1. Phosphorylation on Thr-55 by TAF1 Mediates Degradation of p53
Heng-Hong Li, Andrew G Li, Hilary M Sheppard, Xuan Liu 
Molecular Cell 
Volume 13, Issue 6, Pages (March 2004)
DOI: /S (04)

2. Figure 1 TAF1 Induces Cell G1 Progression in a p53-Dependent Manner
(A) HCT116 p53+/+ or p53−/− cells (HCT116+/+ or HCT116−/−) were transfected with GFP and pCMV-HAhTAF1, empty vector, or a combination of pcDNA-p53. The cell cycle profile was analyzed 42 hr after transfection.
(B) Flow cytometry analysis of cell cycle distribution of U2OS, MCF7, Saos-2, and H1299 cells transfected with GFP and pCMV-HAhTAF1 or empty vector was carried out precisely as in (A). The G1/S ratio of each group of cells transfected with hTAF1-expressing vector is normalized to that of cells transfected with empty vector. The data represent three independent experiments.
(C) HCT116+/+ cells were transfected with pCMV-HAhTAF1 or empty vector and labeled with CFSE. Cell division was monitored with FACS. Blue, parental generation; orange, second generation; green, third generation.
(D) Western blot analysis of TAF1 overexpression.
Molecular Cell  , DOI: ( /S (04) )

3. Figure 2 TAF1 Associates with p53
(A and B) p53 and TFIID reciprocally immunoprecipitate each other in U2OS cells. Cell lysates were immunoprecipated with mouse IgG, anti-p53, anti-TAF1, anti-GST antibodies, and the resulting immunocomplexes were analyzed by immunoblotting with anti-TAF1, anti-TAF5, or anti-Vinculin antibodies (A) or anti-p53 antibody (B), as indicated.
(C) p53 interacts with TAF1 in vitro. TFIID and bacterial-expressed TBP were subjected to SDS-PAGE, transferred to nitrocellulose, renatured, and incubated with 35S-labeled p53 proteins as indicated on the top. The position of TAF1 and TBP are indicated at left.
(D) TAF1 interacts with the C-terminal residues 347–393 in p53. An equivalent amount of mock-infected insect cell extract was added to the reaction as controls.
(E) Purified HA-TAF1 directly interacts with purified p53 in an immunoprecipitation assay using an anti-HA antibody 12CA5. For peptide competition assays, a 100-fold excess of the p53 C-terminal peptide was included in the reaction.
(F) Schematic diagrams of the p53 proteins.
(G) The wild-type conformation of p53 is required for the TAF1-p53 interaction. 35S-labeled p53 was incubated with baculovirus-expressed TAF1, and immunoprecipitation was performed with anti-TAF1 antibody or normal IgG.
Molecular Cell  , DOI: ( /S (04) )

4. Figure 3 TAF1 Phosphorylates p53 at Thr-55 In Vitro
(A) TAF1 phosphorylates p53 in vitro. (Left panel) GST-p53 (200 ng) was tested for phosphorylation by baculovirus-expressed TAF1 (50 ng). In parallel experiments, RAP74 (100 ng) was used as a control. (Right panel) Vaccinia virus-expressed p53 and RAP74 (100 ng of each) were subjected to phosphorylation using purified TFIID complex (2 μl).
(B) Silver staining of the purified TFIID, p53, and partially purified RAP74 and TAF1.
(C) TAF1 is copurified with p53. A total of 2 μg of affinity-purified p53 was subjected to immunoblotting, and associated TAF1 was detected using anti-TAF1 antibody. As a control, the same amounts of insect cell lysates were also subjected to an identical purification with mouse IgG (mock).
(D) Immunodepletion experiment was performed using anti-TAF1 antibody 6B3. Following immunodepletion, proteins were subjected to kinase assay, and p53 phosphorylation was visualized by autoradiography (upper). The amount of the p53 proteins remaining after each immunodepletion was determined by silver staining (bottom).
(E) TFIID phosphorylates p53 at Thr-55. p53 phosphorylation was carried out in the presence or absence of 5 mM ATPγS. After phosphorylation, p53 was immunoprecipitated using the anti-phosphoThr-55 antibody (Ab202), and the proteins were visualized by autoradiography.
(F) TAF1 fails to phosphorylate T55A. A total of 100 ng of vaccinia virus-expressed and purified p53 or T55A was phosphorylated using 50 ng of baculovirus-expressed TAF1 (left) or purified TFIID complex (right).
(G) Baculovirus-expressed TAF1 or mock-infected insect cell lysates were subjected to affinity purification, SDS-PAGE, and denaturation-renaturation. The phosphorylation reaction was carried out in the presence of 1 μg of p53 or T55A.
(H) Silver staining of the purified TAF1 (10 ng), p53 (100 ng), and T55A (100 ng) used in (F) and (G).
Molecular Cell  , DOI: ( /S (04) )

