Volume 35, Issue 4, Pages 442-453 (August 2009) The F Box Protein Fbx6 Regulates Chk1 Stability and Cellular Sensitivity to Replication Stress  You-Wei Zhang, John Brognard, Chris Coughlin, Zhongsheng You, Marisa Dolled-Filhart, Aaron Aslanian, Gerard Manning, Robert T. Abraham, Tony Hunter  Molecular Cell  Volume 35, Issue 4, Pages 442-453 (August 2009) DOI: 10.1016/j.molcel.2009.06.030 Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 1 Fbx6 Regulates Chk1 Stability (A) A549 cells were transfected with 100 nM indicated siRNAs for 48 hr, and blotted with indicated antibodies. (B) A549 cells were infected with control or Fbx6 lentivirus vectors for 48 hr, and the indicated proteins were blotted. (C and D) Cells were transfected with the indicated siRNAs and treated after 48 hr with 160 μM CHX (C), or with 0.5 μM CPT (D), and representative Chk1 expression result is shown in the upper panels. Lower panels show quantitation of the Chk1 blots; data represent mean and standard deviation (SD) from two to four independent experiments. Molecular Cell 2009 35, 442-453DOI: (10.1016/j.molcel.2009.06.030) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 2 Fbx6 Regulates Chk1 Ubiquitination and Degradation (A) Flag-tagged F box proteins and HA-Chk1 were expressed in 293T cells, immunoprecipitated with anti-Flag antibodies and blotted with anti-HA antibodies. The same membrane was stripped and reblotted with anti-Cul1 antibodies. Whole cell extracts (WCE) were blotted for total protein expression. (B) Mutation of human Fbx6. Numbers represent the amino acid residues in human Fbx6. FL, N, C, and FBA represent the full-length, amino terminus, carboxyl terminus, and the FBA domain only of Fbx6, respectively. (C) Myc-tagged Fbx6 and HA-Chk1 were cotransfected into 293T cells, and cell extract was divided into two aliquots for immunoprecipitation with anti-Myc or anti-HA antibodies, followed by immunoblotting with anti-HA and anti-Myc antibodies, respectively. Protein expression was also determined in WCE. (D) WCE from untreated A549 cells were immunoprecipitated with mouse immunoglobulin G or anti-Chk1 antibodies, and blotted with anti-Fbx6 antibodies. (E) Expression of endogenous Chk1 from 293T cells stably transfected with pcDNA3-Flag-F box proteins. (F) HEK293T cells were transfected with HA-Chk1, Myc-tagged Fbx6 FL or mutants with His-ubiquitin for 48 hr. Cells were lysed and blotted with anti-HA antibodies. (G) In vitro ubiquitination assay. 293T cells were transfected with HA-Chk1 with (lane 2) or without His-ubiquitin (lanes 1 and 3–6) for 48 hr, cell extracts were immunoprecipitated with anti-HA (12CA5) antibodies, and the immobilized HA-Chk1 was used as the substrate in reactions containing soluble Myc-Fbx6 FL or mutant proteins as described in Supplemental Experimental Procedures. Reaction products were immunoblotted with rat anti-HA antibodies. Lanes 1 and 2: no addition of either Fbx6 or the in vitro ubiquitination reaction reagents (lane 2 serves as a positive control for ubiquitinated Chk1); lanes 3–5: in vitro ubiquitination with addition of Fbx6 FL, FBA, or the C-terminal mutant C, respectively; lane 6: same as lane 3 except no Fbx6 protein. Molecular Cell 2009 35, 442-453DOI: (10.1016/j.molcel.2009.06.030) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 3 A Degron-like Region in Chk1 (A) Schematic diagram of human Chk1. SQ, the Ser/Gln phosphorylation site cluster that includes S317 and S345; AIR, auto-inhibitory region. Letters on the left side identify the Chk1 fragment, and numbers on the right indicate the amino acid residue site based on the FL polypeptide. (B) 293T cells were transfected with Myc-tagged Chk1 fragments 1–5 for 48 hr, treated with 160 μM CHX, with or without 10 μM MG132, for the indicated times, and expression of proteins was examined. (C) Myc-Chk1 fragments 1-5 were transfected into Fbx6-expressing stable 293T cells, immunoprecipitated with anti-Flag antibody and blotted with anti-Myc antibodies. The same membrane was stripped and re-blotted with anti-Flag antibodies. WCEs were blotted with anti-Myc antibodies. (D) EGFP, EGFP-D1, EGFP-D2, or EGFP-H2B was transfected into 293T cells for 48 hr, cells were treated for 6 hr with 12 μM MG132, and expression of fusion proteins was analyzed. (E) EGFP-D1 or EGFP-D2 was transfected into Fbx6 stably expressing 293T cells, immunoprecipitated with anti-Flag antibodies, and blotted with anti-GFP antibodies. (F) HEK293T cells were infected for 24 hr with control or Fbx6 lentivirus shRNA vector, and then transfected with EGFP-D1 or EGFP-D2 plasmids. After another 48 hr, cells were lysed and blotted with indicated antibodies. The asterisk denotes a non-specific band serving as the loading control. (G) HA-Chk1 WT, S34E, or S345A were transfected into Flag-Fbx6 expressing stable 293T cells, immunoprecipitated with anti-Flag antibodies, and blotted with rat anti-HA antibodies. WCE was blotted to determine total protein expression. Molecular Cell 2009 35, 442-453DOI: (10.1016/j.molcel.2009.06.030) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 4 Regulation of Chk1 Protein Stability (A) 293T cells were transfected with Myc-Chk1 WT or K436R for 48 hr, treated with 320 μM CHX, and blotted with indicated antibodies. (B) Anti-Myc blots as shown in (A) were quantitated from two to four independent experiments and plotted. Data represent mean ± SD. (C) 293T cells were transfected with Myc-Chk1 WT or K436R with or without His-ubiquitin for 48 hr, lysed in 6 M guanidinium hydrochloride, and His-tagged ubiquitinated