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The cyclin-dependent kinase Cdk2 regulates thymocyte apoptosis.

Hakem A, Sasaki T, Kozieradzki I, Penninger JM - J. Exp. Med. (1999)

Bottom Line: Aberrant activation of cell cycle molecules has been postulated to play a role in apoptosis ("catastrophic cell cycle").Cdk2 was found to function upstream of the tumor suppressor p53, transactivation of the death promoter Bax, alterations of mitochondrial permeability, Bcl-2, caspase activation, and caspase-dependent proteolytic cleavage of the retinoblastoma protein.These data provide the first evidence that Cdk2 activity is crucial for the induction of thymocyte apoptosis.

View Article: PubMed Central - PubMed

Affiliation: The Amgen Institute, Department of Medical Biophysics, University of Toronto, Ontario, Canada M5G 2C1.

ABSTRACT
Aberrant activation of cell cycle molecules has been postulated to play a role in apoptosis ("catastrophic cell cycle"). Here we show that in noncycling developing thymocytes, the cyclin- dependent kinase Cdk2 is activated in response to all specific and nonspecific apoptotic stimuli tested, including peptide-specific thymocyte apoptosis. Cdk2 was found to function upstream of the tumor suppressor p53, transactivation of the death promoter Bax, alterations of mitochondrial permeability, Bcl-2, caspase activation, and caspase-dependent proteolytic cleavage of the retinoblastoma protein. Inhibition of Cdk2 completely protected thymocytes from apoptosis, mitochondrial changes, and caspase activation. These data provide the first evidence that Cdk2 activity is crucial for the induction of thymocyte apoptosis.

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P53 is a substrate for Cdk2 activity. (A) Phosphorylation of p53 by Cdk2. p53 phosphorylation was determined after γ-irradiation (500 rads), dexamethasone treatment  (1 μM), or no treatment (Control). Thymocytes were cultured and harvested as described in the legend to Fig. 1. Cdk2  was immunoprecipitated, and Cdk2 activity was determined  using recombinant human p53 as the substrate. The levels of  phosphorylated p53 (top), p53 protein (middle), and Cdk2  protein (bottom) are shown. (B) Coimmunoprecipitation of  p53 and Cdk2. Thymocytes were harvested 2 h after γ-irradiation (500 rads) or after 2 h culture without a death stimulus (Control). Western blot analysis was performed after immunoprecipitation (IP) using an anti-Cdk2 Ab and blotting with anti-p53 and anti-Cdk2.  Similar results were obtained using p53 immunoprecipitations. One result representative of three independent experiments is shown. (C) Levels of p53 protein. Thymocytes were cultured for 2 h after γ-irradiation  (500 rads) or the absence of a death stimulus (Control) in the presence or absence of roscovitine (50 μM,  Rosco). Cells were lysed, and extracts were analyzed by Western blotting using anti-p53 and anti–Bcl-XL  Abs. One result representative of three independent experiments is shown. (D) Induction of Bax mRNA.  Thymocytes were cultured as described in B and harvested after 3 h. 10 μg total RNA was Northern blotted and hybridized to probes for both Bax and β-actin (loading control). Hybridization with the Bax probe  shows two alternatively spliced RNA transcripts of 1.5 and 1.0 kb. The increase in Bax mRNA induced by  γ-irradiation is abolished in the presence of roscovitine (50 μM).
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Figure 5: P53 is a substrate for Cdk2 activity. (A) Phosphorylation of p53 by Cdk2. p53 phosphorylation was determined after γ-irradiation (500 rads), dexamethasone treatment (1 μM), or no treatment (Control). Thymocytes were cultured and harvested as described in the legend to Fig. 1. Cdk2 was immunoprecipitated, and Cdk2 activity was determined using recombinant human p53 as the substrate. The levels of phosphorylated p53 (top), p53 protein (middle), and Cdk2 protein (bottom) are shown. (B) Coimmunoprecipitation of p53 and Cdk2. Thymocytes were harvested 2 h after γ-irradiation (500 rads) or after 2 h culture without a death stimulus (Control). Western blot analysis was performed after immunoprecipitation (IP) using an anti-Cdk2 Ab and blotting with anti-p53 and anti-Cdk2. Similar results were obtained using p53 immunoprecipitations. One result representative of three independent experiments is shown. (C) Levels of p53 protein. Thymocytes were cultured for 2 h after γ-irradiation (500 rads) or the absence of a death stimulus (Control) in the presence or absence of roscovitine (50 μM, Rosco). Cells were lysed, and extracts were analyzed by Western blotting using anti-p53 and anti–Bcl-XL Abs. One result representative of three independent experiments is shown. (D) Induction of Bax mRNA. Thymocytes were cultured as described in B and harvested after 3 h. 10 μg total RNA was Northern blotted and hybridized to probes for both Bax and β-actin (loading control). Hybridization with the Bax probe shows two alternatively spliced RNA transcripts of 1.5 and 1.0 kb. The increase in Bax mRNA induced by γ-irradiation is abolished in the presence of roscovitine (50 μM).

