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• W2080650147 abstract "The tumor suppressor protein p53 is known to undergo cytoplasmic dynein-dependent nuclear translocation in response to DNA damage. However, the molecular link between p53 and the minus end-directed microtubule motor dynein complex has not been described. We report here that the 8-kDa light chain (LC8) of dynein binds to p53-binding protein 1 (53BP1). The LC8-binding domain was mapped to a short peptide segment immediately N-terminal to the kinetochore localization region of 53BP1. The LC8-binding domain is completely separated from the p53-binding domain in 53BP1. Therefore, 53BP1 can potentially act as an adaptor to assemble p53 to the dynein complex. Unlike other known LC8-binding proteins, 53BP1 contains two distinct LC8-binding motifs that are arranged in tandem. We further showed that 53BP1 can directly associate with the dynein complex. Disruption of the interaction between LC8 and 53BP1 in vivo prevented DNA damage-induced nuclear accumulation of p53. These data illustrate that LC8 is able to function as a versatile acceptor to link a wide spectrum of molecular cargoes to the dynein motor. The tumor suppressor protein p53 is known to undergo cytoplasmic dynein-dependent nuclear translocation in response to DNA damage. However, the molecular link between p53 and the minus end-directed microtubule motor dynein complex has not been described. We report here that the 8-kDa light chain (LC8) of dynein binds to p53-binding protein 1 (53BP1). The LC8-binding domain was mapped to a short peptide segment immediately N-terminal to the kinetochore localization region of 53BP1. The LC8-binding domain is completely separated from the p53-binding domain in 53BP1. Therefore, 53BP1 can potentially act as an adaptor to assemble p53 to the dynein complex. Unlike other known LC8-binding proteins, 53BP1 contains two distinct LC8-binding motifs that are arranged in tandem. We further showed that 53BP1 can directly associate with the dynein complex. Disruption of the interaction between LC8 and 53BP1 in vivo prevented DNA damage-induced nuclear accumulation of p53. These data illustrate that LC8 is able to function as a versatile acceptor to link a wide spectrum of molecular cargoes to the dynein motor. 53BP1 1The abbreviations used are: 53BP1, p53-binding protein 1; BRCT, BRCA1 C terminus domain; GFP, green fluorescent protein; GST, glutathione S-transferase; GSH, reduced glutathione; HEK, human embryonic kidney; IC74, dynein intermediate chain; LC8, 8-kDa dynein light chain; HA, hemagglutinin; PBS, phosphate-buffered saline; HIV-1, human immunodeficiency virus, type 1; ADR, adriamycin; nNOS, neuronal nitric-oxide synthase.1The abbreviations used are: 53BP1, p53-binding protein 1; BRCT, BRCA1 C terminus domain; GFP, green fluorescent protein; GST, glutathione S-transferase; GSH, reduced glutathione; HEK, human embryonic kidney; IC74, dynein intermediate chain; LC8, 8-kDa dynein light chain; HA, hemagglutinin; PBS, phosphate-buffered saline; HIV-1, human immunodeficiency virus, type 1; ADR, adriamycin; nNOS, neuronal nitric-oxide synthase. is a large nuclear protein (1972 amino acid residues) that was originally identified as a p53-interacting protein in a yeast two-hybrid screen (1Iwabuchi K. Bartel P.L. Li B. Marraccino R. Fields S. