c-Abl Tyrosine Kinase Selectively Regulates p73 Nuclear Matrix Association
2003; Elsevier BV; Volume: 278; Issue: 36 Linguagem: Inglês
10.1074/jbc.m301051200
ISSN1083-351X
AutoresMerav Ben‐Yehoyada, Israel Ben‐Dor, Yosef Shaul,
Tópico(s)Cancer Research and Treatments
Resumop73 is a structural and functional homologue of the p53 tumor-suppressor protein. Like p53, p73 is activated in response to DNA-damaging insults to induce cell cycle arrest or apoptosis. Under these conditions p73 is tyrosine-phosphorylated by c-Abl, a prerequisite modification for p73 to elicit cell death in fibroblasts. In this study we report that in response to ionizing radiation, p73 undergoes nuclear redistribution and becomes associated with the nuclear matrix. This association is c-Abl-dependent because it was not observed in cells that are defective in c-Abl kinase activation. Moreover, STI-571, a specific c-Abl kinase inhibitor, is sufficient to block significantly p73α nuclear matrix association. The observed c-Abl dependence of nuclear matrix association was recapitulated in the heterologous baculovirus system. Under these conditions p73α but not p53 is specifically tyrosine-phosphorylated by c-Abl. Moreover, the phosphorylated p73α is predominantly found in association with the nuclear matrix. Thus, in response to ionizing radiation p73 is modified in a c-Abl-dependent manner and undergoes nuclear redistribution and translocates to associate with the nuclear matrix. Our data describe a novel mechanism of p73 regulation. p73 is a structural and functional homologue of the p53 tumor-suppressor protein. Like p53, p73 is activated in response to DNA-damaging insults to induce cell cycle arrest or apoptosis. Under these conditions p73 is tyrosine-phosphorylated by c-Abl, a prerequisite modification for p73 to elicit cell death in fibroblasts. In this study we report that in response to ionizing radiation, p73 undergoes nuclear redistribution and becomes associated with the nuclear matrix. This association is c-Abl-dependent because it was not observed in cells that are defective in c-Abl kinase activation. Moreover, STI-571, a specific c-Abl kinase inhibitor, is sufficient to block significantly p73α nuclear matrix association. The observed c-Abl dependence of nuclear matrix association was recapitulated in the heterologous baculovirus system. Under these conditions p73α but not p53 is specifically tyrosine-phosphorylated by c-Abl. Moreover, the phosphorylated p73α is predominantly found in association with the nuclear matrix. Thus, in response to ionizing radiation p73 is modified in a c-Abl-dependent manner and undergoes nuclear redistribution and translocates to associate with the nuclear matrix. Our data describe a novel mechanism of p73 regulation. The p73 polypeptide is a member of the p53 family of proteins (1Kaghad M. Bonnet H. Yang A. Creancier L. Biscan J.C. Valent A. Minty A. Chalon P. Lelias J.M. Dumont X. Ferrara P. McKeon F. Caput D. Cell. 1997; 90: 809-819Abstract Full Text Full Text PDF PubMed Scopus (1539) Google Scholar, 2Jost C.A. Marin M.C. Kaelin Jr., W.G. Nature. 1997; 389: 191-194Crossref PubMed Scopus (903) Google Scholar). There are several p73 isoforms that differ at their N and C termini (3De Laurenzi V. Costanzo A. Barcaroli D. Terrinoni A. Falco M. Annicchiarico-Petruzzelli M. Levrero M. Melino G. J. Exp. Med. 1998; 188: 1763-1768Crossref PubMed Scopus (362) Google Scholar, 4Ueda Y. Hijikata M. Takagi S. Chiba T. Shimotohno K. Oncogene. 1999; 18: 4993-4998Crossref PubMed Scopus (131) Google Scholar, 5De Laurenzi V.D. Catani M.V. Terrinoni A. Corazzari M. Melino G. Costanzo A. Levrero M. Knight R.A. Cell Death Differ. 