Inactivation of DNA-dependent protein kinase by protein kinase Cdelta: implications for apoptosis - PubMed (original) (raw)
. 1998 Nov;18(11):6719-28.
doi: 10.1128/MCB.18.11.6719.
S K Kraeft, M Gounder, P Pandey, S Jin, Z M Yuan, S P Lees-Miller, R Weichselbaum, D Weaver, L B Chen, D Kufe, S Kharbanda
Affiliations
- PMID: 9774685
- PMCID: PMC109255
- DOI: 10.1128/MCB.18.11.6719
Inactivation of DNA-dependent protein kinase by protein kinase Cdelta: implications for apoptosis
A Bharti et al. Mol Cell Biol. 1998 Nov.
Abstract
Protein kinase Cdelta (PKCdelta) is proteolytically cleaved and activated at the onset of apoptosis induced by DNA-damaging agents, tumor necrosis factor, and anti-Fas antibody. A role for PKCdelta in apoptosis is supported by the finding that overexpression of the catalytic fragment of PKCdelta (PKCdelta CF) in cells is associated with the appearance of certain characteristics of apoptosis. However, the functional relationship between PKCdelta cleavage and induction of apoptosis is unknown. The present studies demonstrate that PKCdelta associates constitutively with the DNA-dependent protein kinase catalytic subunit (DNA-PKcs). The results show that PKCdelta CF phosphorylates DNA-PKcs in vitro. Interaction of DNA-PKcs with PKCdelta CF inhibits the function of DNA-PKcs to form complexes with DNA and to phosphorylate its downstream target, p53. The results also demonstrate that cells deficient in DNA-PK are resistant to apoptosis induced by overexpressing PKCdelta CF. These findings support the hypothesis that functional interactions between PKCdelta and DNA-PK contribute to DNA damage-induced apoptosis.
Figures
FIG. 1
Association of DNA-PKcs and PKCδ. (A) Lysates from U-937 cells were subjected to immunoprecipitation with anti-DNA-PK (αDNA-PK), PIRS or anti-PKCδ (αPKCδ). Immunoprecipitates were analyzed by immunoblotting with anti-DNA-PKcs. Whole-cell lysate (Lysate) was used as a positive control for the immunoblot analysis. (B) Soluble proteins from U-937 cells were subjected to immunoprecipitation with anti-PKCδ. Lysates before and after immunoprecipitation were analyzed by immunoblotting with anti-DNA-PKcs (top). The results are expressed as the mean ± standard deviation (SD) of three independent experiments (bottom). (C) U-937 cell lysates were immunoprecipitated with PIRS, anti-PKCδ, or anti-DNA-PKcs. Immunoprecipitates were analyzed by immunoblotting with anti-PKCδ. Lysate was used as a positive control for the immunoblotting. (D) U-937 cells were treated with 20 Gy of IR and harvested at the indicated times. Lysates were immunoprecipitated with anti-DNA-PKcs, and the precipitates were analyzed by immunoblotting with anti-PKCδ.
FIG. 2
Direct interaction of DNA-PKcs with PKCδ. (A) U-937 cell lysate was incubated with GST, GST-PKCδ FL, or GST-PKCδ CF. The protein adsorbates were analyzed by immunoblotting with anti-DNA-PKcs. (B) Purified DNA-PK (1 μg) was incubated with GST, GST-PKCδ FL, or GST-PKCδ CF. After extensive washing, the bound proteins were eluted by boiling in SDS sample buffer and analyzed by immunoblotting with anti-DNA-PK. (C) Purified DNA-PK (1 μg) was resolved by SDS-PAGE and transferred to three nitrocellulose filters. The filters were incubated with GST, GST-PKCδ FL, or GST-PKCδ CF for 1 h at room temperature and then analyzed by immunoblotting with anti-PKCδ antibody.
FIG. 3
PKCδ CF phosphorylates DNA-PKcs and releases DNA-PKcs from Ku-DNA beads. (A) GST-PKCδ FL or GST-PKCδ CF was incubated with purified DNA-PK–Ku complex (top) or myelin basic protein (MBP) (bottom) in the presence of [γ-32P]ATP for 15 min at 30°C. In vitro kinase reactions were analyzed by SDS-PAGE and autoradiography. (B) Purified DNA-PK/Ku was incubated with DNA beads, and the beads were washed and suspended in kinase buffer. Kinase reaction mixtures containing beads, 20 μM wortmannin, ATP, and GST-PKCδ CF or kinase-inactive GST-PKCδ CF K-R were incubated for 15 min at 30°C. The supernatant fraction was obtained by sedimentation of the beads. The beads and supernatant fractions were boiled in SDS sample buffer. Proteins were separated by SDS-PAGE (5% polyacrylamide) and analyzed by immunoblotting with anti-DNA-PKcs.
