Journal: Oncogene

Article Title: FATS is an E2-independent ubiquitin ligase that stabilizes p53 and promotes its activation in response to DNA damage.

doi: 10.1038/onc.2013.494

Figure Lengend Snippet: Figure 1. FATS induces nuclear-localized p53 protein. (a) U2OS cells (1 105) were transfected with 2 mg of Flag-tagged FATS or FATS-N and an empty vector, respectively. After 24 h, lysates were collected and subjected to immunoblotting (IB). FATS expression was analyzed using a monoclonal antibody against Flag. (b) MCF-7 cells (1 105) were transfected with 2 mg of either Flag-FATS-N or an empty vector. The expression of endogenous p53 was examined by immunoblotting. (c) H1299 cells (5 104) were transfected with a p53-expressing vector (0.2 mg) alone or in combination with 2 mg of either Flag-FATS or Flag-FATS-N. GFP (0.1 mg) was co-transfected to avoid transfection artifact and measured as loading control. Cell lysates were analyzed by immunoblotting. FATS expression was probed using an antibody against Flag. (d) U87 cells were transfected with 0.2 mM FATS-shRNA or a non-targeting shRNA, respectively. The expression of endogenous FATS was analyzed by western blotting using a FATS antibody. (e) Immunofluorescence. U2OS cells were transfected with Flag-tagged FATS for 24 h and stained with antibodies against p53 and Flag. Nuclei were stained with DAPI. (f) RNA isolated from transfected U2OS cells was analyzed by RT–PCR. M, marker. (g) The level of p53 mRNA in (f) was analyzed by real time RT–PCR. The normalized value is shown.

Article Snippet: Cell culture and transfection FATS-negative cell lines (MCF-7, U2OS, and H1299) and U87 cells (FATS wild-type, p53 wild-type)24 were obtained from American Type Culture Collection (ATCC).

Techniques: Transfection, Plasmid Preparation, Western Blot, Expressing, Control, shRNA, Staining, Isolation, Reverse Transcription Polymerase Chain Reaction, Marker, Quantitative RT-PCR

Journal: Oncogene

Article Title: FATS is an E2-independent ubiquitin ligase that stabilizes p53 and promotes its activation in response to DNA damage.

doi: 10.1038/onc.2013.494

Figure Lengend Snippet: Figure 2. FATS is required for p53 stabilization in both unstressed and stressed cells. (a) U87 cells were treated with CHX (40 mg/ml) for indicated periods of time after knockdown of endogenous FATS. Lysates were subjected to immunoblotting and analyzed for levels of indicated proteins. Relative p53 levels are indicated. (b) H1299 cells were transfected with p53 and Flag-FATS or an empty vector, respectively. After transfection for 24 h, cells were treated with CHX (40 mg/ml) for indicated periods of time. Protein levels were analyzed by immunoblotting. The relative p53 level was shown in the right panel. (c) Transfected U2OS cells were treated with 25 mM etoposide for 3 h. Phosphorylation of p53 was analyzed by immunoblotting using an antibody against phosphorylated-p53 on Serine 15 (S15), and acetylation of p53 was analyzed by immunoblotting using an antibody against acetylated-p53 on Lysine 382 (K382). (d) U2OS cells were transfected with or without Flag-FATS-N for 24 h. Cells were treated with 25 mM etoposide for indicated periods of time, and box plots show the changes of p53 protein after genotoxic stress. (e) After FATS inhibition by shRNA, U87 cells were treated with 25 mM etoposide for 6 h. Cell lysates were analyzed for levels of indicated proteins.

Article Snippet: Cell culture and transfection FATS-negative cell lines (MCF-7, U2OS, and H1299) and U87 cells (FATS wild-type, p53 wild-type)24 were obtained from American Type Culture Collection (ATCC).

