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The phosphatidylinositol-3-kinase-like kinase ATM (Ataxia – telangiectasia mutated) plays a central role in coordinating the DNA damage responses including cell cycle checkpoint control, DNA repair, and apoptosis. Mutations of ATM cause a spectrum of defects ranging from neurodegeneration to cancer predisposition. However, the mechanism by which DNA damage activates ATM is poorly understood. We show that Cdk5 (cyclin-dependent kinase 5), activated by DNA damage, directly phosphorylates ATM at serine 794 in postmitotic neurons. Phosphorylation at serine 794 precedes and is required for ATM autophosphorylation at serine 1981, and activates ATM kinase activity. Cdk5-ATM signal regulates phosphorylation and function of ATM targets p53 and H2AX. Interruption of Cdk5-ATM pathway attenuates DNA damage-induced neuronal cell cycle reentry and expression of p53 targets PUMA and Bax, protecting neurons from DNA damage-induced death. Thus, activation of Cdk5 by DNA damage serves as a critical signal to initiate ATM response and regulate ATM-dependent cellular processes.
DNA damage signals are transduced to activate a key family of phosphoinositide-3-kinase related kinases (PIKKs) including ATM, which phosphorylates a number of critical proteins to control cell cycle checkpoints, DNA repair, and in the case of excessive DNA damage, cell death1, 2, 3. How ATM activity is regulated by DNA damage signal is not clear. Previous studies indicate that activation of ATM involves autophosphorylation of several serine residues including S19814, 5. The widely accepted model is that autophosphorylation of ATM at S1981 releases it from the inhibitory homodimer structure, leading to its activation and recruitment to the sites of DNA double-strand break (DSB)6, but a recent report questions the role of S1987, the mouse equivalent to S1981, in DNA damage-induced activation of ATM in vivo7,.
Unlike proliferating cells where DNA damage typically triggers cell cycle checkpoints, postmitotic neurons under stress conditions including genotoxic stimulation activate their cell cycle machinery8. This cell cycle re-entry by postmitotic neurons leads to neuronal death and inhibition of such an ectopic cell cycle activity protects neurons9-12. Studies using ATM-/- mice show that ATM is required for the p53-mediated apoptosis of developing postmitotic neurons exposed to ionizing radiation13, 14. Suppression of ATM attenuates DNA damage agent camptothecin-induced cell cycle re-entry and death by postmitotic neurons15. These findings establish the role of ATM in DNA damage-induced neuronal death.
Cdk5, a small serine/threonine kinase regulated by activator p35 but not cell cycle, has been shown to play a role in neuronal stress response16, 17, 18. Examination of the protein sequence of ATM revealed the presence of several putative Cdk5 phosphorylation sites. We performed in vitro Cdk5 kinase assay by incubating purified ATM with purified Cdk5 in complex with its activator p25. Co-incubation of ATM with Cdk5 led to phosphorylation of ATM (Fig. 1a). We tested a set of GST-ATM fusion proteins and showed that GST-ATM4 (amino acid residues 772-1102) was robustly phosphorylated by Cdk5 in vitro (Fig. 1b). Mutation of serine 794 to alanine (S794A) abolished phosphorylation by Cdk5, suggesting that Cdk5 directly recognizes ATM S794 in vitro.
To demonstrate that ATM is a Cdk5 substrate in cells, we raised a phospho specific ATM antibody with a synthetic phospho-peptide corresponding to human ATM residues around S794. This phospho ATM antibody specifically detected wild type ATM when phosphorylated at S794 by Cdk5 in vitro but not S794A mutant (Fig. 1c). We then showed that the phospho ATM antibody detected very low background signal when ATM was expressed alone in HEK293 cells. Co-expression of Cdk5/p25 led to a strong S794 phospho signal (Fig. 1d). Interestingly, mutation of serine 1981 to alanine did not affect phosphorylation at S794, indicating that Cdk5 directly phosphorylates ATM at S794 independent of S1981.
