Quantitative proteomics unveiled: Regulation of DNA double strand break repair by EGFR involves PARP1.

ABSTRACT

BACKGROUND: EGFR inhibition blocks DNA double strand break (DSB) repair but the detailed mechanisms are still unclear. We asked whether EGFR inhibition blocks DSB repair by reducing the X-ray-induced phosphorylation of repair proteins using a phosphoproteomic approach. MATERIALS AND METHODS: Using UT-SCC5 and SAS head and neck cancer cells we established a differential phosphoproteomic approach for quantitative analysis of DNA repair proteins by stable isotope labeling with amino acids. Nuclear phosphoproteins were isolated and analyzed by liquid chromatography/tandem mass spectrometry. Erlotinib, PD98059 and olaparib were used to inhibit EGFR, MEK1/2 and PARP1, respectively. PARP1 was knocked down by siRNA. DSB repair was measured by quantifying residual 53BP1 foci. RESULTS: Over 150 nuclear phosphoproteins were quantified after irradiation, including 24 DNA repair proteins. Two of these, including PARP1, were consistently reduced in both cell lines upon erlotinib treatment. PARP1 inhibition or knock-down and EGFR inhibition resulted in an analog number of residual foci which was not further increased by combination of both strategies. MEK1/2 inhibition with or without blockage of EGFR or PARP1 caused similar effects. CONCLUSION: We have established a powerful, quantitative phosphoproteomic approach to investigate regulatory mechanisms in DSB repair, dependent on protein phosphorylation after irradiation. Using this approach we have identified PARP1 as a mediator of EGFR/MEK-dependent regulation of DSB repair.