1999; 6: 389-390Crossref PubMed Scopus (132) Google Scholar). p73α is the longest isoform containing at its C terminus a sterile-α motif domain, a domain known to mediate protein-protein interactions (6Wang W.K. Proctor M.R. Buckle A.M. Bycroft M. Chen Y.W. Acta Crystallogr. Sect. D. Biol. Crystallogr. 2000; 56: 769-771Crossref PubMed Scopus (15) Google Scholar, 7Thanos C.D. Bowie J.U. Protein Sci. 1999; 8: 1708-1710Crossref PubMed Scopus (126) Google Scholar, 8Chi S.W. Ayed A. Arrowsmith C.H. EMBO J. 1999; 18: 4438-4445Crossref PubMed Scopus (151) Google Scholar). The p73 ΔN isoforms lack the transactivation domain and display dominant negative activity (9Pozniak C.D. Radinovic S. Yang A. McKeon F. Kaplan D.R. Miller F.D. Science. 2000; 289: 304-306Crossref PubMed Scopus (416) Google Scholar, 10Grob T.J. Novak U. Maisse C. Barcaroli D. Luthi A.U. Pirnia F. Hugli B. Graber H.U. De Laurenzi V. Fey M.F. Melino G. Tobler A. Cell Death Differ. 2001; 8: 1213-1223Crossref PubMed Scopus (313) Google Scholar, 11Vossio S. Palescandolo E. Pediconi N. Moretti F. Balsano C. Levrero M. Costanzo A. Oncogene. 2002; 21: 3796-3803Crossref PubMed Scopus (72) Google Scholar, 12Zaika A.I. Slade N. Erster S.H. Sansome C. Joseph T.W. Pearl M. Chalas E. Moll U.M. J. Exp. Med. 2002; 196: 765-780Crossref PubMed Scopus (305) Google Scholar). Thus it is very likely that each of the isoforms has a unique role whose nature remains to be clarified. Like p53, p73 can induce apoptosis in a variety of cell lines and support transcription of genes containing p53-response elements (1Kaghad M. Bonnet H. Yang A. Creancier L. Biscan J.C. Valent A. Minty A. Chalon P. Lelias J.M. Dumont X. Ferrara P. McKeon F. Caput D. Cell. 1997; 90: 809-819Abstract Full Text Full Text PDF PubMed Scopus (1539) Google Scholar, 2Jost C.A. Marin M.C. Kaelin Jr., W.G. Nature. 1997; 389: 191-194Crossref PubMed Scopus (903) Google Scholar). In response to DNA damage signals, these proteins undergo covalent modifications. However, the identity of the upstream effectors was not fully resolved. The c-Abl non-receptor tyrosine kinase was identified as an upstream effector of p73 both in vitro and in vivo (13Agami R. Blandino G. Oren M. Shaul Y. Nature. 1999; 399: 809-813Crossref PubMed Scopus (505) Google Scholar, 14Yuan Z.M. Shioya H. Ishiko T. Sun X. Gu J. Huang Y.Y. Lu H. Kharbanda S. Weichselbaum R. Kufe D. Nature. 1999; 399: 814-817Crossref PubMed Scopus (541) Google Scholar). The level of p73 tyrosine phosphorylation is elevated when cells are exposed to ionizing radiation, a condition whereby c-Abl kinase is activated. Furthermore, p73 and c-Abl are physically associated via the p73 PXXP motif and the c-Abl Src homology 3 domain (13Agami R. Blandino G. Oren M. Shaul Y. Nature. 1999; 399: 809-813Crossref PubMed Scopus (505) Google Scholar). Disruption of c-Abl-p73 interaction rescues cells from irradiation-induced apoptosis (13Agami R. Blandino G. Oren M. Shaul Y. Nature. 1999; 399: 809-813Crossref PubMed Scopus (505) Google Scholar, 14Yuan Z.M. Shioya H. Ishiko T. Sun X. Gu J. Huang Y.Y. Lu H. Kharbanda S. Weichselbaum R. Kufe D. Nature. 1999; 399: 814-817Crossref PubMed Scopus (541) Google Scholar). Interestingly, a p73 point mutant at tyrosine 99 (Y99F), a site that is phosphorylated by c-Abl, behaves as a dominant negative mutant and blocks the apoptotic response to IR 1The abbreviations used are: IR, ionizing radiation; CIP, calf intestinal alkaline phosphatase; DTT, dithiothreitol; PMSF, phenylmethylsulfonyl fluoride; PBS, phosphate-buffered saline; PIPES, 1,4-piperazinediethanesulfonic acid; Gy, gray; PML, promyelocytic leukemia. (14Yuan Z.M. Shioya H. Ishiko T. Sun X. Gu J. Huang Y.Y. Lu H. Kharbanda S. Weichselbaum R. Kufe D. Nature. 