To address how phosphorylation of ATM by Cdk5 may regulate ATM activity, we over-expressed wild type ATM with or without co-expression of Cdk5/activator in HEK293 cells, immunoprecipitated ATM, and determined ATM activity by in vitro kinase assay using a standard commercial substrate PHAS-I. Co-expression of Cdk5 strongly activated wt ATM while S794A mutant lost the response to Cdk5 (Fig. 2a, Supplementary information, Fig. S1a). Compared with wt ATM, the phospho mimetic mutant S794D had higher kinase activity (Fig. 2a, Supplementary information, Fig. S1b). Moreover, over-expression of Cdk5/p35 or Cdk5/p25 in HEK293 cells led to a robust activation of endogenous ATM (Fig. 2b). Thus, phosphorylation at S794 activates ATM kinase activity.
We then tested whether Cdk5 plays a role in the activation of ATM following DNA damage in primary cerebellar granule neurons (CGNs) because of their selective degeneration resulting from ATM deficiency. CGNs cultured for 7 days from postnatal day 6-7 rats were exposed to DNA damage agents known to cause DSBs in postmitotic neurons19-21. Compared to untreated control, all three DNA damage agents caused a robust activation of both Cdk5 and ATM kinase activities (Fig. 2c). In contrast, classic apoptosis inducing agent stausporine, which typically does not cause DSBs, did not activate either Cdk5 or ATM. Tests of more general cell death inducers showed that several of them activate both Cdk5 and ATM (Supplementary information, Fig. S1). Interestingly, serum/potassium (S/K) withdrawal, another widely used model of CGN apoptosis not triggered directly by DNA damage, activated Cdk5 without affecting ATM activity. Subcellular fractionation showed that S/K withdrawal caused a delayed activation of Cdk5 only in the cytoplasm and failed to activate Cdk5 in the nucleus (Supplementary information, Fig. S1d). Together, these findings indicate that activation of Cdk5 and ATM in postmitotic neurons may represent a mode of response to a wider spectrum of signals. Since camptothecin (CPT) is very effective in activating Cdk5 and ATM under our experimental conditions, we used it as the model agent for the subsequent studies.
Because protease calpain is activated by DNA damage22 and involved in the conversion of p35 to the more potent Cdk5 activator p2523, we tested whether CPT activates Cdk5 via increasing p25. CPT treatment caused a rapid and transient increase in p25 level, which correlated with an increase in calpain activity as indicated by the cleavage of α-Fodrin (Fig. 2d). CPT-induced activation of Cdk5 paralleled the increase of p25 and preceded the activation of nuclear ATM (Fig. 2e). Inhibition of calpain with AK295 clearly reduced CPT-induced increase of p25 and activation of both Cdk5 and ATM. Collectively, these findings suggest that calpain-mediated activation of Cdk5 is required for activation of ATM in response to CPT in neurons.
Given our data in Fig. 1d, we tested whether S794 phosphorylation regulates the autophosphorylation at S1981. We found that Cdk5 increased phosphorylation at S1981 of wt ATM but failed to enhance S1981 phosphorylation of S749A mutant (Supplementary information, Fig. S1e). S794D mutant showed higher basal S1981 phosphorylation (Supplementary information, Fig. S1f). We next studied whether Cdk5 affects S1981 phosphorylation of endogenous ATM. Since our phospho ATM S794 antibody was raised using a human ATM immunogen, to achieve maximal antibody effects, we carried out the experiments in neurons differentiated from human SH-SY5Y neuroblastoma cells, known to express both Cdk5/p35 and ATM24. Compared to the low basal signal, CPT induced a rapid and robust ATM phosphorylation at S794 in differentiated SH-SY5Y neuronal cells (Fig. 3a). Closely correlated with this, CPT also caused an increase in ATM phosphorylation at S1981 with a somewhat delayed kinetics. Inhibition of Cdk5 using inhibitor roscovitine or Cdk5 RNAi adenovirus blocked CPT-induced phosphorylation at S794 and S1981 (Fig. 3a-b), indicating that Cdk5-mediated S794 phosphorylation precedes and is required for ATM autophosphorylation at S1981.
