi C.Y. Kim Y.H. Kim Y.O. Park S.J. Kim E.A. Riemenschneider W. Gajewski K. Schulz R.A. Kim Y. J. Biol. Chem. 2005; 280: 21427-21436Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 2Kim Y.H. Choi C.Y. Lee S.J. Conti M.A. Kim Y. J. Biol. Chem. 1998; 273: 25875-25879Abstract Full Text Full Text PDF PubMed Scopus (248) Google Scholar, 3Wang Y. Hofmann T.G. Runkel L. Haaf T. Schaller H. Debatin K. Hug H. Biochim. Biophys. Acta. 2001; 1518: 168-172Crossref PubMed Scopus (21) Google Scholar, 4Moilanen A.M. Karvonen U. Poukka H. Janne O.A. Palvimo J.J. Mol. Biol. Cell. 1998; 9: 2527-2543Crossref PubMed Scopus (100) Google Scholar). HIPK2 interacts with a variety of transcription factors (5Kim E.A. Noh Y.T. Ryu M.J. Kim H.T. Lee S.E. Kim C.H. Lee C. Kim Y.H. Choi C.Y. J. Biol. Chem. 2006; 281: 7489-7497Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 6Zhang Q. Nottke A. Goodman R.H. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 2802-2807Crossref PubMed Scopus (70) Google Scholar, 7D'Orazi G. Cecchinelli B. Bruno T. Manni I. Higashimoto Y. Saito S. Gostissa M. Coen S. Marchetti A. Del Sal G. Piaggio G. Fanciulli M. Appella E. Soddu S. Nat. Cell Biol. 2002; 4: 11-19Crossref PubMed Scopus (570) Google Scholar, 8Hofmann T.G. Moller A. Sirma H. Zentgraf H. Taya Y. Droge W. Will H. Schmitz M.L. Nat. Cell Biol. 2002; 4: 1-10Crossref PubMed Scopus (496) Google Scholar), p300/CBP coactivator (9Aikawa Y. Nguyen L.A. Isono K. Takakura N. Tagata Y. Schmitz M.L. Koseki H. Kitabayashi I. EMBO J. 2006; 25: 3955-3965Crossref PubMed Scopus (109) Google Scholar, 10Kim E.J. Park J.S. Um S.J. J. Biol. Chem. 2002; 277: 32020-32028Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar), and Groucho/TLE corepressor (1Choi C.Y. Kim Y.H. Kim Y.O. Park S.J. Kim E.A. Riemenschneider W. Gajewski K. Schulz R.A. Kim Y. J. Biol. Chem. 2005; 280: 21427-21436Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar), thereby regulating target gene expression in a context-dependent manner. The loss of a functional HIPK2 allele induces a reduction of apoptosis and increased numbers of trigeminal ganglia, whereas HIPK2 overexpression in the developing sensory and sympathetic neurons promotes apoptosis in a caspase-dependent manner (11Wiggins A.K. Wei G. Doxakis E. Wong C. Tang A.A. Zang K. Luo E.J. Neve R.L. Reichardt L.F. Huang E.J. J. Cell Biol. 2004; 167: 257-267Crossref PubMed Scopus (85) Google Scholar, 12Doxakis E. Huang E.J. Davies A.M. Curr. Biol. 2004; 14: 1761-1765Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). The HIPK1 and HIPK2 double-knock-out approach showed that HIPK2 performs overlapping functions with HIPK1 in the mediation of cell proliferation and apoptosis during mouse development (13Isono K. Nemoto K. Li Y. Takada Y. Suzuki R. Katsuki M. Nakagawara A. Koseki H. Mol. Cell Biol. 2006; 26: 2758-2771Crossref PubMed Scopus (91) Google Scholar). A number of key regulatory molecules, including p53, CtBP, Axin, Brn3, Sp100, TP53INP1, and PML, are known to be associated with the function of HIPK2 in the induction of apoptosis (14Rui Y. Xu Z. Lin S. Li Q. Rui H. Luo W. Zhou H.M. Cheung P.Y. Wu Z. Ye Z. Li P. Han J. Lin S.C. EMBO J. 2004; 23: 4583-4594Crossref PubMed Scopus (132) Google Scholar, 15Kanei-Ishii C. Ninomiya-Tsuji J. Tanikawa J. Nomura T. Ishitani T. Kishida S. Kokura K. Kurahashi T. Ichikawa-Iwata E. Kim Y. Matsumoto K. Ishii S. Genes Dev. 2004; 18: 816-829Crossref PubMed Scopus (153) Google Scholar, 16Moller A. Sirma H. Hofmann T.G. Rueffer S. Klimczak E. Droge W. Will H. Schmitz M.L. Cancer Res. 2003; 63: 4310-4314PubMed Google Scholar, 17Moller A. Sirma H. Hofmann T.G. Staege H. Gresko E. Ludi K.S. Klimczak E. Droge W. Will H. Schmitz M.L. Oncogene. 