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6098-6102Crossref PubMed Scopus (354) Google Scholar). The protein was subsequently characterized as an activator of p53-dependent gene transcription (2Iwabuchi K. Li B. Massa H.F. Trask B.J. Date T. Fields S. J. Biol. Chem. 1998; 273: 26061-26068Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar). The C-terminal end of 53BP1 contains two BRCA1 Cterminus (BRCT) domains, a protein interaction module found in a number of proteins implicated in various aspects of cell cycle control, recombination, and DNA repair (3Rappold I. Iwabuchi K. Date T. Chen J. J. Cell Biol. 2001; 153: 613-620Crossref PubMed Scopus (399) Google Scholar, 4Schultz L.B. Chehab N.H. Malikzay A. Halazonetis T.D. J. Cell Biol. 2000; 151: 1381-1390Crossref PubMed Scopus (700) Google Scholar, 5Anderson L. Henderson C. Adachi Y. Mol. Cell. Biol. 2001; 21: 1719-1729Crossref PubMed Scopus (286) Google Scholar, 6Manke I.A. Lowery D.M. Nguyen A. Yaffe M.B. Science. 2003; 302: 636-639Crossref PubMed Scopus (544) Google Scholar, 7Yu X. Chini C.C.S. He M. Mer G. Chen J. Science. 2003; 302: 639-642Crossref PubMed Scopus (679) Google Scholar). The tandem BRCT repeats of 53BP1 are responsible for binding to p53 (Fig. 1, and Refs. 1Iwabuchi K. Bartel P.L. Li B. Marraccino R. Fields S. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6098-6102Crossref PubMed Scopus (354) Google Scholar, 8Joo W.S. Jeffrey P.D. Cantor S.B. Finnin M.S. Livingston D.M. Pavletich N.P. Genes Dev. 2002; 16: 583-593Crossref PubMed Scopus (181) Google Scholar, 9Derbyshire D.J. Basu B.P. Serpell L.C. Joo W.S. Date T. Iwabuchi K. Doherty A.J. EMBO J. 2002; 21: 3863-3872Crossref PubMed Scopus (149) Google Scholar). Upstream of the BRCT repeats resides a Tudor domain, a globular module shown to interact with dimethylated Arg (10Maurer-Stroh S. Dickens N.J. Hughes-Davies L. Kouzarides T. Eisenhaber F. Ponting C.P. Trends Biochem. Sci. 2003; 28: 69-74Abstract Full Text Full Text PDF PubMed Scopus (385) Google Scholar). There are also multiple “(S/T)Q” motifs in the N-terminal region of 53BP1, and some of these (S/T)Q motifs have been shown to be phosphorylation sites of ataxia telangiectasia-mutated kinase (5Anderson L. Henderson C. Adachi Y. Mol. Cell. Biol. 2001; 21: 1719-1729Crossref PubMed Scopus (286) Google Scholar, 11DiTullio 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). It has been proposed that 53BP1 acts as an adaptor protein responsible for recruiting/assembling various proteins in the ataxia telangiectasia-mutated and ataxia telangiectasia-mutated and rad3-related signaling pathways (12Wang B. Matsuoka S. Carpenter P.B. Elledge S.J. Science. 2002; 298: 1435-1438Crossref PubMed Scopus (479) Google Scholar).In response to DNA damage, p53 accumulates in the nucleus (13Kastan M.B. Onyekwere O. Sidransky D. Vogelstein B. Craig R.W. Cancer Res. 1991; 51: 6304-6311PubMed Google Scholar, 14Lane D.P. Nature. 1992; 358: 15-16Crossref PubMed Scopus (4436) Google Scholar, 15Prives C. Cell. 1998; 95: 5-8Abstract Full Text Full Text PDF PubMed Scopus (627) Google Scholar), where it transcriptionally activates a number of genes (e.g. p21 and mdm2) that are involved in growth arrest and apoptosis (16Callebaut I. Mornon J.P. FEBS Lett. 1997; 400: 25-30Crossref PubMed Scopus (484) Google Scholar, 17Levine A.J. Cell. 1997; 88: 323-331Abstract Full Text Full Text PDF PubMed Scopus (6698) Google Scholar, 18el-Deiry W.S. Tokino T. Velculescu V.E. Levy D.B. Parsons R. Trent J.M. Lin D. Mercer W.E. Kinzler K.W. Vogelstein B. Cell. 1993; 75: 817-825Abstract Full Text PDF PubMed Scopus (7890) Google Scholar, 19Miyashita T. Reed J.C. Cell. 1995; 80: 293-299Abstract Full Text PDF PubMed Scopus (303) Google Scholar). The DNA damage-induced nuclear localization of p53 requires the minus end-directed microtubule motor dynein (20Giannakakou P. Sackett D.L. Ward Y. Webster K.R. Blagosklonny M.V. Fojo T. Nat. Cell Biol. 2000; 2: 709-717Crossref PubMed Scopus (303) Google Scholar, 21Giannakakou P. Nakano M. Nicolaou K.C. O'Brate A. Yu J. Blagosklonny M.V. Greber U.F. Fojo T. