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First published online June 25, 2007
doi: 10.1242/10.1242/jcs.03468

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Stabilization and activation of p53 induced by Cdk5 contributes to neuronal cell death

Department of Life Science, Division of Molecular and Life Science, Systems-Biodynamics NCRC, Pohang University of Science and Technology, Pohang, 790-784, Republic of Korea

View larger version (29K): [in this window] [in a new window]   Fig. 1. Cdk5 regulates expression of the p53 tumor suppressor protein in response to genotoxic and oxidative stress. (A) SH-SY5Y cells were treated with 5 µM mitomycin C or 2 mM SNP for the indicated times, and cell lysates were subjected to immunoblotting with the indicated antibodies. (B) SH-SY5Y cells were transfected with Cdk5 targeting or scrambled control siRNA, treated with 5 µM mitomycin C or 2 mM SNP for 9 hours, and then subjected to immunoblotting with the indicated antibodies. (C) SH-SY5Y cells were pretreated with 10 µM roscovitine or Cdk2/5 inhibitor for 30 minutes and then incubated with 5 µM mitomycin C or 2 mM SNP for 9 hours. Cell lysates were subjected to immunoblot analysis with the indicated antibodies. (D) SH-SY5Y cells were treated with 5 µM mitomycin C for 6 hours and then incubated with 60 µg/ml cycloheximide for indicated times. Cdk2/5 inhibitor was added 30 minutes before the incubation with cycloheximide. (i) The amounts of p53 and GAPDH were determined by immunoblotting. (ii) Protein levels were quantified. (E) SH-SY5Y cells were treated as described in C for the indicated times, total RNA was isolated and quantitative real-time RT-PCR was performed. The results are from three independent experiments and are presented as the mean ± s.d. Each experiment was performed in triplicate.

View larger version (40K): [in this window] [in a new window]   Fig. 2. Cdk5 activity increases in the nucleus, and Cdk5 interacts with p53. (A) SH-SY5Y cells were treated with 5 µM mitomycin C for the indicated times, and nuclear fractions were subjected to immunoblotting. Cdk5 kinase activity was determined by autoradiography using histone H1 as a substrate. (B) SH-SY5Y cells were transfected with a vector encoding Flag-Cdk5 and then treated with 5 µM mitomycin C or vehicle for 6 hours. Cell lysates were immunoprecipitated with antibodies against p53 or control IgG. Membranes were immunoblotted with anti-Flag and/or anti-p53 antibodies, with 5% of the cell lysates used as input controls. (C) Lysates from SH-SY5Y cells treated with 5 µM mitomycin C for 3 hours were used for pulldown with GST-p53 or GST, and probed with antibodies against Cdk5 or GST. (D) H1299 cells were transfected with vectors encoding Flag-Cdk5, His-p35 or p53 as indicated. Cell lysates were immunoprecipitated with antibodies against Cdk5 or control IgG, and then immunoblotted with anti-p53, anti-His and/or anti-Flag, with 5% of the cell lysates used as input controls.

View larger version (35K): [in this window] [in a new window]   Fig. 3. Cdk5 stabilizes p53 in the presence of Hdm2. (A) H1299 cells were transiently co-transfected with a fixed amount of vectors encoding p53/Hdm2/Cdk5 (0.5 µg/1.5 µg/2 µg) and the various amounts of vector encoding p25 (0, 2 and 4 µg). Cell lysates were subjected to immunoblotting with the indicated antibodies. (B) In similar experiments as described in A, cells were transfected with a fixed amount of p53/Hdm2/p25 (0.5 µg/1.5 µg/2 µg) as indicated. For the inhibition of Cdk5, cells were transfected with Cdk5(D144N) instead of Cdk5 as indicated. (C) Mouse embryonic fibroblasts derived from double-knockout mice (p53–/–/Mdm2–/–) were transiently co-transfected with a fixed amount of vectors encoding p53/Cdk5 (0.5 µg/2 µg) and the various amounts of vector encoding p25 (0, 2 and 4 µg). Cell lysates were subjected to immunoblotting with the indicated antibodies. (D,E) H1299 cells were transfected with the indicated combinations of vectors encoding p53 (0.5 µg), Hdm2 (1.5 µg), Cdk5-D144N (2 µg) or p35/p25 (2 µg). 20 µM MG132 was added 36 hours after transfection for 8 hours. (i) Total p53 was immunoprecipitated with a polyclonal antibody and detected with a monoclonal antibody. The ubiquitylated form of p53 is indicated. EGFP was used as the transfection control. (ii) For detecting the association of p53 with Hdm2, cell lysates were immunoprecipitated with anti-p53 and then immunoblotted with anti-Hdm2, with 5% of the cell lysates used as input controls.

