Checkpoint-induced phosphorylation and activation of Chk1. (A) HeLa and U20S cells were untreated (−) or were treated (+) with 3 mM HU for 20 h. Cellular lysates containing 150 μg of protein were either resolved directly by SDS-PAGE (lanes 1, 2, 5, and 6) or were treated with 5 U of calf intestinal phosphatase (CIP) in the absence (lane 3) or in the presence (+ INH) of phosphatase inhibitors, including 10 mM β-glycerophosphate and 10 mM NaF (lane 4). Chk1 was detected by Western blotting with Chk1-specific polyclonal antibody (SC7898). (B) HeLa cells were cultured in DMEM alone (lane 1) or DMEM containing 50 μM cisplatin (lane 2), 3 μM etoposide (lane 3), or 3 μM ara-C (lane 4) for 1 h, after which the medium was replaced with fresh DMEM, and then the cells were cultured for an additional 6 h. In addition, HeLa cells were irradiated with 254-nm UV light at a dose of 10 J/m² (lane 5). Cellular lysates containing 150 μg of protein were resolved directly by SDS-PAGE on an 8% polyacrylamide gel. Chk1 was detected by Western blotting with Chk1-specific polyclonal antibody (SC7898). (C) HeLa cells were untreated (−) or were treated (+) with 3 mM HU for 17 h. Clarified cellular lysates were incubated with affinity-purified Chk1 antibodies, and kinase assays were performed in vitro in the presence of 5 μg of GST-Cdc25C_200–256. Reactions were resolved by SDS-PAGE, and proteins were transferred to nitrocellulose. Radiolabeled GST-Cdc25C_200–256was visualized by autoradiography. Levels of Chk1 in each immunoprecipitate were determined by Western blotting with monoclonal antibody specific for Chk1 (SC8408).

Analysis of Chk11 by gel filtration. HeLa cells were either mock infected or infected with recombinant adenovirus encoding Flag-tagged Chk1. After 15 h of infection, cells were either untreated (−HU) or were incubated with 10 mM HU (+HU) for 4 h. Clarified lysates were analyzed directly by SDS-PAGE and Western blotting (Total lysate) or were loaded onto a Superdex 200 10/30 column. For mock-infected cells, 150 μl from fractions 23 to30 was resolved by SDS-PAGE. For adenovirus-infected cells, fractions 23 to 30 were first incubated with anti-Flag–agarose. Chk1 and Flag-Chk1 were visualized by Western blotting with Chk1 polyclonal antibody (SC7898). Fractions containing protein standards of 158 and 44 kDa are indicated. The 44-kDa protein standard eluted primarily in fractions 30 and 31.

Checkpoint-induced phosphorylation of Chk1 is ATM independent but caffeine sensitive. (A) AT⁺ and AT⁻ cells were untreated (lanes 1 and 4) or were treated with 3 mM HU (lanes 2 and 5) or irradiated with 10 Gy of IR (lanes 3 and 6). Cellular lysates containing 150 μg of protein were resolved directly by SDS-PAGE on an 8% polyacrylamide gel. Chk1 was detected by Western blotting with Chk1-specific polyclonal antibody (SC7898). (B) HeLa cells were cultured in DMEM lacking caffeine (lanes 1 and 2) or containing 10 mM caffeine (lanes 3 and 4) for 2.5 h. Cells were cultured for an additional 20 h in the absence (lanes 1 and 3) or presence (lanes 2 and 4) of 3 mM HU. Cellular lysates containing 150 μg of protein were resolved directly by SDS-PAGE on an 8% polyacrylamide gel. Chk1 was detected by Western blotting with Chk1-specific polyclonal antibody (SC7898).

Chk1 is phosphorylated on serine 317 in vivo. (A). The structure of Chk1 is schematically represented to show the location of the kinase domain (amino acids 9 to 265), the SQ-rich region at the C terminus, and trypsin and Asp-N cleavage sites relative to potential phosphorylation sites. (B to D) 293 cells were either mock transfected or transfected with plasmids expressing myc-Chk1 (wild type) or myc-Chk1(S317A). Cells were incubated with ³²P-labeled inorganic phosphate in the presence of HU. Radiolabeled Chk1 was digested with trypsin, and the peptides were subjected to two-dimensional phosphopeptide mapping. Arrows indicate the direction of the first and second dimensions, and the origin is located where the two arrows meet. (E) HeLa cells were untreated (lanes 1 and 5) or were treated with either 3 mM HU (lanes 2, 3, 6, and 7) or 20 J of UV m² (lanes 4 and 8). Lysates were prepared and analyzed as directed by SDS-PAGE (lanes 1 to 4) or were first incubated with the S317 phosphospecific antibody in the absence (lanes 5, 6, and 8) or presence (lane 7) of competing phosphopeptide immunogen. Chk1 was detected by immunoblotting with a monoclonal antibody specific for Chk1 (SC8408).