Next, we tested the effects of inhibiting Cdk5 on ATM-mediated phosphorylation of its two well-characterized substrates, p53 and H2AX. Exposure of CGNs to CPT increased p53 phosphorylation at S15, a known site targeted directly by ATM but not by Cdk5 (Supplementary information, Fig. S1g-h). Inhibition of Cdk5 by roscovitine or RNAi significantly attenuated CPT-induced ATM activity and p53 phosphorylation at S15 (Fig. 3c-d). Immunocytochemistry studies showed that CPT induces robust formation of γ-H2AX foci in CGNs. Both roscovitine and Cdk5 RNAi efficiently reduced the number of CPT-induced γ-H2AX foci (Fig. 3e-f). These data suggest that ATM-mediated phosphorylation of p53 and formation of γ-H2AX foci following DSBs requires Cdk5.
As proline-directed serine/threonine kinase, Cdk5 and other cycle cell related Cdks share the same basic phosphorylation recognition motif. This raised the possibility that other Cdks may also regulate ATM directly. To test this, we re-confirmed the specificity of immunoprecipitation (Supplementary information, Fig. S1i) and showed that maximal activation of Cdk5 occurs 30 min after CPT exposure and precedes ATM activity, which peaks at 60 min (Fig. 4a), and γH2AX foci formation (Supplementary information, Fig. S2). In comparison, while CPT did activate Cdk2 and 6, their activation did not peak until 2 hr after treatment. Furthermore, in vitro kinase assays showed that neither purified Cdk2 nor Cdk6 phosphorylates ATM S794 under the experimental conditions when they robustly phosphorylated control substrate Histone H1 (Fig. 4b). Dominant negative Cdk2 and 6 had little effects on CPT-induced γ-H2AX foci formation in CGNs while dnCdk5 effectively reduced the number of γ-H2AX foci (Fig. 4c). The sequential activation of Cdk5, ATM and Cdk2/6 after DSBs suggests that Cdk2 and 6 do not participate in the initial activation of ATM in CGN model.
Previous studies showed that postmitotic neurons re-express cell cycle markers including Cdk2 and 6 in response to a variety of neuronal death signals including DNA damage8, 10. Our findings in Figure 3a suggested that Cdk5-mediated activation of ATM may be required for stress-induced cell cycle re-entry of postmitotic neurons. Analysis by Real-Time RT-PCR showed that CPT increased Cdk2 and 6 mRNA levels in CGNs while roscovitine clearly blocked such increases as early as 30 min after CPT, which coincides with the peak activity of Cdk5 and precedes significantly the activation of Cdk2 and 6 (Fig. 4a, d, e). This suggests that Cdk5 is responsible for DNA damage-induced re-expression of Cdk2 and 6. Cell cycle analysis by flow cytometry showed that CPT caused a significant number of postmitotic CGNs and differentiated SH-SY5Y neurons to re-enter S phase, which was reduced by roscovitine, Cdk5 RNAi, or ATMi [Fig. 4f, 4g. Supplementary information, Fig. S3A. ATM inhibitors WM and CA had the same effects (data not shown)]. We measured cell cycle using a second method, BrdU labeling, to confirm these findings (Supplementary information, Fig. S3B). Collectively, these results indicate that Cdk5-ATM regulates DNA damage-induced reactivation of cell cycle machinery in postmitotic neurons.
Transcription factor p53 is a key ATM substrate that mediates DNA damage response. Our findings that inhibition of Cdk5 reduces ATM-mediated phosphorylation of p53 suggest that Cdk5 may regulate p53 function via ATM in response to DNA damage. We assessed p53 activity by a luciferase reporter assay in a background without endogenous functional ATM (A-T cells) and in CGNs. Our data showed that DNA damage-induced activation of p53 in CGNs is dependent on Cdk5. ATM mediates Cdk5-induced full activation of p53 activity, which requires S794 but not S1981. These data and related text are included in Supplementary information, Fig. S4.
