The F Box Protein Fbx6 Regulates Chk1 Stability and Cellular Sensitivity to Replication Stress

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1 Molecular Cell, Volume 35 Supplemental Data 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, and Tony Hunter Supplemental Experimental Procedures Plasmid Construction Flag-Fbx6 was kindly provided by Dr. Michele Pagano (NYU). To generate Myc-tagged Chk1 constructs, PCR products flanking different regions of Chk1 were inserted into the EcoR1 site of pcs3-6xmyc vector. Myc-tagged Fbx6 constructs were generated by PCR from the Flag-Fbx6 vector with BamHI and EcoRI sites at 5 and 3, respectively, and inserted into the BglII/EcoRI sites of the pcs3-6xmyc vector. The correct orientation was confirmed by restriction enzyme digest and sequencing. To generate GST-tagged Chk1 carboxyl-terminal constructs, PCR products were inserted into the BamH1/EcoR1 sites of pgex4t-1 vector. To generate the Tetinducible Chk1 construct, the full-length Chk1 WT PCR product with an N-terminal Flag tag was inserted into the EcoR1 site of puhd10-3 (+) vector, and the correct orientation was confirmed by digestion and sequencing. Mutations were introduced with the QuikChange Mutagenesis Kit (Qiagen) and confirmed by sequencing. Primer sequences will be provided upon request. In Vitro Chk1 Activation To prepare crude concentrated HeLa nuclear extracts, 5 x 10 8 cells were suspended in 3 ml buffer A (10 mm HEPES ph 7.9, 1.5 mm MgCl 2, 10 mm KCl, 0.5 mm DTT, and protease inhibitors- PMSF, aprotinin, leupeptin) on ice for 15 min, and lysed by 5 cycles of Dounce homogenization

2 (5 homogenizer strokes performed every 5 min). Nuclei were collected by centrifugation at 1,000 G for 5 min at 4. Then the pellet was resuspended in 1.8 ml buffer B (20 mm HEPES, ph 7.9, 20% glycerol, 420 mm NaCl, 1.5 mm MgCl 2, 0.2 mm EDTA, 0.5 mm DTT, and protease inhibitors), and incubated on ice for 2 h with a Dounce homogenizer every 15 min. The proteins were then centrifuged at 4 for 8 min at 14,000 rpm, aliquoted, and stored at -80. To obtain Chk1 proteins, 293T cells were transfected with Myc-Chk1 WT, and cell extracts were prepared after 48 h. The extracts were immunoprecipitated with anti-myc antibodies, and the immunoprecipitates were incubated for 2 h in TBS buffer containing calf intestinal alkaline phosphatase at 37. The immunoprecipitates were washed 6 times with TBS buffer, and were resuspended in 80 μl TBS to form a 50% slurry. The beads were then divided into two fractions, and one fraction was incubated for 1-4 h with 20 μl HeLa nuclear extracts in the presence of 10 μm okadaic acid, 1 mm ATP, 10 ng/ml creatine kinase, 3 mm phosphocreatine, 10 μm MG132, and 50 ng/μl poly(da/dt) at 30. After 6 additional TBS washes, the Myc-Chk1 protein was stored at -20 until used in experiments. In Vivo Ubiquitination Assay 293T cells were transfected with Myc-tagged Chk1 WT or K436R mutant with or without Hisubiquitin for 48 h, and were treated with 5 μm MG132 during the last 18 h. The cells were lysed in 6 M guanidinium hydrochloride in 0.1 M phosphate buffer (ph 8.0), sonicated for 1 min, and His-ubiquitin-modified proteins were purified with the Talon cobalt affinity resin (Clontech). The eluate was precipitated with 1 g/ml trichloroacetic acid and resuspended in 0.1 M phosphate buffer ph 8.0, followed by addition of one volume of 2X sample buffer. The eluted proteins were resolved by SDS-PAGE, and blotted with anti-myc antibodies.

