Activation of the Kinase Activity of ATM by Retinoic Acid Is Required for CREB-dependent Differentiation of Neuroblastoma Cells
2007; Elsevier BV; Volume: 282; Issue: 22 Linguagem: Inglês
10.1074/jbc.m609628200
ISSN1083-351X
AutoresNorvin D. Fernandes, Yingli Sun, Brendan D. Price,
Tópico(s)Neuroblastoma Research and Treatments
ResumoThe ATM protein kinase is mutated in ataxia telangiectasia, a genetic disease characterized by defective DNA repair, neurodegeneration, and growth factor signaling defects. The activity of ATM kinase is activated by DNA damage, and this activation is required for cells to survive genotoxic events. In addition to this well characterized role in DNA repair, we now demonstrate a novel role for ATM in the retinoic acid (RA)-induced differentiation of SH-SY5Y neuroblastoma cells into post-mitotic, neuronal-like cells. RA rapidly activates the activity of ATM kinase, leading to the ATM-dependent phosphorylation of the CREB protein, extrusion of neuritic processes, and differentiation of SH-SY5Y cells into neuronal-like cells. When ATM protein expression was suppressed by short hairpin RNA, the ATM-dependent phosphorylation of CREB was blocked. Furthermore, ATM-negative cells failed to differentiate into neuronal-like cells when exposed to retinoic acid; instead, they underwent cell death. Expression of a constitutively active CREBVP16 construct, or exposure to forskolin to induce CREB phosphorylation, rescued ATM negative cells and restored differentiation. Furthermore, when dominant negative CREB proteins with mutations in either the CREB phosphorylation site (CREBS133A) or the DNA binding domain (KCREB) were introduced into SH-SY5Y cells, retinoic acid-induced differentiation was blocked and the cells underwent cell death. The results demonstrate that ATM is required for the retinoic acid-induced differentiation of SH-SY5Y cells through the ATM dependent-phosphorylation of serine 133 of CREB. These results therefore define a novel mechanism for activation of the activity of ATM kinase by RA, and implicate ATM in the regulation of CREB function during RA-induced differentiation. The ATM protein kinase is mutated in ataxia telangiectasia, a genetic disease characterized by defective DNA repair, neurodegeneration, and growth factor signaling defects. The activity of ATM kinase is activated by DNA damage, and this activation is required for cells to survive genotoxic events. In addition to this well characterized role in DNA repair, we now demonstrate a novel role for ATM in the retinoic acid (RA)-induced differentiation of SH-SY5Y neuroblastoma cells into post-mitotic, neuronal-like cells. RA rapidly activates the activity of ATM kinase, leading to the ATM-dependent phosphorylation of the CREB protein, extrusion of neuritic processes, and differentiation of SH-SY5Y cells into neuronal-like cells. When ATM protein expression was suppressed by short hairpin RNA, the ATM-dependent phosphorylation of CREB was blocked. Furthermore, ATM-negative cells failed to differentiate into neuronal-like cells when exposed to retinoic acid; instead, they underwent cell death. Expression of a constitutively active CREBVP16 construct, or exposure to forskolin to induce CREB phosphorylation, rescued ATM negative cells and restored differentiation. Furthermore, when dominant negative CREB proteins with mutations in either the CREB phosphorylation site (CREBS133A) or the DNA binding domain (KCREB) were introduced into SH-SY5Y cells, retinoic acid-induced differentiation was blocked and the cells underwent cell death. The results demonstrate that ATM is required for the retinoic acid-induced differentiation of SH-SY5Y cells through the ATM dependent-phosphorylation of serine 133 of CREB. These results therefore define