View larger version (32K): [in this window] [in a new window]   Fig. 4. Cdk5 activity mediates efficient nuclear accumulation of p53. (A) SH-SY5Y cells were pretreated with 10 µM Cdk2/5 inhibitor or an equal volume of vehicle for 30 minutes and then incubated with 5 µM mitomycin C for 6 hours. (i) Cells were double-labeled with Hoechst (blue) and anti-p53 (red) and then analyzed by fluorescence microscopy. (ii) Percentage of cells showing cytoplasmic distribution of p53. Data are the mean ± s.d. from three independent experiments. *P<0.0001. (B) SH-SY5Y cells were transfected with Cdk5-targeting or scrambled control siRNA, and then incubated with 5 µM mitomycin C for 6 hours. (i) Nuclear and cytoplasmic fractions were prepared as described in the Materials and Methods, and subjected to immunoblotting with the indicated antibodies. Lamin B1 and -tubulin were used as nucleus- and cytoplasm-specific controls, respectively. (ii) The results displayed in the bar graphs represent quantitatively analyzed data from three independent experiments and are given as the mean ± s.d. For quantitative analysis, each blot was normalized with respect to the internal controls, lamin B1 and -tubulin. *P<0.05.

View larger version (28K): [in this window] [in a new window]   Fig. 5. Cdk5 activity induces phosphorylation of p53 at residues Ser15, Ser33 and Ser46. (A) GST-p53 protein was incubated with active Cdk5-p35 recombinant protein in a kinase reaction buffer, and phosphorylation of specific residues was determined by western blot analysis using the indicated phosphorylation-specific antibodies. The level of GST-p53 was assessed by immunoblotting with anti-GST. (B) SH-SY5Y cells were treated with 5 µM mitomycin C or 2 mM SNP for the indicated times, and cell lysates were subjected to western blotting with the indicated antibodies. (C) SH-SY5Y cells were transfected with Cdk5-targeting or scrambled control siRNA, and then treated with 5 µM mitomycin C or 2 mM SNP for 9 hours in the presence of 20 µM MG132. Total proteins were subjected to western blotting using the indicated antibodies. (D) SH-SY5Y cells were transiently transfected with mock vector or vectors encoding Cdk5-p35, Cdk5-p25 or Cdk5(D144N)-p25, and the amounts of the indicated proteins were determined by immunoblotting. (E) SH-SY5Y cells were transfected with Cdk5-targeting or scrambled control siRNA, treated with 15 µM wortmannin for 1 hour, and then treated with 5 µM mitomycin C for 6 hours in the presence of 20 µM MG132. (i) The amounts of site-specific p53 phosphorylation were determined by immunoblotting. (ii) The results displayed in the bar graphs represent quantitatively analyzed data from three independent experiments, and are given as the mean ± s.d. For the quantitative analysis, each blot was normalized with respect to the level of total p53. *P<0.05. (F) SH-SY5Y cells were transfected with Cdk5-targeting or scrambled control siRNA, treated with 15 µM wortmannin for 1 hour, and then treated with 5 µM mitomycin C for 6 hours. The amounts of p53 and GAPDH were determined by immunoblotting.

