Karyopherin-α2 Protein Interacts with Chk2 and Contributes to Its Nuclear Import

Abstract

Chk2 is a nuclear protein kinase involved in the DNA damage-induced ataxia telangiectasia mutated-dependent checkpoint arrest at multiple cell cycle phases. Searching for Chk2-binding proteins by a yeast two-hybrid system, we identified a strong interaction with karyopherin-α2 (KPNA-2), a gene product involved in active nuclear import of proteins bearing a nuclear localization signal (NLS). This finding was confirmed by glutathione S-transferase pull-down and co-immunoprecipitation assays. Of the three predicted Chk2 NLSs, located at amino acids 179-182 (NLS-1), 240-256 (NLS-2), and 515-522 (NLS-3), only the latter mediated the interaction with KPNA-2 in the yeast two-hybrid system, and in particular with its C terminus. Unlike mutations in NLS-1 or NLS-2, which left the nuclear localization of Chk2 unaffected, mutations in NLS-3 caused a cytoplasmic relocalization, indicating that the NLS-3 motif acts indeed as NLS for Chk2 in vivo. Finally, co-transfection experiments with green fluorescent protein (GFP)-Chk2 and wild type or mutant KPNA-2 confirmed the role of KPNA-2 in nuclear import of Chk2. Chk2 is a nuclear protein kinase involved in the DNA damage-induced ataxia telangiectasia mutated-dependent checkpoint arrest at multiple cell cycle phases. Searching for Chk2-binding proteins by a yeast two-hybrid system, we identified a strong interaction with karyopherin-α2 (KPNA-2), a gene product involved in active nuclear import of proteins bearing a nuclear localization signal (NLS). This finding was confirmed by glutathione S-transferase pull-down and co-immunoprecipitation assays. Of the three predicted Chk2 NLSs, located at amino acids 179-182 (NLS-1), 240-256 (NLS-2), and 515-522 (NLS-3), only the latter mediated the interaction with KPNA-2 in the yeast two-hybrid system, and in particular with its C terminus. Unlike mutations in NLS-1 or NLS-2, which left the nuclear localization of Chk2 unaffected, mutations in NLS-3 caused a cytoplasmic relocalization, indicating that the NLS-3 motif acts indeed as NLS for Chk2 in vivo. Finally, co-transfection experiments with green fluorescent protein (GFP)-Chk2 and wild type or mutant KPNA-2 confirmed the role of KPNA-2 in nuclear import of Chk2. To prevent genomic instability and cancer, DNA lesions arising from external agents or internal metabolism activate multiple checkpoint pathways that coordinate DNA repair and cell cycle progression. The checkpoint protein Chk2, the mammalian homologue of Saccharomyces cerevisiae Rad53 and Schizosaccharomyces pombe Cds1 (1Matsuoka S. Huang M. Elledge S.J. Science. 1998; 282: 1893-1897Crossref PubMed Scopus (1082) Google Scholar), is a nuclear serine kinase bearing a serine/threonine-(SQ/TQ)-rich region (aa 1The abbreviations used are: aa, amino acids; FHA, forkhead-associated; GST, glutathione S-transferase; GFP, green fluorescent protein; NLS, nuclear localization signal; HA, hemagglutinin; KPNA-2, karyopherin-α2; IBB, importin-β binding; X-gal, 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside.1The abbreviations used are: aa, amino acids; FHA, forkhead-associated; GST, glutathione S-transferase; GFP, green fluorescent protein; NLS, nuclear localization signal; HA, hemagglutinin; KPNA-2, karyopherin-α2; IBB, importin-β binding; X-gal, 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside. 19-69) with several potential phosphorylation sites, a phosphoprotein-interacting FHA domain (aa 115-175), and a catalytic domain (aa 226-486) (2Bartek J. Falck J. Lukas J. Nat. Rev. Mol. Cell. Biol. 2001; 2: 877-886Crossref PubMed Scopus (321) Google Scholar). In response to genotoxic agents, including γ-radiation (IR), Chk2 is phosphorylated on Thr68 by ataxia telangiectasia mutated, the protein mutated in ataxia telangiectasia disease (3Ahn J.Y. Schwarz J.K. Piwnica-Worms H. Canman C.E. Cancer Res. 2000; 60: 5934-5936PubMed Google Scholar, 4Melchionna R. Chen X.B. Blasina A. McGowan C.H. Nat. Cell Biol. 2000; 2: 762-765Crossref PubMed Scopus (262) Google Scholar, 5Matsuoka S. Rotman G. Ogawa A. Shiloh Y. Tamai K. Elledge S.J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10389-10394Crossref PubMed Scopus (683) Google Scholar). Additional steps of phosphorylation, dependent on the expression of a functional Nbs1/Mre11/Rad50 complex (6Buscemi G. Savio C. Zannini L. Miccichè F. Masnada D. Nakanishi M. Tauchi H. Komatsu K. Mizutani S. Khanna K. Chen P. Concannon P. Chessa L. Delia D. Mol. Cell. 