In response to DNA damage and ATM signaling, p53 activates the expression of many critical downstream target genes related to death including puma and bax25. Real-Time RT-PCR analysis showed that CPT leads to a significant increase in the mRNA levels of both puma and bax while roscovitine reduced puma and bax mRNA to near basal levels. Similar results were obtained for additional p53 targets such as PCNA25 (Supplementary information, Fig. S5a-c). Together, these findings indicate that Cdk5 is required for mediating CPT-induced ATM dependent expression of p53 target gene expression.
Our above findings suggest that phosphorylation of ATM by Cdk5 plays a key role in DNA damage-induced neuronal death. We tested this by performing WST-1 survival assays. Our data showed that CPT significantly reduces the survival of CGNs while roscovitine is sufficient to block CPT-induced death of CGNs dose-dependently (Fig. 5a). Roscovitine and KU-5593326 also reduced CPT-induced CGN death in a time-dependent manner (Fig. 5b). In agreement with these, inhibition of calpain also protected CGNs from CPT toxicity (Fig. 5c).
To corroborate these findings, we showed that both knocking down Cdk5 or ATM by RNAi and over-expressing the dnCdk5 or kdATM significantly attenuates CPT-induced neuronal death (Fig. 5d-e, Supplementary information, Fig. S5d). Furthermore, ATM mutant S794A effectively decreased CPT-induced neuronal death in differentiated SH-SY5Y cells as well (Fig. 6f). These findings indicate that phosphorylation of ATM at S794 by Cdk5 directly modulates CPT-induced death process in neurons.
Proper signaling the presence of DSBs requires conversion of the dormant ATM to its highly active form. Our study establishes phosphorylation of ATM at S794 by Cdk5 as a critical step in mediating DNA damage-induced ATM activation in postmitotic neurons. S794 phosphorylation precedes and is required for ATM autophosphorylation at S1981, indicating that S794 phosphorylation is a more upstream initiation event. How phosphorylation at S794 leads to ATM activation is not presently clear. It is conceivable that part of the subsequent changes following S794 phosphorylation may also involve regulation of intermolecular interactions of ATM molecules. Whether S794 phosphorylation regulates the recruitment of ATM to the site of DNA strand break by MRN complex 27-31 and participates DNA repair requires further investigation.
Our studies used postmitotic neurons as a model. Whether phosphorylation of ATM by Cdk5 is limited to neural tissue remains to be addressed. Since both Cdk5 and ATM are widely expressed by a variety of cell types including cancers, it raises the possibility that Cdk5-ATM pathway may be engaged following DNA damage in broad tissue types. Our findings that agents not known to induce DNA damage such as chloroquine or mild osmotic shock can also activate Cdk5-ATM pathway (Supplementary information, Fig. S6) suggests that Cdk5-mediated regulation of ATM may play a role in cellular response to a broad spectrum of signals.
Cdk5 and ATM are detected in both cytoplasmic and nuclear compartments in neurons. It is interesting to note that although CPT activates Cdk5 in both cytoplasm and nucleus, only nuclear ATM exhibits significant activation under the same condition. Similarly, KCl withdrawal induced robust activation of Cdk5 in the cytoplasm. But this failed to cause significant change of ATM in either the cytoplasm or nucleus. Thus, it appears that under the experimental conditions tested, Cdk5 preferentially activates ATM in the nucleus. This may constitute at least part of a process allowing cells to critically distinguish stress signals.