2003; 22: 8731-8737Crossref PubMed Scopus (36) Google Scholar, 18Tomasini R. Samir A.A. Carrier A. Isnardon D. Cecchinelli B. Soddu S. Malissen B. Dagorn J.C. Iovanna J.L. Dusetti N.J. J. Biol. Chem. 2003; 278: 37722-37729Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). UV-induced apoptosis is understood via the action of HIPK2 at the molecular level. HIPK2 is activated and stabilized by UV irradiation and selectively phosphorylates p53 at Ser-46. Thus, the kinase function of HIPK2 increases the expression of p53-target genes and enhances UV-induced apoptosis (7D'Orazi G. Cecchinelli B. Bruno T. Manni I. Higashimoto Y. Saito S. Gostissa M. Coen S. Marchetti A. Del Sal G. Piaggio G. Fanciulli M. Appella E. Soddu S. Nat. Cell Biol. 2002; 4: 11-19Crossref PubMed Scopus (570) Google Scholar, 8Hofmann T.G. Moller A. Sirma H. Zentgraf H. Taya Y. Droge W. Will H. Schmitz M.L. Nat. Cell Biol. 2002; 4: 1-10Crossref PubMed Scopus (496) Google Scholar). Additionally, HIPK2 phosphorylates the Ser-422 of CtBP, and phosphorylated CtBP is degraded via the 26 S proteasome pathway, resulting in apoptosis in p53-deficient cells (19Zhang Q. Yoshimatsu Y. Hildebrand J. Frisch S.M. Goodman R.H. Cell. 2003; 115: 177-186Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar). Endogenous HIPK2 protein is barely detected and is induced by UV irradiation or treatment with the chemotherapeutic drug, cisplatin, whereas the levels of HIPK2 mRNA remained unchanged under identical conditions (20Di Stefano V. Blandino G. Sacchi A. Soddu S. D'Orazi G. Oncogene. 2004; 23: 5185-5192Crossref PubMed Scopus (61) Google Scholar). The levels of p53 and Bax increase concomitantly with the induction of HIPK2 (21Di Stefano V. Rinaldo C. Sacchi A. Soddu S. D'Orazi G. Exp. Cell Res. 2004; 293: 311-320Crossref PubMed Scopus (93) Google Scholar). However, it remains to be determined whether HIPK2 is degraded at the protein level, nor is it understood which molecule is responsible for the tight control of HIPK2 levels in cells under normal conditions. homeodomain-interacting protein kinase 2 Skp1-Cullin-F-box ElonginB/C-Cul2/5-SOCS suppressor of cytokine signaling box green fluorescent protein Myc epitope tag glutathione S-transferase ubiquitin ubiquitin-activating enzyme ubiquitin-conjugating enzyme ubiquitin ligase amino acid(s) hemagglutinin short hairpin RNA. homeodomain-interacting protein kinase 2 Skp1-Cullin-F-box ElonginB/C-Cul2/5-SOCS suppressor of cytokine signaling box green fluorescent protein Myc epitope tag glutathione S-transferase ubiquitin ubiquitin-activating enzyme ubiquitin-conjugating enzyme ubiquitin ligase amino acid(s) hemagglutinin short hairpin RNA. The highly controlled degradation of proteins via the ubiquitin-proteasome pathway represents a key mechanism for a variety of cellular activities, including cell cycle regulation and apoptosis. The ubiquitination of proteins requires three enzymes: E1, ubiquitin-activating enzyme; E2, ubiquitin-conjugating enzyme; and E3, ubiquitin ligase (22Ciechanover A. Nat. Rev. Mol. Cell Biol. 2005; 6: 79-87Crossref PubMed Scopus (809) Google Scholar, 23Hershko A. Ciechanover A. Annu. Rev. Biochem. 1998; 67: 425-479Crossref PubMed Scopus (6825) Google Scholar). Among them, the ubiquitin ligase has specificity and recognizes the target protein. The majority of eukaryotes harbor a single E1 enzyme, several E2 enzymes, and a large and more diverse class of E3 enzymes. Several domains have been associated with E3 enzyme activity. The best known examples are the SCF (Skp1-Cullin-F-box) and the ECS (ElonginB/C-Cul2/5-SOCS) ubiquitin ligase complexes. The SCF ubiquitin ligases are a family of multisubunit RING finger E3 enzymes in which an F-box protein is responsible