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Most recently, additional molecules have been found to interact with Chk2. Among these, the adaptor proteins 53BP1 (12Wang B. Matsuoka S. Carpenter P.B. Elledge S.J. Science. 2002; 298: 1435-1438Crossref PubMed Scopus (481) Google Scholar, 13DiTullio Jr., R.A. Mochan T.A. Venere M. Bartkova J. Sehested M. Bartek J. Halazonetis T.D. Nat. Cell Biol. 2002; 4: 998-1002Crossref PubMed Scopus (355) Google Scholar) and NFBD1/Mdc1 (14Peng A. Chen P.L. J. Biol. Chem. 2003; 278: 8873-8876Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 15Shang Y.L. Bodero A.J. Chen P.L. J. Biol. Chem. 2003; 278: 6323-6329Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 16Lou Z. Minter-Dykhouse K. Wu X. Chen J. Nature. 2003; 421: 957-961Crossref PubMed Scopus (287) Google Scholar) seem to facilitate Chk2 activation, the kinases Plk-3 and Plk-1 phosphorylate Chk2 (17Xie S. Wu H. Wang Q. Kunicki J. Thomas R.O. Hollingsworth R.E. Cogswell J. Dai W. Cell Cycle. 2002; 1: 424-429Crossref PubMed Scopus (65) Google Scholar, 18Bahassi el M. Conn C.W. Myer D.L. Hennigan R.F. McGowan C.H. Sanchez Y. Stambrook P.J. Oncogene. 2002; 21: 6633-6640Crossref PubMed Scopus (135) Google Scholar, 19Tsvetkov L.M. Xu X. Li J. Stern D.F. J. Biol. Chem. 2003; 278: 8468-8475Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar), and PML behaves as a substrate for Chk2 and modulates apoptosis (20Yang S. Kuo C. Bisi J.E. Kim M.K. Nat. Cell Biol. 2002; 4: 865-870Crossref PubMed Scopus (196) Google Scholar). To find out Chk2-interacting proteins, we have undertaken yeast two-hybrid screens using either full-length or deletion forms of Chk2 as baits and a human cDNA library from HeLa cells. Here we report that Chk2 interacts with karyopherin-α2 (KPNA-2), a family member of molecules involved in the nuclear translocation of target proteins that carry a nuclear localization signal (NLS) motif characterized by short stretches of basic amino acids (21Nakielny S. Dreyfuss G. Cell. 1999; 99: 677-690Abstract Full Text Full Text PDF PubMed Scopus (648) Google Scholar, 22Chook Y.M. Blobel G. Curr. Opin. Struct. Biol. 2001; 11: 703-715Crossref PubMed Scopus (418) Google Scholar). NLSs can be monopartite if containing a single stretch of basic amino acids, bipartite if containing two stretches of basic amino acids separated by a spacer region. The nuclear translocation of target proteins by KPNA-2 requires the binding to karyopherin-β, which allows passage of the complex through the nuclear pore (23Gorlich D. Henklein P. Laskey R.A. Hartmann E. EMBO J. 1996; 15: 1810-1817Crossref PubMed Scopus (361) Google Scholar, 24Weis K. Ryder J. Lamond A.I. EMBO J. 1996; 15: 1818-1825Crossref PubMed Scopus (223) Google Scholar). Once in the nucleus, the binding of Ran-GTP to karyopherin-β causes the dissociation with KPNA-2, thereby allowing it to release its cargo and to finally recycle to the cytoplasm via interaction with export proteins (25Moroianu J. Blobel G. Radu A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7059-7062Crossref PubMed Scopus (90) Google Scholar). Here we show that the C terminus of KPNA-2 binds the NLS-3 sequence of Chk2 and that this interaction has a role in the nuclear localization of Chk2 in cells. Cells and Transfections—The lymphoblastoid cell line LCL-N from a normal donor was cultured in RPMI 1640 medium (BioWhittaker, Walkersville, MD) supplemented with 15% heat-inactivated fetal calf serum, penicillin (100 units/ml), and streptomycin (100 μg/ml), whereas the U2OS osteosarcoma cell line was maintained in Dulbecco's modified Eagle's medium with 