3 In Vitro Ubiquitination HA-Chk1 was expressed in 293T cells, pulled down with anti-ha antibodies, and the HA-Chk1- bound beads were used as the ubiquitination acceptor substrate. Extracts from Myc-Fbx6- expressing 293T cells were immunoprecipitated with anti-myc coupled protein A-agarose beads, and proteins were eluted with 0.5 mg/ml Myc peptide. A549 cells were treated with hypotonic buffer (20 mm Tris-HCl, ph 7.2, 2 mm DTT, 0.25 mm EDTA, and protease inhibitors), sonicated for 15 sec, and centrifuged to obtain concentrated cell extracts. The in vitro ubiquitination assay was performed in 30 μl reaction buffer (50 mm Tris-HCl, ph 7.5, 5 mm MgCl 2, 0.5 mm DTT, 2 mm NaF, 10 nm okadaic acid) with 10 μl concentrated A549 cell extracts, 30 μm MG132, 1 μg/ml recombinant Flag-Ub, 20 mm creatine phosphate, 0.3 mg/ml creatine phosphokinase, 2 mm ATP, and an equivalent amount of Myc-Fbx6 eluate. The reaction was run for 30 min at 30 and terminated with 5X sample buffer, resolved on SDS-PAGE, and blotted with anti-ha antibodies. In Vitro Kinase Assay Myc-Chk1 WT or 3RE were expressed in 293T cells, and Myc-Chk1 proteins were purified by immunoprecipitation with anti-myc antibodies. The in vitro kinase assay was run in 30 μl reaction with buffer (20 mm HEPES ph 7.4, 50 mm KCl, 1 mm EGTA, 10 mm MgCl 2, 1 mm DTT), 5 μg/ml GST-Cdc25C (amino acids ), Myc-Chk1 (WT or 3RE beads), and 2 mm ATP at 37 for 5-10 min, followed by addition of 5X sample buffer. After separation by SDS-PAGE, the proteins were blotted with anti-phospho-ser216 of Cdc25C antibodies. For inhibition of Chk1 kinase activity, Myc-Chk1-bound beads were preincubated for 15 min with 500 nm Chk1 inhibitor, PF (Pfizer), and the samples were subjected to the in vitro kinase assay described above.

4 Trypsin Digestion Assay Four μl of Myc-Chk1 bead slurry obtained above was incubated with 15 mm HEPES ph 8.0, 0.5 mm ATP, 0.3 μg trypsin (Sigma), and TBS in 20 μl reaction at 30 for 0, 1, and 5 min. Digestion was stopped with 20 μl of 2X sample buffer, proteins were resolved by SDS-PAGE in a 12.5% polyacrylamide gel, and blotted with anti-p-ser345 Chk1. The membrane was then stripped and sequentially reblotted with anti-chk1 and anti-myc antibodies. RT-PCR Total RNA was extracted from cancer cell lines according to the RNeasy Mini Protocol (Qiagen). The quantity of the RNA was determined using the NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies). RT-PCR was performed using the one-step RT-PCR kit (Qiagen) following the manufacturer s protocol. To determine the linear range for the RT-PCR reactions serial dilutions of RNA were used and 50 ng of RNA and 20 nmol of each primer (described below) were used for each reaction. The cycling conditions for PCR were as follows: cdna synthesis and pre-denaturation (1 cycle at 50 C for 30 min followed by 95 C for 15 min); PCR amplification (28 cycles of denaturing at 95 C for 30 s, annealing at 55 C for 30 s, and extension at 68 C for 60 s) and a final extension at 72 C for 10 min. The PCR products were electrophoresed on 1% agarose gels and visualized with ethidium bromide under UV light. Primers for measuring FBX6, CHK1 and GAPDH were synthesized by Integrated DNA Technologies, which detect 810, 879, and 195 bp products of Chk1, Fbx6, and GAPDH, respectively. The sequence of these primers are: CHK1 sense (5 - GAGCTACCAGGAATTCATGGCAGTGCCCTTTGTGGAAGA-3 ), CHK1 anti-sense (5 - CGGCGGAATTCTCATCACTTGAGGGGTTTGTTGTACC-3 ); FBX6 sense (5 - GTACAACAGGGATCCATGGATGCTCCCCACTCCAAAG-3 ), FBX6 anti-sense (5 -