5. Figure 4 TAF1 Phosphorylates p53 at Thr-55 In Vivo
(A) Overexpression of TAF1 leads to an increase in Thr-55 phosphorylation and p53 degradation. U2OS cells were transfected with an empty vector (vector), TAF1-expressing vector (pCMV-HAhTAF1, left panel), or TAF1 kinase mutant vector (p-LXSN-MT-TAF1N1398 A2/N7Ala, right panel) and assayed for TAF1 protein levels (lanes 1, 2, 7, 8, 9, 10), Thr-55 phosphorylation levels (lanes 3, 4, 11, 12, 13, 14, upper panel), and p53 protein levels (lanes 5, 6, 15, 16, 17, 18, upper panel) using anti-TAF1, anti-p53, and anti-phosphoThr-55 antibody, respectively.
(B) Apigenin inhibits the kinase activity of TAF1 in vitro. p53 and RAP74 were subjected to phosphorylation using purified TAF1 in the presence or absence of 20 μM apigenin.
(C) U2OS cells were treated with 40 μM apigenin, and Thr-55, Ser-15, Ser-20, Ser-46, and Ser-392 phosphorylation as well as p21 and p53 protein levels were measured using specific antibodies at the indicated times.
(D) RNAi-mediated inhibition of endogenous TAF1 reduces Thr-55 phosphorylation in U2OS cells. Cells were harvested at 2 or 3 days after TAF1-RNAi transfection (2d and 3d) and whole-cell extracts were immunoblotted with anti-TAF1, anti-TBP, anti-p53, anti-p21, anti-Mdm2, and anti-Vinculin antibodies (left panel). Thr-55 phosphorylation levels were assayed using anti-phosphoThr-55 antibody (right panel).
(E) A2/N7Ala functions as a dominant-negative mutant under RNAi-mediated TAF1 reduction condition. U2OS cells were transfected with TAF1-specific RNAi with or without the A2/N7Ala mutant and Thr-55 phosphorylation was detected as described.
Molecular Cell  , DOI: ( /S (04) )

6. Figure 5 Thr-55 Phosphorylation Leads to p53 Degradation
Saos-2 cells were transfected with pcDNA-p53 or pcDNA-T55A, in the presence or absence of Mdm2-expressing vector (pCHDM1B).
(A) p53 protein levels were assayed using DO-1 antibody.
(B) As (A), but in the presence or absence of MG132 (Calbiochem).
(C) Half-life assay for p53 and T55A in the presence or absence of TAF1. Protein-synthesis inhibitor cyclohexamide was added 26 hr after transfection. Cells were harvested for Western blot analysis at the indicated times.
(D) Saos-2 cells were transfected with p53, T55A, and Mdm2 in the presence of MG132. The cell lysates were immunoprecipitated using anti-p53 polyclonal antibody (FL-393, Santa Cruz), and the resulting immunocomplexes were analyzed by immunoblotting with anti-Mdm2 antibody.
(E) U2OS cells were transfected with an empty vector (EV) or the TAF1-expressing vector and treated with 40 μM apigenin. Cells were harvested and subjected to immunoprecipitation with anti-p53 polyclonal antibody followed by immunoblotting with anti-Mdm2 antibody.
(F) As (A), but with cotransfection of 0.5 μg of p53 reporter (pRGCE4Luc), and increasing amounts of Mdm2 and luciferase activity were assayed.
(G) Saos-2 cells were transfected with 0.5 μg of GFP and 1.5 μg of pcDNA-p53 or pcDNA-T55A for 42 hr. Cell cycle profile of the GFP-positive cells was analyzed using a FACScan (Becton Dickinson). The data represent three independent experiments. The G1/S ratio was graphed at the right to represent the ability of p53 to induce G1 arrest.
Molecular Cell  , DOI: ( /S (04) )

7. Figure 6 Phosphorylation of Thr-55 Is Reduced Following DNA Damage
U2OS cells, in the presence of MG132, were either untreated (mock) or subjected to UV (100 J/m2) or γ irradiation (10 Gy) and assayed at the time points indicated. Thr-55 phosphorylation levels were assayed by IP with Ab202 followed by Western analysis with DO-1 antibody (A). The p53 and vinculin protein were assayed using either DO-1 or anti-vinculin antibody under the same irradiation conditions in the absence of MG132 (B). (C) CEM cells were either untreated (mock) or subjected to UV irradiation (100 J/m2) at the times indicated, and Thr-55 phosphorylation levels were assayed. (D) TAF1 interacts with p53 in CEM cells. (E) U2OS cells were subjected to UV irradiation. Cell lysates were immunoprecipated with anti-p53 antibody DO-1, and associated TAF1 and TAF5 were detected by immunoblotting with anti-TAF1 or anti-TAF5 antibodies.
Molecular Cell  , DOI: ( /S (04) )