Substantial evidence has shown that overexpression of the inhibitor of apoptosis protein (IAP) Survivin in human tumors correlates significantly with treatment resistance and poor patient prognosis. Survivin serves as a radiation resistance factor that impacts the DNA damage response by interacting with DNA-dependent protein kinase (DNA-PKcs). However, the complexity, molecular determinants and functional consequences of this interrelationship remain largely unknown. By applying co-immunoprecipitation and flow cytometry-based Förster resonance energy transfer assays, we demonstrated a direct involvement of the Survivin baculovirus IAP repeat (BIR) domain in the regulation of radiation survival and DNA repair. This Survivin-mediated activity required an interaction of residues S20 and W67 with the phosphoinositide 3-kinase (PI3K) domain of DNA-PKcs. In silico molecular docking and dynamics simulation analyses, in vitro kinase assays, and large-scale mass spectrometry suggested a heterotetrameric Survivin-DNA-PKcs complex that results in a conformational change within the DNA-PKcs PI3K domain. Overexpression or depletion of Survivin resulted in enhanced PI3K enzymatic activity and detection of differentially abundant phosphopeptides and proteins implicated in the DNA damage response. The Survivin-DNA-PKcs interaction altered the S/T-hydrophobic motif substrate specificity of DNA-PKcs with a predominant usage of S/T-P phosphorylation sites and an increase of DNA-PKcs substrates including Foxo3. These data demonstrate that Survivin differentially regulates DNA-PKcs-dependent radiation survival and DNA double-strand break repair via formation of a Survivin-DNA-PKcs heterotetrameric complex.
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