Cerebellar granule neurons (CGNs) were cultured from Long Evans rats at postnatal day 6 or 7 on plates coated with poly-L-lysine (Sigma- Aldrich) 17. CGNs were maintained in Basal Medium Eagle containing 10% dialyzed FBS, 25 mM KCl, 0.1 mg/ml gentamycin, 2 mM glutamine, and 25 mM glucose. Treatments or transfection performed 7 days after plating. HEK293 cells and AT cells (GM05849C cell line, a human ATM-deficient fibroblasts) were cultured in DMEM supplemented with 10% FBS. Human SH-SY5Y cells were cultured in DMEM/F-12 supplemented with 10% FBS, and differentiated with 10 μM all-trans-retinoic acid (Sigma-Aldrich) for 6 days and then 2nM brain-derived neurotrophic factor (BDNF, Sigma-Aldrich) for another 2 days as described previously 32.
Roscovitine, wortmannin, caffeine, calcium ionopore, glutamate were purchased from Sigma-Aldrich. H2O2 and Hoechst 33258 were from Fisher Scientific. KU-55933 was provided by KuDos. AK-295 was kindly provided by Dr. Jonathan D. Glass. For immunoblotting, rabbit polyclonal anti-p53, mouse monoclonal anti-phospho-p53 (Ser15), rabbit polyclonal anti-actin, rabbit polyclonal anti-Cdk2, and mouse monoclonal anti-Cdk6 are all from Cell signaling; mouse monoclonal anti-ATM is from Sigma-Aldrich; rabbit polyclonal anti-ATM from Calbiochem; rabbit polyclonal anti-p35 (N-20) from Santa Cruz; and mouse monoclonal anti-Cdk5 from Neomakers. For Immunofluorescent staining, mouse monoclonal anti-γH2AX are from Upstate Biotechnology; rabbit polyclonal anti-GFP from Abcam and fluorescent secondary antibodies from Jackson ImmunoResearch.
Rabbit polyclonal phosphospecific antibody against ATM Ser794 was produced by 21st Century Biochemicals with a synthetic 16mer phosphopeptide (KLH-coupled) corresponding to residues surrounding Ser794 of human ATM (Acetyl-CLSNATKpSPNKIASG-amide).
Constructs encoding GST-fusion ATM fragments were kindly provided by Dr. Martin L. Lavin33. A plasmid encoding an N-terminal Flag-tagged ATM protein was a kind gift of Dr. Michael Kastan34. Serine 794 and 1981 mutation to alanine (SA) or glutamine (SD) was performed using QuikChange site-directed II mutagenesis kit (Stratagene). Recombinant GST-fusion proteins were induced and purified from BL21 (DE3) competent cells (Invitrogen).
Primary CGNs were transceintly transfected using Calcium Phosphate ProFection Mammalian Transfection system (Promega) according to the manufacturer’s instructions. HEK293 and A-T cells were transfected using Lipofectamine 2000 reagents (Invitrogen). Drug treatments were performed 24 hours after transfection.
Human CDK5, ATM and control RNAi Adenovirus were purchased from Millipore. Viruses were added to SH-SY5Y cells after RA-BDNF differentiation. Treatments were performed 24 hours after infection.
To construct the 19 nucleotide (corresponding to rat Cdk5 nt positions 639-657) 35 hairpin siRNA cassettes, two complementary DNA oligonucleotides were chemically synthesized in Invitrogen, annealed, and inserted into pSUPER shuttle vector immediately downstream of the H1 RNA pol III promoter: 5’-GATCCCC- GAGGATCTTTCGACTGCTA-TTCAAGAGA-TAGCAGTCGAAAGATCCTC-TTTTTGGAAA-3 ’ a n d 5 ’-AGCTTTTCCAAAAA-GAGGATCTTTCGACTGCTA-TCTCTTGAA- TAGCAGTCGAAAGATCCTC-GGG-3’. The RNAi cassettes with H1 promoter were amplified by PCR and subcloned into pFUGW transfer vector 36. Lentiviruses were produced in HEK293T cells by cotransfection of pFUGW-CDK5i, packaging plasmid p8.91, vesicular stomatitis virus (VSV) envelope expression plasmid (pMD.G). Lentiviruese were concentrated and tittered on HEK293T cells. Serial dilutions of the virus were test to get the highest infectivity. Lentiviruses were applied to CGNs at the 4th day in vitro (DIV) and treatments were performed 72 hours after infection.
