A Spatial and Functional Interaction of a Heterotetramer Survivin–DNA-PKcs Complex in DNA Damage Response

Abstract

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 coimmunoprecipitation and flow cytometry-based Förster resonance energy transfer assays, we demonstrated a direct involvement of the survivin baculovirus IAP repeat 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 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. Significance: These findings provide insight into survivin-mediated regulation of DNA-PKcs kinase and broaden our knowledge of the impact of survivin in modulating the cellular radiation response. See related commentary by Iliakis, p. 2270

Localization matters: nuclear-trapped Survivin sensitizes glioblastoma cells to temozolomide by elevating cellular senescence and impairing homologous recombination

“…It was recently reported that upon ionizing radiation Survivin interacts with DNA-PKcs supporting NHEJ [23]. To analyze whether Survivin interacts with components of HR, we co-immunoprecipitated Survivin in the nuclear fraction of LN229 cells exposed to 50 µM TMZ for 48 h and conducted mass spectrometry-based proteome analysis.…”

“…As TMZ causes cell cycle arrest in the G2-phase, the main repair pathway of TMZ-induced DSBs is HR. A supporting function of Survivin in the repair of ionizing radiationinduced DSBs by NHEJ in glioma cells has been suggested [22] and the physical and spatial interaction of Survivin with DNA-PKcs in irradiated cells recently verified [23]. To elucidate the impact of Surv-GFP and SurvNESmut-GFP on induction and repair of DSBs after TMZ treatment, γH2AX foci formation was analyzed as surrogate marker for DSBs.…”

Dimerization underlies the aggregation propensity of intrinsically disordered coiled‐coil domain‐containing 124

“…CO‐IP assay was performed as reported in detail previously 34 . Briefly, HEK293T cells were co‐transfected with CCDC124‐HA and CCDC124‐flag plasmids using Lipofectamine 2000.…”

Tumor Suppressor Protein p53 and Inhibitor of Apoptosis Proteins in Colorectal Cancer—A Promising Signaling Network for Therapeutic Interventions

“…By this, Survivin accumulates in the nucleus and interacts with a prime non-homologous end joining repair factor DNA-dependent protein kinase (DNA-PKcs) [67,68]. Survivin forms a heterotetramer complex with DNA-PKcs that results in a conformational change on the DNA-PKcs phosphoinositide 3-kinase domain with enhanced enzymatic activity and detection of differentially abundant phosphopeptides and proteins implicated in the DNA damage response [69].…”