a novel mechanism for activation of the activity of ATM kinase by RA, and implicate ATM in the regulation of CREB function during RA-induced differentiation. Ataxia telangiectasia (A-T) 4The abbreviations used are: A-T, ataxia telangiectasia; RA, all-trans-retinoic acid; BDNF, brain-derived neurotrophic factor; RARβ, retinoic acid receptor-β; CREB, cAMP-response element-binding protein; shRNA, short hairpin RNA; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; Ser(P)-1981, phosphoserine 1981; CBP, CREB-binding protein. is an inherited disease characterized by immune deficiencies, premature aging, neurodegeneration, susceptibility to cancer, and extreme sensitivity to ionizing radiation (1.Sedgewick R. Boder E. Vinken P.J. Bruyn G.W. Klavans H.L. Handbook of Clinical Neurology. Vol. 61. Elsevier, Amstersdam1991: 347-423Google Scholar, 2.Meyn M.S. Clin. Genet. 1999; 55: 289-304Crossref PubMed Scopus (157) Google Scholar). The product of the A-T gene, the ATM protein kinase, is a key component of the signal transduction pathway activated by DNA damage (3.Shiloh Y. Nat. Rev. Cancer. 2003; 3: 155-168Crossref PubMed Scopus (2161) Google Scholar, 4.Lavin M.F. Birrell G. Chen P. Kozlov S. Scott S. Gueven N. Mutat. Res. 2005; 569: 123-132Crossref PubMed Scopus (170) Google Scholar). The activity of ATM kinase is activated in response to ionizing radiation-induced DNA damage (5.Bakkenist C.J. Kastan M.B. Nature. 2003; 421: 499-506Crossref PubMed Scopus (2703) Google Scholar), and ATM then phosphorylates multiple proteins involved in the DNA damage response, including nbs1, p53, chk2, and SMC1 (reviewed in Ref. 4.Lavin M.F. Birrell G. Chen P. Kozlov S. Scott S. Gueven N. Mutat. Res. 2005; 569: 123-132Crossref PubMed Scopus (170) Google Scholar). These phosphorylated proteins, in turn, regulate the 2 key responses to DNA damage: the activation of cell cycle checkpoints and the initiation of DNA repair. Consequently, cells lacking functional ATM protein exhibit multiple defects in DNA repair (6.Pandita T.K. Pathak S. Geard C.R. Cytogenet. Cell Genet. 1995; 71: 86-93Crossref PubMed Scopus (165) Google Scholar, 7.Allio T. Preston R.J. Mutat. Res. 2000; 453: 5-15Crossref PubMed Scopus (21) Google Scholar, 8.MacLeod R.A. Buchheim T. Kaufmann M. Drexler H.G. Mutat. Res. 1996; 372: 33-42Crossref PubMed Scopus (6) Google Scholar, 9.Jeggo P.A. Carr A.M. Lehmann A.R. Trends Genet. 1998; 14: 312-316Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar) and loss of cell cycle checkpoints (9.Jeggo P.A. Carr A.M. Lehmann A.R. Trends Genet. 1998; 14: 312-316Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, 10.Cornforth M.N. Bedford J.S. Science. 1985; 227: 1589-1591Crossref PubMed Scopus (244) Google Scholar), resulting in increased sensitivity to ionizing radiation. Following DNA damage, the activity of ATM kinase is rapidly increased, and ATM is autophosphorylated at multiple sites (11.Kozlov S.V. Graham M.E. Peng C. Chen P. Robinson P.J. Lavin M.F. EMBO J. 2006; 25: 3504-3514Crossref PubMed Scopus (221) Google Scholar), including serine 1981 (5.Bakkenist C.J. Kastan M.B. Nature. 2003; 421: 499-506Crossref PubMed Scopus (2703) Google Scholar). Autophosphorylation of ATM is thought to initiate dimer-monomer transition, and release of the active ATM monomer (5.Bakkenist C.J. Kastan M.B. Nature. 2003; 421: 499-506Crossref PubMed Scopus (2703) Google Scholar, 11.Kozlov S.V. Graham M.E. Peng C. Chen P. Robinson P.J. Lavin M.F. EMBO J. 2006; 25: 3504-3514Crossref PubMed Scopus (221) Google Scholar), although the exact contribution of ATM autophosphorylation to ATM activation is still under debate (12.Pellegrini M. Celeste A. Difilippantonio S. Guo R. Wang W. Feigenbaum L. Nussenzweig A. Nature. 