5 GGCGCGGAATTCTCATCAGAAAATCTGGACAACAGCTCG-3 ); GAPDH sense (5 - CCATGGAGAAGGCTGGGG-3 ), and GAPDH anti-sense (5 - CAAAGTTGTCATGGATGACC-3 ). The RT-PCR products were purified by gel extraction and the sequence was confirmed by sequencing. Preparation of Phosphorylated and Nonphosphorylated Chk1 Proteins In order to obtain a purified preparation of non-phosphorylated Chk1, we immunoprecipitated Myc-tagged Chk1 WT protein from transiently-transfected 293T cells, and incubated the beadbound protein in buffer containing calf intestinal protein phosphatase. Immunoblotting with antip-s317 and p S345 Chk1 antibodies revealed no detectable phospho-chk1 in the phosphatasetreated preparation (data not shown). The dephosphorylated Chk1 preparation was then divided into two equal samples. One sample was added to a cell-free Chk1 activation system containing concentrated HeLa nuclear extract, annealed 70-mer poly(da/dt), okadaic acid (a protein phosphatase inhibitor), and MG132. Poly(dA/dT) forms heterogeneous DNA structures, including fork-like double strand DNA/ssDNA junctions and ssdna stretches, which promote the activation of ATR, and, in turn, high-level phosphorylation of endogenous or exogenously added Chk1 (Clarke and Clarke, 2005; Kumagai and Dunphy, 2000). We confirmed that this cell-free system was capable of supporting Chk1 phosphorylation (Supplementary Figure S7A), and maximal phosphorylation of Chk1 was observed after a 4 h incubation in the cell-free system. References Clarke, C.A., and Clarke, P.R. (2005). DNA-dependent phosphorylation of Chk1 and Claspin in a human cell-free system. Biochem. J. 388, Kumagai, A., and Dunphy, W.G. (2000). Claspin, a novel protein required for the activation of Chk1 during a DNA replication checkpoint response in Xenopus egg extracts. Mol. Cell 6,

6 Supplemental Figures Figure S1 (A) A549 cells were transfected with 100 nm sirnas targeting luciferase control or Cul1 for 48 h, expression of Chk1 were examined. (B) Cell cycle profiles of A549 cells after 48 of sirna transfection with indicated sirnas.

7 Figure S2 (A) 293T cells were transfected with Flag-Fbx6 and Myc-Chk1 for 48 h, fractionated into cytoplasm (C), soluble nuclear (N), and chromatin enriched (CE) fractions, immunoprecipitated with anti-myc antibodies and blotted with anti-flag antibodies. (B) 293T cells were infected with lentivirus control or Fbx6 shrna vectors for 48 h, treated with 500 nm CPT for the indicated times, fractionated, and blotted with indicated antibodies. (C) Cell cycle profiles of control, Flag- Fbx6, or Flag-Skp2 stably expressing 293T cells.

8 Figure S3 (A) 293T cells were transfected with the unstable Myc-Chk1 fragment 2 lacking the C-terminal 55 amino acids (Figure 3A) with vectors expressing full-length or mutant Fbx6 for 48 h, and blotted with anti-myc antibodies. (B) Cell cycle profiles of 293T cells expressing full-length or mutant Fbx6 proteins.

9 Figure S4 (A) Schematic diagram of the C-terminal Chk1 and generation of GST mutant a-d. Numbers represent Chk1 amino acid positions for generating fusion proteins. CM, conserved motif. (B) Equal amounts of bacterially expressed and purified GST-fusion proteins were used to pull down Myc-tagged Chk1 kinase domain (fragment 5 in Figure 3A) from HEK 293T cell extracts. The beads were washed extensively and blotted with anti-myc antibodies. (C) Alignment of Chk1 C-terminal conserved motif (CM). The highly conserved CM is underlined. Numbers represent human Chk1 sequence. Arrows define the highly conserved Arg residues mutated to Glu residues to create the Chk1 3RE mutant.

10 Figure S5 (A) 293T cells were transfected with the indicated expression vectors for 48 h, treated with 160 μm CHX for 0, 2, 4 h with or without 10 μm MG132, and blotted with anti-myc antibodies. KD: kinase dead (D148A) mutation of Chk1. (B) 293T cells were transfected with Myc-Chk1 WT or 3RE for 48 h, fractioned into cytoplasm (C), soluble nuclear (N), and chromatin enriched (CE) fractions, and blotted with indicated antibodies. Long and short exposures were presented.

