The CKAD sites of Claspin are specifically phosphorylated by a Drosophila homologue of CK1γ. (A) The GST-tagged Claspin CKAD fragment was expressed in Drosophila S2R+ cells in the absence (lanes 1 and 2) or presence (lanes 3–9) of individual FLAG-tagged Drosophila casein kinases. After 48 h, total cell lysates were prepared and immunoblotted with antibodies against Ser864-phosphorylated Claspin, GST, or the FLAG epitope. In lane 2, cells were treated with APH for 10 min prior to lysis. (B) In vitro kinase assay. Purified GST-CKAD peptide was incubated with the indicated FLAG-tagged Drosophila casein kinases immunoprecipitated from S2R+ cell lysates. The reactions were subjected to SDS–PAGE and immunoblotted with antibodies against Ser864-phosphorylated Claspin, GST, or the FLAG epitope. (C) Quantitation of the data from (B), expressed as a ratio of Ser864-phosphorylated Claspin to the total amount of each kinase.

siRNA knockdown of CK1γ1, but not CK1γ2 or CK1γ3, abolishes phosphorylation of Claspin in human cells. (A) Reduction of CK1γ1 protein levels by siRNA. U2OS cells were transfected with control or CK1γ1 siRNA for 48 h. Whole-cell lysates were prepared and subjected to immunoprecipitation with control (lane 1) or anti-CK1γ1 antibodies (lanes 2–3). The immunoprecipitates were subjected to immunoblotting with anti-CK1γ1 antibodies (Top). The asterisk denotes a nonspecific background band. The total level of α-tubulin in each lysate was also determined by immunoblotting (Bottom). (B) U2OS cells harboring an inducible form of the GST-CKAD peptide were treated with various amounts of CK1γ1 siRNA (0.5–50 nM) for 48 h. Cells were incubated in the absence (lane 1) or presence (lanes 2–9) of APH for 10 min. Expression of the GST-CKAD was induced with doxycycline 16 h before addition of APH. Whole-cell lysates were immunoblotted as indicated. (C) U2OS cells were incubated with no siRNA (lanes 1 and 2), TopBP1 siRNA (lane 3), CK1γ2 siRNA #1 (lane 4), or control siRNA for 48 h, and incubated in the absence (lane 1) or presence of APH for 10 min (lanes 2–5). Expression of the GST-CKAD was induced as described in (B). Whole-cell lysates were immunoblotted as indicated. (D) Same as (C), except CK1γ3 siRNA #2 was used for lane 4. (E) U2OS cells were treated for 48 h with no siRNA (lanes 1 and 2), TopBP1 siRNA (lane 3), CK1γ1 siRNA #1 (lane 4), CK1γ1 #2 (lane 5), CK1γ1 #3 (lane 6), all three CK1γ1 siRNAs (lane 7), or control siRNA (lane 8). Cells were incubated in the absence (lane 1) or presence (lanes 2–8) of APH for 10 min. Expression of the GST-CKAD was induced as described in (B). Total cell lysates were prepared and immunoblotted with the indicated antibodies. Band signals for phospho-Claspin and Claspin were detected and quantitated by using the Odyssey Infrared Imaging System (Bottom). Blotting membranes were also processed for standard ECL with secondary anti-rabbit antibodies (Top). (F) GST pulldown experiments. U2OS cells were treated with no siRNA (lanes 1 and 2), control siRNA (lane 3), or CK1γ1 siRNA #2 (lane 4) for 48 h and then incubated in the absence (lanes 1 and 2) or presence (lanes 3 and 4) of APH for 10 min. Expression of the GST-CKAD was either not induced (lane 1) or induced with doxycycline (lanes 2–4) 16 h before addition of APH. Whole-cell lysates were prepared and incubated with glutathione beads. Bead-associated proteins (top three panels) and whole-cell lysates (bottom two panels) were immunoblotted with the indicated antibodies.