2006; 443: 222-225Crossref PubMed Scopus (160) Google Scholar). Several additional proteins are involved in activation of ATM kinase, including the Mre11-Rad50-Nbs1 complex, and the Tip60 histone acetyltransferase (13.Jiang X. Sun Y. Chen S. Roy K. Price B.D. J. Biol. Chem. 2006; 281: 15741-15746Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 14.Sun Y. Jiang X. Chen S. Fernandes N. Price B.D. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 13182-13187Crossref PubMed Scopus (558) Google Scholar, 15.Lee J.H. Paull T.T. Science. 2004; 304: 93-96Crossref PubMed Scopus (588) Google Scholar, 16.Uziel T. Lerenthal Y. Moyal L. Andegeko Y. Mittelman L. Shiloh Y. EMBO J. 2003; 22: 5612-5621Crossref PubMed Scopus (839) Google Scholar). The Mre11-Rad50-Nbs1 complex contains a specific ATM binding domain that is required for recruitment of ATM to sites of DNA damage (17.Falck J. Coates J. Jackson S.P. Nature. 2005; 434: 605-611Crossref PubMed Scopus (999) Google Scholar). Subsequent activation of ATM depends on the Tip60-dependent acetylation of the ATM protein, which is proposed as the trigger for activating the kinase activity of ATM (13.Jiang X. Sun Y. Chen S. Roy K. Price B.D. J. Biol. Chem. 2006; 281: 15741-15746Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 14.Sun Y. Jiang X. Chen S. Fernandes N. Price B.D. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 13182-13187Crossref PubMed Scopus (558) Google Scholar). In addition to activation of ATM by DNA damage, ATM participates in cellular pathways that are not directly linked to the DNA damage response. For example, compounds that alter chromatin structure, such as trichostatin A or chloroquine, can activate the kinase activity of ATM independently of DNA damage (5.Bakkenist C.J. Kastan M.B. Nature. 2003; 421: 499-506Crossref PubMed Scopus (2703) Google Scholar). Insulin can also up-regulate the kinase activity of ATM (18.Yang D.Q. Kastan M.B. Nat. Cell. Biol. 2000; 2: 893-898Crossref PubMed Scopus (237) Google Scholar), leading to the phosphorylation of insulin target proteins, including protein kinase B and the eIF-4e-binding protein (18.Yang D.Q. Kastan M.B. Nat. Cell. Biol. 2000; 2: 893-898Crossref PubMed Scopus (237) Google Scholar, 19.Viniegra J.G. Martinez N. Modirassari P. Losa J.H. Parada Cobo C. Lobo V.J. Luquero C.I. Alvarez-Vallina L. Ramon y Cajal S. Rojas J.M. Sanchez-Prieto R. J. Biol. Chem. 2005; 280: 4029-4036Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar). The ATM protein can also influence the levels of the insulin-like growth factor-1 receptor in cells (20.Peretz S. Jensen R. Baserga R. Glazer P.M. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 1676-1681Crossref PubMed Scopus (127) Google Scholar). These observations are consistent with the clinical pathology of A-T, which includes mild diabetes and endocrine growth defects (1.Sedgewick R. Boder E. Vinken P.J. Bruyn G.W. Klavans H.L. Handbook of Clinical Neurology. Vol. 61. Elsevier, Amstersdam1991: 347-423Google Scholar, 2.Meyn M.S. Clin. Genet. 1999; 55: 289-304Crossref PubMed Scopus (157) Google Scholar). ATM can therefore participate in multiple growth factor signaling pathways in addition to the DNA damage response. Here, the role of the ATM protein kinase in non-DNA damage-dependent pathways was investigated. In particular, the function of ATM in growth factor-regulated cellular differentiation was examined. A well characterized system for studying differentiation is the retinoic acid (RA)-dependent differentiation of neuroblastoma cells into post-mitotic neuronal-like cells. Exposure of SH-SY5Y cells to RA stimulates RA-dependent gene transcription (reviewed in Ref. 21.Bastien J. Rochette-Egly C. Gene (Amst.). 