Phosphorylation on Thr-55 by TAF1 Mediates Degradation of p53

Molecular Cell, Vol. 13, 867–878, March 26, 2004, Copyright 2004 by Cell Press

Phosphorylation on Thr-55 by TAF1 Mediates Degradation of p53: A Role for TAF1 in Cell G1 Progression Heng-Hong Li,1 Andrew G. Li,1 Hilary M. Sheppard, and Xuan Liu* Department of Biochemistry University of California Riverside, California 92521

Summary The largest subunit of TFIID, TAF1, possesses an intrinsic protein kinase activity and is important for cell G1 progression and apoptosis. Since p53 functions by inducing cell G1 arrest and apoptosis, we investigated the link between TAF1 and p53. We found that TAF1 induces G1 progression in a p53-dependent manner. TAF1 interacts with and phosphorylates p53 at Thr-55 in vivo. Substitution of Thr-55 with an alanine residue (T55A) stabilizes p53 and impairs the ability of TAF1 to induce G1 progression. Furthermore, both RNAimediated TAF1 ablation and apigenin-mediated inhibition of the kinase activity of TAF1 markedly reduced Thr-55 phosphorylation. Thus, phosphorylation and the resultant degradation of p53 provide a mechanism for regulation of the cell cycle by TAF1. Significantly, the Thr-55 phosphorylation was reduced following DNA damage, suggesting that this phosphorylation contributes to the stabilization of p53 in response to DNA damage. Introduction TAF1 is the largest component of transcription factor TFIID that is composed of TATA binding protein (TBP) and multiple TBP-associated factors (TAFs). TAF1 is identical to CCG1, a cell cycle regulatory protein found to be important for progression through the G1 phase and apoptosis in a hamster cell line, ts13 (Sekiguchi et al., 1991, 1995). This cell line contains a single base pair substitution in the gene encoding TAF1, which results in a change of glycine to aspartic acid at position 690 of the hamster protein (ts form of TAF1; Hayashida et al., 1994). These cells grow normally at the permissive temperature (33⬚C), but, at the restrictive temperature (39.5⬚C), they arrest in the G1 stage of the cell cycle and undergo apoptosis (Sekiguchi et al., 1995). The G1-specific cell cycle arrest can be rescued by transfecting human TAF1 into the hamster ts13 cells (Wang and Tjian, 1994). Despite having a mutation in the TAF1, ts13 cells do not exhibit a global defect in gene expression. Analysis of the transcription properties of TFIID containing the mutant form of TAF1 revealed that transcription from the cyclin A (Wang and Tjian, 1994) and cyclin D (Wang et al., 1997) promoters is reduced when ts13 cells are shifted from the permissive to the nonpermissive temperature. Thus, the defects in TAF1 apparently result in *Correspondence: [email protected] 1 These authors contributed equally to this work.

