Two XRCC1-containing complexes in cycling HeLa cells.
To identify proteins that were interacting with aprataxin, we first carried out IP of aprataxin from HeLa nuclear extracts (NE) made from cycling cells and identified the coimmunoprecipitated proteins by mass spectrometry (Fig. ). Four major protein bands with similar staining intensities were identified as aprataxin, XRCC1, DNL3, and a protein of unknown function (labeled with an asterisk). Other minor bands were identified and labeled in Fig. . Since we often find these proteins in our proteomics analysis of protein complexes that are not related, we tentatively determine them to be nonspecific binding proteins. We then immunoprecipitated XRCC1 from HeLa NE and reciprocally identified aprataxin and DNL3, demonstrating that XRCC1 and aprataxin exist within a complex (data not shown). Notably, PNK is also identified in the XRCC1 IP but is absent in the aprataxin IP, suggesting that PNK may not be in a complex with aprataxin.
FIG. 1. IP of the aprataxin complex. HeLa NE were immunoprecipitated with an affinity-purified antibody to aprataxin (α Aprataxin), resolved by SDS-4 to 20% PAGE, and stained with Coomassie blue. The major aprataxin-interacting proteins were identified (more ...)
We carried out IP and Western experiments from HeLa NE to verify our results from mass spectrometric analysis. As shown in Fig. , both aprataxin and XRCC1 coimmunoprecipitate with each other. While PNK and Polβ, two important enzymes in SSBR, coimmunoprecipitate with XRCC1, PNK is not detected and Polβ is detected just above the background level in the aprataxin IP. This finding is consistent with the large-scale IP result in which the level of Polβ is below the detection limit of Coomassie blue. To test whether the aprataxin antibody displaces binding of PNK to XRCC1, we transfected a V5-tagged aprataxin at the C terminus and a FLAG-tagged PNK at the N terminus individually in 293T cells and immunoprecipitated aprataxin and PNK with V5 and M2 antibodies, respectively. While both V5 and M2 antibodies coimmunoprecipitate the endogenous XRCC1, the M2 antibody does not coimmunoprecipitate aprataxin, and the V5 antibody coimmunoprecipitates only background level of PNK (Fig. ). This small amount of PNK that coimmunoprecipitates with the V5 antibody is likely the result of overexpression of FLAG-PNK. Protein-protein interaction data acquired from both endogenous and transfected proteins support a notion that there are two XRCC1-containing complexes in the cycling cells: one is the known XRCC1 complex that contains the essential SSBR enzymes PNK and Polβ and the other is a new complex that contains aprataxin.
FIG. 2. Characterization of interactions of XRCC1 with aprataxin and PNK. (A) HeLa NE were immunoprecipitated with control antibody, antiaprataxin, and anti-XRCC1 antibodies and analyzed by Western blotting. (B) Transfection of 293T cells with V5-aprataxin and (more ...)
To substantiate the idea of two XRCC1-containing complexes, we carried out column fractionation of HeLa NE. Aprataxin was found to dissociate from XRCC1 when proteins were eluted from P11 phosphocellulose or DEAE columns, preventing a biochemical separation of the two XRCC1 complexes (data not shown). The fractionation of HeLa NE on a Superose 6 gel filtration column was not sufficient to separate the aprataxin-XRCC1 and PNK-XRCC1 complexes (data shown). However, if the two XRCC1 complexes are of a comparable size, these complexes would not be separable by size exclusion. About one-half of PNK and aprataxin cofractionate with XRCC1 in the high-molecular-weight complexes, and the other half elutes in lower-molecular-weight complexes that are devoid of XRCC1. Thus, XRCC1 is limiting, and it appears that the abundances of XRCC1-PNK and XRCC1-aprataxin complexes are similar in HeLa NE.