2004; 328: 1-16Crossref PubMed Scopus (607) Google Scholar), up-regulating a diverse array of genes, including TrkB, the receptor for brain-derived neurotrophic factor (BDNF), making the cells responsive to BDNF (22.Ruiz-Leon Y. Pascual A. J. Neurochem. 2001; 79: 278-285Crossref PubMed Scopus (31) Google Scholar, 23.Ruiz-Leon Y. Pascual A. Neuroscience. 2003; 120: 1019-1026Crossref PubMed Scopus (36) Google Scholar). Sequential exposure of SH-SY5Y cells to retinoic acid and BDNF results in growth arrest and differentiation of the SH-SY5Y cells into post-mitotic neuronal-like cells (24.Encinas M. Iglesias M. Liu Y. Wang H. Muhaisen A. Cena V. Gallego C. Comella J.X. J. Neurochem. 2000; 75: 991-1003Crossref PubMed Scopus (588) Google Scholar). The fully differentiated SH-SY5Y cells exhibit widespread neuritic arborization, and express multiple neuronal proteins, including tau, GAP-43, MAP-2, transglutaminase, β-amyloid and others (22.Ruiz-Leon Y. Pascual A. J. Neurochem. 2001; 79: 278-285Crossref PubMed Scopus (31) Google Scholar, 23.Ruiz-Leon Y. Pascual A. Neuroscience. 2003; 120: 1019-1026Crossref PubMed Scopus (36) Google Scholar, 24.Encinas M. Iglesias M. Liu Y. Wang H. Muhaisen A. Cena V. Gallego C. Comella J.X. J. Neurochem. 2000; 75: 991-1003Crossref PubMed Scopus (588) Google Scholar, 25.Irwin N. Chao S. Goritchenko L. Horiuchi A. Greengard P. Nairn A.C. Benowitz L.I. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 12427-12431Crossref PubMed Scopus (50) Google Scholar, 26.Singh U.S. Pan J. Kao Y.L. Joshi S. Young K.L. Baker K.M. J. Biol. Chem. 2003; 278: 391-399Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). Here, we demonstrate that RA rapidly activates the kinase activity of ATM leading to phosphorylation of the CREB protein and differentiation of the SH-SY5Y cells into post-mitotic neuronal-like cells. Inactivation of either ATM or CREB blocks RA-driven differentiation, and results in cell death. The results demonstrate a key role for ATM in the RA-dependent differentiation of neuroblastoma cells. Cell Culture−SH-SY5Y cells (American Type Culture Collection, Manassas, VA) were maintained in a 1:1 mixture of Eagle's minimum essential medium and Ham's F-12 medium supplemented with fetal bovine serum (10%), non-essential amino acids, sodium pyruvate, and HEPES buffer. For differentiation experiments, cells were plated at 30% confluence and allowed to grow for 24 h. All-trans-retinoic acid (Biomol, PA) was added to 15 μm and cells were maintained in RA for 5 days, with media change every day. Cells were then washed in serum-free Dulbecco's modified Eagle's medium to remove serum and RA, and incubated in Dulbecco's modified Eagle's medium supplemented with BDNF (50 ng/ml) for 7 days. Phase-contrast photography of cell lines was carried out using a light microscope and a Nikon Cool-pix digital camera. For shRNA experiments, plasmid pBS/U6ATM601 (described in Ref. 27.Chen S. Wang G. Makrigiorgos G.M. Price B.D. Biochem. Biophys. Res. Commun. 2004; 317: 1037-1044Crossref PubMed Scopus (26) Google Scholar), which contains an shRNA sequence targeting nucleotides 601-618 of ATM (GGGAGCTGATTGTAGCAACATAAGCTTATGTTGCTACAATCAGCTCCC) was transfected into SH-SY5Y cells using FuGENE 6 reagent (Roche) according to the manufacturer's protocol and individual clones selected using hygromycin (300 μg/ml). An ATM cDNA with 3 base changes in the shRNA targeting site (nucleotides 601-618 of ATM, base changes underlined: AGCCGATTGCAGTAACAT), which altered codon usage but did not change the amino acid sequence, was constructed. Individual clones derived from the shRNA experiment were then transfected with shRNA-resistant ATM, and cells selected with G418 (400 μg/ml). pCMV-CREB, pCMV-CREBS133A, and pCMV-KCREB vectors were obtained from BD Biosciences, and cell lines selected with G418 (450 μg/ml). Immunoprecipitation, Western