Mentions: Therefore, we tested whether p53 is a target for Cdk2 activity during thymocyte apoptosis after γ-irradiation. In vitro kinase assays using immunoprecipitated Cdk2 from γ-irradiated and dexamethasone-treated thymocytes showed that Cdk2 can phosphorylate p53 (Fig. 5 A). Moreover, p53 was found to associate with Cdk2 in thymocytes (Fig. 5 B). To test the effect of Cdk2 activity on p53 expression, we analyzed the levels of p53 protein in γ-irradiated thymocytes in the presence or absence of Cdk2 inhibitors. Although p53 protein accumulated to significant levels after treatment of cells with γ-irradiation alone, little p53 accumulation was observed when cells were treated with γ-irradiation in the presence of Cdk2 blockers (Fig. 5 C). Irradiation-induced p53 protein accumulation was caused by enhanced p53 protein stability but not by p53 gene transactivation (not shown).


The cyclin-dependent kinase Cdk2 regulates thymocyte apoptosis.

Hakem A, Sasaki T, Kozieradzki I, Penninger JM - J. Exp. Med. (1999)

P53 is a substrate for Cdk2 activity. (A) Phosphorylation of p53 by Cdk2. p53 phosphorylation was determined after γ-irradiation (500 rads), dexamethasone treatment  (1 μM), or no treatment (Control). Thymocytes were cultured and harvested as described in the legend to Fig. 1. Cdk2  was immunoprecipitated, and Cdk2 activity was determined  using recombinant human p53 as the substrate. The levels of  phosphorylated p53 (top), p53 protein (middle), and Cdk2  protein (bottom) are shown. (B) Coimmunoprecipitation of  p53 and Cdk2. Thymocytes were harvested 2 h after γ-irradiation (500 rads) or after 2 h culture without a death stimulus (Control). Western blot analysis was performed after immunoprecipitation (IP) using an anti-Cdk2 Ab and blotting with anti-p53 and anti-Cdk2.  Similar results were obtained using p53 immunoprecipitations. One result representative of three independent experiments is shown. (C) Levels of p53 protein. Thymocytes were cultured for 2 h after γ-irradiation  (500 rads) or the absence of a death stimulus (Control) in the presence or absence of roscovitine (50 μM,  Rosco). Cells were lysed, and extracts were analyzed by Western blotting using anti-p53 and anti–Bcl-XL  Abs. One result representative of three independent experiments is shown. (D) Induction of Bax mRNA.  Thymocytes were cultured as described in B and harvested after 3 h. 10 μg total RNA was Northern blotted and hybridized to probes for both Bax and β-actin (loading control). Hybridization with the Bax probe  shows two alternatively spliced RNA transcripts of 1.5 and 1.0 kb. The increase in Bax mRNA induced by  γ-irradiation is abolished in the presence of roscovitine (50 μM).
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Figure 5: P53 is a substrate for Cdk2 activity. (A) Phosphorylation of p53 by Cdk2. p53 phosphorylation was determined after γ-irradiation (500 rads), dexamethasone treatment (1 μM), or no treatment (Control). Thymocytes were cultured and harvested as described in the legend to Fig. 1. Cdk2 was immunoprecipitated, and Cdk2 activity was determined using recombinant human p53 as the substrate. The levels of phosphorylated p53 (top), p53 protein (middle), and Cdk2 protein (bottom) are shown. (B) Coimmunoprecipitation of p53 and Cdk2. Thymocytes were harvested 2 h after γ-irradiation (500 rads) or after 2 h culture without a death stimulus (Control). Western