Techniques: Knockdown, Western Blot, Transfection, Plasmid Preparation, Phospho-proteomics, Inhibition, shRNA

Journal: Oncogene

Article Title: FATS is an E2-independent ubiquitin ligase that stabilizes p53 and promotes its activation in response to DNA damage.

doi: 10.1038/onc.2013.494

Figure Lengend Snippet: Figure 3. p53 protein interacts with N-terminal domain of FATS. (a) U2OS cells were transfected with 2 mg of Flag-tagged FATS/ FATS-N and an empty vector, respectively. Lysates were immunoprecipitated (IP) using a mouse monoclonal antibody against Flag for indicated proteins. The co-eluted antibody (IgG) heavy chain was detected in the left panel after immunoblotting using a mouse monoclonal antibody against p53. The interference of IgG heavy chain was eliminated in the right panel when a rabbit polyclonal antibody against p53 (FL-393) was used for immunoblotting. FATS expression in lysates was detected by immunoblotting using a monoclonal antibody against Flag. (b) MCF-7 cells were treated as in (a), and immunoprecipitates were analyzed for indicated proteins. (c) Lysates from transfected H1299 cells as indicated were immunoprecipitated with a rabbit polyclonal antibody against p53 or a rabbit IgG control and analyzed for Flag-tagged FATS using a mouse monoclonal antibody against Flag. (d) Lysates from U87 cells were immunoprecipitated using a rabbit polyclonal antibody against FATS or a rabbit IgG control, and co-immunoprecipitation of endogenous FATS and p53 was analyzed by immunoblotting using a mouse monoclonal antibody against p53. (e) GST-tagged FATS-N was incubated with in vitro translated p53, and pulled-down proteins were blotted by an antibody against p53. (f) GST pulldown assay was performed using truncated FATS-N mutants, and their binding activities to p53 were summarized.

Article Snippet: Cell culture and transfection FATS-negative cell lines (MCF-7, U2OS, and H1299) and U87 cells (FATS wild-type, p53 wild-type)24 were obtained from American Type Culture Collection (ATCC).

Techniques: Transfection, Plasmid Preparation, Immunoprecipitation, Western Blot, Expressing, Control, Incubation, In Vitro, GST Pulldown Assay, Binding Assay

Journal: Oncogene

Article Title: FATS is an E2-independent ubiquitin ligase that stabilizes p53 and promotes its activation in response to DNA damage.

doi: 10.1038/onc.2013.494

Figure Lengend Snippet: Figure 4. FATS suppresses Mdm2 binding to p53. (a) U2OS cells were transfected with HA-tagged Mdm2 alone or in combination with Flag-FATS, respectively. After 24 h, cells were treated with 20 mM MG132 for 2 h, and cell lysates were subjected to immunoprecipita- tion with a p53-specific antibody, followed by immunoblotting using a HA antibody. (b) Purified GST-Mdm2 protein was incubated with in vitro translated FATS or p53 and subjected to in vitro binding assay. (c) U2OS cells were transfected with Mdm2-shRNA alone or in combination with Flag-FATS for 24 h. Protein levels of p53 in cells were analyzed by western blot. b-actin was used as loading control.

Article Snippet: Cell culture and transfection FATS-negative cell lines (MCF-7, U2OS, and H1299) and U87 cells (FATS wild-type, p53 wild-type)24 were obtained from American Type Culture Collection (ATCC).

Techniques: Binding Assay, Transfection, Western Blot, Incubation, In Vitro, shRNA, Control

Journal: Oncogene

Article Title: FATS is an E2-independent ubiquitin ligase that stabilizes p53 and promotes its activation in response to DNA damage.