Chk1 is also phosphorylated on serine 345 in vivo. 293 cells were transfected with plasmids expressing myc-Chk1 (wild type), myc-Chk1(S317A), myc-Chk1(S345A), and myc-Chk1(S357A, S366A). Cells were incubated with ³²P-labeled inorganic phosphate in the presence of HU. Radiolabeled Chk1 (wild type and mutants) was digested with trypsin followed by Asp-N endopeptidase, and peptides were subjected to two-dimensional phosphopeptide mapping. Arrows indicate the direction of the first and second dimensions, and the origin is located where the two arrows meet.

HU-induced activation of Chk1 requires phosphorylation of serines 317 and 345. (A) HeLa cells were infected with recombinant adenovirus encoding Flag-Chk1 (wild type), Flag-Chk1(S317A), Flag-Chk1(S345A), or Flag-Chk1(S317A, S345A). After 15 h of infection, cells were incubated with 10 mM HU for 4 h. Clarified lysates were loaded onto a Superdex 200 10/30 column. Fractions 26 and 27 were pooled. Fractions 26/27, 28, 29, and 30 were incubated with anti-Flag–agarose. Precipitates were monitored for their ability to phosphorylate GST-Cdc25C_200–25. Kinase reactions were resolved by SDS-PAGE, and proteins were transferred to nitrocellulose. Radiolabeled GST-Cdc25C_200–256 was visualized by autoradiography, Chk1 was visualized by Western blotting with Chk1-specific polyclonal antibody (SC7898).

Phosphorylation site mutants retain intrinsic protein kinase activity. (A) Chk1, Chk1(D130A), Chk1(S317A), Chk1(S345A), and Chk1(S317A, S345A) were purified as GST-fusion proteins in bacteria and tested for their ability to phosphorylate GST-Cdc25C_200–256 in vitro. Reaction products were resolved by SDS-PAGE, and proteins were transferred to nitrocellulose. Phosphorylated Cdc25C_200–256 was detected by autoradiography, and levels of GST-Chk1 (wild type and mutants) were visualized by Western blotting with Chk1-specific polyclonal antibody (SC7898). (B) HeLa cells were infected with recombinant adenoviruses encoding GFP (lane 1), Flag-Chk1 (lane 2), Flag-Chk1(S317A, S345A) (lane 3), and Flag-Chk1(D130A). Lysates were prepared and incubated with Flag-agarose. Precipitates were tested for their ability to phosphorylate GST-Cdc25C_200–256 in vitro. Reaction products were resolved by SDS-PAGE, and proteins were transferred to nitrocellulose. Phosphorylated Cdc25C_200–256 was detected by autoradiography, and levels of GST-Chk1 (wild type and mutants) were visualized by Western blotting with Chk1-specific polyclonal antibody (SC7898).

Interactions between ATR and Chk1 in vitro and in vivo. (A) 293 cells were transfected with plasmids encoding kinase-active (ATR⁺) and -inactive (ATR⁻) forms of Flag-tagged ATR. Lysates were prepared and incubated with anti-Flag–agarose. Precipitates were monitored for their ability to phosphorylate bacterially produced kinase-inactive Chk1 fused to GST (GST-Chk1D130A). Kinase reactions were resolved by SDS-PAGE, proteins were transferred to nitrocellulose, and radiolabeled Chk1 was visualized by autoradiography. (B to E) 293 cells were transfected with plasmid encoding Flag-tagged ATR. Lysates were prepared and incubated with anti-Flag–agarose. Kinase reactions were carried out in the presence of bacterially produced GST-Chk1(D130A), GST-Chk1(D130A, S317A), and GST-Chk1(D130A, S345A). Radiolabeled Chk1 proteins were digested with trypsin followed by Asp-N endopeptidase, and peptides were subjected to two-dimensional phosphopeptide mapping. The first and second dimensions of separation are indicated, and the origin is located where the two arrows meet. Phosphopeptides from proteolyzed GST-Chk1(D130A) were mixed with unlabeled phosphopeptide (LVQGISFpSQPTCP) prior to mapping (B), or the phosphopeptide was resolved alone (C) and visualized by staining with ninhydrin. The arrows indicate the position of the unlabeled phosphopeptide. (F) 293 cells were transfected with plasmids encoding kinase-active (ATR⁺) and -inactive (ATR⁻) forms of Flag-tagged ATR. Transfected cells were untreated (lanes 1, 3, 5, and 7) or were treated with 20 J of UV m² (lanes 2, 4, 6, and 8). Lysates were prepared and analyzed directed by SDS-PAGE (lanes 1 to 4) or were first incubated with the S317 phosphospecific antibody prior to SDS-PAGE (lanes 5 to 8). Chk1 was detected by immunoblotting with a monoclonal antibody specific for Chk1 (SC8408).