1999; 399: 814-817Crossref PubMed Scopus (541) Google Scholar). 2M. Ben-Yehoyada, I. Ben-Dor, and Y. Shaul, unpublished data. Accumulating evidence indicates that p73 is essential for apoptosis induced by many chemotherapeutic agents and IR. These treatments often lead to p73 accumulation. However, some cell lines are refractory to certain agents (1Kaghad M. Bonnet H. Yang A. Creancier L. Biscan J.C. Valent A. Minty A. Chalon P. Lelias J.M. Dumont X. Ferrara P. McKeon F. Caput D. Cell. 1997; 90: 809-819Abstract Full Text Full Text PDF PubMed Scopus (1539) Google Scholar, 15Irwin M.S. Kondo K. Marin M.C. Cheng L.S. Hahn W.C. Kaelin W.G. Cancer Cell. 2003; 3: 403-410Abstract Full Text Full Text PDF PubMed Scopus (366) Google Scholar, 16Bergamaschi D. Gasco M. Hiller L. Sullivan A. Syed N. Trigiante G. Yulug I. Merlano M. Numico G. Comino A. Attard M. Reelfs O. Gusterson B. Bell A.K. Heath V. Tavassoli M. Farrell P.J. Smith P. Lu X. Crook T. Cancer Cell. 2003; 3: 387-402Abstract Full Text Full Text PDF PubMed Scopus (386) Google Scholar) suggesting the involvement of additional effectors in this process. A recent study showed that the induction of apoptosis by p53 requires the presence of p73 (17Flores E.R. Tsai K.Y. Crowley D. Sengupta S. Yang A. McKeon F. Jacks T. Nature. 2002; 416: 560-564Crossref PubMed Scopus (725) Google Scholar). However, p73 can induce apoptosis in the absence of p53 (15Irwin M.S. Kondo K. Marin M.C. Cheng L.S. Hahn W.C. Kaelin W.G. Cancer Cell. 2003; 3: 403-410Abstract Full Text Full Text PDF PubMed Scopus (366) Google Scholar). An interesting interplay between p73 and p53 mutants became recently evident. Several p53 mutants, in particular of the p53Arg-72 variant, are potent inhibitors of p73 function (15Irwin M.S. Kondo K. Marin M.C. Cheng L.S. Hahn W.C. Kaelin W.G. Cancer Cell. 2003; 3: 403-410Abstract Full Text Full Text PDF PubMed Scopus (366) Google Scholar, 16Bergamaschi D. Gasco M. Hiller L. Sullivan A. Syed N. Trigiante G. Yulug I. Merlano M. Numico G. Comino A. Attard M. Reelfs O. Gusterson B. Bell A.K. Heath V. Tavassoli M. Farrell P.J. Smith P. Lu X. Crook T. Cancer Cell. 2003; 3: 387-402Abstract Full Text Full Text PDF PubMed Scopus (386) Google Scholar). Although not directly demonstrated, it is possible that their physical interaction (18Di Como C.J. Gaiddon C. Prives C. Mol. Cell Biol. 1999; 19: 1438-1449Crossref PubMed Scopus (382) Google Scholar, 19Marin M.C. Jost C.A. Brooks L.A. Irwin M.S. O'Nions J. Tidy J.A. James N. McGregor J.M. Harwood C.A. Yulug I.G. Vousden K.H. Allday M.J. Gusterson B. Ikawa S. Hinds P.W. Crook T. Kaelin Jr., W.G. Nat. Genet. 2000; 25: 47-54Crossref PubMed Scopus (471) Google Scholar, 20Strano S. Munarriz E. Rossi M. Cristofanelli B. Shaul Y. Castagnoli L. Levine A.J. Sacchi A. Cesareni G. Oren M. Blandino G. J. Biol. Chem. 2000; 275: 29503-29512Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar) accounts for the inhibitory function of p53 mutants. Although an enzyme-substrate relationship between c-Abl and p73 has been established, it is still unclear how p73 modification by c-Abl influences p73 ability to induce apoptosis. It has been demonstrated that the p73 half-life is prolonged by treatment with the DNA-damaging agent cisplatin, and the accumulated p73 activates certain downstream targets to induce apoptosis (21Gong J.G. Costanzo A. Yang H.Q. Melino G. Kaelin Jr., W.G. Levrero M. Wang J.Y. Nature. 