To test whether interaction between XRCC1 and aprataxin or PNK is altered in the presence of DNA SSB, we treated cells with MMS at 300 μg/ml and examined the interaction of XRCC1 with aprataxin or PNK. No induced interaction was observed (Fig. ), suggesting that these XRCC1 complexes are preformed constitutively.
To investigate whether mutations in AOA1 affect XRCC1 and aprataxin interaction, we cotransfected plasmids encoding FLAG-XRCC1 with the wild-type (WT) V5-aprataxin and V263G and P206L mutations that are located in the central HIT domain in 293T cells, immunoprecipitated XRCC1 with a M2 antibody, and detected coimmunoprecipitated aprataxin with Western blotting by using a V5 antibody. As shown in Fig. , mutations in the aprataxin HIT domain do not seem to abolish interaction with XRCC1. Therefore, a defect in the interaction of aprataxin with XRCC1 may not account for the mechanism of AOA disease.
XRCC1 is phosphorylated in vivo and in vitro by CK2.
We also analyzed XRCC1 phosphorylation in vivo by using endogenous XRCC1 purified from cycling HeLa cells by IP and SDS-PAGE. Using mass spectrometry, we found two tryptic phosphopeptides (phosphopeptide 1 [P1] [aa 459 to 494] and P2 [aa 503 to 546]) (Fig. ) encompassing the linker region between the BRCT1 and BRCT2 domains. P1 was observed to be phosphorylated in vivo from one to four phosphates, and P2 is phosphorylated from three to four phosphates. The very large phosphopeptides prevented mass spectrometric sequencing to identify the exact phosphorylation sites (19
). Inspection of the phosphopeptide sequences revealed that they contain CK2 phosphorylation consensus sites. To identify the exact phosphorylation sites in vivo, we made phospho-specific antibodies against individually phosphorylated pS461 and pS475, doubly phosphorylated pS485/pT488, and triply phosphorylated pS518/pT59/pT523. Among these sites, S461, S475, and S518 do not conform to CK2 consensus sites.
FIG. 3. Characterization of XRCC1 phosphorylation. (A) Sequence of tryptic phospho-specific XRCC1 peptides identified by mass spectrometry analysis of XRCC1 immunoprecipitated from HeLa NE. The underlined amino acids are the sites of XRCC1 phosphorylation. P1 (more ...)
As shown in Fig. , phospho-specific antibodies against pS461, pS475, and doubly phosphorylated pS485/pT488 recognize XRCC1 in a phosphorylation-dependent manner. Thus, these sites can be phosphorylated in vivo in HeLa cells. Since P2 contains three phosphorylation sites (S518, T519, and T523) that are fully phosphorylated in vivo, and the rest of the sites are not fully phosphorylated, we concentrated our biochemical and functional analysis on the triple-phosphorylation sites.
To verify that the phospho-specific antibody against triply phosphorylated pS518/pT59/pT523 is sequence specific, we generated stable EM9-WT, EM9-3A, and EM9-GS cell lines that are complemented with XRCC1-WT, XRCC1-3A (where S518, T519, and T523 are mutated to Ala), and an empty vector, respectively. This triple-phospho-specific antibody is specific to the three phosphorylation sites since it recognizes only WT XRCC1 and not the 3A mutant (Fig. ); it also recognizes XRCC1 from cycling HeLa cells but does not recognize the dephosphorylated XRCC1, demonstrating that it is phosphorylation specific (data not shown). These results also show that S518, T519, and T523 are phosphorylated in vivo in both EM9 and HeLa cells.
To test whether CK2 phosphorylates XRCC1 in vitro, we immunoprecipitated the CK2 kinase from HeLa cells and carried out an immunocomplex kinase assay with a recombinant His-tagged XRCC1 protein purified from Escherichia coli. Western blotting with the phospho-specific antibodies showed that XRCC1 is phosphorylated at six sites by CK2 in an ATP-dependent manner in vitro (Fig. ), despite the fact that S475 and S518 do not strictly conform to CK2 consensus sites. Phosphorylation of S461, which does not conform to the CK2 site and is farther away from the CK2 consensus site cluster, is not phosphorylated by CK2.
