Pin1 Links the Activities of c-Abl and p300 in Regulating p73 Function Fiamma Mantovani, Silvano Piazza, Monica Gostissa, Sabrina Strano, Paola Zacchi, Roberto Mantovani, Giovanni Blandino, Giannino Del Sal  Molecular Cell  Volume 14, Issue 5, Pages 625-636 (June 2004) DOI: 10.1016/j.molcel.2004.05.007

Figure 1 Pin1 Regulates the Apoptotic Functions of p73 (A) H1299 cells expressing inducible p73α and either GFP-Pin1 (p73α/GFP-Pin1) or GFP alone (p73α/GFP) were treated with cisplatin (CDDP), either inducing p73α expression with Ponasterone A (PonA) or not as indicated. After 48 hr, cell death was evaluated by trypan blue staining. The histogram represents the mean results of three independent experiments, with standard deviations. The lower panels show parallel experiments, where caspase-processed PARP p85 fragment and p73 expression were analyzed by Western blot of cell lysates. Anti-actin staining was used as loading control. (B) p73α-inducible H1299 cells were transfected with either Pin1-specific or control scrambled (C) siRNA oligonucleotides and treated with CDDP, either inducing p73α with Pon A or not as indicated. Protein lysates were analyzed by Western blot for caspase-processed PARP p85 fragment and for p73α, PIG3, and Pin1 protein levels. (C) H1299-p73α cells were treated as in (B), fixed, stained with Hoechst, and apoptotic nuclei were then scored by fluorescence microscopy. The histogram shows the mean results of six independent experiments, with standard deviations. (D) Shown are typical examples of the nuclear staining observed in the experiment described in (C). p73 expression was induced in all samples before treatment with CDDP where indicated. Nuclei exhibiting condensed chromatin on fluorescence microscopy analysis were scored as apoptotic. Molecular Cell 2004 14, 625-636DOI: (10.1016/j.molcel.2004.05.007)

Figure 2 Pin1 Controls the Stability of p73 Protein in Both Normal and Stress Conditions (A) HCT116/3 cells were transfected with either Pin1 or control siRNA (C). After 72 hr, protein synthesis was blocked with cycloheximide (CHX) and cells were harvested at the indicated time points. The amount of p73 protein remaining was analyzed by Western blot and densitometric scanning. The results of three independent experiments are summarized in the graph, where the amount of p73 protein at time 0 was set as 100%. Standard deviations are indicated. Pin1 expression and equal loading were checked by Western blot. (B) The experiment described in (A) was performed by adding CDDP to growth medium for 18 hr before CHX block. Anti-p53 staining was also performed as control. The graph summarizes the results of three independent experiments. (C) wt and Pin1−/− mouse embryo fibroblasts (MEF) were first infected with a retroviral vector expressing p73α and subsequently treated with CDDP for 18 hr. After CHX treatment, cells were processed as in (A). Molecular Cell 2004 14, 625-636DOI: (10.1016/j.molcel.2004.05.007)

Figure 3 Pin1 Regulates p73 Transcriptional Function (A) Luciferase activity asssays were performed on lysates of SaOS-2 cells transfected with 1 μg of either Bax-luc or p21-luc reporter constructs, together with a fixed amount (0.1 μg) of p73α expression vector and increasing amounts (0.5 and 1.5 μg) of Pin1 expression vector. pRL-TK (50 ng) were also introduced to allow normalization of transfection efficiency. The histograms represent the mean results of three independent experiments, and standard deviations are indicated. The expression of p73 and Pin1 proteins is shown in the panels below. (B) Wild-type and Pin1−/− MEF were transfected with 1 μg of Bax-luc reporter construct and with increasing amounts (0.1 and 0.3 μg) of p73α expression construct or empty plasmid as indicated. Luciferase activity was measured 48 hr later, normalizing for transfection efficiency based on pRL-TK reporter activity. (C) H1299 cells expressing inducible p73α were transfected with different p73-responsive reporters (Bax-luc, PIG3-luc, and p53AIP1-luc), together with either Pin1-specific or scrambled siRNA (C) oligonucleotides as a control. The expression of p73α was induced by Pon A for 12 hr, luciferase activity was measured, and the results normalized for transfection efficiency as in (A), 48 hr after transfection. The histograms represent the mean results of three independent experiments, and standard deviations are indicated. The expression of p73 and Pin1 proteins is shown in the panels below. (D) p73α-inducible H1299 cells were transfected with either Pin-1 or control siRNA as in (C). After 48 hr, p73α was induced and the cells were either treated or not with CDDP for 18 hr. The levels of p73α, Pin1, PIG3, Bax, p21, and actin proteins were assessed by Western blot. Molecular Cell 2004 14, 625-636DOI: (10.1016/j.molcel.2004.05.007)