FIG. 4
Association of PKCδ CF with specific DNA-PKcs protein fragments. (A) Positions of the DNA-PKcs protein fragments. Protein fragments from various regions of the DNA-PKcs gene were prepared by PCR and in vitro transcription-translation as described in the text. (B) GST-PKCδ CF bound to glutathione-Sepharose was mixed with in vitro-translated products from DNA-PKcs regions to allow binding. After being washed, samples were separated by SDS-PAGE (10% polyacrylamide) and analyzed by autoradiography. (C) GST-PKCδ CF or GST bound to glutathione-Sepharose was mixed with DNA-PKcs fragment 6, 8, or 9. After being washed, the bound proteins were analyzed by SDS-PAGE and autoradiography.
FIG. 5
Caspase 3 cleaves DNA-PKcs and partially inhibits DNA-PKcs activity. (A) Purified DNA-PK was incubated with recombinant caspase 3 (2.5 μg/ml) (left) or with recombinant ICE (right) at room temperature for 1 h or 30 min, respectively. The reaction products were subjected to SDS-PAGE and analyzed by immunoblotting with anti-DNA-PKcs. (B and C) Purified DNA-PK/Ku in the presence of DNA-beads was incubated with recombinant caspase 3 for 1 h at room temperature. In vitro kinase reactions containing [γ-32P]ATP were performed in the absence (B) or presence (C) of GST-p53 as the substrate. The reactions were stopped by the addition of SDS sample buffer, and the products were analyzed by SDS-PAGE and autoradiography. DNA-PK CL1 and CL2, DNA-PK-cleaved fragments 1 and 2.
FIG. 6
Phosphorylation and inactivation of DNA-PKcs by PKCδ CF. (A and B) Purified DNA-PK–Ku in the presence of DNA-beads was incubated with recombinant caspase 3 for 1 h at room temperature. In vitro phosphorylation of the cleaved fragments of DNA-PK was then performed in the presence of [γ-32P]ATP and GST-PKCδ CF or GST-PKCδ CF K-R for 15 min at 30°C. After phosphorylation of DNA-PKcs and removal of PKCδ-CF or PKCδ CF K-R by sedimentation, kinase reactions were performed in the absence (A) or presence (B) of GST-p53 for an additional 15 min at 30°C. The reactions were stopped by the addition of SDS sample buffer, and the products were analyzed by SDS-PAGE and autoradiography. Lanes: 1, DNA-PK–Ku with DNA; 2, DNA-PK–Ku without DNA; 3, DNA-PK–Ku with DNA and caspase 3; 4, DNA-PK–Ku with DNA caspase 3, and GST-PKCδ CF; 5, DNA-PK–Ku with DNA, caspase 3, and GST-PKCδ CF K-R; 6, DNA-PK–Ku with DNA and GST-PKCδ CF; 7, GST-p53; 8, buffer with [γ-32P]ATP. (C) The percent inhibition of DNA-PKcs-mediated GST-p53 phosphorylation is expressed as the mean ± SD of four independent experiments.
FIG. 7
Transient overexpression of PKCδ CF in SCH8-1 (DNA-PK+/+) and ScSV3 (DNA-PK−/−) cells. GFP-tagged PKCδ CF was transiently transfected in SCH8-1 (DNA-PK+/+) and ScSV3 (DNA-PK−/−) cells. The cells were stained with DAPI, and the GFP-positive cells were analyzed by confocal microscopy. The results are shown as overlay photographs of DAPI and GFP staining. Arrows indicate apoptotic cells. Bar, 10 μm.
FIG. 8
Transient overexpression of PKCδ CF in DNA-PK−/− and DNA-PK+/+ CHO cells. GFP-tagged PKCδ CF was transiently transfected in CHO (DNA-PK+/+) and CHO V-3 (DNA-PK−/−) cells. The cells were stained with DAPI, and the GFP-positive cells were analyzed by confocal microscopy. The results are shown as overlay photographs of DAPI and GFP staining. Arrows indicate apoptotic cells. Bar, 10 μm.
FIG. 9
Transient overexpression of PKCδ CF and not PKCδ CF K-R in DNA-PK+/+ cells is associated with induction of apoptosis. GFP-tagged PKCδ CF or PKCδ CF K-R mutant was transiently transfected in SCH8-1 (DNA-PK+/+) (lane 1, GFP; lane 2, GFP-PKCδ CF; lane 3, GFP-PKCδ CF K-R) or ScSV3 (DNA-PK−/−) (lane 4, GFP; lane 5, GFP-PKCδ CF; lane 6, GFP-PKCδ CF K-R) cells. As controls, cells were transfected with GFP-expressing empty vector. The GFP-positive cells were sorted by FACScan analysis and analyzed for DNA content by flow cytometry. The results are expressed as the percentage (mean ± SD of three independent experiments, each performed in duplicate) of cells with sub-G1 DNA content.
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