Fig 2: Serines 558/561 and 688/689 are phosphorylated by CK2. (a) CK2 phosphorylates MRE11 in vitro. FLAG MRE11FLAG MRE11 ?N WT or M1, M2, M3, M23 and M123 proteins were immunoprecipitated from HEK293T cells using anti-FLAG M2 affinity gel. Beads were washed with 1m NaCl to eliminate contamination with protein kinases and incubated with 10 U of CK2 holoenzyme complex (a2/ß Biaffin GmbH, Kassel, Germany) in 30 µl of kinase buffer (20 mM Tris-HCl (pH 8.0); 50 mM KCl; 10 mM MgCl2) supplemented with 33 µM ATP and [?-32P]ATP (5 µCi) and incubated for 30 min at 37 °C. Reaction was stopped by the addition of 4 × Laemmli buffer (200 mM Tris (pH 6.8), 400 mM dithiothreitol (DTT), 8% sodium dodecyl sulfate (SDS), 0.4% bromophenol blue, 40% (v/v) glycerol) and phosphorylation of MRE11 was detected by autoradiography. Amount of the FLAG MRE11 was determined by immunoblotting. (b) MRE11 pS688/689 antibody specifically recognizes phosphorylated MRE11. We raised a rabbit polyclonal antibody against synthetic VSKGVDFEpSpSEDDDDD peptide, where pSpS represent phosphorylated serines corresponding to pS688/9 of human MRE11 and performed a two-step affinity purification using non-phosphorylated and phosphorylated peptide (Davids Biotechnologie GmbH, Regensburg, Germany). U2OS cells were transfected with siRNA against MRE11 (siMRE11) or with plasmids coding for GFP MRE11 WT or GFP MRE11 S688/9A. Whole-cell lysates were analyzed by immunoblotting with indicated antibodies. Antibody against tubulin (GeneTex; GTX112141) was used as a loading control. (c) Phosphorylation of MRE11 on serine 688/689 does not change in the presence of genotoxic stress. DNA damage was induced in U2OS cells by exposure to ionizing radiation (10 Gy 1 h, generated by X-ray instrument T-200; Wolf-Medizintechnik, St Gangloff, Germany), UVC (10 J/m2 for 1 h) or hydroxy urea (2 mM for 16 h) and whole-cell lysates were analyzed by immunoblotting. (d) CK2 inhibition decreases phosphorylation of MRE11 S688/689. U2OS cells were treated with dimethyl sulfoxide (DMSO) or CK2 inhibitor (5–30 µm, CX-4945; MedChemExpress, Monmouth Junction, NJ, USA) for 1 or 24 h and whole-cell lysates were probed with indicated antibodies. Antibody recognizing a general phospho-CK2 substrate (Cell Signaling; no. 8738) was used as the control of CK2i efficiency. (e) CK2 knockdown decreases phosphorylation of MRE11 S688/689. U2OS cells were treated with siRNA targeting luciferase (siLUC) and siRNA targeting CK2 subunit a, ß or both for 4 days. Whole-cell lysates were probed with indicated antibodies. Antibody recognizing CK2a was from GeneTex (GTX107897). (f) CK2 promotes the interaction between PIH1D1 and MRE11. Purified FLAG MRE11 ?N was dephosphorylated with ? phosphatase and rephosphorylated by recombinant CK2 as indicated and incubated with GST or GST PIH1D1. Bound proteins were analyzed by immunoblotting. Recombinant full-length GST PIH1D1 is at mark 55 kDa, and other bands are degradation products.