blot analysis was performed after immunoprecipitation (IP) using an anti-Cdk2 Ab and blotting with anti-p53 and anti-Cdk2. Similar results were obtained using p53 immunoprecipitations. One result representative of three independent experiments is shown. (C) Levels of p53 protein. Thymocytes were cultured for 2 h after γ-irradiation (500 rads) or the absence of a death stimulus (Control) in the presence or absence of roscovitine (50 μM, Rosco). Cells were lysed, and extracts were analyzed by Western blotting using anti-p53 and anti–Bcl-XL Abs. One result representative of three independent experiments is shown. (D) Induction of Bax mRNA. Thymocytes were cultured as described in B and harvested after 3 h. 10 μg total RNA was Northern blotted and hybridized to probes for both Bax and β-actin (loading control). Hybridization with the Bax probe shows two alternatively spliced RNA transcripts of 1.5 and 1.0 kb. The increase in Bax mRNA induced by γ-irradiation is abolished in the presence of roscovitine (50 μM).
Mentions: Therefore, we tested whether p53 is a target for Cdk2 activity during thymocyte apoptosis after γ-irradiation. In vitro kinase assays using immunoprecipitated Cdk2 from γ-irradiated and dexamethasone-treated thymocytes showed that Cdk2 can phosphorylate p53 (Fig. 5 A). Moreover, p53 was found to associate with Cdk2 in thymocytes (Fig. 5 B). To test the effect of Cdk2 activity on p53 expression, we analyzed the levels of p53 protein in γ-irradiated thymocytes in the presence or absence of Cdk2 inhibitors. Although p53 protein accumulated to significant levels after treatment of cells with γ-irradiation alone, little p53 accumulation was observed when cells were treated with γ-irradiation in the presence of Cdk2 blockers (Fig. 5 C). Irradiation-induced p53 protein accumulation was caused by enhanced p53 protein stability but not by p53 gene transactivation (not shown).

Bottom Line: Aberrant activation of cell cycle molecules has been postulated to play a role in apoptosis ("catastrophic cell cycle").Cdk2 was found to function upstream of the tumor suppressor p53, transactivation of the death promoter Bax, alterations of mitochondrial permeability, Bcl-2, caspase activation, and caspase-dependent proteolytic cleavage of the retinoblastoma protein.These data provide the first evidence that Cdk2 activity is crucial for the induction of thymocyte apoptosis.

View Article: PubMed Central - PubMed

Affiliation: The Amgen Institute, Department of Medical Biophysics, University of Toronto, Ontario, Canada M5G 2C1.

ABSTRACT
Aberrant activation of cell cycle molecules has been postulated to play a role in apoptosis ("catastrophic cell cycle"). Here we show that in noncycling developing thymocytes, the cyclin- dependent kinase Cdk2 is activated in response to all specific and nonspecific apoptotic stimuli tested, including peptide-specific thymocyte apoptosis. Cdk2 was found to function upstream of the tumor suppressor p53, transactivation of the death promoter Bax, alterations of mitochondrial permeability, Bcl-2, caspase activation, and caspase-dependent proteolytic cleavage of the retinoblastoma protein. Inhibition of Cdk2 completely protected thymocytes from apoptosis, mitochondrial changes, and caspase activation. These data provide the first evidence that Cdk2 activity is crucial for the induction of thymocyte apoptosis.

Show MeSH
Related in: MedlinePlus