Blot Analysis, Luciferase Assay, and Kinase Assay−Cells were lysed in 50 mm Tris, pH 7.4, 150 mm NaCl, 0.2% Tween 20, 1.5 mm MgCl2, 1 mm EGTA, 2 mm dithiothreitol, 50 mm NaF, 500 μm Na3VO4, 1 mm phenylmethylsulfonyl fluoride, 3 μg/ml aprotinin, 3 μg/ml leupeptin, 1× phosphatase inhibitor mixture set 1 (Calbiochem). All immunoprecipitates were washed five times in lysis buffer. For kinase assays, immunoprecipitates were washed in kinase buffer (10 mm HEPES, pH 7.4, 10 mm MgCl2, 50 mm NaCl, 10 mm MnCl2), and incubated in kinase buffer (50 μl) supplemented with 50 μm ATP, p53 peptide (2 μg: EPPLSQEAFADL-WKK), and 10 μCi of [γ-32P]ATP for 30 min at 30 °C. Reactions were terminated as described by us (14.Sun Y. Jiang X. Chen S. Fernandes N. Price B.D. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 13182-13187Crossref PubMed Scopus (558) Google Scholar, 28.Fernandes N. Sun Y. Chen S. Paul P. Shaw R.J. Cantley L.C. Price B.D. J. Biol. Chem. 2005; 280: 15158-15164Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). SH-SY5Y cells were transiently transfected with CRE-luciferase reporter plasmid (3 μg: Stratagene) using FuGENE 6 (Roche) and allowed to recover for 24 h. Following treatment, cell lysates were prepared and analyzed for luciferase activity using the Luciferase Assay Kit (Stratagene, CA). Antibodies used were: ATM antibodies, PC116 (Oncogene Science, CA), 5C2 and 2C1 (Genetex, TX), and Ser(P)-1981 ATM (Rockland Immunochemicals, PA). β-Actin (Cell Signaling), CREB and phospho-CREB (Upstate Biotech, NY), TrkB and VP-16 (Santa Cruz Biotechnology, CA) were also used. Immunofluorescence−Cells were seeded onto LabTek II chamber slides (Nunc, NY), fixed in paraformaldehyde (14.Sun Y. Jiang X. Chen S. Fernandes N. Price B.D. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 13182-13187Crossref PubMed Scopus (558) Google Scholar, 28.Fernandes N. Sun Y. Chen S. Paul P. Shaw R.J. Cantley L.C. Price B.D. J. Biol. Chem. 2005; 280: 15158-15164Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar), incubated with Ser(P)-1981 ATM antibody (Rockland Biochemicals), washed, and incubated with IgG-Texas Red (Santa Cruz Biotechnology). Slides were mounted with Fluoromount-G (Southern Biotech, AL) and visualized with a Nikon Eclipse TE 2000 microscope. Cell Survival Assays−MTT assays were carried out using the CellTiter Cell Proliferation Assay Kit (Promega). 5 × 103 cells were exposed to RA (15 μm) for specified time points and then incubated with MTT at 37 °C in 5% CO2 for 1.75 h. Absorbance was recorded at 490 nm (using 670 nm as the reference filter) using a Benchmark Plus microplate spectrophotometer (Bio-Rad). Background absorbance was subtracted. KU-55933 was purchased from Sigma. To evaluate the role of ATM in the signaling pathways required for differentiation of SH-SY5Y neuroblastoma cells, ATM expression was silenced by stable expression of an ATM-specific shRNA vector (27.Chen S. Wang G. Makrigiorgos G.M. Price B.D. Biochem. Biophys. Res. Commun. 2004; 317: 1037-1044Crossref PubMed Scopus (26) Google Scholar). Supplementary Fig. 1A demonstrates robust suppression of ATM in multiple individual SH-SY5Y clonal cell lines expressing ATM shRNA. Two of these clones, SA2 and SA6, were selected for further analysis. To control for clonal variation and off-gene regulation by the shRNA, an ATM cDNA construct was engineered to contain silent point mutations in the region targeted by the ATM-specific shRNA. Fig. 1A demonstrates the successful re-expression of the shRNA-resistant ATM in SA2 and SA6 cells (Fig. 1, lanes 2 and 4), although the reintroduced ATM was expressed at slightly lower levels than the endogenous ATM protein (Fig. 1A). Exposure of SH-SY5Y cells containing a nonspecific shRNA (Fig. 1B, shVector) to retinoic acid for 5 days induced growth arrest, followed by the extension of neuritic processes. Subsequent removal of RA and incubation in serum-free media supplemented with BDNF results in elongation of these neuritic outgrowths, forming a complex network of neuritic processes (Fig. 1B), as previously reported (24.Encinas M. Iglesias M. Liu Y. Wang H. Muhaisen A. Cena V. Gallego C. Comella J.X. J. Neurochem. 