Identification of an ATR-associated protein, Cep164, and its interaction with ATR and ATRIP. (A) A diagrammatic representation of domains and motifs in Cep164 sequence. Cep164 consists of 1455 amino acids. M26 and M4 are GST-Cep164 fusion peptides used as antigens for the production of antibodies. The corresponding antibodies are designated anti-M26 and anti-M4, respectively. N11 is a monoclonal antibody isolated using GST-Cep1641-194 fusion protein as antigen. The 14-amino-acid peptide and phospho-Ser186 peptide of Cep164 used to raise polyclonal anti-Cep164 and anti-phospho-Ser186 (anti-p Ser186) antibodies are shown. The asterisks indicate potential substrate sites (SQ/TQ) of ATR/ATM kinases; the encircled asterisk specifies Ser186 of Cep164. The triangle and black and gray rectangles depict the WW domain, Rad26 homologous domain, and coiled-coil region, respectively. (B) Specificity of Cep164 antibodies. Monoclonal anti-Cep164 antibody (M26) was used to immunoprecipitate endogenous or overexpressed GFP-Cep164. The same antibody was used for immunoblotting to detect Cep164 in HeLa cells lysate or in vitro translated product, or in the immunoprecipitates. (C) Nuclear localization of endogenous Cep164 or GFP-Cep164. HeLa cells were fixed and stained with anti-Cep164 (M4) and DAPI. HeLa cells were visualized under a fluorescence microscope 8 h after transfection.

Interaction of Cep164 with ATR and ATM complexes. (A) Interaction between Cep164 and ATR in vivo. HeLa cell lysates were incubated with anti-Cep164, anti-ATR, and anti-ATRIP antibodies, respectively, in the presence (lanes 2–5) or absence (lanes 6–8) of ethidium bromide. Immunoprecipitates were separated by SDS-PAGE followed by immunoblotting using monoclonal anti-Cep164 (M26) and anti-ATR (2B5) antibodies. (B) Direct interaction of Cep164 with ATRIP. (Top panel) The indicated GST fusion peptides were subjected to pull-down assay with HeLa cell lysate and blotted with ATRIP antibody. ³⁵S-Methinonine-labeled ATRIP by in vitro transcription/translation was subjected to the indicated GST fusion peptide of Cep164 and the samples were electrophoresed; the gel was dried and exposed to X-ray film. (C) Effects of DNA damage on interaction between Cep164 and ATR or ATM. HeLa cells exposed to IR or UV were incubated for 1 h, and the Cep164 immunoprecipitates were prepared. The samples were analyzed by immunoblotting using ATM, ATR, and Cep164 antibodies. (D) Fractionation of HeLa cell lysate. HeLa cell lysates were fractionated using a Superdex-200 column, and samples were analyzed by immunoblotting.

Modification of Cep164 in response to DNA damage or replicative stress. (A) Modification of Cep164 upon UV irradiation. HeLa cells were treated with the indicated doses of UV, cells were lysed 1 h later, and lysates were analyzed by immunoblotting. (Top panel) Mitosin p84 is expressed at a constant level and serves as a loading control. UV-irradiated (20 J/M²) cells were lysed at the indicated time points post-UV irradiation. (Bottom panel) The samples were immunoblotted with the specified antibody. (B) UV-induced Cep164 foci formation. HeLa cells were exposed to UV at 20 J/M² and incubated for 2 min. Cells were fixed and stained with anti-Cep164 (M4) antibodies. DNA was visualized by DAPI staining. (C) Modification of Cep164 upon IR. HeLa cells were treated with indicated doses of IR, and lysates were prepared 4 h later. Lysates were analyzed as described previously. IR-irradiated (20 Gy) cells were analyzed at different time points post-IR. (D) IR-induced Cep164 foci formation. HeLa cells were exposed to UV at 20 Gy and incubated for 2 min. Cells were fixed and stained with anti-Cep164 (M4) antibodies. DNA was visualized by DAPI staining. (E) Modification of Cep164 upon replication stress. HeLa cells were treated with 1 mM hydroxyurea or 2.5 μg/mL aphidicolin for the indicated period of time, and cell lysates were analyzed as described. (F) Replication stress-induced Cep164 foci formation. HeLa cells were exposed to 1 mM hydroxyurea for an indicated time, and cells were fixed and stained with anti-Cep164 (M4) antibodies and DAPI.

Mapping of a phosphorylation site in Cep164. (A) The effect of caffeine on the phosphorylation of Cep164. HeLa cells were treated with 2 mM caffeine for 1 h and irradiated with UV at 20 J/M². The treated and mock-treated cells were lysed at the indicated time points and analyzed as described. (B) Phosphatase treatment reverts the slowly migrating modified Cep164. HeLa cells were UV-irradiated with 20 J/M² and incubated for 1 h. The nuclear lysates were prepared and treated with or without alkaline phosphatase and subjected to immunoblotting with anti-Cep164 antibody. (C) In vitro ATR kinase assays using GST-Cep164 substrate. The GST-Cep164^wt (amino acids 174–194) and GST-Cep164^S186A (Ser186 to Ala186) polypeptides were subjected to in vitro kinase assays using the ATR immunoprecipitates from lysates of UV-irradiated HeLa cells. Samples were gel-electrophoresed and immunoblotted with phospho-Ser186 antibody (top panel) or stained with Coomassie blue (bottom panel). (D) In vitro ATM kinase assays using GST-Cep164 substrate. Experiments were as described in C, except immunoprecipitated ATM was used in the assay. (E) Specificity of phospho-Ser186 antibody. HeLa cells were treated with 20 J/M² of UV, and the cells were lysed at different time points as indicated. The samples were immunoblotted using phospho-Ser186, Cep164, and p84 antibodies, respectively.

