CK2 phosphorylation-dependent interaction between aprataxin and MDC1 in the DNA damage response

Abstract

Aprataxin, defective in the neurodegenerative disorder ataxia oculomotor apraxia type 1, resolves abortive DNA ligation intermediates during DNA repair. Here, we demonstrate that aprataxin localizes at sites of DNA damage induced by high LET radiation and binds to mediator of DNA-damage checkpoint protein 1 (MDC1/NFBD1) through a phosphorylation-dependent interaction. This interaction is mediated via the aprataxin FHA domain and multiple casein kinase 2 di-phosphorylated S-D-T-D motifs in MDC1. X-ray structural and mutagenic analysis of aprataxin FHA domain, combined with modelling of the pSDpTD peptide interaction suggest an unusual FHA binding mechanism mediated by a cluster of basic residues at and around the canonical pT-docking site. Mutation of aprataxin FHA Arg29 prevented its interaction with MDC1 and recruitment to sites of DNA damage. These results indicate that aprataxin is involved not only in single strand break repair but also in the processing of a subset of double strand breaks presumably through its interaction with MDC1.

Figure 1.

Co-localization of aprataxin with γH2AX and MDC1 at sites of DSB. (A) Methodology employed to study the dynamic recruitment of GFP-tagged fusion proteins (aprataxin and MDC1) to localized DNA damage induced by heavy ion irradiation. Transfection of GFP constructs and irradiation procedures are described in Supplementary Data. (B) Recruitment of GFP-aprataxin to uranium (4.2 MeV/nucleon LET 14925 keV/µm) ions-induced DNA DSB in HeLa cells. DSB are visualized by γH2AX immunostaining and nuclei by ToPro3 staining. (C) Co-localization of GFP-aprataxin with MDC1 along particle tracks after uranium ions irradiation in HeLa cells. (D) Recruitment of endogenous aprataxin to DSB and co-localization with MDC1 along particle tracks after krypton ions irradiation (5.4 MeV/nucleon; LET 5060 keV/µm) revealed by immunostaining with anti-aprataxin and anti-MDC1 antibodies in human fibroblasts.

Figure 2.

Direct interaction between aprataxin and MDC1. (A) Co-IP of MDC1 and aprataxin using an anti-aprataxin antibody followed by immunoblotting with the respective antibodies. Non-specific serum (Ig) is used as a negative control and the amounts of protein in WCE are shown. (B) Co-IP of aprataxin with an anti-MDC1 antibody. Non-specific serum (Ig) is included and WCE is also shown. (C) Diagram of aprataxin domains and GST-fragments used in the pull-down assays. (D) Protein extracts from control LCL (C3ABR) were either incubated with GST alone or GST-aprataxin fragments. Bound proteins were separated on SDS–PAGE and detected by anti-MDC1 antibodies. Coomassie staining shows equivalent loading of GSTs. (E) Direct binding of aprataxin to MDC1. Aprataxin FHA pull downs in the absence or presence of Benzonase^TM, a potent nuclease that degrades both RNA and DNA.

Figure 3.

CK2-mediated phosphorylation-dependent binding of MDC1 to the aprataxin FHA domain. (A) Diagram of aprataxin GST fusion used in the pull-down assays. (B) Effect of protein phosphatase activity on MDC1 binding to the aprataxin FHA domain. Whole cell extracts from HeLa cells were mock treated or treated with alkaline phosphatase (Alk PPase: 2500 U) for 1 h at room temperature and subsequently used for pull-down assays using GST only, and GST-aprataxin FHA domain. (C) Reduced binding of MDC1 to the aprataxin FHA domain following CK2 inhibitor treatment. HeLa cells were treated for 8 h with increasing concentrations of CK2 inhibitor TBB, lysed and WCE were used for GST pull-down assays. (D) Schematic of MDC1 protein sequence showing the GST fusion protein (150–350) used in the pull-down assays. In vitro binding of CK2-phosphorylated (150–350) region of MDC1 with HeLa WCE. Binding endogenous aprataxin to CK2-phosphorylated MDC1 (150–350) fragment was revealed by immunoblotting.

Figure 4.

Aprataxin binds to a conserved di-phosphorylated CK2 motif in MDC1. (A) MDC1 contains six conserved motifs with a core SDTD motif that is di-phosphorylated in vivo (48–50). (B) ITC binding isotherms for interaction of wild-type aprataxin FHA with pSDTD and SDpTD phosphopeptides (C, D) ITC binding isotherms for the titration of pSDpTD phosphopeptide into wild-type and K38A aprataxin FHA, respectively. (E) Diagram of GFP-MDC1 mutants used in the pull-down assays. (F) Aprataxin FHA GST pull downs from HeLa cells expressing wild-type GFP-MDC1, a mutant containing a deletion of the SDTD region (ΔSDTD), and a mutant where the Thr residues contained in the SDTD sequence were replaced by Ala (SDAD)₂.

Figure 5.

Structural comparison of the aprataxin and PNK FHA domains and modelling of the aprataxin–MDC1 interaction. (A) Superposition of Cα backbone structures of the aprataxin FHA domain (green) with that of the PNK FHA/XRCC4 phosphopeptide complex (grey). (B) Sequence alignment of the core FHA domains residues from aprataxin and PNK. Identical positions are shown in red with the highly conserved arginine and serine residues that make canonical pT contacts in all available FHA-phosphopeptide structures are boxed. Black dots highlight the five Arg/Lys residues conserved between the two proteins. (C) Modelling of the aprataxin FHA–pSDpTD MDC1 phospho-motif complex (left) based on the crystal structure of the aprataxin FHA domain and the PNK–XRCC4 complex structure (right).

Figure 6.

Key role of aprataxin FHA domain in targeting aprataxin to sites of DNA damage in vivo. (A) Substitution of Arg29 and Lys38 to Ala abrogated the interaction between aprataxin and MDC1 as determined by pull-down assays as described earlier. (B) Effect of Arg29Ala and Lys38Ala mutations on the recruitment of aprataxin to high LET heavy ion-induced DSB in HeLa cells. HeLa cells were transiently transfected with wild-type GFP-aprataxin, GFP-aprataxin R29A or GFP-aprataxin K38A mutants, and cells were subjected to nickel ions irradiation (6.0 MeV/nucleon; LET 3430 keV/µm). DSB are visualized by γH2AX immunostaining and nuclei by ToPro3 staining.

Figure 7.

Normal repair of IR-induced DSB in AOA1 cells. (A) γH2AX foci repair kinetic after low LET ionizing radiation (IR; 2 Gy). AOA1 fibroblasts (FD105) were transfected with either GFP-aprataxin (wild-type) or GFP-aprataxin (R29A) mutant. Untransfected cells were used as control. Graph represents average numbers of foci per cell ± SD. Foci were counted in 50–60 individual cells per time point. DSB repair kinetics were similar regardless of the presence of absence of aprataxin indicating that aprataxin is not required for low LET IR-induced DSB repair. (B) Repair kinetics of DSB (γH2AX foci) after low LET irradiation (IR) in wild-type and Mdc1^−/− cells. Graph represents average number of foci per cell ± SD. Foci were counted in 50–60 individual cells per time points a significant difference was observed at 6 and 8 h post-irradiation (*P < 0.05, Student’s t-test).

Figure 8.

A model for the dual role of aprataxin in SSB and DSB repair. The FHA domain of aprataxin, through which it interacts with MDC1, XRCC1 and XRCC4, is shown three times to represent a series of dynamic interactions with these three proteins or to represent individual aprataxin molecules.