10% serum. Cells were transfected by the calcium phosphate method. Irradiations were performed in an IBL437CO instrument equipped with a 137Ce source emitting a dose of 8 grays/min. Yeast Two-hybrid Screening—The screening in yeast was performed as described (26Benetti R. Del Sal G. Monte M. Paroni G. Brancolini C. Schneider C. EMBO J. 2001; 20: 2702-2714Crossref PubMed Scopus (101) Google Scholar). Briefly, Chk2 c-DNA deleted of aa 76-213 (obtained by digestion of full-length Chk2 c-DNA with AocI) was cloned in the EcoRI-SalI sites of pLexA vector in-frame with the Lex A DNA binding domain and used as a bait to transform the EGY48 yeast strain. A selected clone was then transformed with a HeLa c-DNA library cloned in the pJG4-5 vector in-frame with the B42 transactivation domain. About 2 × 106 clones were obtained and tested for their capacity to grow in a medium without leucine and by the X-gal filter method. Plasmid DNA was then extracted from positive clones, and the c-DNA inserts were amplified by PCR and analyzed by sequencing. To map the regions of interaction between Chk2 and KPNA-2, deletion mutants for the two proteins were prepared by PCR and cloned in the pLexA and in pJG4-5 vector. The constructs were then used to transform the EGY48 strain and tested for the interaction using a medium containing X-gal or without leucine. GST Pull-down Assays—The GST-Chk2 recombinant protein was purified from bacteria transformed with the full-length Chk2 cDNA cloned in the EcoRI-NotI sites of pGEX-4T-1 vector. LCL-N cells were lysed in ice-cold ELB buffer (150 mm NaCl, 50 mm Hepes, pH 7.5, 5 mm EDTA, 0.1% Nonidet P-40), and 1 mg of cell extracts were incubated for 3 h at 4 °C with 10 μg of GST-Chk2 or GST together with glutathione-Sepharose. The beads were extensively washed in lysis buffer, size-fractionated by SDS-PAGE, and immunoblotted for KPNA-2 using a specific monoclonal antibody (Clone 2, Transduction Laboratories). Co-immunoprecipitations—LCL-N, unirradiated or irradiated with 10 grays of IR, and U2OS cells transiently transfected with pCDNA3-HA-Chk2 and pCDNA3-FLAG-KPNA-2 were lysed in ELB buffer. After preclearing with protein A/G-coupled Sepharose beads, lysates were immunoprecipitated with an anti-Chk2 polyclonal antibody (27Tominaga K. Morisaki H. Kaneko Y. Fujimoto A. Tanaka T. Ohtsubo M. Hirai M. Okayama H. Ikeda K. Nakanishi M. J. Biol. Chem. 1999; 274: 1463-1467Abstract Full Text Full Text PDF Scopus (76) Google Scholar) or with the anti-FLAG M2 monoclonal antibody (Sigma) and extensively washed. Immunoprecipitates were analyzed by Western blot with monoclonal antibodies anti-KPNA-2 (Clone 2, Transduction Laboratories) and anti-Chk2 (Clone 44D4-21, generated in our laboratory). Site-directed Mutagenesis—Mutations in the full-length Chk2 cDNA cloned in the EcoRI-NotI sites of pCDNA3-FLAG vector were introduced by the Gene Editor kit (Promega). To mutagenize the lysine and arginine residues to alanine, the oligonucleotide primers used were 5′-AGCTGGCTTTCGAGGCGGCAACATGTAAGAAAG-3′ (Chk2 bp 704-737) and 5′-AGATCATCAGCAAAGCGGCGTTTGCTATTGGTT-3′ (Chk2 bp 746-779) for the bipartite NLS (NLS-2) and 5′-CTTCTACTAGTCGAGCGGCGCCCCGTGAAGGGG-3′ (Chk2 bp 1544-1577) for the NLS-3. The sequences were always verified by automatic DNA sequencing. Immunofluorescence Analysis—U2OS cell lines were transiently transfected with pCDNA3-carrying wild type or mutant forms of FLAG-tagged Chk2. After 48 h, the cells were cytocentrifuged on glass slides, fixed with 3.7% paraformaldehyde, permeabilized in 0.2% Triton X-100, and immunostained with the anti-FLAG-M2 monoclonal antibody (Sigma). Binding of primary antibodies was revealed with a fluorescein isothiocyanate-labeled F(ab′)2 anti-mouse antibody (Jackson Laboratories). Nuclei were stained with 4′,6 diamidino-2-phenylindole, and coverslips were mounted with an anti-fade solution. Slides were analyzed using a Zeiss Axioskop (Oberkochen, Germany) fluorescence microscope and digital imaging. GFP-Chk2/FLAG-KPNA-2 Co-expression—To establish the role in vivo of Chk2/KPNA-2 interaction, Chk2 cDNA was