Depletion of CK1γ1 results in abnormal cell growth, increased DNA damage, and failure in checkpoint responses. (A) Cell numbers in cultures of U2OS cells were determined at the indicated times following transfection with control siRNA or CK1γ1 siRNA #2. (B) Cell cycle distribution. Asynchronous culture of U2OS cells treated with control siRNA or CK1γ1 siRNA #2 for 48 h were stained with 7-AAD, and DNA content was analyzed by flow cytometry. Percentages of cells in each phase of the cell cycle were quantified with the FlowJo program (Ashland, OR). (C) DNA replication. U2OS cells treated with control siRNA or CK1γ1 siRNA #2 for 48 h, pulse-labeled with EdU for 45 min, stained with 7-AAD, and analyzed by flow cytometry. Percentages of EdU-positive cells are indicated. (D) Assay for damaged DNA. U2OS cells were treated with control siRNA, CK1γ1 siRNA #2, CK1γ2 siRNA #1, or Chk1 siRNA for 48 h. Cells were pulse-labeled with EdU for 45 min. Immunofluorescence staining was performed by incubating cells with Hoechst dye to detect DNA, Click-iT reagent to detect incorporated EdU, and antibodies against γ-H2AX to detect DNA damage foci. Scale bar: 30 μm. (E) Colony survival assay. U2OS cells were treated with control siRNA, CK1γ1 siRNA #2, CK1γ2 siRNA #1, or Chk1 siRNA for 48 h, and then incubated with the indicated doses of HU for 24 h. The cells were washed, trypsinized, and then replated at low density. Surviving colonies were stained with crystal violet and counted after 8 d. (F) Killing curve. U2OS cells treated with control siRNA or CK1γ1 siRNA #2 were grown in the presence of different doses of APH. Cells were stained with crystal violet after 6 d. Staining intensities were measured with the Odyssey Imaging System, and the relative percentages of surviving cells were plotted. (G) S-phase checkpoint. In the experimental scheme summarized on top, U2OS cells transfected with control siRNA or CK1γ1 siRNA #2 were first treated with APH for 10 min, allowed to recover for 60 min, and finally labeled with EdU for 30 min. Representative images showing the incorporation of EdU before and after treatment with APH are shown in the bottom. Scale bar: 50 μm. (H) Percentages of EdU-positive cells in (G) were counted and plotted. (I) G2/M checkpoint. U2OS cells transfected with control siRNA or CK1γ1 siRNA #2 were irradiated with UV (20 J/m²), and then incubated for 1 h before fixation. Cells were stained with anti-phospho-histone H3 antibodies conjugated with Alexa Fluor 488 dye and with 7-AAD. (J) Percentages of mitotic cells from (I) were determined by flow cytometry.

Assay of DNA damage checkpoint responses in Drosophila S2R+ cells. (A) Diagram showing the Xenopus Chk1 reporter construct used in Drosophila S2R+ cells. MT, metallothionein promoter; NLS, nuclear localization signal. (B) Expression of the Chk1-GST fusion protein was induced for 16 h and cells were treated in the absence or presence of either APH (10 μg/ml for 10 min) or HU (2 mM for 16 h). Whole-cell lysates were prepared and immunoblotted with antibodies against the Ser344-phosphorylated form of Xenopus Chk1 (Top) or GST (Bottom). (C) The Drosophila reporter cells and human U2OS cells were treated with APH for the indicated length of times. Whole-cell lysates were immunoblotted with the indicated antibodies against phospho-Chk1, GST, and human Chk1. (D and E) Phosphorylation of Chk1 in Drosophila cells is dependent on ATR and Claspin. Drosophila reporter cells were incubated with dsRNAs against Mei-41 (M) or Claspin (Cl) for 96 h. Subsequently, cells were treated with APH or HU, as in (B). Whole-cell lysates were prepared and analyzed by immunoblotting. dsRNA against firefly luciferase (F) was used as a control.

