Characterization of new interaction partners of the DNA double-strand break repair protein DNA-PKcs

Unrepaired DNA double-strand breaks can lead to apoptosis or
tumorigenesis. In mammals double-strand breaks are repaired mainly
by nonhomologous end joining mediated by the DNA-PK complex. The
core protein of this complex, DNA-PKcs, is a DNA-dependent
serine/threonine kinase that phosphorylates protein targets as well
as itself. To identify new proteins, which contribute to
double-strand break repair by nonhomologous end joining, we
previously performed a yeast two-hybrid screen with fragments of
human DNA-PKcs as bait. From the identified putative interaction
partners of DNA-PKcs we chose two for further characterization:
Protein phosphatase 5 (PP5) and Ku70 binding protein 3 (Kub3). With
PP5 we have identified the first protein phosphatase with a
function in doublestrand break repair. We show that protein
phosphatase 5 interacts with DNA-PKcs and dephosphorylates with
surprising specificity at least two functional sites of it. Cells
with either hypo or hyperphosphorylation of DNA-PKcs at these sites
show increased radiation sensitivity. For the characterization of
Kub3 we describe its correct reading frame and a putative
metalloprotease domain. Using a rabbit polyclonal antibody against
human Kub3 we demonstrate that Kub3 is a nuclear protein which
co-precipitates with DNA-PKcs. When Kub3 is overexpressed in HeLa
cells, DNA-PKcs phosphorylation at T2609 is increased after
ionizing radiation. However, when Kub3 is knocked down in
drosophila cells by RNAi, cell survival after ionizing radiation is
not affected. In summary, with PP5 and Kub3 we have characterized
two new interaction partners of DNA-PKcs. While PP5 clearly
functions in double-strand break repair, further experiments are
necessary to confirm such a role for Kub3.