1999; 399: 806-809Crossref PubMed Scopus (837) Google Scholar). However, sole p73 accumulation might not be sufficient in this process. Recently, it has been reported that DNA damage induces p73 acetylation by the acetyltransferase p300, a modification that gives rise to selective transactivation of proapoptotic target genes (22Costanzo A. Merlo P. Pediconi N. Fulco M. Sartorelli V. Cole P.A. Fontemaggi G. Fanciulli M. Schiltz L. Blandino G. Balsano C. Levrero M. Mol. Cell. 2002; 9: 175-186Abstract Full Text Full Text PDF PubMed Scopus (280) Google Scholar). This process requires functional c-Abl, suggesting that p73 tyrosine phosphorylation may trigger the whole pathway. This is also the case in other p73 pathways. For example, p73 threonine phosphorylation by p38 mitogen-activated protein kinase requires functional c-Abl (23Sanchez-Prieto R. Sanchez-Arevalo V.J. Servitja J.M. Gutkind J.S. Oncogene. 2002; 21: 974-979Crossref PubMed Scopus (97) Google Scholar). It appears therefore that p73 tyrosine phosphorylation by c-Abl sensitizes p73 to undergo additional modifications. It is evident from the study of p53 that, in addition to protein modification and stabilization, subcellular localization plays a critical role in regulation of p53 function (24Jimenez G.S. Khan S.H. Stommel J.M. Wahl G.M. Oncogene. 1999; 18: 7656-7665Crossref PubMed Scopus (167) Google Scholar, 25Vousden K.H. Biochim. Biophys. Acta. 2002; 1602: 47-59Crossref PubMed Scopus (304) Google Scholar, 26Nikolaev A.Y. Li M. Puskas N. Qin J. Gu W. Cell. 2003; 112: 29-40Abstract Full Text Full Text PDF PubMed Scopus (325) Google Scholar). Little is known about p73 subcellular localization, but it appears that this may play an important role in p73 function as well. Both p73 and c-Abl colocalize in the cytoplasm of spermatogonia and spermatocytes suggesting a tissue-specific regulation of these proteins by the mechanism of subcellular localization (27Hamer G. Gademan I.S. Kal H.B. de Rooij D.G. Oncogene. 2001; 20: 4298-4304Crossref PubMed Scopus (49) Google Scholar). The Yes-associated protein is a p73 transcriptional coactivator (28Strano S. Munarriz E. Rossi M. Castagnoli L. Shaul Y. Sacchi A. Oren M. Sudol M. Cesareni G. Blandino G. J. Biol. Chem. 2001; 276: 15164-15173Abstract Full Text Full Text PDF PubMed Scopus (358) Google Scholar). It has been reported recently (29Basu S. Totty N.F. Irwin M.S. Sudol M. Downward J. Mol. Cell. 2003; 11: 11-23Abstract Full Text Full Text PDF PubMed Scopus (654) Google Scholar) that cytoplasmic sequestration of Yes-associated protein reduces p73-mediated induction of Bax expression and p73-mediated apoptosis, following DNA damage. However, the possibility that under this condition p73 translocates to the cytoplasm was not investigated. Finally, HIPK2, a homeodomain interacting protein kinase 2, has been found to bind to p73 (30Kim 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). HIPK2 is a member of a novel family of nuclear serine/threonine kinases previously found to bind and activate p53 (31Wang Y. Debatin K.M. Hug H. BMC Mol. Biol. 2001; 2: 8Crossref PubMed Scopus (38) Google Scholar, 32Hofmann T.G. Moller A. Sirma H. Zentgraf H. Taya Y. Droge W. Will H. Schmitz M.L. D'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: 1-10Crossref PubMed Scopus (503) Google Scholar) via direct p53 phosphorylation at Ser-46 (33D'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 (578) Google Scholar). HIPK2 localizes with p53 and PML-3 in the nuclear bodies (also named promyelocytic leukemia (PML) bodies). Interestingly, similar to p53, p73 colocalizes with HIPK2 in PML. These data define functional interaction between p73 and HIPK2 that results in the targeted subcellular localization of p73. In this study we followed p73 cellular distribution under conditions whereby p73 is tyrosine-phosphorylated by c-Abl. We provide evidence for p73α translocation to the nuclear matrix fraction in a c-Abl-dependent manner. This process requires a functional c-Abl kinase domain. Interference with