(a) Phosphorylation of purified ATM protein by Cdk5/p25 in vitro. (b) Phosphorylation of ATM by Cdk5 at S794 in vitro. Purified recombinant GST-ATM4 (aa772 ~aa1102) and S794 to Alanine mutant (GST-ATM4-S794A) were phosphorylated by purified Cdk5/p25 in vitro (top panel). The same membrane was probed with α-GST antibody as loading control (bottom panel). (c) Characterization of phosphospecific ATM S794 antibody. GST-ATM4 and GST-ATM4-S794A either unphosphorylated or phosphorylated with cold ATP by purified Cdk5/p25 in vitro were probed with preimmune serum (top panel) and ATM S794 phosphospecific antibody (middle panel). Coomassie blue staining of substrate shows loading control (bottom panel). (d) Phosphorylation of ATM by Cdk5 at S794 in cells. Cdk5/p25 and ATM [wild type (wt) or S794A, S1981A, S794A/S1981A (AA) mutants] were transfected into HEK293 cells as indicated. Phospho-S794 levels in the lysates were determined by immunoblotting using phospho-specific antibody (top panel). The same membrane was re-probed for total ATM (bottom panel).

(a) Activation of ATM by Cdk5 mediated phosphorylation. HEK293 cells were transfected with Cdk5/p25 and ATM (wt, S794A and S794D mutants) as indicated. After 24 hours, ATM kinase activity was measured. The bottom panel shows the equal expression of ATM used in the lysates. The numbers are relative values with control set to one (same hereafter). (b) Activation of endogenous ATM by Cdk5/p25 or Cdk5/p35. Cdk5, p25 and p35 were overexpressed in HEK293 cells as indicated. After 24 hours, the levels of overexpressed proteins (top two panels) and ATM kinase activities (bottom panel) were determined. (c) Activation of Cdk5 and ATM by DNA damage. CGNs were treated with 10 μM camptothecin (CPT), 10 μM etoposide (Etop), 100 μg ml-1 bleomycin (Bleo), or 2 μM stausporine (STS) for 1 hour. Cdk5 and ATM kinase activity were measured using Histone H1 and PHAS-I as substrate, respectively. (d) CPT-induced p35 degradation. CGNs were treated with 10 μM CPT for the indicated periods of time. Level of cleaved α-fodrin, p35, p25 and Cdk5 were measured by immunoblotting. (e) Effects of inhibiting calpain on DNA damage-induced Cdk5 and ATM activation. CGNs were treated with or without 50 μM calpain inhibitor AK295 for 30 min and then with 10 μM CPT for the indicated periods of time. Cytoplamic and nuclear lysates were used for the determination of the levels of p35 and p25, and Cdk5 and ATM kinase activities. The membranes were reprobed for c-Raf, NeuN and actin as cytoplasmic fraction, nuclear fraction and loading controls, respectively.

(a) Inhibition of CPT-induced ATM phosphorylation at S794 and S1981 by roscovitine. Neurons differentiated from SH-SY5Y cells were pretreated with or without 10 μM roscovitine (Ros) for 30 min and followed with 10 μM CPT for the indicated periods of time. Phospho-S794, phospho-S1981 and total ATM were determined by immunoblotting. (b) Inhibition of CPT-induced ATM phosphorylation at S794 and S1981 by knocking down Cdk5. Neurons differentiated from SH-SY5Y cells were infected with control or Cdk5 RNAi adenovirus for 24 hours, and then treated with 10 μM CPT for the indicated periods of time. Cdk5, phospho-S794, phospho-S1981, total ATM and actin were determined by immunoblotting. (c) Inhibition of CPT-induced ATM activation and p53 phosphorylation by roscovitine. CGNs were treated with or without 10 μM Roscovitine for 30 min and then with 10 μM CPT treatment for 1 hour. ATM kinase activity, phospho-S15, total p53 and actin were measured in the same set of lysates. (d) Inhibition of CPT-induced ATM activation and p53 phosphorylation by knocking down Cdk5. CGNs were infected with Cdk5i or scrambled RNAi lentivirus for 72 hours and then treated with 10 μM CPT treatment for 1 hour. Cdk5 and ATM kinase activities and levels for Cdk5, phospho-S15, total p53 and actin were measured in the same set of lysates. (e) Reduction of CPT-induced γ-H2AX foci formation by roscovitine. CGNs were treated with or without 10 μM Roscovitine (Ros) for 30 min and then exposed to 10 μM CPT for 1 hour. γ-H2AX (green) was detected by immunocytochemistry, and nuclei were labeled with Hoechst (blue). The average numbers of foci/cell counted blindly are control, 0.26; CPT, 3.91; CPT+Ros, 1.08 (p< 0.001). (f) Reduction of CPT-induced γ-H2AX foci formation by knocking down Cdk5. CGNs were infected with CDK5 RNAi or control lentivirus for 72 hours and then exposed to 10 μM CPT for 1 hour. γ-H2AX (red) and GFP (green) were detected by immunocytochemistry, and nuclei were labeled with Hoechst (blue). The scale bars represent 10 μm. The average numbers of foci/cell counted blindly are control, 0.21; control+CPT, 3.00; Cdk5i, 0.13; Cdk5i+CPT, 1.22 (p< 0.001).