cloned in the EcoRI-SalI sites of pEGFP-C vector, whereas full-length or deletion mutants of KPNA-2 cDNAs were cloned in the EcoRI-XhoI sites of pcDNA3-FLAG vector. FLAG-KPNA-2 and GFP-Chk2 constructs were co-transfected in U2OS cells at a ratio of 6:1, respectively, and 48 h the localization of GST-Chk2 was analyzed by fluorescence In Chk2 were performed using a active GST-Chk2 recombinant protein incubated for at °C with 1 μg of either or substrate latter used as positive in of kinase buffer mm mm 5 mm mm EDTA, 2 mm 50 and of The were separated by and The were then stained with to the of substrate Yeast Two-hybrid for Chk2 yeast two-hybrid was to for Chk2 The bait of Chk2 deleted of aa 76-213 full-length Chk2 a and in the latter the and the of yeast two were by the X-gal filter and for their capacity to grow without leucine. Of positive clones, the full or of the karyopherin-α2 the of which aa KPNA-2 (21Nakielny S. Dreyfuss G. Cell. 1999; 99: 677-690Abstract Full Text Full Text PDF PubMed Scopus (648) Google Scholar, 22Chook Y.M. Blobel G. Curr. Opin. Struct. Biol. 2001; 11: 703-715Crossref PubMed Scopus (418) Google Scholar) is a nuclear of proteins bearing an NLS, characterized by an terminus importin-β binding domain for nuclear involved in the binding and of NLSs, and a domain Chk2 with KPNA-2 in and in interaction between Chk2 and KPNA-2 was in pull-down in which extracts from normal lymphoblastoid cells were incubated with recombinant the KPNA-2 GST-Chk2 GST the interaction in To this interaction in we Chk2 with The that Chk2 from LCL-N cells with KPNA-2, and the of this finding was by the of a KPNA-2 signal with a antibody Chk2 is involved in the response to DNA (1Matsuoka S. Huang M. Elledge S.J. Science. 1998; 282: 1893-1897Crossref PubMed Scopus (1082) Google Scholar, J. Falck J. Lukas J. Nat. Rev. Mol. Cell. Biol. 2001; 2: 877-886Crossref PubMed Scopus (321) Google Scholar), we tested the of on the interaction between Chk2 and In cells with 10 grays of and 3 h Chk2 KPNA-2 to the as in cells Chk2 protein be in KPNA-2 the by the monoclonal anti-KPNA-2 antibody in the region binding to Chk2, we transiently co-transfected U2OS cell lines with FLAG-KPNA-2 and constructs and analyzed Chk2 in KPNA-2 obtained with anti-FLAG we were to find protein with KPNA-2 an interaction between Chk2 and KPNA-2 in human cells. of the of between Chk2 and map the region of interaction between Chk2 and KPNA-2, the full-length and deleted of proteins were tested in the yeast two-hybrid three Chk2 were the region the FHA domain the kinase domain only or the C terminus full-length Chk2, and with full-length KPNA-2 indicating that the KPNA-2 binding is located at aa of Chk2. the KPNA-2 sequence found in the two-hybrid screening the amino acids, it was that this sequence mediated the interaction with Chk2. to the region of interaction, mutants deleted of the domain or domain the two were tested in yeast full-length Chk2. with Chk2 it can be that aa of KPNA-2 for binding to Chk2. The expression of and proteins in yeast was by Western blot with an Of mutant proteins in to full-length KPNA-2, with the anti-KPNA-2 antibody that aa the anti-KPNA-2 binding of Chk2 KPNA-2 the nuclear import of proteins the NLS sequence Y.M. Blobel G. Curr. Opin. Struct. Biol. 2001; 11: 703-715Crossref PubMed Scopus (418) Google Scholar), we the of an NLS of Chk2 in the interaction with A of Chk2 sequence identified three NLSs, a monopartite NLS-1 aa a bipartite aa and a monopartite NLS-3 aa To the of NLSs, the localization of Chk2 NLS mutants in transiently transfected U2OS cells was by full-length deleted of aa 76-213 of was in the nucleus, that NLS-1 is for Chk2 nuclear a cytoplasmic localization the lysine and arginine of NLS-3 were with to the in motif on Chk2 nuclear localization and In with KPNA-2 to interact in yeast with Chk2 mutated in NLS-3 the role of NLS-3 in the interaction with KPNA-2 and nuclear localization of mutants in the wild in a of the Chk2/KPNA-2 in the in vivo of Chk2/KPNA-2 we the of KPNA-2 on the nuclear translocation of Chk2. U2OS cells