for substrate recognition. F-box proteins interact with the core SCF complexes via their common N-terminal F-box motif and harbor a C-terminal protein-protein interaction motif, typically WD40 repeats or leucine-rich repeats (24Nakayama K.I. Nakayama K. Semin. Cell Dev. Biol. 2005; 16: 323-333Crossref PubMed Scopus (290) Google Scholar, 25Vodermaier H.C. Curr. Biol. 2004; 14: R787-R796Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar). The ECS complex displays striking overall similarities with the SCF complex. The SOCS (suppressor of cytokine signaling) box-containing proteins function as adaptors that link substrates to the ElonginB/C-Cullin complex (26Heuze M.L. Guibal F.C. Banks C.A. Conaway J.W. Conaway R.C. Cayre Y.E. Benecke A. Lutz P.G. J. Biol. Chem. 2005; 280: 5468-5474Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 27Kamura T. Maenaka K. Kotoshiba S. Matsumoto M. Kohda D. Conaway R.C. Conaway J.W. Nakayama K.I. Genes Dev. 2004; 18: 3055-3065Crossref PubMed Scopus (364) Google Scholar). In addition to the SOCS box, SOCS box proteins harbor a variety of protein-protein interaction domains, including the β-domain, WD40 repeats, and ankyrin motifs that bind to substrates. Thus far, at least twenty proteins have been determined to harbor a C-terminal SOCS box. These proteins fall into five classes based on the protein motifs found in the N-terminal region of the SOCS box (28Hilton D.J. Richardson R.T. Alexander W.S. Viney E.M. Willson T.A. Sprigg N.S. Starr R. Nicholson S.E. Metcalf D. Nicola N.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 114-119Crossref PubMed Scopus (610) Google Scholar). WSB-1 and WSB-2 have been classified as part of a new family; members of this family harbor seven WD40 repeats and an SOCS box in the N terminus and C terminus of a protein, respectively. Although the domain organization and the interactions of WSB-1 with ElonginB/C are very well understood, investigators have only recently undertaken studies to determine whether WSB-1 can function as an E3 ubiquitin ligase, and to investigate the potential target molecules of WSB-1 (29Dentice M. Bandyopadhyay A. Gereben B. Callebaut I. Christoffolete M.A. Kim B.W. Nissim S. Mornon J.P. Zavacki A.M. Zeold A. Capelo L.P. Curcio-Morelli C. Ribeiro R. Harney J.W. Tabin C.J. Bianco A.C. Nat. Cell Biol. 2005; 7: 698-705Crossref PubMed Scopus (178) Google Scholar, 30Erkeland S.J. Aarts L.H. Irandoust M. Roovers O. Klomp A. Valkhof M. Gits J. Eyckerman S. Tavernier J. Touw I.P. Oncogene. 2007; 26: 1985-1994Crossref PubMed Scopus (20) Google Scholar, 31Zeold A. Pormuller L. Dentice M. Harney J.W. Curcio-Morelli C. Tente S.M. Bianco A.C. Gereben B. J. Biol. Chem. 2006; 281: 31538-31543Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). In this study, we show that WSB-1 is an E3 ubiquitin ligase that specifically targets HIPK2 for degradation via the 26 S proteasome pathway. We determined that WSB-1 promotes the ubiquitination and degradation of HIPK2, a process in which both WD40 repeats and the SOCS box are required for the recognition and degradation of HIPK2. WSB-1-mediated degradation of HIPK2 was blocked in DNA-damaged cells, which provide regulatory mechanisms by which the HIPK2 levels are tightly controlled in cells under normal conditions, and DNA damage stresses overcome the degradation of HIPK2 by WSB-1. Cell Culture and Transfection–U2OS, RKO, and HEK293 cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. For immunoblot analysis, HEK293 cells were seeded onto 6-well plates, and DNA transfection was carried out using the N,N-bis-(2-hydroxyethyl)-2-aminoethanesulfonic acid-buffered saline version of the calcium phosphate procedure. U2OS and RKO cells were