1. Yap1 Phosphorylation by c-Abl Is a Critical Step in Selective Activation of Proapoptotic Genes in Response to DNA Damage 
Dan Levy, Yaarit Adamovich, Nina Reuven, Yosef Shaul 
Molecular Cell 
Volume 29, Issue 3, Pages (February 2008)
DOI: /j.molcel
Copyright © 2008 Elsevier Inc. Terms and Conditions

2. Figure 1 c-Abl Stabilizes Yap1 in a Kinase-Dependent Manner
(A) HEK293 cells were transfected with Flag-Yap1 alone or together with increasing amounts of constitutively active c-Abl Δ1–81. Cell lysates were subjected to western blotting analysis using the indicated antibodies.
(B) HEK293 cells were transfected with Flag-Yap1 alone or together with c-Abl Δ1–81 without or with pSUPER c-Abl encoding c-Abl-specific siRNA. Forty-eight hours after transfection, cells were harvested and subjected to western blot analysis as in (A).
(C) The same as for (A) except that analysis was done with WT and kinase dead mutant (km).
(D) HEK293 cells were transfected with plasmids expressing Flag-Yap1 with or without WT c-Abl. Twenty-four hours after transfection, cells were treated with 20 μg/ml cycloheximide for different time points. Equal amounts of total protein lysates were subjected to western blotting analysis with the indicated antibodies.
(E) The same as (D) except that analysis was done with the constitutively active c-Abl Δ1–81.
(F) HEK293 cells were transfected with Flag-Yap1 together with c-Abl Δ1–81 or with an empty vector. Twenty-four hours posttransfection, cells were labeled for 1 hr with [35S]methionine for pulse-chase analysis, immunoprecipitated with anti-Flag, and visualized by autoradiography.
(G) HEK293 cells were transfected with Flag-Yap1 and were treated with 10 μM STI571. Twenty-four hours posttreatment, cells were subjected to western blot analysis with the indicated antibodies.
(H and I) Endogenous Yap1 protein level (H) and mRNA level by RT (I) were measured in the paired HCT116-2(1) and HCT116-3(6) cell lines.
(J) The paired MCF-7 cell lines stably expressing empty pRetroSuper or pRetroSUPER c-Abl encoding c-Abl-specific siRNA were subjected to western blotting analysis.
Molecular Cell  , DOI: ( /j.molcel )
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3. Figure 2
c-Abl Binds Yap1 and Phosphorylates It on Y357 In Vitro and In Vivo
(A) The domain structure of Yap1 is proline-rich region (PRR), WW domain (WW), coiled-coil domain (CC), and conserved region (CR). Also, the motifs interacting with , SH3, and PDZ are shown. Phosphorylated residues by Akt (S127) and c-Abl (Y357) are indicated by black triangles.
(B) HEK293 cells were transfected with Flag-Yap1 alone or together with c-Abl WT. Cells were subjected to IP and analyzed by western blot, using the indicated antibodies.
(C) HEK293 cells were transfected with HA-Yap1 and HA-Yap1 Y357F alone or together with c-Abl Δ1–81 and were subjected to IP using anti-HA antibody and analyzed as in (B).
(D) HEK293 cells were transfected with Flag-Yap1 alone or together with c-Abl Δ1–81 WT or siRNA-resistant clone along with pSUPER c-Abl encoding c-Abl-specific siRNA or nonspecific pSuper pX, as a control. Forty-eight hours after transfection, cells were harvested and subjected to western blot analysis with the indicated antibodies.
(E) HEK293 cells were transfected with Flag-Yap1 and c-Abl Δ1–81, as indicated, and were treated with 10 μM STI571 for 24 hr. Cell extracts were subjected to IP and treated as in (B).
(F) HEK293 cells were transfected with Flag-Yap1 alone or together with c-Abl Δ1–81 WT or STI571-resistant clone. Twenty-four hours after transfection, cells were treated with 5 μM of STI571 for 5 hr, harvested, and analyzed as in (B).
(G) Flag-Yap1 and Flag-Yap1 Y357F were immunoprecipitated from HEK293 cells and were subjected to an in vitro kinase assay using a recombinant c-Abl (Recom.) in the presence or absence of ATP. Phosphorylated Yap1 was detected with the specific anti-pY357 antibody.
(H) HEK293 cells were transfected with Flag-Yap1 WT and Y357F alone or together with different amounts of WT c-Abl. Cell lysates were subjected to western blotting analysis using the indicated antibodies.
(I) HEK293 cells were transfected with plasmids expressing HA-Yap1 Y357F with or without c-Abl Δ1–81. Twenty-four hours after transfection, cells were treated with 20 μg/ml cycloheximide for different time points. Equal amounts of total protein lysates were subjected to western blotting with the indicated antibodies.
(J) H1299 cells were transfected with Flag-Yap1 or Flag-Yap1 Y357F together with c-Abl Δ1–81 or with an empty vector. Twenty-four hours posttransfection, cells were labeled for 1 hr with [35S]methionine for pulse-chase analysis, immunoprecipitated with anti-Flag, and visualized by autoradiography.
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 3
DNA Damage Induces Yap1 Phosphorylation and Stabilization by c-Abl
(A) HEK293 cells were transfected with Flag-Yap1 alone or together with WT c-Abl. Twenty-four hours after transfection, cells were irradiated (20 Gy) and treated with 10 μM STI571 as indicated. After 4 hr, cells were harvested and subjected to western blot analysis.
