1. Volume 11, Issue 1, Pages 11-23 (January 2003)
Akt Phosphorylates the Yes-Associated Protein, YAP, to Induce Interaction with and Attenuation of p73-Mediated Apoptosis 
Subham Basu, Nicholas F Totty, Meredith S Irwin, Marius Sudol, Julian Downward 
Molecular Cell 
Volume 11, Issue 1, Pages (January 2003)
DOI: /S (02)

2. Figure 1 Proteins Binding 14-3-3τ in an Akt-Dependent Manner
(A) Tandem affinity purification (TAP) of proteins binding to dual epitope-tagged τ. Cos-7 cells were transiently transfected with either pCDNA-3-TAP or pCDNA τ-TAP as indicated. Cells were harvested 24 hr after transfection and subjected to TAP purification as described in Experimental Procedures. Duplicate aliquots of fractions were resolved by one-dimensional SDS-PAGE on 4%–12% gradient gels (I, 1/2000 input lysate; FT, 1/2000 post-IgG column flow through; TC, 1/30 TEV, cleavage eluate from IgG column; CM, 1/30 calmodulin column eluate, final purified fraction). Proteins were detected either by colloidal Coomassie staining or Western blotting with anti antibody.
(B) binding proteins exhibit increased Akt-dependent phosphorylation. Cos-7 cells were transiently transfected with pCDNA τ-TAP as in (A). 16 hr after transfection, cells were serum starved for a further 24 hr. Cells were treated as indicated with 50 ng/ml EGF and 50 μM LY for 4 hr and TAP purified. Concentrated purified fractions were resolved by 2D gel electrophoresis using pH 4–7 strips and 4%–12% gradient gels. Samples were analyzed by Western blotting with anti-phosphorylated Akt-substrate antibody.
Molecular Cell  , 11-23DOI: ( /S (02) )

3. Figure 2 Identification of YAP as a 14-3-3 Binding Protein
(A) Large-scale purification of binding proteins. Cos-7 cells were transfected with TAP, serum starved, and treated with EGF as in Figure 1. Duplicates of concentrated purified fractions were resolved by 2D gel electrophoresis using pH 4–7 strips and 10% SDS-PAGE. One duplicate gel was transferred onto PVDF membrane, initially stained with SYPRO Ruby and subsequently analyzed by Western blotting with anti-phosphorylated Akt-substrate antibody as in Figure 1. Second gel was stained with colloidal Coomassie. Staining was similar to SYPRO Ruby (data not shown).
(B and C) Mass spectrometric analysis of candidate proteins. Spots on colloidal Coomassie-stained gel which correlated with spots detected with anti-phosphorylated Akt-substrate antibody were excised and digested as described in Experimental Procedures. Resultant peptides were subject to MALDI (B) or LC-MS/MS (C). Arrows in (A) indicate spots positively identified by both analyses. Asterisks in (B) indicate mass spectra positive for YAP.
Molecular Cell  , 11-23DOI: ( /S (02) )

4. Figure 3 YAP Is a Substrate for Akt Phosphorylation
(A) Western blot analysis of binding proteins with anti-phosphorylated Akt-substrate antibody and anti-YAP antibody confirms YAP to be phosphorylated by Akt. Membrane from Figure 2A was stripped of antibody and reprobed with anti-YAP antibody.
(B) YAP binding to is induced by EGF in a PI 3-kinase-dependent manner. Membrane from Figure 1C was stripped of antibody and reprobed with anti-YAP antibody as in (A).
(C) YAP is phosphorylated in response to Akt activation. hTert-RPE 1 mAktER cells were seeded, grown for 24 hr in growth media, serum starved for 16 hr, and then treated for 4 hr with 100 nM 4-hydroxytamoxifen (4-OHT) or 1:5000 ethanol as a vehicle control as indicated. Cells were lysed, and 1/4 sample aliquots were resolved by 2D gel electrophoresis as in Figure 1C. Membranes were sequentially analyzed by Western blot with anti-phosphorylated Akt substrate antibody and anti-YAP antibody as in Figure 3A and then stained by Ponceau S.
Molecular Cell  , 11-23DOI: ( /S (02) )