doi: 10.1038/onc.2013.494

Figure Lengend Snippet: Figure 5. N-terminal domain of FATS stimulates non-proteolytic polyubiquitination of p53 in vivo. (a) U2OS cells (2 105) were transfected with His-tagged ubiquitin (His-Ub) alone or cotransfected with Flag-FATS, respectively. After 24 h, cells were either untreated or treated with MG132 (20 mM) for 6 h. His-tagged and ubiquitin-conjugated proteins were purified by Ni-NTA beads, followed by immunoblotting with a p53 antibody (DO1). The levels of indicated proteins in cell lysates are shown in the lower panel. The expression of FATS was examined by immunoblotting using a Flag antibody. (b) At 24 h post-transfection, U2OS cells were either untreated or treated with etoposide (25 mM) for 6 h. His-Ub-conjugated proteins were purified, and ubiquitinated p53 was detected by western blotting using a p53 antibody (Ab-6). (c) U2OS cells were transfected with Flag-FATS-N for 24 h and subsequently treated with etoposide (25 mM) for the indicated time. His-Ub-conjugated proteins were purified, followed by western blotting using a p53 antibody (FL-393). (d) U2OS cells were transfected with indicated expression vectors for 24 h. Cells were treated with MG132 (20 mM) for 4 h before collecting protein lysates. K48-linked polyubiquitination of p53 was detected by Western blotting using a p53 antibody (Ab-6). (e) U2OS cells were transfected with indicated expression vectors for 24 h. FATS-mediated non-proteolytic polyubiquitination of p53 before and after knockdown of Mdm2 was examined.

Article Snippet: Cell culture and transfection FATS-negative cell lines (MCF-7, U2OS, and H1299) and U87 cells (FATS wild-type, p53 wild-type)24 were obtained from American Type Culture Collection (ATCC).

Techniques: In Vivo, Transfection, Ubiquitin Proteomics, Western Blot, Expressing, Knockdown

Journal: Oncogene

Article Title: FATS is an E2-independent ubiquitin ligase that stabilizes p53 and promotes its activation in response to DNA damage.

doi: 10.1038/onc.2013.494

Figure Lengend Snippet: Figure 6. N-terminal domain of FATS is sufficient to exhibit E3 activity independently of E2. (a) Purified E1 (40 ng), UbcH5b (E2, 100 ng), ubiquitin (Ub, 2 mg) and 1mg of GST or GST-tagged FATS-N in reaction buffer were incubated at 30 1C for 90 min, followed by immunoblotting using an antibody against ubiquitin. FATS-catalyzed assembly of poly-ubiquitin was detected. (Ub)n, poly-ubiquitin. (b) In vitro ubiquitination assay was performed using reaction buffer with or without ATP. FATS-catalyzed assembly of poly-ubiquitin is shown. (c) FATS C-terminal fragment was used as a negative control for in vitro ubiquitination assay. The E3 activity of FATS-N was tested in the absence or presence of another E2 UbcH5c. (d) Ubiquitin ligase activity of FATS-N was analyzed using ubiquitin mutant Ub-K48R or Ub-K63R. FATS-catalyzed assembly of poly-ubiquitin is shown. The quantity of GST-tagged FATS-N in each reaction is shown in the lower panel. (Ub)n, poly-ubiquitin. (e) In vitro ubiquitination assay was performed using ubiquitin mutants that contain only one lysine residues in each ubiquitin protein. FATS-catalyzed assembly of poly-ubiquitin consisting of single ubiquitin-linkage is shown. (f) FATS-N formed thioester complexes with ubiquitin. Reactions were incubated with the same buffer for in vitro ubiquitination assay in the absence or presence of a reducing agent DTT (100 mM). (g) In vitro ubiquitination assay was performed in the presence of in vitro translated p53 and in the absence of E2. Polyubiquitinated p53 protein was detected by immunoblotting using a p53 antibody. The level of p53 protein in each reaction is shown in lower panel. (h) FATS acted as a HECT-type E3 with a catalytic Cys residue. Site-specific mutagenesis was introduced to FATS-N, and in vitro ubiquitination assay was performed.

Article Snippet: Cell culture and transfection FATS-negative cell lines (MCF-7, U2OS, and H1299) and U87 cells (FATS wild-type, p53 wild-type)24 were obtained from American Type Culture Collection (ATCC).