Cep164 is an in vivo substrate of ATR. (A) Phosphorylation of Cep164 in cells expressing ATR^KD. Cells with tetracycline-regulated ATR^KD expression vector were grown in the presence or absence of doxycycline for 72 h. Cells were harvested at different time points after UV irradiation. Lysates were analyzed using immunoblotting. (B) Phosphorylation of Cep164 in cells with ATR knockdown. HeLa cells were transfected with either ATRi or GFPi and were irradiated with 20 J/M² of UV 72 h post-transfection. Lysates were prepared at the indicated time points and analyzed using Western blotting. (C) UV-induced phosphorylation of Cep164 in AT cells. AT lymphoblastoid cells and ATM-complemented cells were irradiated with 20 J/M² and lysed at the indicated time points. Cell lysates were analyzed as described. (D) IR-induced phosphorylation of Cep164 in cells with ATR knockdown. HeLa cells were transfected with either ATRi or GFPi; cells were irradiated with 20 Gy of IR 72 h post-transfection. Cell lysates were analyzed using Western blotting. (E) IR-induced phosphorylation of Cep164 in AT cells. AT lymphoblastoid cells and ATM-complemented cells were irradiated with 20 Gy of IR. Cell lysates were analyzed using Western blotting. (F) ATR kinase activity and Cep164 foci. HeLa cells were knocked down with GFP RNAi or ATRIP RNAi, and the cells were irradiated with IR or UV. The cells were subjected to immunostaining with ATRIP and Cep164 antibody.

Cep164, MDC1, and RPA in ATR/ATM signaling pathways. (A) The effect of RPA70 knockdown on the phosphorylation of Cep164. HeLa cells were transfected with either RPA70i or GFPi; cells were irradiated with the indicated doses of UV 48 h post-transfection and lysed 1 h post-UV irradiation. Lysates were analyzed by Western blotting as indicated. (B) The effect of MDC1 knockdown on the phosphorylation of Cep164. HeLa cells were transfected with either MDC1i or GFPi; cells were irradiated with 20 J/M² of UV 72 h post-transfection. Cell lysates were analyzed by immunoblotting as indicated. (C) The effects of Cep164 knockdown on the ATR signal pathway. HeLa cells were transfected with either Cep164i or GFPi; cells were irradiated with the indicated doses of UV 72 h post-transfection. Cells were harvested 1 h post-UV irradiation; samples were analyzed by immunoblotting. (D) HeLa cells were transfected with either control RNAi or Cep164 RNAi and irradiated with IR at 10 Gy or UV at 10 J/M² 48 h post-transfection, followed by immunostaining with ATRIP or Cep164 antibody at the time points as indicated in the bottom panel. Percentage of ATRIP foci was scored from 200 cells for each time point. Only cells with more than eight foci were counted. (E) G2/M checkpoint in cells with Cep164 knockdown. HeLa cells were transfected with Cep164i, ATRi, or GFPi and irradiated at 20 J/M² 48 h post-transfection. The cell cycle distributions are based on FACS analyses of GFPi- or Cep164i-transfected cells prior to radiation. (Top figure) Samples were collected at the indicated time points. (Bottom figure) The RNAi-transfected cells were irradiated with the indicated doses of UV, and the samples were collected 2 h after UV irradiation. These cells were fixed and stained with DAPI or costained with phospho-histone 3^S10 antibody. The cells with condensed nuclear or positively stained with phospho-histone 3^S10 antibody were considered as in G2/M phase. The percentage of cells in G2/M phase was determined. The G2/M percentage in control cells (without UV treatment) was artificially set as 100%, and all other percentages shown on both the top and bottom panels are relative ratios compared with the control. (F) Nuclear morphology of cells with Cep164 knockdown. HeLa-H2B-EGFP cells were transfected with Cep164 or Luciferase RNAi. Forty-eight hours after transfection, cells were fixed and the nuclear morphology was compared under the microscope. (Top) Nuclear morphology of cells transfected with Luciferase RNAi. (Middle) Nuclear morphology of cells transfected with Cep164 RNAi. (Bottom) The percentage of cells harboring multinuclei. Bars: top and middle figures, 10 μm.

Cep164 and the ATM/ATR signal pathways. ATM and ATR serve as sensors responding to DNA damage induced by IR or UV, respectively. Mre11 nuclease of the MRN complex processes damaged DNA to generate ssDNA overhang. ATR phosphorylates CHK1 at Ser317 and activates the G2/M checkpoint. MDC1 and H2AX are upstream mediators whose phosphorylation is required for CHK1 phosphorylation and activation. The phosphorylation of Cep164 and MDC1 was compromised in cells with MDC1 or Cep164 knockdown, respectively, suggesting that these two proteins are mutually dependent for their proper regulation. RPA phosphorylation requires Cep164. See the text for details. (↓) Activation. (*) RPA is required for UV-induced Chk1 activation, but whether RPA phosphorylation is required has yet to be shown.