(a) Kinetics of CPT-induced activation of Cdks and ATM. CGNs were treated with 10 μM CPT for the indicated periods of time. Cdk5, ATM, Cdk2 and Cdk6 kinase activities were measured following immunoprecipitation. The lower graph is the quantification of various kinase activities. Data were shown as mean ± SD (n = 3. All Ns represent independent experiments throughout). (b) Phosphorylation of ATM by Cdks in vitro. Purified Cdk2, 5, and 6 were incubated with the recombinant GST-ATM4 protein in vitro in the presence of [γ-³²P]-ATP under manufacturer’s recommended conditions (top panel) or with the standard control substrate Histone H1 (bottom panel). (c) The effects of dominant negative (dn) Cdks on CPT-induced γ-H2AX foci formation. CGNs were transfected with constructs for GFP and dnCdks. After 24 hours, cells were treated with 10 μM CPT for 1 hour. γ-H2AX (red) and GFP (green) were detected by immunocytochemistry, and nuclei were labeled with Hoechst (blue). The scale bar represents 5 μM. The average number of foci counted blindly are: GFP control, 0.22; CPT+GFP, 4.81; CPT+dnCdk2, 4.56; CPT+dnCdk5, 3.25, p<0.001; and CPT+dnCdk6, 4.44. (d, e) Effects of Cdk5 inhibition on the levels of Cdk2 and 6 transcripts. CGNs were treated with 10 μM CPT for the indicated times with or without 10 μM roscovitine. Cdk2 and Cdk6 mRNA levels were quantified by qRT-PCR. Data are shown as mean ± SD (n = 3). (f) Inhibition of CPT-induced cell cycle re-entry by roscovitine. CGNs were treated by 10 μM CPT with or without 10 μM roscovitine (Ros) for 8 hours. The percentages of S phase neurons were measured by flow cytometry. (g) Inhibition of CPT-induced cell cycle re-entry by knocking down Cdk5. CGNs were infected with Cdk5 RNAi or control lentivirus for 72 hours. The infected cells were treated by 10 μM CPT for 8 hours. The percentages of S phase neurons were measured by flow cytometry. For both f and g, data are shown as mean ± SD (n = 3. *, P< 0.05. **, P<0.01).

(a) Inhibition of CPT-induced neuronal death by roscovitine. CGNs were exposed to 10 μM CPT and different concentrations of roscovitine for 24 hours. Neuronal viability was measured by WST-1 assay. (b) Time dependent effect of roscovitine on CPT-induced neuronal death. CGNs were treated 10 μM CPT alone or with 10μM roscovitine (Ros) or 10 μM Ku-55933 (KU) for the different periods of time. WST-1 data are mean ± SD (n =4. **, p < 0.01). (c) Inhibition of CPT-induced neuronal death by calpain inhibitor AK295. CGNs were exposed to 10 μM CPT for 24 hours with 50 μM AK295. Neuronal viability was measured by WST-1 assay. (d) Effect of knocking down Cdk5 on CPT- induced neuronal death. CGNs were infected with Cdk5 RNAi or control lentivirus for 72 hours and then treated by 10 μM CPT for 24 hours. Neuronal viability was measured by WST-1 assay. WST-1 data are mean ± SD (n =4. *, p < 0.05; **, p < 0.01). (e) Effect of inhibiting Cdk5 or ATM on CPT-induced neuronal death. CGNs were co-transfected with vectors for GFP and dnCdk5 or kinase-dead ATM (kdATM) as indicated. After 24 hours, cells were treated by 10 μM CPT for 24 hours, and stained by EthD1. Cell death was scored blindly and calculated as described in methods. (f) Effect of ATM-S794A on CPT-induced neuronal death. Neurons differentiated from SH-SY5Y cells were co-transfected with vectors for GFP and ATM-S794A as indicated. Following CPT treatment, single cell survival assay was performed as described in (e). For e and f, data are shown as mean ± SD (n=4. ** p<0.01).