2000; 75: 991-1003Crossref PubMed Scopus (588) Google Scholar, 29.Miloso M. Villa D. Crimi M. Galbiati S. Donzelli E. Nicolini G. Tredici G. J. Neurosci. Res. 2004; 75: 241-252Crossref PubMed Scopus (83) Google Scholar). In contrast, both SA2 and SA6 cells, in which ATM was silenced, detached from the dish and subsequently underwent cell death when exposed to RA (Fig. 1B). When ATM was re-expressed in the SA2 and SA6 cells (using an shRNA resistant version of the ATM gene), the ability of the cells to differentiate into post-mitotic neuronal-like cells was restored, demonstrating that this phenotype was due to loss of ATM protein rather than a nonspecific effect of the shRNA vector. This effect was quantitated in Fig. 1C, where exposure of SH-SY5Y cells to retinoic acid inhibited cell proliferation, but did not result in detectable changes in cell viability (Fig. 1C). In contrast, SA2 cells exhibited >90% loss of viability following 72 h exposure to retinoic acid (Fig. 1C). However, re-expression of ATM in SA2 cells rescued the cells from retinoic acid-induced cell death (Fig. 1C) and allowed the cells to differentiate. Similar results were seen with SA6 cells (data not shown). These results were further confirmed by treating SH-SY5Y cells with the ATM-specific kinase inhibitor KU-55933 (30.Hickson I. Zhao Y. Richardson C.J. Green S.J. Martin N.M. Orr A.I. Reaper P.M. Jackson S.P. Curtin N.J. Smith G.C. Cancer Res. 2004; 64: 9152-9159Crossref PubMed Scopus (996) Google Scholar). KU-55933 on its own had no significant impact on SH-SY5Y cell viability (supplementary Fig. 1B). However, combined treatment of SH-SY5Y cells with the ATM kinase inhibitor KU-55933 and retinoic acid induced rapid cell death and failure to differentiate (supplementary Fig. 1, B and C). Thus, both pharmacological and genetic inactivation of ATM in retinoic acid-treated SH-SY5Y cells leads to cell death rather than differentiation. Furthermore, the ability of KU-55933 to inhibit the differentiation of SH-SY5Y cells indicates that it is the kinase activity of ATM that is essential for this process. A key step in the differentiation of SH-SY5Y cells is the RA-dependent up-regulation of growth factor receptors, including RARβ and TrkB, the receptor for BDNF (23.Ruiz-Leon Y. Pascual A. Neuroscience. 2003; 120: 1019-1026Crossref PubMed Scopus (36) Google Scholar, 24.Encinas M. Iglesias M. Liu Y. Wang H. Muhaisen A. Cena V. Gallego C. Comella J.X. J. Neurochem. 2000; 75: 991-1003Crossref PubMed Scopus (588) Google Scholar, 31.Canon E. Cosgaya J.M. Scsucova S. Aranda A. Mol. Biol. Cell. 2004; 15: 5583-5592Crossref PubMed Scopus (154) Google Scholar, 32.Aggarwal S. Kim S.W. Cheon K. Tabassam F.H. Yoon J.H. Koo J.S. Mol. Biol. Cell. 2006; 17: 566-575Crossref PubMed Scopus (105) Google Scholar). Up-regulation of TrkB levels is required for BDNF to induce terminal differentiation of SH-SY5Y cells (23.Ruiz-Leon Y. Pascual A. Neuroscience. 2003; 120: 1019-1026Crossref PubMed Scopus (36) Google Scholar, 24.Encinas M. Iglesias M. Liu Y. Wang H. Muhaisen A. Cena V. Gallego C. Comella J.X. J. Neurochem. 