c-Abl kinase activity either by using c-Abl kinase-incompetent cells or STI-571, a c-Abl kinase pharmacological inhibitor, disrupts p73α nuclear matrix association. Finally, we utilized the insect cells to overexpress p73 and c-Abl. Under these conditions p73α is efficiently tyrosine-phosphorylated with its concomitant translocation to the nuclear matrix fraction. These data provide evidence for a novel mechanism of p73 regulation. Cell Cultures and Baculovirus Infection—Human colon carcinoma lines HCT116, HCT116-3(6) (34Boyer J.C. Umar A. Risinger J.I. Lipford J.R. Kane M. Yin S. Barrett J.C. Kolodner R.D. Kunkel T.A. Cancer Res. 1995; 55: 6063-6070PubMed Google Scholar), and monkey kidney fibroblast COS-1 were cultured in standard medium that was supplemented with 10% fetal bovine serum and antibiotics. H5 insect cells were grown in Grace's insect medium (Invitrogen) supplemented with 10% heat-inactivated fetal bovine serum, 50 units/ml penicillin, 50 μg/ml streptomycin, and 50 ng/ml gentamicin at 27 °C. Expression of c-Abl and p73 in insect cells was performed as described in the Bac-to-Bac baculovirus expression system manual (Invitrogen). c-Abl cDNA (nucleotides 1–4113) was ligated into the BamHI-StuI sites of the pFASTBAC HTb expression vector encoding an N-terminal histidine tag. Simian p73 cDNA (nucleotides 1–1887) was introduced into the BamHI-HindIII sites of pFASTBAC HTc also encoding a histidine tag. Bacmid DNA corresponding to each plasmid was transfected into H5 insect cells, and the viruses were amplified for three additional times. The p53 baculovirus was kindly provided by Dr. V. Rotter. Materials and Antibodies—Propidium iodide and micrococcal nuclease were purchased from Sigma; genistein was from Calbiochem; cisplatin was from ABIC Israel; calf intestinal alkaline phosphatase (CIP) was from New England Biolabs, and DNase I was from Roche Applied Science. STI-571 was kindly provided by Novartis Pharmaceuticals. IR was done with Co60 source. Antiserum to simian p73 was raised in rabbits immunized with bacterially produced, SDS-PAGE-purified p73 (amino acids 1–667). PY99 antibodies were raised in rabbits immunized with phosphorylated peptide (CVPTHSPY(PO3H2)AQP-NH2). Rabbit antiserum was further purified on a protein-A/G column. The phosphorylated antibody was characterized, and it recognizes specifically phosphorylated p73 at tyrosine 99 (data not shown). Antibody 1801, a p53 monoclonal antibody (35Banks L. Matlashewski G. Crawford L. Eur. J. Biochem. 1986; 159: 529-534Crossref PubMed Scopus (486) Google Scholar), was kindly provided by Dr. M. Oren. Goat polyclonal anti-lamin B (M-20), rabbit polyclonal anti-c-Abl (K-12), mouse monoclonal p73 (H-79), and mouse monoclonal anti-phosphotyrosine (PY20) were purchased from Santa Cruz Biotechnology. Protein Extraction and Immunoprecipitation—Forty eight hours post-infection, H5 insect cells were collected and resuspended in RIPA buffer (50 mm Tris-HCl, pH 8, 150 mm NaCl, 1% Nonidet P-40 (v/v), 0.5% deoxycholate (v/v), 0.1% SDS (v/v), 1 mm dithiothreitol (DTT), 1 μg/ml each of leupeptin, aprotinin, pepstatin, and 1 mm PMSF (Sigma mixture)). Insoluble pellets were discarded, and protein concentrations were determined using the Bio-Rad protein assay. Immunoprecipitation was performed by incubation of the extract with protein-A/G-Sepharose beads (Santa Cruz Biotechnology) and 5 μg of anti-Tyr(P), anti-c-Abl, or anti-p73 (H79) for 4 h at 4 °C. Next the beads were washed five times with RIPA buffer and boiled in Laemmli sample buffer. Whole cell lysates or immunoprecipitates were separated on 10% SDS-polyacrylamide gels. Immunoblotting of the proteins was performed using the indicated antibodies. For the CIP