Deciphering phenotypic variance in different models of DNA-PKcs deficiency

Abstract

DNA-PKcs deficiency has been studied in numerous animal models and cell culture systems. In previous studies of kinase inactivating mutations in cell culture systems, ablation of DNA-PK's catalytic activity results in a cell phenotype that is virtually indistinguishable from that ascribed to complete loss of the enzyme. However, a recent compelling study demonstrates a remarkably more severe phenotype in mice harboring a targeted disruption of DNA-PK's ATP binding site as compared to DNA-PKcs deficient mice. Here we investigate the mechanism for these divergent results. We find that kinase inactivating DNA-PKcs mutants markedly radiosensitize immortalized DNA-PKcs deficient cells, but have no substantial effects on transformed DNA-PKcs deficient cells. Since the non-homologous end joining mechanism likely functions similarly in all of these cell strains, it seems unlikely that kinase inactive DNA-PK could impair the end joining mechanism in some cell types, but not in others. In fact, we observed no significant differences in either episomal or chromosomal end joining assays in cells expressing kinase inactivated DNA-PKcs versus no DNA-PKcs. Several potential explanations could explain these data including a non-catalytic role for DNA-PKcs in promoting cell death, or alteration of gene expression by loss of DNA-PKcs as opposed to inhibition of its catalytic activity. Finally, controversy exists as to whether DNA-PKcs autophosphorylates or is the target of other PIKKs; we present data demonstrating that DNA-PK primarily autophosphorylates.

Keywords: DNA dependent protein kinase; DNA-PKcs; Non-homologous end joining (NHEJ).

Figure 1.. ATM expression is substantially reduced in cultured cells ablated for DNA-PKcs.

(A). DNA-PKcs and ATM protein expression levels were examined by western blotting of whole cell extracts obtained from wild type or SCID ear fibroblasts (left panel), and patient derived fibroblasts (right panel). A non-specific band detected by the ATM antibody (left panel) or Ku86 (right panel) serve as loading controls. (B) ATM protein expression levels were assessed in wild type and DNA-PKcs deficient 293T cells, HCT116 cells, and MO59K/J cells. Protein levels from indicated amounts of whole cell extract were assessed by immunoblotting. A non-specific band detected by the ATM antibody serves as a loading control. (C) ATM expression was assessed from 293T cells ablated for other c-NHEH components as indicated.(D) Expression of the components of the TTT complex were assessed by immunoblotting in wild type 293T cells, or in cells lacking DNA-PKcs, or harboring a kinase inactivating mutation of DNA-PKcs as indicated. White line indicate removal of lanes not presented; figure is derived from a single blot. (E) DNA-PKcs and ATM expression in wild type or DNA-PKcs deficient 293T cells 72 hours after transfection with a DNA-PKcs expression construct or vector alone. A non-specific band detected by the ATM antibody serves as a loading control. (F) GFP and ATM expression levels in wild type or DNA-PKcs deficient 293T cells transfected with either or both GFP-DNA-PKcs or GFP-ATM as indicated.

Figure 2.. Human and murine DNA-PKcs complement radiomimetic drug sensitivity in DNA-PKcs deficient hamster cells to a similar extent, and kinase-inactivating mutations impart similar radiosensitivity as DNA-PKcs deficiency in both V3 cells and 293T cells; non-transformed SCID fibroblasts expressing kinase-inactivating DNA-PKcs mutants are substantially more radiosensitive than DNA-PKcs deficient cells.