(B) H1299 cells were untreated (−) or were γ irradiated (+). Cells were harvested after 4 hr and subjected to western blot analysis with the indicated antibodies.
(C) HEK293 and H1299 cells were treated with 25 μM cisplatin (for 10 hr) with or without 10 μM STI571 and were then treated as in (B).
(D) HeLa cells treated with 25 μM cisplatin for 10 hr or untreated were subjected to subcellular fractionation. Equivalent cell lysates from cytoplasmic (C) and nuclear (N) fractions were run on SDS-PAGE and analyzed by western blotting.
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 4 p73 Preferentially Binds to Phosphorylated Yap1
(A and B) HEK293 cells were transfected with the indicated plasmids. Twenty-four hours after transfection, cell extracts were subjected to IP and western blot analysis with the indicated antibodies.
(C) HEK293 cells were transfected with the indicated plasmids. Cell extracts were immunoprecipitated with anti-HA antibody, and endogenous p73 protein was detected.
(D) Same as (A), except that cell extracts were immunoprecipitated with antibodies recognizing endogenous p73.
(E) 35S-labeled Flag-Yap1 Y357F or Flag-Yap1 Y357E was subjected to IP with 35S-labeled HA-p73 and was analyzed by SDS-PAGE and phosphoimager.
(F) Cells were treated as in (A), except that 10 μM STI571 was added where indicated.
(G) HEK293 cells were transfected with the indicated plasmids, treated with 25 μM cisplatin and 10 μM STI571, and subjected to IP as in (A).
(H and I) H1299 cells were treated 1 hr prior to irradiation with 10 μM STI571, where indicated. Cells were γ irradiated and collected 4 hr post-IR. Cell extracts were immunoprecipitated with endogenous Yap1 (H) or p73 (BL906) (I) antibodies and were subjected to western blot analysis with the indicated antibodies.
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 5
Phosphorylated Yap1 Discriminates between p73 and Runx in Binding
HEK293 cells were transfected with the indicated plasmids. Twenty-four hours after transfection, cell extracts were subjected to IP using anti-HA antibody. Coprecipitating proteins were detected with the indicated antibodies.
Molecular Cell  , DOI: ( /j.molcel )
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7. Figure 6
Yap1 Phosphorylation Controls Gene Target Specificity Transactivation
(A) Crosslinked chromatin derived from HEK293T (upper panel) or H1299 (lower panel) cells treated for 24 hr with 25 μM cisplatin and 10 μM STI571 was immunoprecipitated with the indicated antibodies and was analyzed by PCR for the indicated promoters. IP using only the protein A/G beads served as a control. Nonimmunoprecipitated crosslinked chromatin served as the input.
(B) γ irradiated HEK293T cells were collected 10 hr post-IR, chromatin immunoprecipitated with the indicated antibodies, and subjected to quantitative real-time PCR using primers for the Bax promoter.
(C) The pair of MCF-7 cell lines stably expressing a control shRNA plasmid or a c-Abl-specific shRNA were γ irradiated and then were treated as in (B).
(D and E) Crosslinked chromatin derived from HEK293T cells treated with 25 μM cisplatin was immunoprecipitated with the indicated antibodies and treated as in (A). OC, Osteocalcin promoter.
Molecular Cell  , DOI: ( /j.molcel )
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8. Figure 7
Cisplatin-Induced Apoptosis by Yap1 Is Regulated by c-Abl Phosphorylation
(A) HEK293T cells were transiently transfected with control, Yap1, and c-Abl-specific shRNA-encoding plasmids and were treated with 25 μM cisplatin for 24 hr. RT-PCR was done using Bax-specific primers or β-actin-specific primers as controls.
(B) H1299 cells stably expressing a control (cont.) or Yap1 (Yap1) siRNA were treated with 25 μm cisplatin for 24 hr. The number of cells undergoing apoptosis (sub-G1 population) were determined by FACS analysis.
(C) HEK293T cells were transiently transfected with increasing amounts of plasmid encoding WT and Y357F Yap1 and were treated as in (A).
(D) H1299 cells were transfected with p73 alone or together with Yap1 WT or Y357F mutant and the Bax-luciferase reporter plasmid and were treated with 25 μM cisplatin for 24 hr. Cell extracts were prepared 36 hr later and subjected to determination of luciferase activity. Results are represented as fold induction of luciferase activity compared with the control cells transfected with an empty expression vector. SD is calculated from at least three independent experiments.
(E) H1299 cells were transfected with p73 alone or together with Yap1 WT or Y357E mutant, and the p21-luciferase reporter activity was measured as in (D).
(F) H1299 cells were transfected as indicated with WT Yap1 or Yap1 Y357F mutant together with an H2b-GFP (GFP) plasmid, and were treated with 25 μm cisplatin (cisp) for 24 hr. The number of cells undergoing apoptosis (sub-G1 population) were determined by FACS analysis 48 hr posttransfection. Bars represent SD for at least three independent experiments.
(G) The pair of MCF-7 cell lines expressing control shRNA (gray bars) or c-Abl shRNA (black bars) were transfected with wild-type Yap1 (WT) or mutant Yap1 Y357F and were treated as in (E). SD is calculated from at least three independent experiments.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2008 Elsevier Inc. Terms and Conditions