Cytoplasmic and nuclear fractionation was performed using EZ Nuclei Islation Kit (Sigma-Aldrich) according to manufacturer’s protocol, which involves three cycles of thorough cell lysis and washing. Only the first cytoplasmic lysate and the last nuclear lysate were kept and used in experiments.
Lysates were generally prepared with NP-40 lysis buffer (20 mM Tris, pH 8.0, 135 mM NaCl, 1 mM MgCl2, 0.1 mM CaCl2, 10% glycerol, 1% NP-40, 0.1 mM Na3VO4, 50 mM ß-glycerol phosphate, 10 mM NaF, and protease inhibitors from Roche). Fifty to one hundred micrograms of protein were used for immunoblotting by SDS-PAGE analysis and one hundred to five hundred micrograms of protein were used for immunoprecipitation to incubate with 1 to 2 μg corresponding antibody at 4°C overnight. The immune complexes were collected with protein G Plus/Protein A-Agarose (Calbiochem) and washed three times with NP-40 lysis buffer.
Active Cdk5/p25, Cdk2/cyclin A, or Cdk6/cyclin D3 kinases were purchased from Upstate Biotechnology. In vitro Cdk kinase assay were performed following the manufacturer’s instructions. Briefly, two microgram purified ATM or GST-ATM4 fragment recombinant proteins were incubated with active Cdk kinase in Cdk kinase buffer (8 mM MOPS/NaOH pH7.0, 200 nM EDTA) containing 20 μM ATP, 10 μCi of [γ-32P]-ATP for 15 min at 30°C. To determine kinase activity of ATM or Cdks in cells, corresponding immunoprecipitates were washed twice with either ATM kinase buffer (50 mM HEPES, pH 7.5, 150 mM NaCl, 4 mM MnCl2, 10% glycerol, 1 mM dithiothreitol, and 100 μM Na3VO4) or Cdk kinase buffer, and incubated in the kinase buffer containing [γ-32P]-ATP and 1 μg of substrate [PHAS-I for ATM kinase assay (Calbiochem) and histone H1 for Cdk kinase assay (Upstate)] for 30 min at 30°C. The reaction was stopped with SDS sample buffer and 5 min boiling. Phosphorylation of substrates after SDS-PAGE was analyzed by autoradiography.
Cells were harvested by trypsinization, fixed, and stained with propidium iodide. Total DNA content was analyzed on a FACSCalibur flow cytometer (Becton Dickinson) using the programs BD CompBeads plus BD FACSDiva and FlowJo.
WST-1 assay (Roche) was performed following the manufacturer’s instructions. Briefly, cells were cultured in 24 well plates. After treatments, incubate cells with WST-1 substrate, a ready-to-use substrate which measures the metabolic activity of viable cells, for 1–4 hours, followed by spectrophotometric assay of colored product. Single-cell survival assays were performed using Viability/Cytotoxicity Kit (Invitrogen). Briefly, CGNs were stained with ethidium homodimer-1 (EthD-1) without permeabilization. GFP-positive cells with or without EthD-1 staining were counted using Olympus IX51 fluorescence microscope in a blind manner. Two hundred or more transfected cells were counted for each treatment. Rates of dead CGNs were calculated as percentage of cells with co-localization of EthD1 and GFP in the total number of GFP-positive cells counted.
Transient transfections were performed by the calcium phosphate transfection method (Sigma-Aldrich) as described by the manufacturer with PG13pyLuc (wild-type p53 reporter) and its corresponding mutant MG13pyLuc (mutant p53 reporter) 37. The amount of DNA transfected was equalized by addition of a control vector. Treatments were performed 24 h posttransfection. Cell lysates were analyzed using Enhanced Luciferase Assay Kit (BD Bioscience) according to the manufacturer’s instruction.
