The C-terminal conserved domain of DNA-PKcs, missing in the SCID mouse, is required for kinase activity - PubMed (original) (raw)
The C-terminal conserved domain of DNA-PKcs, missing in the SCID mouse, is required for kinase activity
H J Beamish et al. Nucleic Acids Res. 2000.
Abstract
DNA-PKcs, the catalytic subunit of DNA-dependent protein kinase (DNA-PK), has a phosphoinositol 3-kinase (PI 3-K) domain close to its C-terminus. Cell lines derived from the SCID mouse have been utilised as a model DNA-PKcs-defective system. The SCID mutation results in truncation of DNA-Pkcs at the extreme C-terminus leaving the PI 3-K domain intact. The mutated protein is expressed at low levels in most SCID cell lines, leaving open the question of whether the mutation abolishes kinase activity. Here, we show that a SCID cell line that expresses the mutant protein normally has dramatically impaired kinase activity. We estimate that the residual kinase activity typically present in SCID fibroblast cell lines is at least two orders of magnitude less than that found in control cells. Our results substantiate evidence that DNA-PKcs kinase activity is required for DSB rejoining and V(D)J recombination and show that the extreme C-terminal region of DNA-PKcs, present in PI 3-K-related protein kinases but absent in bona fide PI 3 lipid kinases, is required for DNA-PKcs to function as a protein kinase. We also show that expression of mutant DNA-PKcs protein confers a growth disadvantage, providing an explanation for the lack of DNA-PKcs expression in most SCID cell lines.
Figures
Figure 1
Survival of V-3 and V-3 YAC fusion hybrids following γ-irradiation. Closed circles, AA8; closed inverted triangles, V-3 cells containing YAC 155 and expressing wild-type DNA-PKcs protein; open circles, V-3; closed squares, V-3 clones containing YAC D3941E that did not express DNA-PKcs protein; open inverted triangles, a V-3 clone containing D3941E YAC and expressing DNA-PKcs protein. Survival of V-3, AA8 and a complemented clone represent the means of a minimum of three survival experiments. Five additional complemented clones gave a similar level of resistance. The results from the non-expressing mutant clones represent the means of 10 clones analysed. The expressing mutant clone represents the results of a single experiment carried out on a single clone; similar survival results were obtained with a second expressing mutant clone.
Figure 2
Analysis of DNA-PKcs protein and kinase activity from pre-B bcl2 SCID+ and SCID– cells. (A) Lanes 1 and 2, western blot analysis of WCE (100 µg protein) from a large scale preparation (5 × 107 cells) of SCID+ and SCID– cells using anti-DNA-PKcs antibody 18-2. The origin of the band representing DNA-PKcs was verified using purified DNA-PKcs and since it was absent or severely diminished in extracts from V-3 and mouse SCID fibroblast cells (data not shown). Lanes 3 and 4, western blot analysis of proteins microfractionated from WCE (250 µg) using DNA–cellulose beads. Following microfractionation, the beads were boiled in 2× SDS loading buffer and an aliquot of the detached proteins used for western blot analysis. Lanes 5 and 6, assay of the proteins microfractionated using DNA-cellulose beads for DNA-PK activity. The remainder of the same samples used for the analysis in lanes 3 and 4 were utilised for DNA-PK assays using a p53-derived peptide as a substrate. Following incubation, 2× SDS loading buffer was added, the samples boiled, the beads removed by centrifugation and the samples subjected to PAGE electrophoresis to separate the p53 peptide (lanes 5 and 6). The band representing the phosphorylated p53 peptide is highlighted. Two minor phosphorylation products were also obtained which do not represent the specific p53 phosphorylation event under study. The microfractionation and p53 peptide phosphorylation assay has been utilised extensively by us and others to demonstrate the defect in DNA-PKcs defective cell lines such as V-3 and irs-20, demonstrating that the major kinase phosphorylating p53 in this assay is DNA-PK (see for example 31,38). (B) The results of a similar DNA-PK assay carried out as described in (A) using a smaller number of cells and less extract (10 µg WCE for 1BR3 and 50 µg WCE for pre-B SCID+ and pre-B SCID–; only half the microfractionated proteins were utilised for the kinase assay in this experiment). 1BR3 is a primary human fibroblast cell line used as a control. As reported previously, rodent cells have ~50-fold lower levels of DNA-PK activity compared with human cells. The -peptide lane represents the assay carried out in the absence of p53 peptide
Figure 3
‘Grow-back’ survival experiment following exposure to ionising radiation. (A) Pre-B bcl2 SCID+ cells. (B) Pre-B bcl2 SCID– cells. Closed circles, unirradiated cells; inverted triangles, cells irradiated with 1 Gy; closed squares, cells irradiated with 2 Gy; open diamonds, cells irradiated with 3 Gy. (C) Survival of SCID+ and SCID– cells after ionising radiation estimated by ‘grow-back’ experiments. Open circles, pre-B bcl2 SCID+; closed circles, pre-B bcl2 SCID–. The results represent the means of three ‘grow-back’ experiments. No observable recovery of the SCID– cells after 3 Gy was seen in most experiments even after 6 days post-irradiation incubation [see for example (B)]. This point has therefore been omitted from the figure. The survival, however, was clearly lower than that observed with the SCID+ cells. The results of the single cloning survival experiment were 37.6 and 15.3% survival for SCID+ cells and 11.9 and 1.4% survival for SCID– cells following exposure to 1 and 2 Gy, respectively.
Figure 4
Repair of DNA DSBs in SCID+ and SCID– cells. Cells were exposed to 20 Gy γ-rays and DSB repair measured by PFGE. Open circles, pre-B bcl2 SCID+; closed circles, pre-B bcl2 SCID–. FAR (fraction of [14C] radioactivity released) is the fraction of activity migrating out of the well relative to the total activity loaded (activity in the well and the gel). The results are expressed as a percentage of the value obtained for an irradiated sample that was not allowed time to repair. The background activity in unirradiated samples treated in parallel is subtracted from that present in the irradiated samples.
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