2000; 75: 991-1003Crossref PubMed Scopus (588) Google Scholar). Western analysis demonstrated that RA increased the levels of RARβ and TrkB receptors in both SH-SY5Y and in the ATM-deficient SA2 and SA6 (supplementary Fig. 2A). The inability of SA2 and SA6 cells to differentiate is therefore not due to a failure to up-regulate RA driven transcription of the RARβ and TrkB genes. Overall, Fig. 1 demonstrates a central role for the ATM protein in the retinoic acid-induced differentiation of SH-SY5Y neuroblastoma cells into post-mitotic neuronal-like cells. The requirement for ATM in RA signaling implies that RA activates the kinase activity of ATM. The up-regulation of the kinase activity of ATM by DNA damage causes autophosphorylation of serine 1981, which can be monitored by immunofluorescence using a phospho-specific antibody to detect autophosphorylation of serine 1981 of ATM (28.Fernandes N. Sun Y. Chen S. Paul P. Shaw R.J. Cantley L.C. Price B.D. J. Biol. Chem. 2005; 280: 15158-15164Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). In Fig. 2B, exposure of ATM negative cells to ionizing radiation (A-T) did not result in phosphorylation of serine 1981 of ATM as detected by immunofluoresence, whereas re-expression of ATM in these cells (A-TATM; described by us in Refs. 14.Sun Y. Jiang X. Chen S. Fernandes N. Price B.D. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 13182-13187Crossref PubMed Scopus (558) Google Scholar and 28.Fernandes N. Sun Y. Chen S. Paul P. Shaw R.J. Cantley L.C. Price B.D. J. Biol. Chem. 2005; 280: 15158-15164Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar)) allows detection of autophosphorylated ATM in irradiated, but not control cells. Thus, the phospho-serine 1981 antibody can be used to monitor ATM autophosphorylation in vivo, as shown by other groups (5.Bakkenist C.J. Kastan M.B. Nature. 2003; 421: 499-506Crossref PubMed Scopus (2703) Google Scholar, 16.Uziel T. Lerenthal Y. Moyal L. Andegeko Y. Mittelman L. Shiloh Y. EMBO J. 2003; 22: 5612-5621Crossref PubMed Scopus (839) Google Scholar, 17.Falck J. Coates J. Jackson S.P. Nature. 2005; 434: 605-611Crossref PubMed Scopus (999) Google Scholar). Addition of RA to SH-SY5Y cells resulted in rapid ATM autophosphorylation within 15 min of RA addition, and ATM remained autophosphorylated throughout the 5-day exposure to RA (Fig. 2A). Subsequent withdrawal of RA led to loss of ATM autophosphorylation (Fig. 2A) and the cells subsequently underwent cell death (data not shown). However, addition of BDNF (in the absence of RA) maintained ATM in the active, autophosphorylated form (Fig. 2A), and the cells remained in the growth arrested, differentiated state for up to 2 weeks in culture (Fig. 1B). Supplementary Fig. 2B demonstrates that ATM protein levels do not significantly change during the differentiation of SH-SY5Y cells, indicating that the increased phosphoserine 1981 autophosphorylation signal is due to ATM autophosphorylation rather than increased ATM protein levels. Fig. 2A therefore demonstrates increased ATM autophosphorylation, implying increased ATM kinase activity is maintained throughout the RA/BDNF-induced differentiation of SH-SY5Y cells. To determine whether RA increased ATM autophosphorylation in other neuroblastoma cell lines, SK-N-BE (which undergoes retinoic acid-induced differentiation (33.Choi S.Y. Choi B.H. Suh B.C. Chae H.D. Kim J.S. Shin M.J. Kang S.S. Negishi M. Kim K.T. J. Neurochem. 2001; 79: 303-310Crossref PubMed Scopus (26) Google Scholar)) and SK-N-AS (which does not respond to retinoic acid (34.Gaetano C. Matsumoto K. Thiele C.J. Cell Growth & Differ. 1991; 2: 487-493PubMed Google Scholar)) were examined. RA increased ATM autophosphorylation in differentiation proficient SK-N-BE cells, but not in RA refractive SK-N-AS cells (Fig. 2C). Because ATM is activated in response to DNA damage, it is possible that retinoic acid may induce ATM activation through production of DNA damage. Activation of ATM by DNA damage results in the recruitment of ATM to specific sites of DNA damage, referred to as ionizing radiation-induced foci (5.Bakkenist C.J. Kastan M.B. Nature. 