5. Figure 4
YAP Is Phosphorylated at Serine 127 within an Unusual Akt Substrate Motif
(A) Antibody generated against a phosphopeptide including phosphoserine 127 within the candidate Akt phosphorylation site in YAP detects increased phosphorylation of endogenous YAP in an EGF-inducible manner. Cos-7 cells were seeded and treated with EGF or LY as in Figure 1 for the indicated times. Cells were lysed, and duplicate aliquots were resolved by 8% SDS-PAGE and analyzed by Western blot using the antibodies indicated.
(B) Phospho-YAP antibody specifically detects increased phosphorylation of endogenous YAP in cells in which Akt is activated upon 4-OHT treatment. MCF-7 mAktER cells were incubated with either YAP siRNA or EGFP siRNA oligonucleotides and were further treated with 4-OHT as in Figure 3C. Cells were lysed and resolved as in (A) and analyzed by Western blotting with the antibodies indicated.
(C) Phospho-YAP antibody detects increased phosphorylation of endogenous YAP in cells exhibiting greater endogenous Akt activation. MDA 231 and MDA 468 were seeded and harvested after 36 hr. Cells were lysed and resolved as in (A) and analyzed by Western blotting with the antibodies indicated.
(D) Mutation at serine 127 abolishes recognition by anti-phosphorylated Akt substrate and anti-phospho YAP antibodies in EGF-stimulated cells. Cos-7 cells were seeded as in (A), transiently transfected with pEGFP-YAP or pEGP-S127A YAP as indicated, and treated with EGF as in Figure 1. Cells were lysed, and triplicate aliquots were resolved as in (A) and analyzed by Western blot with the antibodies indicated.
(E) Cos-7 cells were seeded as in Figure 1 and transiently transfected with pCDNA τ-TAP and either pEGFP-YAP or pEGFP-S127A YAP as indicated and treated with EGF as in Figure 1. Equivalent aliquots of TAP purification fractions from input (I) and final purified eluate (P) were resolved as in (A) and analyzed by Western blot with YAP antibody.
(F) Mutation at serine 127 abolishes recognition by anti-phosphorylated Akt substrate and anti-phospho YAP antibodies in cells in which Akt is activated. H1299 mAktER cells were seeded and transfected as in (D) and further treated as in Figure 3C as indicated. Akt was activated by 4-OHT treatment for 4 hr. Cells were lysed and duplicate aliquots were resolved as in (A) and analyzed by Western blot with the antibodies indicated.
(G) Akt directly phosphorylates YAP. In vitro kinase assay employing recombinant Akt and either Bad, GST, or GST-YAP was carried out, and phosphorylated proteins were detected using anti-phosphorylated Akt substrate antibody.
Molecular Cell  , 11-23DOI: ( /S (02) )

6. Figure 5 Akt Phosphorylation Regulates YAP Subcellular Localization
H1299 mAktER cells were transfected with either pEGFP-YAP or pEGFP-S127A YAP and were further treated with 100 nM 4-OHT or vehicle control as indicated. Cells were harvested and GFP-protein expression visualized by confocal fluorescence microscopy as described in Experimental Procedures. Images shown are representative of more than 100 cells analyzed for each condition.
Molecular Cell  , 11-23DOI: ( /S (02) )