transfected with Fugene6 reagent (Roche Molecular Biochemicals) as described above, on 6-well plates. Plasmid Construction and Site-directed Mutagenesis–The full-length human WSB-1 EST clone was purchased from Invitrogen (clone ID 4865448). The full open-reading frames of WSB-1 were PCR-amplified from human WSB-1 with specific primers, and the DNA fragment was inserted into EcoRI and XhoI sites of pEntr3C. Various WSB-1 deletion mutants were constructed by insertion of each PCR-amplified DNA fragments into the EcoRI and XhoI sites of pEntr3C. Various HIPK2 deletion constructs and GFP-HIPK2 plasmid have been described previously (5Kim E.A. Noh Y.T. Ryu M.J. Kim H.T. Lee S.E. Kim C.H. Lee C. Kim Y.H. Choi C.Y. J. Biol. Chem. 2006; 281: 7489-7497Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 32Choi C.Y. Kim Y.H. Kwon H.J. Kim Y. J. Biol. Chem. 1999; 274: 33194-33197Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar, 33Kim Y.H. Choi C.Y. Kim Y. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12350-12355Crossref PubMed Scopus (142) Google Scholar). The pET3E-Ubc5C plasmid expressing the His-Ubc5C was kindly provided by Yue Xiong (University of North Carolina). The Myc-WSB-1, GFP-WSB-1, His-WSB-1, and GST-WSB-1 plasmids were constructed by Gateway Technology (Invitrogen) with pEntr-WSB-1. The point mutant of WSB-1 for the WD40 domain was generated using the QuikChange mutagenesis kit (Stratagene) according to the recommendations of the manufacturer. The mutations were verified by DNA sequencing. Mutagenesis was conducted on the pEntr-derived WSB-1 plasmid, and GST-WSB-1 mutant expression plasmid was generated using Gateway Technology (Invitrogen). WSB-1 shRNA plasmid was constructed by inserting double-stranded oligonucleotides, which harbor the WSB-1 sequence (5′-GCTGTAAAGTGCAAGGAAATT-3′), into the BglII and HindIII sites of pSUPER (OligoEngine), in accordance with the manufacturer's recommendations. Yeast Two-hybrid Screening–For bait construction, a DNA fragment encoding amino acids 503–1189 of HIPK2 was subcloned into the EcoRI and SalI sites of pGBKT7 (Clontech) and transformed into the yeast strain, AH109, followed by mating with yeast Y187 cells pretransformed with mouse embryonic day 11 cDNA library (Clontech). Approximately 107 transformants were screened, and the positive colonies were confirmed with β-galactosidase colony lift assays. The yeast plasmids were isolated and transformed into Escherichia coli, DH5α. The isolated yeast DNA was co-transformed with a bait plasmid into AH109 to verify the interactions between HIPK2 and the interacting proteins. The specific interaction and binding strengths of the two fusion proteins were verified by the streaking of several colonies onto synthetic drop-out agar plates lacking TL (tryptophan and leucine) or TLH (tryptophan, leucine, and histidine). In Vitro Ubiquitination–For the in vitro ubiquitination assay, affinity-purified GST-WSB-1 was mixed with in vitro translated Myc-HIPK2 as a substrate, and mixtures were added to the ubiquitin ligation reaction (final volume, 30 μl) containing 40 mm Tris-HCl, pH 7.6, 5 mm MgCl2, 2 mm dithiothreitol, 2 mm ATP, 8 μg of purified ubiquitin (Sigma), 0.5 μg of E1 (Boston Biochem), and 1 μg of His-Ubc5c. The reactions were incubated for 90 min at 37 °C and were terminated by boiling for 7 min in SDS sample buffer. The reaction mixtures were resolved by 6% SDS-PAGE and transferred to polyvinylidene difluoride membranes. The ubiquitinated HIPK2 proteins were detected by Western blotting using anti-Myc antibody. In Vitro Pull-down Assays–The GST-fusion proteins were expressed in E. coli. BL21(DE3) cells, and were purified with glutathione-Sepharose beads according to the