We used siRNA to demonstrate that XRCC1 phosphorylation depends on CK2 in vivo. Since there are two independent kinase catalytic subunits (α and α′) in HeLa cells, we transfected HeLa cells with siRNA specific to these two subunits. Down-regulation of CK2 attenuates phosphorylation of XRCC1 at these sites significantly, including S475, which is not a CK2 consensus site (Fig. ). Residual CK2 activity after RNA interference most likely can account for the remaining phosphorylation. We conclude that CK2 phosphorylates XRCC1 both in vitro and in vivo.
To test whether MMS treatment modulates XRCC1 phosphorylation, we treated cells with various doses of MMS and examined phosphorylation by Western blotting. Phosphorylation at the triply phosphorylated sites S518, T519, and T523 as well as the other three in vivo CK2-dependent sites does not change in response to MMS (Fig. and data not shown), in agreement with previously published observations that CK2 is constitutively active (18
FIG. 4. Phosphorylation of XRCC1 at S518/T519/T523 in response to DNA damage caused by MMS and its requirement for cellular survival. (A) Phosphorylation of XRCC1 at S518/T519/T523 is not induced by MMS treatment. Whole-cell extracts made from HeLa cells that (more ...)
We used the colony formation assay to test MMS sensitivity of the stable EM9-WT, EM9-3A, and EM9-GS cell lines to evaluate the functional consequence of XRCC1 phosphorylation. The EM9-3A cell line exhibits MMS sensitivity similar to that of EM9-WT, while the EM9-GS cell line is hypersensitive (Fig. ). Therefore, the phosphorylation of XRCC1 at S518, T519, and T523 is not required for cellular survival in response to MMS. In agreement with this conclusion, the EM9-WT and EM9-3A cell lines display similar SSBR capacity when tested with the Comet assay (data not shown).
Because S518, T519, and T523 are phosphorylated by CK2, we investigated the requirement of CK2 for cellular survival in response to MMS. As shown in Fig. , CK2 is required for survival. Down-regulation of CK2 does not significantly change XRCC1 and aprataxin protein levels (Fig. ), ruling out the possibility that the requirement of CK2 for survival is met through the regulation of XRCC1 and aprataxin protein levels.
The FHA domain of aprataxin binds the phosphorylated S518, T519, and T523 of XRCC1.
To test whether the FHA domain of aprataxin binds XRCC1 in a phosphorylation-dependent manner, we made a GST fusion of the N terminus of aprataxin containing the FHA domain and used it to pull down XRCC1 from whole-cell lysates made from EM9-WT and EM9-3A. As shown in Fig. , only the WT XRCC1, not the XRCC1-3A mutant, can be pulled down by the GST-N-aprataxin, and dephosphorylation of XRCC1 diminishes the interaction to the same extent as the 3A mutant (Fig. ).
FIG. 5. Interactions between XRCC1 and aprataxin or PNK are mediated by phosphorylation of XRCC1 at S518/T519/T523. (A) The interaction between the N terminus of aprataxin and XRCC1 is phosphorylation dependent. The purified GST-N-aprataxin was mixed with whole-cell (more ...)
To demonstrate that the FHA domain is responsible for binding, we mutated two conserved residues, I27 and R29, in the FHA domain of GST-N-aprataxin to Ala. This FHA mutant significantly diminishes interaction with XRCC1 (Fig. ). Thus, phosphorylation of XRCC1 at S518, T519, and T523 regulates binding to aprataxin through the FHA domain in vitro.