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 10855-10860Crossref PubMed Scopus (178) Google Scholar). Disruption of the motor activity of dynein by microinjection of an antibody against the intermediate chain (IC74) of the motor or overexpressing dynamitin (p50) results in impaired accumulation of p53 in the nucleus following DNA damage (20Giannakakou P. Sackett D.L. Ward Y. Webster K.R. Blagosklonny M.V. Fojo T. Nat. Cell Biol. 2000; 2: 709-717Crossref PubMed Scopus (303) Google Scholar). The molecular basis of dynein-mediated p53 nuclear trafficking is poorly understood, because p53 is not known to bind directly to the dynein motor complex. One would expect that certain adaptor protein(s) can act as a linker to couple p53 to the dynein motor. Identification of such adaptor protein(s) should provide insights into the dynein-mediated p53 nuclear trafficking. Recently, it was shown that hsp90/immunophilin complex might act as an adaptor to link p53 to a subunit of dynactin within the dynein motor complex in human colon cancer cells (22Galigniana M.D. Harrell J.M. O'Hagen H.M. Ljungman M. Pratt W.B. J. Biol. Chem. 2004; 279: 22483-22489Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar).Cytoplasmic dynein is a multisubunit protein complex (∼1.2 MDa) composed of two globular heads joined by flexible stalk domains to a common base. The base of the motor complex contains two 74-kDa intermediate chains (IC74), four light intermediate chains (52–61 kDa), and several light chains (10–25 kDa) (23Holzbaur E.L. Vallee R.B. Annu. Rev. Cell Biol. 1994; 10: 339-372Crossref PubMed Scopus (325) Google Scholar, 24Vallee R.B. Sheetz M.P. Science. 1996; 271: 1539-1544Crossref PubMed Scopus (252) Google Scholar). The 8-kDa light chain (LC8, also named DLC8, PIN, and LC1) is a stoichiometric component of the cytoplasmic dynein complex (25King S.M. Barbarese E. Dillman III, J.F. Patel-King R.S. Carson J.H. Pfister K.K. J. Biol. Chem. 1996; 271: 19358-19366Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar). LC8 has been highly conserved throughout evolution and has been shown to bind to a large array of proteins with diverse cellular functions. For example, LC8 was found to bind specifically to neuronal nitric oxide synthase (26Jaffrey S.R. Snyder S.H. Science. 1996; 274: 774-777Crossref PubMed Scopus (423) Google Scholar), to the proapoptotic member of the Bcl-2 family proteins Bim and Bmf (27Puthalakath 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 (904) Google Scholar, 28Puthalakath H. Villunger A. O'Reilly L.A. Beaumont J.G. Coultas L. Cheney R.E. Huang D.C. Strasser A. Science. 2001; 293: 1829-1832Crossref PubMed Scopus (497) Google Scholar), to the product of the Drosophila swallow gene (29Schnorrer F. Bohmann K. Nusslein-Volhard C. Nat. Cell Biol. 2000; 2: 185-190Crossref PubMed Scopus (207) Google Scholar), to transcriptional regulator IκB (30Crepieux P. Kwon H. Leclerc N. Spencer W. Richard S. Lin R. Hiscott J. Mol. Cell. Biol. 1997; 17: 7375-7385Crossref PubMed Google Scholar), to guanylate kinase domain-associated protein (31Naisbitt S. Valtschanoff J. Allison D.W. Sala C. Kim E. Craig A.M. Weinberg R.J. Sheng M. J. Neurosci. 2000; 20: 4524-4534Crossref PubMed Google Scholar), to viral phosphoproteins (32Jacob Y. Badrane H. Ceccaldi P.E. Tordo N. J. Virol. 2000; 74: 10217-10222Crossref PubMed Scopus (198) Google Scholar, 33Raux H. Flamand A. Blondel D. J. Virol. 