Figure 4 p73 Interacts with Pin1 in Unstressed Cells through Three S/T-P Sites in its C Terminus (A) Lysates of untreated HCT116/3 cells were subjected to GST- or GST-Pin1 pull-down followed by Western blot with anti-p73 antibody. An aliquot of the lysate was treated with calf intestine phosphatase (CIP) prior to pull-down assay. Efficient dephosphorylation of Pin1 binding sites by CIP treatment was verified by Western blot of total lysates with pS/pT-P-specific MPM-2 antibody. The GST protein inputs were also checked by anti-GST Western blot. (B) Expression of endogenous p73 protein was increased in U2OS ER/E2F-1 cells by treatment with 4-OHT for 18 hr. Protein extracts were then subjected to precipitation with anti-Pin1 antiserum or nonimmune serum (NIS) as a control and finally checked by Western blot with anti-p73 antibody. (C) (Left) Schematic representation of p73 isoforms generated by differential mRNA splicing at the 3′ end. TA, transactivation domain; PP, polyproline region; DBD, DNA binding domain; OD, oligomerization domain; SAM, sterile alpha motif. Black dots indicate putative Pin1 binding sites (S/T-P). The C-terminal PP domain, containing Pin1-consensus sites at residues 412, 442, and 482 (indicated by white dots), is enlarged in the right panel. M1, M2, and M3 are p73α proteins bearing Ser/Thr to Ala substitutions at either one, two, or all these sites, respectively, as indicated. (D) (Upper panels) p73 isoforms α, β, γ, δ, and ϵ were transiently overexpressed in 293T cells, and protein lysates were then subjected to GST-Pin1 pull-down followed by Western blot with anti-p73 antibody. (Lower panels) wt p73α and M1, M2, and M3 point mutants were subjected to GST-Pin1 pull-down assay as above, including the truncated form p73δ as a control. (E) SaOS-2 cells were transfected with 1 μg of Bax-luc reporter construct together with 0.1 μg of construct expressing either wt p73α, M1, M2, or M3 point mutants and with 0.5 μg of Pin1 expression vector or empty plasmid as indicated. pRL-TK reporter was also introduced for normalization of transfection efficiency. The truncated form p73δ was included as a control. The mean results of three independent experiments are summarized in the histogram, while the expression levels of p73 and Pin1 proteins are shown in the Western blots below. Molecular Cell 2004 14, 625-636DOI: (10.1016/j.molcel.2004.05.007)

Figure 5 c-Abl Mediates Augmented Interaction of p73 with Pin1 upon Genotoxic Stress, Thereby Inducing p73 Apoptotic Function (A) p73 expression was increased in U2OS E2F-1/ER cells by 4-OHT, and cells were then either left untreated (−) or treated with CDDP or adriamycin (Adria) for 18 hr. Cell lysates were normalized for p73 content and subsequently subjected to GST-Pin1 pull-down followed by Western blot with anti-p73 antibody. Input levels of p73 are shown in the panel below. (B) (Upper panels) Wild-type (wt) and Y99F mutant p73α were overexpressed in 293T cells, either alone or together with HA-c-Abl, or with HA-p38 and MKK6 kinases. Cells were treated with CDDP for 18 hr where indicated, and expression of p73, c-Abl, and p38 was controlled by Western blot. (Lower panels) Upon normalizing for the amount of p73 protein, the above lysates were subjected to GST-Pin1 pull-down followed by Western blot with anti-p73 antibody. (C) (Upper panels) U2OS cells were transfected with either c-Abl-specific or control siRNA oligonucleotides and then either left untreated or treated with CDDP. p73 and c-Abl protein levels were analyzed by Western blot. (Lower panels) Upon normalizing for the amount of p73 protein, the above lysates were subjected to GST-Pin1 pull-down followed by Western blot with anti-p73 antibody. (D) p73α was overexpressed in SaOS-2 cells either alone or with HA-c-Abl, together with either Pin1-specific or control siRNA as indicated. After 72 hr, protein synthesis was blocked with CHX and the amount of p73 remaining after 6 hr was checked by Western blot. Protein loading was assessed by anti-actin staining. The Western blots in the lower panels show the expression of HA-c-Abl and Pin1. (E) wt and Pin1−/− MEF were transiently transfected with Bax-luc reporter and with p73α and c-Abl expression constructs as indicated. Luciferase activity was measured 48 hr later, normalizing for transfection efficiency based on pRL-TK reporter activity. (F) HA-tagged c-Abl and p73α were overexpressed in SaOS-2 cells either alone or in combination, including GFP expression plasmid to identify transfected cells. Parallel experiments were performed by transfecting either Pin1-specific or control siRNA (C). After 72 hr, cells were fixed and stained with Hoechst to screen for GFP-positive apoptotic nuclei. The histogram represents the mean results of three independent experiments with standard deviations. Expression levels of c-Abl, p73, and Pin1 are shown in the Western blots below. Molecular Cell 2004 14, 625-636DOI: (10.1016/j.molcel.2004.05.007)