the downregulation of key cell cycle proteins that may be responsible for the G1 arrest phenotype. Human TAF1 possesses intrinsic protein kinase activity (Dikstein et al., 1996), histone acetyltransferase activity (HAT; Mizzen et al., 1996), and ubiquitin-activating and conjugating activity (E1/E2; Pham and Sauer, 2000). The TAF1 kinase is bipartite, consisting of N- and C-terminal kinase domains. TAF1 is capable of autophosphorylation as well as specific phosphorylation of the 74 kDa subunit (RAP74) of transcription factor IIF (Dikstein et al., 1996) and the ␤ subunit of transcription factor IIA (Solow et al., 2001). Kinase-deficient forms of TAF1 have a significantly reduced ability to rescue ts13 cells, suggesting that the kinase activity of TAF1 is important for the progression through the G1 phase (O’Brien and Tjian, 1998). It has been shown that the retinoblastoma protein Rb interacts directly with TAF1 and inhibits the kinase activity of TAF1 (Siegert and Robbins, 1999), suggesting that the kinase activity of TAF1 may play a significant role in tumor suppression. Despite the progress made in analyzing TAF1 kinase activity and in demonstrating the significance of TAF1 in cell cycle regulation, little is known about its kinase properties in relation to its effect upon cell cycle control. p53 is a tumor suppressor, which functions by inducing G1 cell cycle arrest and apoptosis in response to DNA damage (Vogelstein et al., 2000; Sharpless and DePinho, 2002). An important mechanism used to control p53 function is the regulation of its protein levels, principally through stabilization of the protein. Following DNA damage, as a result of posttranscriptional modification, the p53 protein is protected from rapid degradation and acquires transcription-activating function. Phosphorylation of p53 plays a critical role in the regulation of p53 stabilization (Appella and Anderson, 2001). p53 has been described as a substrate for many kinases, including DNA-dependent protein kinase (DNA-PK) and ATM on Ser-15 (Banin et al., 1998; Canman et al., 1998; Shieh et al., 1997), Chk2 on Ser-20 (Hirao et al., 2000; Shieh et al., 2000; Chehab et al., 2000), cyclin D-associated kinase on Ser-315 (Price et al., 1995), and casein kinase II on Ser-392 (Fiscella et al., 1994). Activation of the p53 protein as a transcription factor allows it, in turn, to regulate the expression of genes whose products promote G1 cell cycle arrest. Since both TAF1 and p53 carry out important functions in cell cycle control, we investigated whether TAF1 could function through p53. We found that TAF1 induces G1 progression in p53⫹/⫹ but not in p53⫺/⫺ cell lines. To investigate the link between TAF1 and p53, we demonstrated that p53 interacts with TAF1 in the TFIID complex and TAF1 directly phosphorylates p53 at Thr-55 in vivo. The phosphorylation of Thr-55 results in p53 degradation and leads to a decrease in cell G1 arrest. The treatment of cells with apigenin, a naturally occurring flavonoid that was reported to cause a p53-mediated p21 induction (McVean et al., 2000), leads to an inhibition of the kinase activity of TAF1 and a decrease in Thr-55 phosphorylation. Furthermore, RNAi-mediated inhibition of endogenous TAF1 also reduced Thr-55 phos-

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phorylation. Introducing wild-type p53 but not a phosphorylation mutant back into p53⫺/⫺ cells rescues the TAF1 G1 progression phenotype. These results reveal that TAF1 induces G1 progression, at least in part, in a p53-dependent manner. Thus, phosphorylation and the resultant destabilization of p53 provide a mechanism for regulation of the cell cycle by TAF1. Significantly, the Thr-55 phosphorylation was reduced following DNA damage, suggesting that this phosphorylation contributes to the stabilization of p53 in response to DNA damage. Results TAF1 Induces Cell G1 Progression through p53 To investigate whether TAF1 and p53 could be functionally linked, we first tested the ability of TAF1 to induce G1 progression in p53⫹/⫹ and p53⫺/⫺ cells. We considered the possibility that different cell lines might differ in ways other than p53 status and therefore chose the HCT116 cell line, with targeted deletions of both p53 alleles (⫺/⫺), and its parental wild-type p53 cell line (⫹/⫹) (Bunz et al., 1998). Compared with cells transfected with the empty CMV vector, overexpression of TAF1 in HCT116⫹/⫹ cells resulted in more cells in the S phase (from 34% to 51%) and fewer in the G1 phase (from 56% to 45%; Figure 1A, top profiles). In contrast, overexpression of TAF1 in HCT116⫺/⫺ cells did not affect cell cycle profile (Figure 1A, middle profiles). These results indicate that overexpression of TAF1 leads to a cell G1 phase progression or an S phase arrest in wildtype p53 cells. To distinguish these phenotypes, we measured cell division and growth using a 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE) labeling approach (Lyons and Parish, 1994). Because the CFSE label is divided equally between two daughter cells upon cell division, this analysis allows us not only to quantify overall cell growth but also to monitor the division history of individual cells. Compared with controls, HCT116 cells overexpressing TAF1 showed enhanced growth activity (Figure 1C). The presence of overexpressed TAF1 during the analysis was confirmed by immunoblotting analysis (Figure 1D). These results suggest that overexpression of TAF1 leads to a cell G1 progression. To assay directly the role of p53 in TAF1-mediated G1 progression, we reintroduced wild-type p53 into HCT116⫺/⫺ cells (Figure 1A, the bottom profiles). As anticipated, cells transfected with p53 exhibit a G1 arrest phenotype, with about 61% of the cells in the G1 phase as compared with 49% of the cells transfected with the control vector in the G1 phase. Significantly, overexpression of TAF1 in p53-transfected HCT116⫺/⫺ cells partially recovered the G1 progression phenotype with more cells in the S phase and fewer cells in the G1 phase. These results suggest that reintroducing p53 in HCT116⫺/⫺ cells, at least in part, recovered the TAF1mediated G1 progression phenotype. To confirm these results, we compared the ability of TAF1 to induce G1 progression in several other p53⫹/⫹ (U2OS and MCF7) and p53⫺/⫺ cell lines (Saos-2 and H1299). We used the G1/S ratio to represent the ability of TAF1 to induce G1 progression. As presented in Fig-