2000; 74: 10212-10216Crossref PubMed Scopus (259) Google Scholar), and to a number of proteins with unknown functions (34Lo K.W. Naisbitt S. Fan J.S. Sheng M. Zhang M. J. Biol. Chem. 2001; 276: 14059-14066Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar). It has been suggested that LC8 acts as a versatile adaptor that links cargo proteins to the dynein motor (34Lo K.W. Naisbitt S. Fan J.S. Sheng M. Zhang M. J. Biol. Chem. 2001; 276: 14059-14066Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar, 35DiBella L.M. Benashski S.E. Tedford H.W. Harrison A. Patel-King R.S. King S.M. J. Biol. Chem. 2001; 276: 14366-14373Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 36Fan J.S. Zhang Q. Tochio H. Li M. Zhang M. J. Mol. Biol. 2001; 306: 97-108Crossref PubMed Scopus (122) Google Scholar, 37Fan J.S. Zhang Q. Tochio H. Zhang M. J. Biomol. NMR. 2002; 23: 103-114Crossref PubMed Scopus (29) Google Scholar).Here we report that LC8 specifically binds to 53BP1. Biochemical dissection studies reveal that 53BP1 contains two distinct LC8-binding motifs that are arranged in tandem. Formation of the LC8·53BP1·p53 and 53BP1·LC8·dynein complexes suggests that 53BP1 can function to link p53 to the dynein complex. Disruption of the interaction between LC8 and 53BP1 in vivo prevented DNA damage-induced nuclear accumulation of p53. The data described in this work provide a distinct mechanism for dynein-mediated p53 nuclear trafficking.MATERIALS AND METHODSYeast Two-hybrid Screening—Two-hybrid screening was performed as described previously (31Naisbitt S. Valtschanoff J. Allison D.W. Sala C. Kim E. Craig A.M. Weinberg R.J. Sheng M. J. Neurosci. 2000; 20: 4524-4534Crossref PubMed Google Scholar). Briefly, LC8 was cloned into pBHA (LexA fusion vector) and used to screen ∼1 × 106 clones of a human cDNA library constructed in pGAD10 (GAL4 activation vector, Clontech).Expression Constructs—The plasmid containing the full-length 53BP1 with an N-terminal hemagglutinin (HA) epitope and a histidine tag (pCMH6K53BP1) were provided by Dr. Kuniyoshi Iwabuchi. Truncation and deletion mutants of 53BP1 were generated by PCR cloning. The plasmid encoding GFP-fused p53 (GFP-p53) was provided by Dr. Randy Poon.For bacterial expression of 53BP1 and its truncation mutants, the corresponding DNA fragments were individually cloned into the EcoRI/XhoI sites of the pGEX-4T1 expression vector (Amersham Biosciences). The GST-fused LC8 bacterial expression vector was generated by inserting LC8-coding DNA into the pGEX-4T1 vector by PCR cloning. The histidine-tagged p53 truncation mutant was constructed by inserting the p53 encoding gene into the EcoRI/BamHI sites of an in-house-modified pET32a vector.Expression and Purification of Fusion Proteins—Bacterial expression of recombinant proteins was carried out using Escherichia coli BL21(DE3) as the host cells. To express GST-LC8 (or GST-53BP1), host cells containing the expression plasmid were grown in LB medium at 37 °C to an A600 of ∼0.8. Expression of the fusion protein was induced by the addition of isopropyl-1-thio-β-d-galactopyranoside to a final concentration of ∼0.25 mm. Protein expression continued for ∼3 h at 37 °C before harvesting by centrifugation. The fusion proteins were expressed in soluble forms and purified using GSH-Sepharose affinity columns (Amersham Biosciences) following the manufacturer's instructions. The eluted proteins were dialyzed against phosphate-buffered saline (PBS) to remove the residual GSH. The dialyzed proteins were directly used for binding assay experiments. Expression of His6-p53 proteins followed the procedure described for the expression of GST-LC8. His6-p53 was purified by passing the cell lysate through a Ni2+-nitrilotriacetic acid affinity column (Novagen). The N-terminal His tag of the fusion protein was cleaved by thrombin digestion. The cleaved His tag and other contaminating proteins were removed by passing the digestion mixture through a gel-filtration column. Preparation of purified, untagged LC8 has been described in our earlier work (38Fan J.S. Zhang Q. Li