title: 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.
  
  
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Laboratory of Radiobiology & Experimental Radiooncology, University Medical Center Hamburg - Eppendorf, Germany.
authors.forename: Laura
authors.initials: L
authors.lastname: Myllynen
authors.affiliation: Institute of Clinical Chemistry, University Medical Center Hamburg - Eppendorf, Germany.
authors.forename: Marcel
authors.initials: M
authors.lastname: Kwiatkowski
authors.affiliation: Laboratory of Radiobiology & Experimental Radiooncology, University Medical Center Hamburg - Eppendorf, Germany.
authors.forename: Lisa
authors.initials: L
authors.lastname: Gleißner
authors.affiliation: Laboratory of Radiobiology & Experimental Radiooncology, University Medical Center Hamburg - Eppendorf, Germany.
authors.forename: Britta
authors.initials: B
authors.lastname: Riepen
authors.affiliation: Laboratory of Radiobiology & Experimental Radiooncology, University Medical Center Hamburg - Eppendorf, Germany.
authors.forename: Konstantin
authors.initials: K
authors.lastname: Hoffer
authors.affiliation: Institute of Clinical Chemistry, University Medical Center Hamburg - Eppendorf, Germany.
authors.forename: Marcus
authors.initials: M
authors.lastname: Wurlitzer
authors.affiliation: Laboratory of Radiobiology & Experimental Radiooncology, University Medical Center Hamburg - Eppendorf, Germany.
authors.forename: Cordula
authors.initials: C
authors.lastname: Petersen
authors.affiliation: Laboratory of Radiobiology & Experimental Radiooncology, University Medical Center Hamburg - Eppendorf, Germany.
authors.forename: Ekkehard
authors.initials: E
authors.lastname: Dikomey
authors.affiliation: Laboratory of Radiobiology & Experimental Radiooncology, University Medical Center Hamburg - Eppendorf, Germany.
authors.forename: Kai
authors.initials: K
authors.lastname: Rothkamm
authors.affiliation: Institute of Clinical Chemistry, University Medical Center Hamburg - Eppendorf, Germany.
authors.forename: Hartmut
authors.initials: H
authors.lastname: Schlüter
authors.affiliation: Laboratory of Radiobiology & Experimental Radiooncology, University Medical Center Hamburg - Eppendorf, Germany. Electronic address: m.kriegs@uke.uni-hamburg.de.
authors.forename: Malte
authors.initials: M
authors.lastname: Kriegs
mesh_terms: D002294:Carcinoma, Squamous Cell / Q000235:genetics; D004247:DNA / Q000235:genetics; D053903:DNA Breaks, Double-Stranded; D004260:DNA Repair / Q000502:physiology*; D004268:DNA-Binding Proteins / Q000235:genetics; D004791:Enzyme Inhibitors / Q000494:pharmacology; D066246:ErbB Receptors / Q000037:antagonists & inhibitors / Q000502:physiology*; D000069347:Erlotinib Hydrochloride / Q000494:pharmacology; D005419:Flavonoids / Q000494:pharmacology; D006258:Head and Neck Neoplasms / Q000235:genetics; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics; D010766:Phosphorylation / Q000528:radiation effects; D010793:Phthalazines / Q000494:pharmacology; D010879:Piperazines / Q000494:pharmacology; D000071137:Poly (ADP-Ribose) Polymerase-1; D011065:Poly(ADP-ribose) Polymerases / Q000502:physiology*; D040901:Proteomics; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000494:pharmacology; D000077195:Squamous Cell Carcinoma of Head and Neck; D014407:Tumor Cells, Cultured; D000071857:Tumor Suppressor p53-Binding Protein 1
chemical_list: D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D005419:Flavonoids; D047908:Intracellular Signaling Peptides and Proteins; D010793:Phthalazines; D010879:Piperazines; D034741:RNA, Small Interfering; C498301:TP53BP1 protein, human; D000071857:Tumor Suppressor p53-Binding Protein 1; D004247:DNA; D000069347:Erlotinib Hydrochloride; C499320:PARP1 protein, human; D000071137:Poly (ADP-Ribose) Polymerase-1; D011065:Poly(ADP-ribose) Polymerases; D066246:ErbB Receptors; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one; C531550:olaparib
keywords: DNA repair proteins; EGFR inhibition; PARP1; Phosphorylation; Quantitative proteomics; SILAC