Figure 6 Pin1 Induces Binding and Acetylation of p73 by p300, Thereby Stabilizing and Activating It (A) p73α was overexpressed in wt and Pin1−/− MEFs, and cells were then treated with adriamycin, 1 μM TSA, and 5 mM nicotinamide for 18 hr. p73 was immunoprecipitated from cell lysates, and acetylation was analyzed by Western blot with anti-acetylated lysine antibody. (B) Partial subtilisin cleavage was performed on in vitro-translated, radioactively labeled, His-tagged p73α protein after incubation with either GST, GST-Pin1, or GST-Pin1(C109A) protein, as well as upon p73 dephosphorylation with CIP as a control. The reaction products were then analyzed by SDS-PAGE and autoradiography. (C) In vitro-translated, radioactively labeled, HA-tagged p73α protein was immunoprecipitated with anti-HA antibody (NIS, nonimmune serum used as control) and bound to protein A-Sepharose. Beads were incubated with either GST-Pin1 or GST, washed, and then subjected to binding with purified full-length p300 protein. Bound p300 was then revealed by anti-p300 Western blot (upper right panel), while the amounts of immunoprecipitated p73 in each sample were assessed by SDS-PAGE and autoradiography (lower right panel). Input protein levels are shown in the left panels. (D) p300 was overexpressed in H1299 p73α/GFP and p73α/GFP-Pin1 cells, and protein lysates were immunoprecipitated with either anti-p73 or nonimmune serum (NIS) as a control. The amount of coimmunoprecipitated p300 was then assessed by Western blot. Lower panels confirm equal expression of p73 and p300 in lysates before IP. (E) 293T cells were transfected with HA-p73α, HA-p300, and HA-Pin1 expression plasmids in the indicated combinations, treated as in (A), and p73 acetylation was then assessed. Expression levels of p73, p300, and Pin1 in total cell lysates were checked by anti-HA Western blot. (F) Pin1 expression was ablated by RNA interference in SaOS-2 cells transiently transfected with Bax-luc reporter and with p73α and p300 as indicated. Luciferase activity was measured 48 hr later, normalizing for transfection efficiency based on pRL-TK reporter activity. Histograms represent the mean results of four independent experiments, and standard deviations are indicated. (G) p73α was overexpressed in SaOS-2 cells either alone or with HA-tagged p300, with either Pin1-specific or control siRNA (C) as indicated. After 72 hr, protein synthesis was blocked with CHX and the amount of p73 protein remaining at different times as well as expression of HA-p300 and Pin1 were then checked by Western blot. Protein loading was controlled by anti-actin staining. Molecular Cell 2004 14, 625-636DOI: (10.1016/j.molcel.2004.05.007)

Figure 7 Apoptosis Induced by Endogenous p73 Requires Pin1 (A) SaOS-2 cells were transfected with either Pin1- or p73-specific siRNA or with control siRNA and then treated with CDDP. After 48 hr, expression of p73 and its apoptotic target PUMA was analyzed by Western blot of cell lysates. Anti-actin staining was used as loading control. (B) Cells were treated as in (A), then trypsinized and incubated with trypan blue to quantitate cell death. The histogram represents the mean results of three independent experiments. (C) Crosslinked chromatin derived from U2OS/E2F-ER cells, transfected with either Pin1-specific or control siRNA, and treated with CDDP was immunoprecipitated with p73 antiserum or nonimmune serum (NIS) as a control and analyzed by PCR with specific primers for the indicated regulatory regions. Input, nonimmunoprecipitated crosslinked chromatin. Molecular Cell 2004 14, 625-636DOI: (10.1016/j.molcel.2004.05.007)