instructions of the manufacturer. The domains of Myc-HIPK2 proteins were synthesized by using the coupled TnT in vitro transcription-translation system (Promega, Madison, WI). Synthesized proteins were incubated with GST-WSB-1 (aa 284–421) at 4 °C for 1 h in binding buffer (50 mm Tris-HCl, pH 8.0, 100 mm NaCl, 0.05% Triton X-100) and washed three times with phosphate-buffered saline with 0.5% Triton X-100. After washing, the bound proteins were resolved by SDS-PAGE and detected by Western blotting with anti-Myc antibody. Co-immunoprecipitation and Western Blotting–The co-immunoprecipitation of endogenous proteins was performed after the lysis of 2 × 107 cells in high salt lysis buffer (50 mm Hepes, 350 mm NaCl, 10% glycerol, 1% Nonidet P-40, 1 mm EDTA). After incubation on ice for 10 min and centrifugation for 10 min at 4 °C, equal volumes of protein were diluted with lysis buffer lacking NaCl (dilution buffer), then incubated overnight with antibody and protein A/G-Sepharose beads at 4 °C on a rotating wheel. The beads were washed three times with lysis buffer. The whole cell lysates and immunoprecipitates were separated by SDS-PAGE and transferred onto polyvinylidene difluoride membranes. The membranes were immunoblotted with anti-Myc (Invitrogen), anti-HA (Invitrogen), anti-WSB-1 (Abnova, Taiwan), and anti-HIPK2 antibody (Aviva Systems Biology, San Diego, CA). After the priming antibodies were washed, the membranes were incubated with anti-mouse or anti-rabbit secondary antibodies conjugated with horseradish peroxidase (Amersham Biosciences), followed by detection with ECL Western blotting detection solutions (Pierce). Immunocytochemistry–Immunostaining of RKO cells was performed as described previously (5Kim E.A. Noh Y.T. Ryu M.J. Kim H.T. Lee S.E. Kim C.H. Lee C. Kim Y.H. Choi C.Y. J. Biol. Chem. 2006; 281: 7489-7497Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). In brief, U2OS cells were grown on coverslips and transfected with 0.05 μg of EGFP-C2 plasmid and 1.5 μg of the WSB-1 shRNA expression plasmid. Thirty-six hours after transfection, the cells were fixed with 100% methanol for 5 min at –20 °C and incubated with anti-HIPK2 antibody after the blocking of cells with 1× phosphate-buffered saline containing 1% bovine serum albumin. Fluorescence microscopy was conducted with a Zeiss Axioskop 2 microscope, using excitation wavelengths of 543 nm (rhodamine red) and 488 nm (GFP). The acquired images were processed with Adobe Photoshop. Identification of WSB-1 as HIPK2-interacting Protein–It is known that the levels of HIPK2 protein are increased as the result of UV irradiation and cisplatin treatment, both of which are genotoxic stresses that induce DNA damage (7D'Orazi G. Cecchinelli B. Bruno T. Manni I. Higashimoto Y. Saito S. Gostissa M. Coen S. Marchetti A. Del Sal G. Piaggio G. Fanciulli M. Appella E. Soddu S. Nat. Cell Biol. 2002; 4: 11-19Crossref PubMed Scopus (570) Google Scholar, 8Hofmann T.G. Moller A. Sirma H. Zentgraf H. Taya Y. Droge W. Will H. Schmitz M.L. Nat. Cell Biol. 2002; 4: 1-10Crossref PubMed Scopus (496) Google Scholar, 20Di Stefano V. Blandino G. Sacchi A. Soddu S. D'Orazi G. Oncogene. 2004; 23: 5185-5192Crossref PubMed Scopus (61) Google Scholar). However, it remains to be determined whether HIPK2 is stabilized in protein levels by escaping from proteolysis, and which molecule is involved in this process. In an effort to ascertain the degradation of HIPK2 in cells under normal conditions, the cells expressing GFP-HIPK2 were exposed to UV, and the levels of HIPK2 were observed by GFP fluorescence. The intensity of GFP fluorescence from GFP-HIPK2 was increased via UV irradiation in a time-dependent manner (Fig. 1A, upper panel). The