7. Figure 6
YAP Mediates p73-Dependent Transcription and Is Regulated by Akt Phosphorylation
(A) Bax protein expression induced by both cisplatin (CDDP) treatment and YAP expression is dramatically reduced by treatment with human p73 siRNA (hp73) but not control siRNA (cont). Treatment with YAP siRNA also dramatically reduces cisplatin-induced Bax expression. U2OS cells were incubated with control, human p73, or human YAP siRNA oligonucleotides as described in Experimental Procedures, transiently transfected with pEGFP (GFP) or pEGFP-YAP (YAP), and further treated with 2 μg/ml cisplatin for 24 hr as indicated. Cells were lysed and lysates analyzed by Western blot for Tubulin (control) and Bax. Western blot signals were quantified by densitometry and the Bax signal normalized to the accompanying Tubulin signal. Bax expression levels are shown as a fold increase over the normalized signal from cells transfected with control siRNA and GFP without cisplatin treatment. Data shown are representative of three independent experiments.
(B) Akt phosphorylation decreases YAP-activated p73-mediated transcription. MCF-7 mAktER cells were transiently transfected with pRenilla and pGL3-Bax in all cases, plus pCDNA-3-HA or pCDNA-3-HA-p73α and either pEGFP, pEGFP-YAP, or pEGFP-S127AYAP as indicated. Luciferase activity was quantified and normalized to internal renilla reporter signal. Means of three independent experiments are shown with error bars indicating standard error of the mean (SEM).
Molecular Cell  , 11-23DOI: ( /S (02) )

8. Figure 7 Akt Phosphorylation Regulates YAP-Induced Apoptosis
(A) RNA interference with YAP siRNA in MCF7 cells results in dramatic reduction in YAP protein. MCF-7 cells were incubated with either YAP siRNA or EGFP siRNA oligonucleotides. Cells were lysed, and duplicate aliquots were analyzed by Western blot with antibodies indicated.
(B) Reduction in endogenous YAP protein expression results in attenuation of cisplatin-induced apoptosis in MCF-7 cells. MCF-7 cells were treated as in (A) and further treated with cisplatin (CDDP) at the indicated doses. Cells were harvested and apoptosis quantified.
(C) RNA interference with YAP siRNA in H1299 cells results in reduction in YAP protein expression. H1299 cells were treated and lysates analyzed as in (A).
(D) Reduction in endogenous YAP protein expression in H1299 cells results in attenuation of cisplatin-induced apoptosis only in cells expressing p73. H1299 cells were treated as in (A), transiently transfected with either pCDNA-3-HA or pCDNA-3-HA-p73α (0.4 μg each) as indicated, and further treated with 100 μM cisplatin. Cells were harvested and apoptosis quantified as in (B).
(E) Expression of YAP lacking the Akt phosphorylation site sensitizes cells to p73-induced apoptosis. H1299-mAktER cells were transiently transfected with equal amounts (0.4 μg) of either pEGFP-YAP or pEGP-S127A YAP and with either pCDNA-3-HA or pCDNA-3-HA-p73α (0.2 μg) as indicated. Cells were harvested, gated for GFP-expression, and apoptosis was quantified as in (B).
(F) Differences in apoptosis in (E) were not due to differences in amount of expressed p73 or either YAP construct. Lysates from equivalent aliquots of cells from (E) were analyzed by Western blot with indicated antibodies.
(G) Cisplatin treatment enhances apoptosis induced by YAP plus p73. H1299 mAktER cells were transiently transfected as in (E) and further treated with 25 μM cisplatin. Cells were harvested and apoptosis quantified as in (E).
(H) Akt activation protects cells expressing p73 plus wild-type, but not S127A, YAP from cisplatin-induced apoptosis. H1299-mAktER cells were transfected as in Figure 7G but with less pEGFP-S127A YAP (0.1 μg) than pEGFP-YAP (0.2 μg). Cells were further treated with 25 μM cisplatin and 100 nM 4-OHT as indicated and apoptosis quantified as in (B).
(I) YAP does is not required for ultraviolet radiation-induced apoptosis. MCF-7 cells were treated as in (A) and UV irradiated at the indicated doses. Means of three independent experiments are shown with error bars indicating SEM.
Molecular Cell  , 11-23DOI: ( /S (02) )