M. Tochio H. Yamazaki T. Shimizu M. Zhang M. J. Biol. Chem. 1998; 273: 33472-33481Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar).Cell Cultures and Transfection—HEK293 and H1299 cells were cultured in Dulbecco's modified essential medium containing 10% fetal bovine serum (Invitrogen) and penicillin/streptomycin at 37 °C in a humidified, 5% CO2 incubator. For transient transfection, cells were plated on a 10-cm Petri dish (Falcon) to ∼70% confluence and then were transfected with an appropriate amount of DNA using a Lipofectamine transfection kit (Invitrogen) following the manufacturer's instructions. Transient expression of transfected proteins was allowed to continue for 24–48 h.Immunoblot Analysis—Anti-HA monoclonal antibody was purchased from Sigma, Anti-IC74 antibody was provided to us by Dr. K. Kevin Pfister, and anti-p150Glued antibody was from BD Biosciences. SDS-PAGE gel-resolved proteins were electrotransferred to a nitrocellulose membrane in a transferring buffer. Membranes were incubated with 10% skim milk, 0.1% Tween 20 in Tris-buffered saline and then probed with respective antibodies. Protein bands on Western blots were visualized by an enhanced chemiluminescent detection kit (ECL, Amersham Biosciences).Pull-down Experiments—GST-LC8 (10 μg each) was mixed with HEK293 cell lysates (100 μg of total protein) containing HA-tagged full-length 53BP1 or HA-tagged 53BP1 truncation mutants. The GST-LC8-containing complexes were precipitated with fresh GSH-Sepharose beads. The pelleted beads were washed extensively with PBS buffer and subsequently boiled with 2× SDS-PAGE sample buffer. The proteins were first resolved by SDS-PAGE and subsequently analyzed by immunoblot analysis. The assay of the direct binding between LC8 and various purified GST fusion proteins followed the same method as described in our earlier work (34Lo K.W. Naisbitt S. Fan J.S. Sheng M. Zhang M. J. Biol. Chem. 2001; 276: 14059-14066Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar).HIV-1 Tat Fusion Peptide Experiments—The following peptides were synthesized and HPLC-purified. The underlined sequences of the peptides correspond to the protein transduction domain of HIV-1 Tat amino acid 47–57 (Tat). YGRKKRRQRRRSYSKSTQTT (Tat-KSTQTT) and YGRKKRRQRRRSYSKSAAAT (Tat-KSAAAT). H1299 cells transfected with GFP-p53 were washed with PBS and replenished with fresh Dulbecco's modified essential medium. Adriamycin (ADR, 0.4 μg/ml) and different concentrations of Tat-peptides were added into the medium and incubated at 37 °C for 60 min. Fresh fetal bovine serum was added to the cells to a final concentration of 10%, and cells were cultured for another 2 h. At this point, the cells were washed twice with PBS, fixed with 4% paraformaldehyde/PBS, and permeabilized with 0.2% Triton X-100/PBS. The nuclei were stained with 4′,6-diamidino-2-phenylindole and then examined by fluorescence microscopy.RESULTSIdentification of 53BP1 as a LC8-binding Protein—A yeast two-hybrid screen of a human brain cDNA library using LC8 as the bait was carried out to identify potential LC8-binding proteins. Among many positive clones identified (34Lo K.W. Naisbitt S. Fan J.S. Sheng M. Zhang M. J. Biol. Chem. 2001; 276: 14059-14066Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar), one encodes a fragment of 53BP1 (residues 1047–1313, Fig. 1A). The interaction between LC8 and 53BP1 was confirmed in an in vitro pull-down experiment (Fig. 1B). When expressed in HEK293 cells, the 53BP1 fragment isolated from the yeast two-hybrid screen was shown to interact robustly with GST-LC8 (Fig. 1B, lane 1 in the second panel). In addition, HA-tagged, full-length 53BP1 could be specifically and robustly pelleted by purified recombinant GST-LC8 