CGNs were cultured on cover glasses coated with poly-L-lysine. Cells were fixed with 3.7% neutral buffered formalin and permeabilized with 0.3% Triton X-100 in PBS, blocked in 5% normal goat serum, and were stained with anti-γH2AX or anti-GFP followed by corresponding fluorescent secondary antibodies (Jackson ImmunoResearch). Cells were counterstained with Hoechst 33258, mounted with Vectashield mounting medium (Vector Laboratories), and then analyzed with an Olympus BX51 fluorescence microscope. Numbers of γ-H2AX foci in two hundred or more CGNs were counted.
For real-time RT-PCR, the total RNA was extracted using TRIzol reagent (Invitrogen). Total RNA (1 μg) of each sample was reversely transcribed using SuperScript™ First-Strand Synthesis SuperMix for qRT-PCR (Invitrogen) in a 20 ml volume. The amplification was carried out in a total volume of 50 μL containing 1ml of each primer, 25 ml Platinum SYBR Green qPCR SuperMix-UDG (invitrogen) 1ml ROX reference dye, and 2 μL of 1:10 diluted cDNA using ABI PRISM 7000 Real-Time PCR Systems according to the manufacturer’s instructions. In each experiment, the 18S ribosome RNA (18S rRNA) was amplified as a reference standard. Primer sets were designed using OligoPerfect program (Invitrogen). 18S rRNA primers were 18S rRNA-F, 5’-AACGGCTACCACATCCAA-3 ’ ; 1 8 S r R N A-R, 5’-GACTCATTCCAATTACAGGGC-3’. Other target gene primers were BAX-F, 5’-TGTTGCTGATGGCAACTTC-3’; BAX-R, 5 ’-GATCAGCTCGGGCACTTTAG-3’; PUMA-F, 5 ’- GCGGAGACAAGAAGAGCAAC-3 ’ ; P U M A-R, 5 ’-CTCCAGGATCCCTGGGTAAG-3’; Cdk2-F, 5’- CATTCCTCTTCCCCTCATCA-3’; Cdk2-R, 5 ’-TACCAGAGGGTCACCACCTC-3 ’ ; C d k 6-F, 5 ’-TGTTTCAGCTTCTCCGAGGT-3’; Cdk6-R, 5’-GGCTTTCTGCGAAACAGTTC-3’; p21-F, 5 ’- TGGACAGTGAGCAGTTGAGC-3 ’ ; P 2 1-R, 5 ’-ACACGCTCCCAGACGTAGTT; PCNA-F, 5’- TTGGAATCCCAGAACAGGAG-3’; PCNA-R, 5’- AGAAAACTTCACCCCGTCCT-3’. All PCR reactions were performed in duplicate. Standard curves (cycle threshold values versus template concentration) were prepared for each target gene and for the endogenous reference (18S rRNA) in each sample. To confirm the specificity of the PCR reaction, PCR products were electrophoresed on a 1.2% agarose gel.
The results were analyzed using by two-tailed Student’s T test where appropriate.
We thank Drs. Bert Vogelstein, Richard Gatti, Michael Kastan, Martin Lavin, and Shuki Mizutani for p53 reporter constructs, purified ATM protein, ATM mammalian expression constructs, GST-ATM expression constructs, and A-T cell lines, respectively. This work was partially supported by NIH grants R01 NS048254 (Z. M.) and R01 AG023695 (Z. M.) and The Robert W. Woodruff Health Sciences Center Fund (Z. M.).
AUTHOR CONTRIBUTION Z. M. coordinated the overall project and planned the original experimental design. B. T. and Q. Y. conducted the experiments. Z. M. and B. T. analyzed the data and wrote the paper.
COMPETING FINANCIAL INTERESTS The authors declare that they have no competing financial interests.