TIE2-mediated tyrosine phosphorylation of H4 regulates DNA damage response by recruiting ABL1

Abstract

DNA repair pathways enable cancer cells to survive DNA damage induced after genotoxic therapies. Tyrosine kinase receptors (TKRs) have been reported as regulators of the DNA repair machinery. TIE2 is a TKR overexpressed in human gliomas at levels that correlate with the degree of increasing malignancy. Following ionizing radiation, TIE2 translocates to the nucleus, conferring cells with an enhanced nonhomologous end-joining mechanism of DNA repair that results in a radioresistant phenotype. Nuclear TIE2 binds to key components of DNA repair and phosphorylates H4 at tyrosine 51, which, in turn, is recognized by the proto-oncogene ABL1, indicating a role for nuclear TIE2 as a sensor for genotoxic stress by action as a histone modifier. H4Y51 constitutes the first tyrosine phosphorylation of core histones recognized by ABL1, defining this histone modification as a direct signal to couple genotoxic stress with the DNA repair machinery.

Keywords: ABL1; ANG1; Cell biology; DNA repair; NHEJ; TIE2; oncogenes.

Fig. 1. Nuclear TIE2 localization is associated with the resistance of glioma to IR.

(A and B) Cell viability assay to determine the response to IR of (A) TIE2-expressing GSCs (GSC-13 and GSC-20), TIE2-nonexpressing GSC (GSC-17), and (B) TIE2 isogenic U251 cultures in a time-point experiment. (C) Colony-forming assay of isogenic U251 cells upon IR treatment. (D) TIE2 silencing results in the radiosensitization of GSCs and U251.Tie2 cells. ntsiRNA, nontargeting siRNA. (E and F) TIE2 localizes in the nucleus of U251 cells upon IR, as assessed by (E) immunofluorescence and confocal microscopic analysis and (F) Western blot analysis. (G) TIE2 localizes in the nucleus of GSCs upon in vivo IR of intracranial xenografts. (H) Schematic representation of Tie2 constructs with mutations within the NLS sequence. WT, wild type. (I and J) NLS mutations jeopardize TIE2 (I) nuclear translocation upon IR, and (J) U251 glioma radioresistance. Data represent means ± SD; **P ≤ 0.01, ***P ≤ 0.001. EV, empty vector.

Fig. 2. ANG1 induces TIE2 nuclear localization.

(A) Increase of total ANG1 protein levels in U251.Tie2 cells in response to IR. (B) ANG1 protein levels of expression upon in vivo IR treatment of GSC-20–derived intracranial xenografts. HPF, high-power field. (C) Decrease of cell membrane–bound TIE2 upon ANG1 exposure in U251.Tie2 cultures. IgG, immunoglobulin G. (D) ANG1 exposure results on TIE2 nuclear translocation. (E) TIE2 localizes in the nucleus of human umbilical vein endothelial cells (HUVECs) upon ANG1 exposure, as assessed by immunofluorescence and confocal microscopic analysis. DAPI, 4′,6-diamidino-2-phenylindole. (F) TIE2 protein levels in cytoplasmic and nuclear U251.Tie2 cellular compartments after exposure to several ligands. bFGF, basic fibroblast growth factor; VEGF, vascular endothelial growth factor. (G and H) Soluble TIE2 (sTIE2) (G) jeopardizes IR-induced TIE2 nuclear translocation and (H) sensitizes GSCs to IR. (I) NLS mutations jeopardize TIE2 nuclear translocation upon ANG1 exposure. Data represent means ± SD; **P ≤ 0.01, ***P ≤ 0.001.

Fig. 3. Nuclear TIE2 regulates DNA repair through a NHEJ mechanism.

(A) γH2AX persistence in U251.EV, U251.Tie2, and U251.Tie2SS/AA cells in response to IR. (B) Formation of TIE2/DNA complexes in U251.Tie2 in response to IR, analyzed after DNA/protein cross-linking and protein elution from precipitated DNA. (C) γH2AX/TIE2 colocalization in HUVECs after IR treatment, as assessed by immunofluorescence and confocal microscopy. (D) TIE2 complexes in HUVECs in response to ANG1, ANG2, and IR stimuli. IP, immunoprecipitation; WCL, whole-cell lysate. (E) Increased NHEJ efficiency in TIE2-expressing cells. GFP, green fluorescent protein. (F) Increased recovery of NHEJ reporter plasmids in TIE2-expressing cells. (G) TIE2 silencing results in decreased NHEJ efficiency. (H) Silencing endogenous TIE2 in U87 MG cells results in decreased NHEJ efficiency. (I) NHEJ efficiency is jeopardized in cytoplasmic-sequestered TIE2. Data represent means ± SD; **P ≤ 0.01, ***P ≤ 0.001.

Fig. 4. ABL1 is a reader of the TIE2-modified H4Y51 histone mark.

(A) rTIE2 potentially phosphorylates core histones. (B) rTIE2 potentially phosphorylates core histones at tyrosine residues. WB, Western blotting. (C) rTIE2 potentially phosphorylates rH4 but not rH3. (D) rTIE2 phosphorylates H4 as assayed using a phospho-tag gel. APase, alkaline phosphatase. (E) Cross-linked chromatin contains TIE2/H4 complexes upon ANG1 or IR stimulus. (F) Detection of phosphorylation of H4Tyr51 in the nucleosomes isolated from HEK293.Tie2 cells after ANG1 and IR exposure. Mass spectrometric analysis of a tryptic fragment at m/z mass/charge ratio of 630.7990 (mass error: 2.71 ppm) matched to the doubly charged phosphopeptide ISGLIpyEETR, suggesting that Y6 was phosphorylated. Mascot ion score was 52, with an expectation value of 6.5 × 10⁻⁵. Phosphotyrosine-containing peptide fragments are shown in dotted circles. MS/MS, tandem mass spectrometry. (G and H) Tyrosine phosphorylation of H4 analyzed using H4pY51 (G) and pTyr (H) antibodies with purified H4 mutant proteins. (I) Colocalization of TIE2 and H4pY51 in HEK293.Tie2 cells upon ANG1 and IR stimuli, as assessed by confocal microscopy. Colocalization was quantified with Olympus FluoView version 3.1a software. (J) rH4pY51 peptide binds to specific SH2 domain–containing proteins. (K) H4pY51 complexes with a panel of DNA repair proteins, including ABL1, in U251.Tie2-myc cells after IR exposure. (L) TIE2-induced NHEJ DNA repair is jeopardized by inhibiting ABL1 but not ABL2. Data represent means ± SD; **P ≤ 0.01, ***P ≤ 0.001.