attached to GSH-Sepharose beads (Fig. 1B, lane 1 in the first panel). When the two proteins were co-expressed in HEK293 cells, 53BP1 and LC8 formed a tight complex that could be efficiently immunoprecipitated from cell lysates (Fig. 1C, lane 1).The LC8-binding fragment of 53BP1 isolated from the yeast two-hybrid screen partially overlapped with the KLR domain of the protein. Removal of the amino acid residues that overlap with the KLR domain (residues 1178–1313) generated a 65-residue peptide fragment corresponding to amino acid residues 1113–1177 of 53BP1. This 65-residue fragment was found to bind robustly to LC8 (Fig. 1B, lane 1 in the third panel). Deletion of this 65-residue fragment from the full-length 53BP1 completely abolished its binding to LC8 (Fig. 1B, lane 1 in the bottom panel). Taken together, the data in Fig. 1 demonstrate that 53BP1 can specifically bind to LC8, and the 65-residue fragment N-terminal to the KLR domain is necessary and sufficient for 53BP1 to bind to LC8.53BP1 Contains Two Distinct LC8-binding Motifs—In our earlier work, we showed that LC8 binds to multiple target proteins via a consensus (R/K)XTQT motif (where “X” is variable amino acids) (Fig. 2A and Ref. 34Lo K.W. Naisbitt S. Fan J.S. Sheng M. Zhang M. J. Biol. Chem. 2001; 276: 14059-14066Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar). Inspection of the amino acid sequence reveals that the 1169AATQT1173 sequence within the 65-residue LC8-binding fragment of 53BP1 resembles this consensus LC8-binding motif (Fig. 2C). To determine whether the 1169AATQT1173 sequence is indeed the LC8-binding motif of 53BP1, we deleted three residues (TQT) from the 65-residue LC8-binding fragment of 53BP1 (Fig. 2C). Earlier biochemical and structural studies indicated that deletion of the TQT cassette from the consensus (R/K)XTQT motif was sufficient to disrupt its LC8-binding capacity (34Lo K.W. Naisbitt S. Fan J.S. Sheng M. Zhang M. J. Biol. Chem. 2001; 276: 14059-14066Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar, 36Fan J.S. Zhang Q. Tochio H. Li M. Zhang M. J. Mol. Biol. 2001; 306: 97-108Crossref PubMed Scopus (122) Google Scholar). To our surprise, the 1171ΔTQT1173 deletion mutant of 53BP1 could still bind to LC8 (Fig. 2D, second lane). This outcome suggested that either the 1169AATQT1173 sequence is not the authentic LC8-binding motif or this sequence is not the only LC8-binding motif. It is known that some LC8 target proteins do not contain the (R/K)XTQT motif in their LC8-binding domains (31Naisbitt S. Valtschanoff J. Allison D.W. Sala C. Kim E. Craig A.M. Weinberg R.J. Sheng M. J. Neurosci. 2000; 20: 4524-4534Crossref PubMed Google Scholar, 34Lo K.W. Naisbitt S. Fan J.S. Sheng M. Zhang M. J. Biol. Chem. 2001; 276: 14059-14066Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar, 36Fan J.S. Zhang Q. Tochio H. Li M. Zhang M. J. Mol. Biol. 2001; 306: 97-108Crossref PubMed Scopus (122) Google Scholar, 39Liang J. Jaffrey S.R. Guo W. Snyder S.H. Clardy J. Nat. Struct. Biol. 1999; 6: 735-740Crossref PubMed Scopus (143) Google Scholar, 40Rodriguez-Crespo I. Yelamos B. Roncal F. Albar J.P. Ortiz de Montellano P.R. Gavilanes F. FEBS Lett. 2001; 503: 135-141Crossref PubMed Scopus (85) Google Scholar). For example, the 239GIQVD243 sequence of neuronal nitric-oxide synthase (nNOS) plays a central role in the binding of the enzyme to LC8 (36Fan J.S. Zhang Q. Tochio H. Li M. Zhang M. J. Mol. Biol. 2001; 306: 97-108Crossref PubMed Scopus (122) Google Scholar, 39Liang J. Jaffrey S.R. Guo W. Snyder S.H. Clardy J. Nat. Struct. Biol. 1999; 6: 735-740Crossref PubMed Scopus (143) Google Scholar). We also found that another LC8-binding protein, GKAP, contains two GIQVD-like motifs (Fig. 2B) and that both motifs are involved in LC8-binding. 