XLF is a newly discovered member of the NHEJ pathway. Since the protein kinase activity of DNA-PKcs is required for NHEJ, here we asked whether XLF is a substrate for DNA-PK. We show that XLF is phosphorylated in vitro
by DNA-PK at two major sites, serines 245 and 251. Serine 245 is followed by leucine and therefore conforms to the non-SQ/TQ motif found in several other DNA-PK substrates (reviewed in [3
]). In contrast, serine 251 is followed by a glutamine and is therefore an SQ site (). DNA-PK also phosphorylates serine 263 and threonine 266 and possibly threonine 223 in vitro
(data not shown), however, these represent minor sites. All of the major in vitro
phosphorylation sites are located in the C-terminal 60 amino acids of XLF (amino acids 240–299), in a region that is outside of the characterized head and stalk domains of XLF and is predicted to be largely unstructured [8
]. Interestingly, the in vitro
DNA-PK phosphorylation sites in the related protein XRCC4, which like XLF also contains characteristic head and stalk domains, are also located in the extreme C-terminus [18
],inaregion that isalso predicted to be unstructured [18
]. Similarly, the in vitro
DNA-PK phosphorylation sites in both Ku70 and Ku80 are also located in regions that are predicted to be unstructured and/or are highly flexible in solution [33
]. It is likely that such flexible regions may be more accessible to the active site of the large DNA-PKcs polypeptide.
Summary of identified in vitro and in vivo phosphorylation sites in XLF
We also show that XLF is highly phosphorylated in vivo
, that in vivo
phosphorylation of XLF is largely PIKK-dependent and that the identified in vitro
sites, serines 245 and 251, represent the major in vivo
phosphorylation sites in XLF. Serine 203, which does not conform to a known protein kinase consensus sequence, was also phosphorylated in vivo
. Some phosphorylation of S132 was detected by MS/MS, but this was not confirmed by in vivo
labeling (). Other SQ sites, such as serine 55 and serine 132, which are highly conserved in vertebrate species (see in [8
]), were not identified as major sites of phosphorylation either in vitro
or in vivo
, possibly because they are located in globular domains that are either not accessible or are involved in dimerization or other protein–protein interactions.
Using phosphospecific antibodies, we show that serine 245 is phosphorylated in vivo in both unirradiated and irradiated transfected cells. Moreover, phosphorylation at this site was abrogated by inhibition of DNA-PK, suggesting that serine 245 is a DNA-PK-dependent phosphorylation site in vivo. In untransfectedcells, endogenous XLF was only phosphorylated at serine 245 in irradiated cells, suggesting that the observed DNA-PK-dependent phosphorylation of XLF at this site in unirradiated, transfected cells might be a result of transient transfection. Serine 251 was also phosphorylated in irradiated, non-transfected human cells and was phosphorylated in response to IR in XLF-transfected human cells. Interestingly, phosphorylation at this site was ATM-dependent, indicating that both DNA-PKcs and ATM can phosphorylate XLF in vivo.
The C-terminal ~70 amino acids of human XLF have been predicted to contain DNA-binding activity [8
]. Because the major in vitro
phosphorylation sites in XLF were located in the C-terminal portion of XLF, we tested whether this region of XLF did indeed bind directly to DNA in vitro
and whether phosphorylation by DNA-PK affected this interaction. Our experiments confirm that the C-terminus of human XLF does indeed possess the ability to bind to DNA, and as shown previously for full-length XLF, DNA binding of the C-terminal fragment was highly dependent on both the length of the DNA and protein concentration [12
]. Interestingly, the C-terminal ~70 amino acids of the Saccharomyces cerevisiae
homologue of XLF, Nej1p, also contains DNA binding activity and is phosphorylated in response to DNA damage in vivo
]. However, phosphorylation of the C-terminal fragment of human XLF, at least at the sites identified in this study, did not have a significant effect on its ability to interact with DNA in vitro
or in vivo.
We have also used three separate approaches to determine whether phosphorylation of XLF is required for DSB repair in vivo
. Using a UV laser to introduce DNA damage in living cells, we show that phosphorylation of the C-terminus of XLF is not required for the initial recruitment of XLF to DSBs in vivo
. These results are consistent with recent findings from Chen and colleagues that show that the protein kinase activity of DNA-PK is not required for recruitment of XLF to DNA after damage [21
]. However, a modest decrease in the retention of the phosphorylation mutant at sites of laser-induced DNA damage was observed, suggesting that phosphorylation of XLF might contribute to the release of XLF from DSBs in vivo
The kinetics of phosphorylation of histone H2AX is widely regarded as a surrogate for the kinetics of DSB repair in vivo
]. Our results show that both wt-XLF and XLF in which up to six of the identified in vivo
phosphorylation sites are mutated to alanine (6A-XLF) are able to complement the repair defect of XLF-deficient 2BN cells, and that the kinetics of γ-H2AX formation and dissipation are virtually identical in cells expressing either wt- or phosphorylation- defective XLF. Likewise, in colony formation assays, 6A-XLF was shown to complement survival after exposure to IR in 2BN cells. Together, these studies suggest that phosphorylation of XLF at the identified in vivo
sites is unlikely to be required for DSB repair.
We also conclude that, like the related protein, XRCC4, which is highly phosphorylated by DNA-PK in vitro
, XLF is not likely a physiologically relevant target of DNA-PK. However, we caution that these studies were carried out on transfected cells in which the expression of both wt- and phosphorylation-defective XLF protein was increased approximately eight-fold over the level of XLF in normal human fibroblasts (Supplementary Fig. 6
). It is therefore possible that over-expression could have masked subtle effects of phosphorylation on XLF function. It is also possible that by mutating multiple phosphorylation sites in XLF, differences in the effects of ATM-dependent versus DNA-PK-dependent phosphorylation events may have been missed. It is also possible that IR-induced phosphorylation of XLF affects other functions of the protein that were not examined here. However, we cannot exclude the possibility that, as has been suggested previously [38
], not all IR-induced phosphorylation events have functional significance, and that some may simply be a consequence of exposing cells to large amounts of DNA damage.