11 Figure S6 (A) U2-OS Tet/On cells grown on glass cover slides were transiently transfected with the Tet-inducible Flag-Chk1 WT construct for 36 h, and 0.5 μg/ml doxycycline was then added for 16 h. During the last 4 h, cells were incubated with 40 μm BrdU. Then cells were fixed and stained with anti-flag and anti- BrdU antibodies. The arrow defines a Chk1-expressing cell with reduced BrdU incorporation. (B) 293T cell were transfected with Flag-Chk1 for 48 h and blotted with anti-cdc25a antibodies. (C) U2-OS Tet/On cells were treated the same as in A, but blotted with anti-flag antibodies. (D) U2-OS cells were transfected with Flag-Chk1 WT or 3RE for 48 h. During the last 16 h, cells were treated with 1 μg/ml doxycycline. The cells were then released into doxycycline-free medium for the indicated times. 40 μm BrdU was added during the last 4 h for each sample group, as denoted by arrows. One sample that was not released served as the control. The cells were fixed and stained with anti-brdu and anti-flag antibodies.

12 (E) U2-OS Tel/On cells were transfected with Flag-Chk1 WT or 3RE, treated as in D except that cells were collected and lysed at different time points, and blotted with anti-flag antibodies. The two non-specific bands at around 35 and 100 kda serve as the protein loading controls.

13 Figure S7 (A) Cell-free Chk1 activation. Concentrated HeLa nuclear extract (15 μl) was incubated with 10 μm okadaic acid, 1 mm ATP, 10 ng/ml creatine kinase, 3 mm phosphocreatine, and 50 ng/μl poly(da/dt) at 30 for 30 min, added 15 μl of 2X sample buffer and heated for 5 min, resolved on 10% SDS- PAGE gel, and blotted with anti-p- Ser317 or anti-p-ser345 Chk1 antibodies. (B) Phospho- and non-phospho-chk1 proteins were incubated with 1μg/ml trypsin for the indicated times, and blotted with anti-chk1 antibodies. (C) Myc-Chk1 (WT, S317E, S345E) were transfected into 293T cells, proteins were immunoprecipitated with anti-myc antibodies and treated with alkaline phosphatase for 2 h, performed the trypsin digestion assay, and blotted with anti-chk1 antibodies.

14 Figure S8. Roles of Chk1 in Drug Resistance (A) Asynchronous A549 and TK-10 cells were treated for 8 h with the indicated concentrations of CPT. Protein expression levels were 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 panel A were cultured for 48 h in fresh medium. Cell death was determined by staining with trypan blue. The data are plotted as mean +/- standard deviation from 3 independent trials. (C) Scanned images of the entire gel shown in Figure 6E. Equal amount of total proteins from growing A549, MDA-MB-231, and TK-10 cells were blotted with purified rabbit anti-fbx6

15 antibody first, then stripped and sequentially re-blotted with mouse anti-chk1 and anti-tubulin antibodies. Numbers at the left indicate standard molecular weight.

16 Figure S9. Overexpression of Fbx6 Reduces the Level of Chk1 and Increases Sensitivity to CPT-Induced Cell Death in MDA-MB-231 Cells (A and B) MDA-MB-231 cells were grown on glass cover slides and transfected with Flag-Fbx6 for 48 h, treated with 500 nm CPT for 8 h, released into drug-free medium for 12 h, fixed and stained with mouse anti-flag (green) and rabbit anti-chk1 (red, A) or anti-caspase cleavage product (red, B). Cells a and b are Flag-Fbx6 positive and negative cells, respectively. (C) A549 cells were plated into 12-well plates and infected at 24 and 48 h with lentivirus targeting control or Fbx6 shrna freshly transfected in 293T cells. At 36 h, the cells were transfected with sirna targeting luciferase control or Chk1 with or without Flag-Fbx6 vector. At 60 h, cells were treated with 500 nm CPT for 8 h, and cultured in drug-free medium for

17 additional 24 h, and cell death was measured by trypan blue assay. Data represent mean and standard deviation from three wells.

18 A B Figure S10 Total RNA from asynchronously growing cells representing mixed cancer cell lines (A), nonsmall cell lung cancer (B), glioblastoma (C), and breast (D) were harvested and a semiquantitative RT-PCR was performed to analyze expression of indicated genes using specific primers as described in the Experimental Procedures.

19 Figure S11 (A and B) Asynchronously growing cells derived from (A) glioblastoma or (B) breast were lysed and blotted with anti-chk1 and anti-fbx6 antibodies, and the same membranes were stripped and re-blotted with anti-tubulin and anti- PCNA antibodies, respectively. (C) Asynchronously growing A549 cells were treated with 500 nm CPT for the indicated times, and blotted with the indicated antibodies.