treatment, 2 units of CIP were added to cell lysates and incubated for 15 min at 37 °C. The reaction was terminated by adding Laemmli sample buffer. Nuclear Matrix Fractionation—Nuclear matrix fractionation was performed as described (36Reyes J.C. Muchardt C. Yaniv M. J. Cell Biol. 1997; 137: 263-274Crossref PubMed Scopus (200) Google Scholar). In brief, cells were seeded at a density adjusted to reach 70% confluency at the end of the experiment. Cells were washed two times with ice-cold phosphate-buffered saline (PBS). Cell pellet was resuspended in three packed cell volumes of cytoskeleton buffer (CSK) (10 mm PIPES, pH 6.8, 100 mm NaCl, 300 mm sucrose, 3 mm MgCl2, 1 mm EGTA, 1 mm DTT, 0.5% (v/v) Triton X-100, 1 μg/ml each of leupeptin, aprotinin, pepstatin, and 1 mm PMSF (Sigma mixture)). After 5 min at 4 °C, the cytoskeletal framework was separated from soluble proteins by centrifugation at 5,000 × g for 3 min. Chromatin was digested with 1 mg/ml DNase I in 2 volumes of CSK buffer for 15 min at 37 °C. Then ammonium sulfate was added from a 1 m stock solution in CSK buffer to a final concentration of 0.25 m and after 5 min at 4 °C and spun down. The pellet was further extracted with 2 m NaCl in CSK buffer for 5 min at 4 °C and centrifuged. This treatment removed the entire DNA and histones from the nucleus. The remaining pellet was solubilized in urea buffer (8 m urea, 0.1 m NaH2PO4, and 0.01 m Tris, pH 8), and regarded as the nuclear matrix containing fraction. Chromatin Fractionation—Chromatin fractionation was performed as described (37Mendez J. Stillman B. Mol. Cell Biol. 2000; 20: 8602-8612Crossref PubMed Scopus (760) Google Scholar). In brief, for chromatin isolation, cells were resuspended in buffer A (10 mm HEPES, pH 7.9, 10 mm KCl, 1.5 mm MgCl2, 0.34 m sucrose, 10% glycerol, 1 mm DTT, 1 μg/ml each of leupeptin, aprotinin, pepstatin, and 1 mm PMSF (Sigma mixture)). Triton X-100 (0.1%) was added, and the cells were incubated for 5 min on ice. Nuclei were collected by low speed centrifugation (4 min for 1,300 × g). The supernatant was further clarified by high speed centrifugation (10 min for 20,000 × g) to remove cell debris and insoluble aggregates. Nuclei were washed once in buffer A and then lysed in buffer B (3 mm EDTA, 0.2 mm EGTA, 1 mm DTT, protease inhibitors as described above). Insoluble chromatin was collected by centrifugation (4 min for 1,700 × g), washed once in buffer B, and centrifuged again under the same conditions. The final chromatin pellet was resuspended in Laemmli buffer and sonicated for 15 s. To release chromatin-bound proteins by nuclease treatment, cell nuclei were resuspended in buffer A plus 1 mm CaCl2 and 0.5 units of micrococcal nuclease. After incubation for 5 min at 37 °C, the nuclease reaction was stopped by the addition of 1 mm EGTA. Nuclei were collected by low speed centrifugation and lysed according to the chromatin isolation protocol described above. Cell Cycle Analysis—For fluorescence-activated cell sorter analysis cells were harvested 2–40 h post-IR, washed twice with PBS, and fixed in 70% ethanol. After fixation, the cells were washed with PBS and resuspended in 50 μg/ml RNase A and 25 μg/ml propidium iodide in PBS. In each assay 10,000 cells were collected by FACScan and analyzed with the Cellquest program (BD Biosciences). p73α Associates with the Nuclear Matrix in Irradiated Cells—Protein post-translational modifications such as phosphorylation and sumoylation regulate protein spatial compartmentalization (38Mancini M.A. Shan B. Nickerson A.J. Lee W. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 418-422Crossref PubMed Scopus (274) Google Scholar, 39Fogal 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 (321) Google Scholar, 40Kwek S.S. Derry J. Tyner A.L. Shen Z. Gudkov A.V. Oncogene. 