Human CK1γ1 phosphorylates the Claspin CKAD sites both in vitro and in human cells. (A) Tagged versions of human CK1γ1, CK1γ2, and CK1γ3 proteins were purified from Sf9 insect cells with nickel agarose beads, resolved by SDS–PAGE, and stained with Coomassie Brilliant Blue (CBB). (B) In vitro kinase assays. Purified GST-CKAD was incubated in vitro with no added kinase (lane 1), the indicated forms of CK1γ (lanes 2–4), or Xenopus Chk1-His6-GST (lane 5), as described in Materials and Methods. The samples were subjected to SDS–PAGE and immunoblotted with antibodies against Ser864-phosphorylated Xenopus Claspin (Top) or stained with Coomassie Brilliant Blue (Bottom). (C) In vitro kinase assays were performed as described in (B) with no substrate (lanes 1–3), wild-type GST-CKAD (lanes 4–6), or a mutant of the substrate in which Ser864 and Ser895 were changed to alanine (2AG; lanes 7–9). (D) In vitro kinase assays were performed as described in (B). Purified wild-type GST-CK1γ1 (WT) and its kinase-dead (KD) mutant were incubated in the absence (lanes 1–2) or presence (lanes 4–5) of the GST-CKAD substrate. For lane 3, the incubation contained substrate, but not any recombinant kinase. (E) Only CK1γ1 phosphorylates Claspin well in human cells. CK1γ1, CK1γ2, and CK1γ3 were coexpressed with the GST-CKAD in human U2OS cells. Cells were incubated in the absence or presence of APH for 10 min as indicated. Cell lysates were prepared and immunoblotted with anti-P-Ser864, anti-GST, and anti-FLAG antibodies.

CK1γ1 is required for the damage-induced phosphorylation of Chk1. (A) U2OS cells were incubated for 48 h with no siRNA (lanes 1 and 2), control siRNA (lanes 3, 4, and 9), TopBP1 siRNA (lane 5), CK1γ1 siRNA #2 (lane 6), CK1γ2 siRNA #1 (lane 7), and CK1γ3 siRNA #2 (lane 8). Cells were incubated in the absence (lanes 1 and 3) or presence (lanes 2 and 4–9) of APH for 10 min. Whole-cell lysates were immunoblotted with antibodies that detect phospho-Chk1 Ser345, phospho-Chk1 Ser317, Chk1 protein, and tubulin. (B) U2OS cells were treated for 48 h with no siRNA (lanes 1 and 2), TopBP1 siRNA (lane 3), CK1γ1 siRNA #1 (lane 4), CK1γ1 #2 (lane 5), CK1γ1 #3 (lane 6), all three CK1γ1 siRNAs (lane 7), or control siRNA (lane 8). Cells were incubated in the absence (lane 1) or presence (lanes 2–8) of APH for 10 min. Whole-cell lysates were prepared and immunoblotted with antibodies that detect phospho-Chk1 Ser317, Chk1 protein, and tubulin. Band signals for phospho-Chk1 and Chk1 were detected and quantitated by using the Odyssey Infrared Imaging System (Bottom). Blotting membranes were also processed for standard ECL, with secondary anti-rabbit or anti-mouse antibodies (Top).

Contribution of multiple CK1γ1 isoforms to the phosphorylation of Claspin. (A) Distinct localizations of various isoforms of CK1γ1 in human cells. U2OS cells expressing FLAG-tagged CK1γ1 isoforms were processed for indirect immunofluorescence with anti-FLAG antibodies. Scale bar: 30 μm. (B) Differential phosphorylation of Claspin by CK1γ1 isoforms. The CKAD fragment of Claspin was coexpressed with different isoforms of CK1γ1 in human U2OS cells. Whole-cell lysates were prepared and analyzed by immunoblotting. (C) Isoform-specific siRNAs. U2OS cells expressing the FLAG-tagged CK1γ1 isoform C were treated with a control siRNA, CK1γ1 siRNA #2, or two isoform-specific siRNAs (#71 and #156). Note that siRNA #156, which targets the coding/3′-UTR junction region of CK1γ1 isoform C, only partially pairs with the FLAG-tagged version of CK1γ1 isoform C in the expression plasmid. (D and E) Knockdown of CK1γ1 isoform C expression only partially compromises phosphorylation of Claspin and activation of Chk1. U2OS cells, transfected with the different siRNAs as indicated, were treated with APH, and whole-cell lysates were then prepared and analyzed by immunoblotting. (F and G) Rescue experiment. U2OS cells were concurrently infected with three lentiviruses encoding siRNA-resistant forms of CK1γ1 isoforms A, B, and C. After siRNA transfection and treatment with APH as indicated, whole-cell lysates were prepared and analyzed by immunoblotting. Quantitation of the band intensities was performed with the Odyssey Imaging System. Relative levels of phosphorylation of Claspin and Chk1 were calculated and plotted in (G). (H) Model for the role of CK1γ1 in mediating the activation of Chk1.