To examine whether phosphorylation at S518, T519, and T523 regulates binding to aprataxin in vivo, V5-XRCC1-WT or V5-XRCC1-3A was transfected into 293T cells, and IP with antibodies against V5 or aprataxin was carried out. Endogenous aprataxin can coimmunoprecipitate only with V5-XRCC1-WT but not V5-XRCC1-3A; in the reciprocal experiment, V5-XRCC1-WT, but not V5-XRCC1-3A, can coimmunoprecipitate aprataxin (Fig. ), demonstrating that phosphorylation of XRCC1 at S518, T519, and T523 regulates its binding to aprataxin in vivo. Surprisingly, XRCC1-3A also coimmunoprecipitates significantly less endogenous PNK than XRCC1-WT does, indicating that phosphorylation of XRCC1 at the triple sites may also strengthen the interaction with PNK. However, PNK seems able to bind XRCC1 with a higher affinity than that of aprataxin when the triple-phosphorylation sites are mutated.
We estimated binding affinity by using fluorescence polarization (Fig. ). A peptide where only T523 (pT1) is phosphorylated binds to the aprataxin FHA domain with an apparent Kd of approximately 210 nM. Because the FHA domain has been reported to prefer acidic amino acids at the P+3 position (three amino acid residues C terminal to the phosphorylation sites), we synthesized a mutant pT1 peptide where the amino acid residue E at the P+3 position was substituted to A (pT1-EA). This mutation completely abolishes the binding of pT1 to aprataxin, indicating that the amino acid E is important for aprataxin FHA domain recognition. Significantly, additional phosphorylation of the two upstream residues (S518 and T519) dramatically enhances the binding of the pT1 peptide to the FHA domain. This was reflected by both a twofold decrease in the apparent Kd value (~90 nM) and a fivefold increase in maximum binding.
To further demonstrate that CK2 phosphorylation of XRCC1 is important for aprataxin binding, we treated HeLa cells with a CK2 inhibitor (26
), 4,5,6,7-tetrabromo-2-azabenzamidazole (TBB), that results in the attenuation of XRCC1 phosphorylation in a dose-dependent manner (Fig. ). Reduction in XRCC1 phosphorylation leads to dissociation of XRCC1 from aprataxin (Fig. ). Thus, CK2 kinase activity is required for XRCC1 and aprataxin binding.
An acute loss of aprataxin results in a lower steady-state protein level of XRCC1.
Since the binding of aprataxin to XRCC1 does not seem to be required for cellular survival in response to MMS treatment (Fig. ), we tested whether aprataxin itself is required. We transfected HeLa cells with siAPTX and XRCC1 siRNA and tested their sensitivity to MMS by using the colony formation assay. An acute loss of APTX and XRCC1 renders HeLa cells sensitive to MMS (Fig. ), suggesting a functional link between aprataxin and XRCC1.
Western blotting of cell lysates from HeLa cells transfected with different siRNA revealed that an acute loss of aprataxin leads to a reduced protein level of XRCC1 (Fig. ). A loss of XRCC1, however, has a minimal effect on aprataxin; neither the loss of aprataxin nor the loss of XRCC1 has an effect on the protein level of PNK (Fig. ). We measured XRCC1 protein stability when cells were transfected with different siRNA. When protein synthesis is inhibited with 80 μM of cycloheximide, XRCC1 protein is degraded much faster in siAPTX-transfected cells than in siVimentin-transfected cells (Fig. ). Therefore, aprataxin is required to stabilize XRCC1 in HeLa cells. This finding suggests an indirect mechanism for the requirement of aprataxin for cellular survival in response to MMS treatment in which aprataxin is required to maintain the steady-state protein level of XRCC1, which is essential for SSBR.
To substantiate this idea, we overexpressed XRCC1 after transfection of siAPTX in HeLa cells and measured MMS sensitivity. Exogenously expressed XRCC1 rescues MMS sensitivity of HeLa cells transfected with siAPTX to a significant degree (Fig. ). This result supports the notion that MMS sensitivity of HeLa cells after an acute loss of aprataxin may be due to the reduced protein level of XRCC1.