2001; 20: 2587-2599Crossref PubMed Scopus (113) Google Scholar). To investigate the effect of IR and cisplatin on p73 nuclear redistribution, we employed the high salt nuclear matrix extraction procedure to separate the various nuclear fractions (36Reyes J.C. Muchardt C. Yaniv M. J. Cell Biol. 1997; 137: 263-274Crossref PubMed Scopus (200) Google Scholar). First, soluble proteins were extracted using non-ionic detergent (fraction I) followed by DNase I digestion and ammonium sulfate precipitation of the chromatin associated proteins (fraction II). Fraction III was obtained by washing the pellet with 2 m NaCl. The remaining pellet was solubilized in 8 m urea to obtain fraction IV which is contained with the structural nuclear matrix and the associated proteins (Fig. 1A) (41Hakes D.J. Berezney R. J. Biol. Chem. 1991; 266: 11131-11140Abstract Full Text PDF PubMed Google Scholar). The obtained fractions were SDS-PAGE separated and immunoblotted with the indicated antibodies. In the non-treated COS-1 cells p73α is detected predominantly in the nucleocytoplasm fraction (Fig. 1B). Interestingly, following exposure to IR, a significant increase in p73 level was detected in the nuclear matrix fraction (fraction IV). The effectiveness of the fractionation protocol was verified by specific detection of the lamin B nuclear matrix protein in this fraction. In COS-1 cells cisplatin did not increase p73 levels, and no nuclear matrix accumulation was observed. As a control we monitored the level of p53 in the obtained fractions. Notably, under these conditions the amount of p53 remained unchanged in response to IR and only slightly increased upon cisplatin treatment. The poor p53 accumulation is possibly due to the effect of the SV40 large T antigen that is constitutively expressed in these cells. In addition, p53 unlike p73α did not translocate to the nuclear matrix fraction. These data suggest that in response to IR, p73α selectively translocates to the nuclear matrix. p73α Association with the Nuclear Matrix Depends on c-Abl Kinase Activation—Activation of c-Abl and p73 by cisplatin requires a functional MLH1 gene as well as c-Abl activation (21Gong J.G. Costanzo A. Yang H.Q. Melino G. Kaelin Jr., W.G. Levrero M. Wang J.Y. Nature. 1999; 399: 806-809Crossref PubMed Scopus (837) Google Scholar). The mismatch-repair-defective human cell line HCT116 lacks the MLH1 gene on chromosome 3, whereas the matched HCT116-3(6) cells have acquired the functional MLH1 gene by transfer of chromosome 3 (34Boyer J.C. Umar A. Risinger J.I. Lipford J.R. Kane M. Yin S. Barrett J.C. Kolodner R.D. Kunkel T.A. Cancer Res. 1995; 55: 6063-6070PubMed Google Scholar). The acquisition of MLH1 gene has recovered c-Abl kinase activation by cisplatin. We have confirmed this finding and found that this is not specific to cisplatin-treated cells but also true for irradiated cells (data not shown). These paired cell lines were used to test the possible role of c-Abl kinase activity in p73 nuclear redistribution following IR exposure and cisplatin treatment. In non-treated HCT116 and HCT116-3(6) cells, p73α is equally distributed in the nucleocytoplasm, chromatin, and nuclear matrix fractions (Fig. 2A). Remarkably, 40 h following IR, p73α was translocated to the nuclear matrix fraction only in the kinase-competent c-Abl HCT116-3(6) cells but not in the matched HCT116 cell line (Fig. 2A, lanes 4 and 8). In contrast, cisplatin treatment caused p73 accumulation, in agreement with a previous report (21Gong J.G. Costanzo A. Yang H.Q. Melino G. Kaelin Jr., W.G. Levrero M. Wang J.Y. Nature. 