were transiently co-transfected with GFP-Chk2 and wild type or mutant KPNA-2 and by fluorescence Of the fluorescent cells in with GFP-Chk2 and an vector, a nuclear whereas the in a cytoplasmic was in the nuclear After co-transfection with wild type KPNA-2, the of cells with nuclear GFP-Chk2 to whereas co-transfection with KPNA-2 deleted of either the Chk2-interacting region or the domain to to the the to indicating a role for KPNA-2 in the nuclear translocation of Chk2 kinases their nuclear import by phosphorylation of KPNA-2 K. 2001; PubMed Scopus Google Scholar), we KPNA-2 is a target substrate for Chk2 this in kinase were performed using active recombinant GST-Chk2 and full-length as a whereas GST-Chk2 as Cdc25C, it to phosphorylate KPNA-2 a in the interaction of This finding is with the of a Chk2 substrate motif KPNA-2 by and with the normal nuclear localization of Chk2 Two-hybrid in yeast have to a and functional interaction between Chk2 kinase and KPNA-2, a family member of proteins involved in the active from the cytoplasm to the of cargo proteins containing an NLS (21Nakielny S. Dreyfuss G. Cell. 1999; 99: 677-690Abstract Full Text Full Text PDF PubMed Scopus (648) Google Scholar, 22Chook Y.M. Blobel G. Curr. Opin. Struct. Biol. 2001; 11: 703-715Crossref PubMed Scopus (418) Google Scholar). of NLS by the a short stretch of basic amino acids and the in two of basic amino acids separated by a spacer region of amino acids To the nuclear and its KPNA-2 has to karyopherin-β (23Gorlich D. Henklein P. Laskey R.A. Hartmann E. EMBO J. 1996; 15: 1810-1817Crossref PubMed Scopus (361) Google Scholar, 24Weis K. Ryder J. Lamond A.I. EMBO J. 1996; 15: 1818-1825Crossref PubMed Scopus (223) Google Scholar). In the nucleus, complex including the allowing the of to the cytoplasm (25Moroianu J. Blobel G. Radu A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7059-7062Crossref PubMed Scopus (90) Google Scholar). confirmed the interaction in yeast between Chk2 and KPNA-2 in pull-down between recombinant GST-Chk2 and KPNA-2 from cell have that Chk2 from unirradiated or irradiated the latter Chk2 (1Matsuoka S. Huang M. Elledge S.J. Science. 1998; 282: 1893-1897Crossref PubMed Scopus (1082) Google Scholar, J. Falck J. Lukas J. Nat. Rev. Mol. Cell. Biol. 2001; 2: 877-886Crossref PubMed Scopus (321) Google Scholar), The by of KPNA-2 in extracted from cells to we were to Chk2 with anti-KPNA-2 the that it the region of interaction with Chk2 we have in Western of KPNA-2 deletion mutants in and be to Chk2, we Chk2 in FLAG-KPNA-2 immunoprecipitated with an anti-FLAG antibody from cells FLAG-KPNA-2 and confirmed the interaction between two we this was mediated by the the of Chk2 cDNA sequence three NLS at aa 179-182 aa 240-256 aa 515-522 we in the two-hybrid deletion mutants of Chk2 and KPNA-2 and found that only the aa of Chk2, which with To the functional role in vivo of the three NLSs, the of FLAG-tagged Chk2 constructs transiently transfected in U2OS cells was analyzed by have that mutations in in NLS-1 or NLS-2, a cytoplasmic of Chk2. This together with the that NLS-3 mutations the interaction with KPNA-2 in yeast the of NLS-3 for the binding to KPNA-2 and nuclear translocation of Chk2. KPNA-2 an IBB, involved in and binding to NLSs, and a domain Y.M. Blobel G. Curr. Opin. Struct. Biol. 2001; 11: 703-715Crossref PubMed Scopus (418) Google Scholar). have that the aa of KPNA-2 for binding to Chk2. This is in with the finding that the the binding to NLS Y.M. Blobel G. Curr. Opin. Struct. Biol. 2001; 11: 703-715Crossref PubMed Scopus (418) Google Scholar), with that proteins as and the export protein interact with the region of the family S.A. J. R. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, A. M. N. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, T. N. K. T. Y. EMBO J. PubMed Scopus Google Scholar, A. R. H. J. Cell Biol. 1998; PubMed Scopus Google Scholar). is that the of region for allows to at the to a for protein T. N. K. T. Y. EMBO J. PubMed Scopus Google Scholar). 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