2K. W.-H. Lo, H.-M. Kan, and M. Zhang, our unpublished data. The 65-residue LC8-binding fragment of 53BP1 also contains a GIQVD-like motif (1153GIQTM1157). This motif is located slightly upstream of the 1169AATQT1173 motif (Fig. 2, B and C). To test if the 1153GIQTM1157 motif of 53BP1 plays an active role in LC8 binding, we deleted the 1153GIQ1155 cassette from the 65-residue LC8-binding segment of 53BP1 and assayed the LC8-binding capacity of the mutant. Again, based on earlier structural and mutagenesis studies, deletion of the GIQ-cassette would abrogate the GIQVD motif-mediated LC8 binding (36Fan J.S. Zhang Q. Tochio H. Li M. Zhang M. J. Mol. Biol. 2001; 306: 97-108Crossref PubMed Scopus (122) Google Scholar, 39Liang J. Jaffrey S.R. Guo W. Snyder S.H. Clardy J. Nat. Struct. Biol. 1999; 6: 735-740Crossref PubMed Scopus (143) Google Scholar). In contrast to our prediction, the 1153GIQ1155-deletion mutant of 53BP1 also interacted with LC8 (Fig. 2D, first lane), indicating the involvement of other regions of 53BP1 in LC8 binding. We next generated a double deletion mutant with both the 1153GIQ1155 and the 1171TQT1173 cassettes removed. This double mutant showed no detectable LC8 binding (Fig. 2D, third lane). Taken together, we conclude that 53BP1 contains two distinct LC8-binding motifs with amino acid sequences of 1153GIQTM1157 and 1169AATQT1173.Fig. 253BP1 contains two distinct LC8-binding motifs. A, amino acid sequence alignment of LC8-binding domains from selected target proteins containing the consensus (K/R)XTQT motif. The (K/R)XTQT-like sequence of 53BP1 is shown at the bottom. B, sequence alignment of GIQVD motifs of selected LC8-binding proteins. The GIQVD-like motif of 53BP1 is indicated at the bottom of the panel. C, schematic diagram showing the GST-53BP1 deletion mutants used to map the LC8 binding sequences of the protein. The amino acid sequences of the two potential LC8-interacting motifs are included in the figure. D, Coomassie Blue staining of SDS-PAGE gel showing the interactions between various GST-53BP1 deletion mutants and LC8. The lane labeled “GST” serves as a negative control.View Large Image Figure ViewerDownload (PPT)We expect that the 1169AATQT1173 motif of 53BP1 binds to LC8 in an essentially identical manner as does the (R/K)XTQT motif, because we earlier showed that mutation of the positively charged Arg/Lys in the motif to an Ala introduced very limited changes to LC8 binding (34Lo K.W. Naisbitt S. Fan J.S. Sheng M. Zhang M. J. Biol. Chem. 2001; 276: 14059-14066Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar). Therefore, we chose not to characterize the interaction between the 1169AATQT1173 motif of 53BP1 and LC8 in further detail. In contrast, the nature of the interaction between the 1153GIQTM1157 motif of 53BP1 and LC8 remains uncertain because of the sequence divergence between this motif and the LC8-binding domain of nNOS (the only well characterized LC8-binding protein without an (R/K)XTQT motif). We next studied the interaction between the 1153GIQTM1157 motif of 53BP1 and LC8 in more detail. To test the binding of the GIQTM motif to LC8, we used a 13-residue synthetic peptide corresponding to 1149QNNIGIQTMECSL1161 of 53BP1 (called the GIQ-peptide here), and titrated 15N-labeled LC8 with the peptide using NMR spectroscopy. In the presence of sub-stoichiometric amount of the GIQ-peptide, we observed two distinct sets of backbone amide peaks in the 1H,15N HSQC spectra of LC8/peptide mixtures. These two sets of resonances correspond to the peptide-bound form and the free form of LC8. The slow exchange of the peptide-bound form and the free form of LC8 observed in the NMR sample tube indicate that the