2003; 421: 499-506Crossref PubMed Scopus (2703) Google Scholar). Exposure of SH-SY5Y cells to ionizing radiation gives rise to the characteristic punctate Ser(P)-1981 ATM staining pattern (Fig. 2D), corresponding to the recruitment of active ATM to sites of DNA damage (5.Bakkenist C.J. Kastan M.B. Nature. 2003; 421: 499-506Crossref PubMed Scopus (2703) Google Scholar, 28.Fernandes N. Sun Y. Chen S. Paul P. Shaw R.J. Cantley L.C. Price B.D. J. Biol. Chem. 2005; 280: 15158-15164Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). In contrast, exposure to RA results in a diffuse staining for phosphorylated ATM (Fig. 2D). This diffuse distribution of activated ATM is similar to the pattern observed when ATM is activated by non-DNA damaging agents (such as trichostatin A (5.Bakkenist C.J. Kastan M.B. Nature. 2003; 421: 499-506Crossref PubMed Scopus (2703) Google Scholar, 28.Fernandes N. Sun Y. Chen S. Paul P. Shaw R.J. Cantley L.C. Price B.D. J. Biol. Chem. 2005; 280: 15158-15164Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar)). We interpret this diffuse nuclear ATM staining pattern to indicate that retinoic acid treatment does not activate ATM through generation of DNA damage. Furthermore, we note that the retinoic acid-activated ATM is located exclusively within the nucleus of the cells (Fig. 2, A and D), with minimal signal associated with the cytoplasmic fraction. Fig. 2 therefore demonstrates specific and rapid autophosphorylation of ATM in response to RA. To examine more clearly how RA regulates ATM, we sought to confirm that the increased autophosphorylation of ATM seen in Fig. 2 was due to increased intrinsic ATM kinase activity. In Fig. 3A, cell extracts immunoprecipitated with IgG displayed minimal ATM kinase activity (Fig. 3A). The activity of ATM kinase was increased 2-fold by the radiomimetic agent bleomycin. This 2-fold increase of the kinase activity of ATM in response to DNA damage is similar to that previously reported (14.Sun Y. Jiang X. Chen S. Fernandes N. Price B.D. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 13182-13187Crossref PubMed Scopus (558) Google Scholar, 35.Chen S. Paul P. Price B.D. Oncogene. 2003; 22: 6332-6339Crossref PubMed Scopus (20) Google Scholar). When cells were incubated with RA for 20 min, the kinase activity of ATM was increased to the same levels seen following DNA damage (Fig. 3A), consistent with the rapid autophosphorylation of ATM seen in vivo in Fig. 2A. Figs. 2A and 3C therefore demonstrate that the kinase activity of ATM is increased when SH-SY5Y cells are exposed to RA. Retinoids are potent inducers of neuronal differentiation and activate the CREB transcriptional regulatory protein (31.Canon E. Cosgaya J.M. Scsucova S. Aranda A. Mol. Biol. Cell. 2004; 15: 5583-5592Crossref PubMed Scopus (154) Google Scholar, 36.Lonze B.E. Ginty D.D. Neuron. 2002; 35: 605-623Abstract Full Text Full Text PDF PubMed Scopus (1757) Google Scholar). CREB plays a key role in cellular differentiation, with a particularly important function in regulating neuronal function, including neuronal survival (37.Mantamadiotis T. Lemberger T. Bleckmann S.C. Kern H. Kretz O. Martin Villalba A. Tronche F. Kellendonk C. Gau D. Kapfhammer J. Otto C. Schmid W. Schutz G. Nat. Genet. 2002; 31: 47-54Crossref PubMed Scopus (576) Google Scholar). The ability of CREB to regulate neuronal survival suggested that ATM may promote differentiation of SH-SY5Y cells through activation of the CREB transcriptional regulator. CREB activation involves the phosphorylation of serine 133 of CREB, which creates a binding site for the CBP hi
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