1999; 399: 806-809Crossref PubMed Scopus (837) Google Scholar), but not nuclear matrix association. The level of p53 was induced in both cell lines upon IR and cisplatin treatment, suggesting that p53 stabilization is likely to be c-Abl kinase-independent. Here again, no significant p53 nuclear redistribution was obtained. These results hint at the possibility that the selective p73α translocation from the nucleocytoplasm to the nuclear matrix fraction is c-Abl kinase-dependent. To substantiate the finding that p73α associates with the nuclear matrix and to rule out the possibility that this is the direct outcome of the high salt extraction procedure, we used an alternative chromatin fractionation protocol (37Mendez J. Stillman B. Mol. Cell Biol. 2000; 20: 8602-8612Crossref PubMed Scopus (760) Google Scholar). In this procedure, cells are lysed with non-ionic detergent in a sucrose-rich buffer, and nuclei are then collected by low speed centrifugation, washed, and lysed in no salt buffer. A second centrifugation step separates the remaining soluble nuclear proteins from the insoluble fraction that contains proteins bound to chromatin or to the nuclear matrix. To distinguish between the chromatin and the nuclear matrix-bound proteins, micrococcal nuclease treatment was employed to release the chromatin fraction (Fig. 2B). In the untreated HCT116-3(6) cells, p73α is predominantly found in the fraction containing the soluble proteins (Fig. 2C, lane 1) with a minute amount in the soluble nuclear protein fraction (lanes 2 and 3) but not in the chromatin fraction. Upon IR treatment, the p73α level in the soluble protein fraction decreased with concomitant accumulation in the chromatin and nuclear matrix fractions. Because p73α was not solubilized after micrococcal nuclease treatment, we concluded that p73α is preferentially associated with the nuclear matrix and not with the chromatin fraction. In contrast to p73, p53 was accumulated upon IR treatment in the chromatin but not the nuclear matrix fraction, because it was solubilized almost completely by micrococcal nuclease (Fig. 2C, lower panel, lanes 4 and 5). These data indicate that in response to IR, p73α, but not p53, translocates from soluble nuclear fraction to the nuclear matrix. Inhibition of c-Abl Kinase Activity Blocks p73α Association with the Nuclear Matrix—To substantiate the role of c-Abl kinase in p73α nuclear matrix translocation, we used the c-Abl kinase inhibitor STI-571 (42Druker B.J. Tamura S. Buchdunger E. Ohno S. Segal G.M. Fanning S. Zimmermann J. Lydon N.B. Nat. Med. 1996; 2: 561-566Crossref PubMed Scopus (3167) Google Scholar, 43Schindler T. Bornmann W. Pellicena P. Miller W.T. Clarkson B. Kuriyan J. Science. 2000; 289: 1938-1942Crossref PubMed Scopus (1630) Google Scholar). The proficient c-Abl HCT116-3(6) cells were irradiated in the presence or absence of STI-571, and nuclear fractions were analyzed. Here again, irradiation of HCT116-3(6) cells triggered p73α association with nuclear matrix (Fig. 3, lane 3). Interestingly, no p73α accumulation in the nuclear matrix fraction was evident when c-Abl kinase activity was inhibited by STI-571 (lane 4). The proficiency of the fractionation protocol was confirmed by using anti-lamin B antibody, a nuclear matrix protein. This finding indicates that in response to IR c-Abl kinase regulates p73α association with the nuclear matrix. p73α Nuclear Redistribution Is a Slow, Gradual, and IR Dose-dependent Process—By having demonstrated that in response to IR, p73α becomes associated with the nuclear matrix in a c-Abl kinase-dependent manner, we next measured IR dose response and time kinetics of this process. For IR dose response the paired HCT cells were u
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