GIQ-peptide binds to LC8 with high affinity. The LC8 dimer was saturated upon addition of a two molar ratio of the GIQ-peptide (Fig. 3A), indicating that each LC8 dimer binds to two molecules of the GIQ-peptide. The specificity of the binding of the GIQ-peptide to LC8 was further demonstrated by the does-dependent competition between the GIQ-peptide and the full-length 53BP1 for binding to LC8 (Fig. 4A).Fig. 3The GIQ-peptide of 53BP1 binds to LC8 with high affinity. A, superposition plot of the 1H,15N HSQC spectra of free (black) and the GIQ-peptide saturated (red) LC8. The assignment of the free form LC8 is labeled with each amino acid residue name and number. B, plot as a function of the residue number of combined 1H and 15N chemical shift changes of LC8 induced by the GIQ-peptide binding. The combined 1H and 15N chemical shift changes are defined as: Δppm = [(ΔδHN)2 + (ΔδN × αN)2]1/2, where ΔδHN and ΔδN represent chemical shift differences of amide proton and nitrogen chemical shifts of free LC8 and the protein in complex with the GIQ-peptide, respectively. The scaling factor (αN) used to normalize the 1H and 15N chemical shifts is 0.17. The secondary structure of LC8 is indicated at the top of the plot. The inset shows the amplitude (in pseudo-color scale) of the GIQ-peptide-induced chemical shift changes of LC8 mapped on to the three-dimensional structure of the LC8 dimer.View Large Image Figure ViewerDownload (PPT)Fig. 4Synthetic peptides containing either class of LC8-binding motifs specifically compete with 53BP1 for binding to LC8. A, the GIQ-peptide competes with 53BP1 for LC8 in a dose-dependent manner. Due to the limited solubility of the GIQ-peptide in the aqueous buffer, a peptide stock solution was prepared by dissolving the sample in Me2SO. The volume of Me2SO was kept to <5% of the total volume and remained the same in each assay mixture. B, the KSTQT-peptide competes for binding to with 53BP1 for LC8 in a similar dose-dependent manner as the GIQ-peptide. C, substitution of the critical TQT cassette with the AAA cassette in the KSTQT-peptide completely abolished the LC8-binding capacity of the peptide.View Large Image Figure ViewerDownload (PPT)We next investigated which regions of LC8 are involved in the GIQ-peptide binding. We used the well established minimum chemical shift perturbation approach to map the GIQ-peptide-binding region on LC8 by taking the advantage of complete chemical shift assignment of the free form of LC8 (36Fan J.S. Zhang Q. Tochio H. Li M. Zhang M. J. Mol. Biol. 2001; 306: 97-108Crossref PubMed Scopus (122) Google Scholar). Fig. 3B summarizes the chemical shift changes of LC8 induced by the GIQ-peptide binding. The inset in Fig. 3B shows the GIQ-peptide-induced chemical shift changes mapped on to the backbone structure of the LC8 dimer (36Fan J.S. Zhang Q. Tochio H. Li M. Zhang M. J. Mol. Biol. 2001; 306: 97-108Crossref PubMed Scopus (122) Google Scholar). The data clearly show that amino acid residues in the β2-strand, β1/β2-loop, β2/β3-loop, and the N-terminal part of α2-helix are involved in the GIQ-peptide binding. The GIQ motif peptide-induced chemical shift perturbation profile of LC8 is highly similar to that induced by the LC8-binding peptide derived from nNOS (36" @default.
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• W2080650147 date "2005-03-01" @default.
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• W2080650147 title "The 8-kDa Dynein Light Chain Binds to p53-binding Protein 1 and Mediates DNA Damage-induced p53 Nuclear Accumulation" @default.
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• W2080650147 doi "https://doi.org/10.1074/jbc.m411408200" @default.
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