proteins were recovered on Co2+ beads, and eluates were blotted with anti-Myc antibodies. (D) Myc-Chk1 WT or 3RE mutant was expressed in 293T cells for 48 hr, treated as described in Figure 3B, and protein expression was examined. (E) The anti-Myc blots in (B) from two or three independent experiments were quantitated and normalized to the control sample. Data represent mean ± SD. (F) HEK293T cells were transfected with the indicated siRNAs for 24 hr, then transfected with Myc-Chk1 3RE mutant, and protein expression was examined after an additional 48 hr. (G) Myc-Chk1 WT or 3RE was transfected into Fbx6 expressing 293T cells for 48 hr, treated for 4 hr with 12.5 μM MG132 to allow accumulation of Chk1 3RE, cell extracts were immunoprecipitated with anti-Flag antibodies, and then blotted with anti-Myc antibodies. The same membrane was stripped and reblotted with anti-Flag antibodies. Total protein levels were blotted in WCE. (H) In vitro kinase assay. Equal amount of immunoprecipitated Chk1 WT or 3RE mutant was added into the kinase reaction, and immunoblotted with a specific phospho-S216-Cdc25C antibody. Parallel samples without ATP are shown in the lower panel. Molecular Cell 2009 35, 442-453DOI: (10.1016/j.molcel.2009.06.030) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 5 Chk1 Degradation and Checkpoint Recovery U2OS Tet/On cells transiently expressing Tet-inducible Flag-Chk1 WT or 3RE mutants were monitored for expression of Flag-Chk1 and cumulative BrdU incorporation as described. The fluorescence intensities (pixels) of Flag- and BrdU-positive cells were quantified with the Photoshop Histogram analysis program, and normalized to the 0 hr and preinduction samples for Flag and BrdU, respectively. (A) Relative Flag-Chk1 WT and 3RE expression levels. (B) Relative BrdU incorporation. Data represent mean ± SD from 50–100 cells. (C) In vitro trypsin digestion assay was performed as described in Supplemental Experimental Procedures. (D) Equal amounts of phosphorylated and non-phosphorylated Myc-Chk1 beads were used to perform in vitro kinase assays as described in Figure 4F legend. Indicated samples were pretreated for 15 min with 500 nM Chk1 kinase inhibitor PF-003946901 (Pfizer) on ice. Molecular Cell 2009 35, 442-453DOI: (10.1016/j.molcel.2009.06.030) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 6 Chk1 Degradation and CPT Sensitivity (A) Asynchronous A549 and MDA-MB-231 cells were treated for 8 hr with the indicated concentrations of CPT. Protein expression was determined by immunoblotting with the indicated antibodies. Numbers at the top of each sample lane represent the relative Chk1 protein level, normalized to that obtained in the no-drug control. (B) Cells treated as described in (A) were cultured for 48 hr in fresh medium. Cell death was determined by staining with trypan blue. The data are plotted as mean ± SD from three independent trials. (C) A549 and MDA-MB-231 cells were transfected for 48 hr with the indicated siRNAs and treated with CPT. Upper panel shows expression level of Chk1, and lower panel shows cell death after 36 hr of release from CPT treatment. The data are presented as mean ± SD from three independent experiments. (D) Asynchronous A549 and MDA-MB-231 cells were treated with 500 nM CPT or 15 Gy IR. After 8 hr, cell extracts were prepared and immunoblotted with the indicated antibodies. Numbers at the top of each sample lane represent relative Chk1 protein levels, normalized to that obtained in the nontreated control sample for each cell type. (E) Equal amounts of total proteins from exponentially growing A549, MDA-MB-231, and TK-10 cells were blotted with purified rabbit anti-Fbx6 antibody first, then stripped and sequentially reblotted with mouse anti-Chk1 and anti-tubulin antibodies. (F) MDA-MB-231 cells grown on glass cover slides were transfected with control vector, Flag-Fbx6 only, or Flag-Fbx6 plus Myc-Chk1 expression vectors. After 48 hr, cells were treated for 8 hr with 500 nM CPT, and were released into drug-free medium for another 12 hr; cells were then fixed and stained with anti-Flag and anti-Chk1 or anti-activated caspase 3 antibodies. The caspase-3-positive cells in control or Flag-Fbx6-expressing cells were counted; data represent mean ± SD from two independent experiments. At least 50 positive cells were counted in each experimental setting. Molecular Cell 2009 35, 442-453DOI: (10.1016/j.molcel.2009.06.030) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 7 Inverse Correlation between Chk1 and Fbx6 Expression (A and B) Asynchronously growing cells derived from (A) various tumor types and (B) non-small-cell lung cancer were lysed and blotted with anti-Chk1 and anti-Fbx6 antibodies, and the same membranes were stripped and reblotted with anti-tubulin and anti-PCNA antibodies, respectively. (C) Representative staining of Chk1 and Fbx6 in breast tumor sections. (D) The breast tumor staining data were analyzed and quantitated as described in Experimental Procedures. (E) Model for Chk1-regulated replication checkpoint activation and termination. The distal C terminus of Chk1 interacts with the kinase domain likely through adaptor proteins (marked with “X”). After the phosphorylation-induced conformational change, the CM (conserved motif) and part of the regulatory domain form the degron-like region of Chk1. For simplicity, the spatial regulation of Chk1 is omitted in this model. See text for details. Molecular Cell 2009 35, 442-453DOI: (10.1016/j.molcel.2009.06.030) Copyright © 2009 Elsevier Inc. Terms and Conditions