administration of DNA damage reagents, such as Adriamycin or cisplatin, to cells also resulted in induction of the endogenous HIPK2 protein level (Fig. 1, A (middle panel) and B, lanes 1–4). Furthermore, the treatment of cells with the proteasome inhibitor, MG132, resulted in the accumulation of HIPK2 (Fig. 1B, lane 6). Taken together, our results show that HIPK2 may be degraded by proteolysis in cells under normal conditions. A yeast two-hybrid screening was conducted in an effort to identify potential cellular proteins that target HIPK2 for degradation. First, HIPK2 domains for proteolytic degradation were determined to construct a bait plasmid for yeast two-hybrid screening. Expression plasmids for either wild-type HIPK2, N-terminal HIPK2, or C-terminal HIPK2 were transfected into the HEK293 cells, and the protein levels were determined either prior to or after treatment with the proteasome inhibitor, MG132. The protein levels of wild-type HIPK2 and C-terminal HIPK2, but not N-terminal HIPK2, were increased as the result of MG132 treatment (Fig. 2A, lanes 2 and 6). This result indicates that HIPK2 is degraded by proteasome via its C terminus. Therefore, the C terminus of HIPK2-(503–1189) was utilized as bait for the screening of mouse embryonic match-maker cDNA libraries. Among the several cellular proteins that were identified and sequenced, a clone showed identity to WSB-1, a subunit of E3 ubiquitin ligase (Fig. 2B). The isolated WSB-1 clone harbored a C-terminal domain (aa 284–421) that included two WD40 repeats and a SOCS box. The specific interactions between WSB-1 and HIPK2 were verified in the yeast two-hybrid and co-immunoprecipitation assays in cultured cells (Fig. 2, B and C). GST pull-down analysis showed that WSB-1 interacted physically with HIPK2 (Fig. 2D). The association of endogenous HIPK2 with WSB-1 was also verified via co-immunoprecipitation with anti-HIPK2 antibody followed by Western blotting using anti-WSB-1 antibody (Fig. 2E). Collectively, our results show that WSB-1 is an HIPK2-interacting protein in vitro and in vivo and is associated with HIPK2 in cultured cells. Ubiquitination and Degradation of HIPK2 by WSB-1–In an effort to determine whether WSB-1 functions as an E3 ubiquitin ligase against HIPK2, Myc-HIPK2 expression plasmids were transfected into HEK293 cells either with or without the WSB-1 expression plasmid. Transfected cells were chased with cycloheximide to inhibit de novo protein synthesis. At the indicated times after the addition of cycloheximide, the levels of the HIPK2 proteins were analyzed by Western blot analysis. The stability of HIPK2 was markedly reduced by the coexpression of WSB-1 (Fig. 3A). Because the C terminus of HIPK2 was utilized as bait for yeast two-hybrid screening for the identification of WSB-1, HIPK2 domain for degradation by WSB-1 was determined. As had been expected, the C terminus (aa 503–1189) of HIPK2, but not the N terminus, was degraded by the coexpression of WSB-1 (Fig. 3B, lane 6). To ascertain the functional interaction of WSB-1 with HIPK2, ubiquitinations of HIPK2 by WSB-1 were explored under in vitro and in vivo conditions. HIPK2 was efficiently polyubiquitinated when HA-WSB-1 was expressed in cells along with HA-Ub (Fig. 3C, lane 3). Consistent with the above observations, affinity-purified GST-WSB-1 ubiquitinated Myc-HIPK2 only when all of E1, E2, ubiquitin, and WSB-1 were contained in the reaction mixture (Fig. 3D, lane 5). These results indicate that WSB-1 could function as an E3 ubiquitin ligase against HIPK2. Knockdown of Endogenous WSB-1 Increases HIPK2 Stability–Next, we assessed the effects of WSB-1 knockdown on the stability of HIPK2. RKO cells were transfected with increasing quantities