(A) Left, V3 clonal transfectants expressing wild type human DNA-PKcs (hWt), wild type murine DNA-PKcs (mWt), the catalytically inactive K3753>R mutant of human DNA-PKcs (hK>R), the catalytically inactive D3921>A mutant of human DNA-PKcs (hD>A), the catalytically inactive D3922>A mutant of murine DNA-PKcs (mD>A), the ABCDE>ala cluster mutant (ABCDE>A), or no DNA-PKcs (vector) were plated at cloning densities into complete medium with increasing doses of Zeocin. Colonies were stained after seven days, and percent survival was calculated. Error bars represent the standard error of the means for six independent experiments. Right, immunoblot analyses of 50ug whole cell extracts assessed first for ATM and then for DNA-PKcs expression as indicated; non-specific band serves as a loading control. (B) Left; Wild type 293T cells, or 293T cells with targeted ablation of DNA-PKcs, or a kinase inactivating targeted mutation resulting in the K3752>R mutation were assessed for Zeocin sensitivity by MTS staining. Error bars represent the standard error of the means for six independent experiments Right, immunoblot analyses of 50ug whole cell extracts assessed first for ATM and then for DNA-PKcs as indicated. White line indicate removal of lanes not presented; figure is derived from a single blot. (C) Left, Sf19 transfectants expressing DNA-PKcs constructs described in A. were analyzed for Zeocin sensitivity as described in A. Error bars represent the standard error of the means for four independent experiments. Right, immunoblot analyses of 50ug whole cell extracts assessed first for ATM and then for DNA-PKcs expression as indicated; non-specific band serves as a loading control.

Figure 3.. Kinase inactive DNA-PKcs does not alter end-joining deficits in DNA-PKcs deficient cells.

(A) The fluorescent substrate ISceRFP/CFP (left panel) was utilized to detect joining of I-Sce1 induced DSBs. Percent recombination of episomal fluorescent I-Sce1 end joining substrate in Sf19 transfectants expressing wild type DNA-PKcs, vector alone, or the D3922>A mutation. The fluorescent substrate telN-CFP (right panel) was utilized to detect CFP expression from uncut (U) or cut substrate (C), co-transfected with dsRED to assess transfection efficiency. TelN cleavage generates a DNA break with hairpin termini. Percent CFP/RFP is presented. Error bars indicate SEM from three independent experiments. Ns, P value, not significant, in two-tailed unpaired t test. (B) Diagram of region of murine AICDA locus targeted by two gRNAs and position of primers utilized to detect chromosomal deletions. Ethidium bromide staining of PCR amplifications of DNA isolated from Sf19 transfectants expressing wild type DNA-PKcs, vector only, or D3922A mutant DNA-PKcs as indicated. (C) Summary of sequenced joints. P= 0.1916; (not significant) in two-tailed unpaired t test comparing nucleotide loss between sequences from cells expressing vector only versus the D3922>A mutant.

Figure 4.. DNA-PKcs primarily autophosphorylates the ABCDE and PQR clusters.

(A) Left, V3 transfectants expressing wild type, vector alone, ABCDE>Ala, or PQR>Ala DNA-PKcs were treated or not with zeocin and okadaic acid for one hour. 50ug whole cell extracts were analyzed by immunoblotting (left panel) with the S2056 phospho-specific antibody (top panel) or a DNA-PKcs specific antibody (second panel). On a separate gel (right panel), 50ug whole cell extracts were analyzed by immunoblotting with the T2609 phospho-specific antibody (top panel) or a DNA-PKcs specific antibody (second panel). On a third gel, 10ug whole cell extracts were analyze for phosphorylated γH2AX or actin. (B) 293T cells of the indicated genotypes were treated or not with zeocin for one hour. 50ug whole cell extracts were analyzed by immunoblotting with the S2056 phospho-specific antibody (top panel) or a T2609 phospho-specific antibody (second panel), or a DNA-PKcs specific antibody (third panel). 10ug whole cell extracts were analyze for phosphorylated γH2AX (forth panel) or actin (bottom panel). (C) 293T cells of the indicated genotypes were treated or not with zeocin with or without the ATM inhibitor KU55933 as indicated for one hour. Whole cell extracts were analyzed by immunoblotting with the S2056 phospho-specific antibody or a DNA-PKcs specific antibody. (D) Wild type 293T cells were treated or not with zeocin and Okadaic acid for one hour. Whole cell extracts were analyzed by immunoblotting with the S2056 phospho-specific antibody, T2609 phospho-specific antibody, or a DNA-PKcs specific antibody.