View larger version (36K): [in this window] [in a new window]   Fig. 6. Cdk5 induces p53 stabilization by phosphorylation. (A) GST-p53 or GST-p53-SDM (a mutant p53 with alanine substituted for Ser15, Ser33 and Ser46) proteins were incubated with active Cdk5-p35 recombinant protein in a kinase reaction buffer, with roscovitine added to inhibit Cdk5 activity. Levels of p53 phosphorylation and Cdk5 kinase activity were examined by autoradiography, and the amount of GST-p53 was assessed by Coomassie Blue staining. (B) H1299 cells were transfected with the indicated plasmids, as described in Fig. 3D,E. At 36 hours post-transfection 20 µM MG132 was added and cultures were incubated for an additional 8 hours. (i) Total p53 was immunoprecipitated with a polyclonal antibody and detected with a monoclonal antibody. The ubiquitylated form of p53 is indicated. EGFP was used as the transfection control. (ii) To detect the association of p53 with Hdm2, cell lysates were immunoprecipitated with anti-p53 and then immunoblotted with anti-Hdm2, with 5% of the cell lysates used as input controls. (C) SH-SY5Y cells were transfected with Cdk5-targeting or scrambled control siRNA, and then treated with 5 µM mitomycin C or an equal volume of vehicle for 8 hours in the presence of 20 µM MG132. (i) Total p53 was immunoprecipitated with a polyclonal antibody and detected with a monoclonal antibody. The ubiquitylated form of p53 is indicated, and 5% of the cell lysates was used as input controls. (ii) For detecting the association of p53 with Hdm2, cell lysates were immunoprecipitated with anti-p53 and then immunoblotted with anti-Hdm2, with 5% of cell lysates used as input controls.

View larger version (43K): [in this window] [in a new window]   Fig. 7. Cdk5 induces acetylation of p53 and its cooperation with p300. (A) SH-SY5Y cells were treated with 5 µM mitomycin C or 2 mM SNP for the indicated times. The amounts of acetyl-p53 () and GAPDH were determined by immunoblotting. (B) SH-SY5Y cells were transfected with Cdk5-targeting or scrambled control siRNA, and then treated with 5 µM mitomycin C or 2 mM SNP for 9 hours in the presence of 20 µM MG132. Total proteins were subjected to western blotting using the indicated antibodies. (C) SH-SY5Y cells were transiently transfected with vectors encoding Flag-p53-WT or Flag-p53-SDM (a mutant p53 substituted with alanine at Ser15, Ser33 and Ser46), and then treated with 5 µM mitomycin C or 2 mM SNP for 9 hours. Total proteins were subjected to western blotting using the indicated antibodies. (D) SH-SY5Y cells were transfected with vectors encoding Flag-p53-WT or Flag-p53-SDM and then treated with 5 µM mitomycin C or vehicle for 6 hours. Cell lysates were immunoprecipitated with antibodies against Flag-p53, and membranes were immunoblotted with anti-p300 and/or anti-Flag, with 5% of the cell lysates used as input controls.

View larger version (42K): [in this window] [in a new window]   Fig. 8. Cdk5-mediated p53 transactivation induces mitochondria-mediated neuronal apoptosis in response to cellular stress. (A) SH-SY5Y cells were transfected with Cdk5-targeting or scrambled control siRNA, and then treated with 5 µM mitomycin C for 9 hours. Total RNA was isolated, and quantitative real-time RT-PCR was performed. The presented results are from three independent experiments and are given as the mean ± s.d. Each experiment was performed in triplicate. *P<0.005. (B) SH-SY5Y cells were pretreated with 10 µM Cdk2/5 inhibitor or an equal volume of vehicle for 30 minutes and then incubated with 5 µM mitomycin C for 12 hours. Cells were triple-labeled with Hoechst (blue), anti-cytochrome C (red) and MitoTracker (green) and then analyzed by fluorescence microscopy. (C) SH-SY5Y cells were transiently transfected with vectors encoding EGFP-Cdk5(D144N) or EGFP, treated with 5 µM mitomycin C or 2 mM SNP for 12 hours and fixed and stained with antibodies against cleaved caspase-3. (i) Representative image of EGFP-Cdk5(D144N)-positive cells. (ii) Percentage of EGFP- or EGFP-Cdk5(D144N)-expressing cells positive for cleaved caspase-3. The data represent the mean ± s.d. from three independent experiments. *P<0.0001. (D) SH-SY5Y cells were transiently transfected with mock vector or vectors encoding Cdk5(D144N), and then treated with 5 µM mitomycin C or 2 mM SNP for 12 hours. Cell lysates were subjected to immunoblotting with the indicated antibodies. (E) SH-SY5Y cells were transiently transfected with mock vector or vectors encoding Cdk5(D144N), and then treated with 5 µM mitomycin C, 2 mM SNP or an equal volume of vehicle. Cell viability was measured 24 hours later by MTT assay. The data represent the mean ± s.d. from quadruplicate determinations. Similar results were obtained in three independent experiments. *P<0.0001. (F) Proposed model for Cdk5-mediated regulation of p53 and subsequent mitochondria-mediated apoptosis.