of expression plasmids for shRNA against WSB-1, and WSB-1 levels were analyzed by Western blotting with anti-WSB-1 antibody. As shown in Fig. 4A, endogenous WSB-1 was silenced efficiently by the expression of shRNA against WSB-1 in a dose-dependent manner. The knockdown of endogenous WSB-1 concomitantly resulted in the elevation of endogenous HIPK2 levels (Fig. 4B, middle panel). Furthermore, HIPK2 stabilization was assessed in a single cell. To this end, U2OS cells were transfected with WSB-1 shRNA expression plasmids coupled with the GFP expression plasmid, and the expression levels of endogenous HIPK2 were observed by immunostaining using anti-HIPK2 antibody. The WSB-1 shRNA-expressing cells could be monitored indirectly by co-transfection of the GFP expression plasmid at a high ratio, of 1.5 μg (WSB-1 shRNA) to 0.05 μg (EGFP-C2). As shown in Fig. 4C, the GFP-positive cells expressed endogenous HIPK2 at a high level (middle panel, arrows), whereas the cells expressing either control shRNA (upper panel) or no GFP/shWSB-1 (middle panel, arrowhead) displayed relatively low HIPK2 levels. The administration, however, of MG132 to the cells resulted in a high level of endogenous HIPK2 expression, regardless of WSB-1 shRNA expression (lower panel). Taken together, these results showed that WSB-1 could degrade HIPK2 constantly in the cells under normal conditions. WSB-1 Interacts with HIPK2 through WD40 Repeats–WSB-1 is composed of seven WD40 repeats and an SOCS box in the C terminus (Fig. 5A). WD40 repeats have been shown to participate in diverse cellular functions, and the SOCS box is known to be involved in the protein degradation in the ECS ubiquitin ligase complex (34Smith T.F. Gaitatzes C. Saxena K. Neer E.J. Trends Biochem. Sci. 1999; 24: 181-185Abstract Full Text Full Text PDF PubMed Scopus (1012) Google Scholar, 35Chen G. Courey A.J. Gene (Amst.). 2000; 249: 1-16Crossref PubMed Scopus (324) Google Scholar, 36Kile B.T. Schulman B.A. Alexander W.S. Nicola N.A. Martin H.M. Hilton D.J. Trends Biochem. Sci. 2002; 27: 235-241Abstract Full Text Full Text PDF PubMed Scopus (360) Google Scholar). To determine whether the WD40 domain and/or SOCS box of WSB-1 are required for its interaction with and degradation of HIPK2, a series of WSB-1 deletion mutants were constructed (Fig. 5A) and utilized for the domain analysis of WSB-1. GST pulldown analysis with GST-HIPK2-(503–860) and Myc-tagged WSB-1 mutants showed that all of the deletion mutants of WSB-1 containing the WD40 repeat interacted with HIPK2 (Fig. 5B). The binding of WSB-1 was specific to GST-HIPK2, as we noted no binding of WSB-1 to the GST protein (middle panel). Consistently, both N-terminal WSB-1 (aa 1–279, 1–5th WD40 repeats) and C-terminal WSB-1 (aa 200–421, 4–7th WD40 repeats and SOCS box) were associated with full-length HIPK2 in cultured cells (Fig. 5C, lanes 5–8). However, the SOCS box of WSB-1 did not bind to HIPK2 in vitro (Fig. 5B, lower panel, lane 5) and yeast two-hybrid analysis (data not shown). These results indicate that WD40 repeats are responsible for the interaction of WSB-1 with HIPK2. WSB-1 harbors seven WD40 repeats, spanning majority of the protein (Fig. 5A). Six of the WD40 repeats (second to seventh WD40) contain a few mismatches from the consensus sequence (37Neer E.J. Schmidt C.J. Nambudripad R. Smith T.F. Nature. 1994; 371: 297-300Crossref PubMed Scopus (1287) Google Scholar), whereas the most N-terminal WD40 motif is more div" @default.
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• W2018304918 title "Ubiquitination and Degradation of Homeodomain-interacting Protein Kinase 2 by WD40 Repeat/SOCS Box Protein WSB-1" @default.
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