Techniques: Activity Assay, Ubiquitin Proteomics, Incubation, Western Blot, In Vitro, Negative Control, Mutagenesis, Residue

Journal: Oncogene

Article Title: FATS is an E2-independent ubiquitin ligase that stabilizes p53 and promotes its activation in response to DNA damage.

doi: 10.1038/onc.2013.494

Figure Lengend Snippet: Figure 7. The E3 activity of FATS is required for promoting p53 transcriptional activity and p53-depedent cell cycle arrest. (a) Wild-type (WT) or p53 / MEF cells were transfected with pGL2-p21-luc and pRL-TK (Promega) or cotransfected with FATS, respectively. pRL-TK was used as transfection control. Dual luciferase assay was performed after 24 h, and relative luciferase activities are shown. Data are means± s.d. (b) MEF cells were transfected with FATS-N or an empty vector, respectively. The expression of p21 and GAPDH was determined by semi-quantitative RT–PCR. (c) The transfected MEF cells were subjected to chromatin immunoprecipitation (ChIP) assay to analyze the binding of endogenous p53 to p21 promoter. (d) U2OS cells were transfected with His-Ub alone or cotransfected with Flag-tagged FATS-N or FATS-N-C211G, respectively. In vivo ubiquitination assay was performed to examine ubiquitinated p53 using a p53 antibody (Ab-6). (e) H1299 cells were transfected with pGL2-p21-luc, pRL-TK and indicated expression vectors. After 24 h, cells were harvested and assayed for firefly (FL) and Renilla (RL) luminescence. Results are presented as average of FL/RL ratio ± s.d. (n ¼ 3). (f) H1299 cells were transfected with indicated constructs for 24 h, and cell-cycle profiles were determined by flow cytometry. The G1/S ratios are shown. (g) FATS-N induces p53-dependent G1 arrest in an E3-dependent manner. Wild-type and p53-null MEF cells were transfected with 0.5 mg of Flag-tagged FATS-N and FATS-N-C211G, respectively. After transfection for 24 h, cell-cycle profiles were determined by flow cytometry. The relative values of G1/S ratios are shown as means± s.d.

Article Snippet: Cell culture and transfection FATS-negative cell lines (MCF-7, U2OS, and H1299) and U87 cells (FATS wild-type, p53 wild-type)24 were obtained from American Type Culture Collection (ATCC).

Techniques: Activity Assay, Transfection, Control, Luciferase, Plasmid Preparation, Expressing, Quantitative RT-PCR, Chromatin Immunoprecipitation, Binding Assay, In Vivo, Ubiquitin Proteomics, Construct, Cytometry

Journal: Oncogene

Article Title: FATS is an E2-independent ubiquitin ligase that stabilizes p53 and promotes its activation in response to DNA damage.

doi: 10.1038/onc.2013.494

Figure Lengend Snippet: Figure 8. The catalytic function of FATS is required for promoting p53 stability and activation in response to DNA damage. (a) In vitro binding assay using purified proteins including an E3-defective mutant of FATS (C211G). (b) U2OS cells were transfected with Flag-tagged FATS-N, FATS-N-C211G and an empty vector, respectively. After transfection for 24 h, cells were treated with CHX (40 mg/ml) for the indicated time. The endogenous p53 protein was measured by immunoblotting. The relative protein level of p53 is shown in the lower panel. (c) After transfection for 24 h, U2OS cells were treated with 20 mM etoposide for the indicated time. The accumulation of p53 protein and its phosphorylation and acetylation in response to DNA damage were examined. Long exposure (exp.) photographs are also shown. (d) U2OS cells were transfected with Flag-tagged FATS-N, FATS-N-C211G and an empty vector, respectively. After transfection for 24 h, cells were treated with 20 mM etoposide for 6 h and analyzed for indicated proteins. FATS-N and FATS-N-C211G expression was analyzed by immunoblotting using a Flag antibody.

Article Snippet: Cell culture and transfection FATS-negative cell lines (MCF-7, U2OS, and H1299) and U87 cells (FATS wild-type, p53 wild-type)24 were obtained from American Type Culture Collection (ATCC).

Techniques: Activation Assay, In Vitro, Binding Assay, Mutagenesis, Transfection, Plasmid Preparation, Western Blot, Phospho-proteomics, Expressing