ure 1B, a G1 progression was observed in p53⫹/⫹ but not in p53⫺/⫺ cells. Taken together, our results suggest that TAF1 induces cell G1 progression in a p53-dependent manner. TAF1 Physically Interacts with p53 Because the kinase activity of TAF1 is important for the progression through the G1 phase (O’Brien and Tjian, 1998), our finding that TAF1 induces cell G1 progression in a p53-dependent manner prompted us to investigate whether TAF1 and p53 could be functionally linked via phosphorylation. As many protein kinases associate with their substrates, we first sought to determine whether this was so for TAF1. U2OS cell lysates were immunoprecipitated with anti-p53 antibody (DO-1), and the resulting immunocomplexes were analyzed by immunoblotting with anti-TAF1 antibody (6B3). As indicated in Figure 2A, immunoprecipitation of p53 resulted in coprecipitation of TAF1. In contrast, no TAF1 was detected when control antibodies (anti-GST antibody or IgG) were used. As an additional control, complexes between p53 and TAF1 were not detected in Saos-2 cells (lacking p53). We also performed the immunoprecipitation experiment with the anti-TAF1 antibody and Western blotting with the anti-p53 antibody. Since endogenous p53 protein levels are very low, this experiment was performed using lysates from cells treated with a proteasome inhibitor, MG132. As shown in Figure 2B, endogenous p53 and TAF1 can reciprocally coimmunoprecipitate each other in vivo. To determine whether p53 interacts with TAF1 in the TFIID complex, Western blotting of the resulting immunocomplexes with an antiTAF5 antibody revealed that TAF5 is in the immunocomplex (Figure 2A). Thus, our results suggest that endogenous p53 is likely to associate with the assembled TFIID complex in vivo. To ensure that p53 directly interacts with TAF1, the TFIID complex and bacterial-expressed TBP were subjected to SDS-PAGE followed by far-Western analysis with 35S-labeled p53 protein. Labeled p53 bound to TAF1 (Figure 2C, lane 1). The binding was also observed to both bacterial-expressed TBP (lane 2) and TBP present in the purified TFIID complex (lane 1). Although TAF1 was the only TAF observed to bind p53 in the far-Western analysis, other potential interactions might also present but not be observed because of the renaturation condition. Nevertheless, our results indicated that p53 physically interacts with TAF1. We next mapped the domain of p53 that interacts with TAF1 and showed TAF1 interacted with full-length p53, p53⌬N92, and p53⌬N160 but not p53⌬C292 (Figure 2C), indicating that binding of TAF1 to p53 required the C-terminal region. To confirm the mapping results, we performed immunoprecipitation experiments with baculovirus-expressed TAF1 and in vitro-translated p53 deletion mutants (Figure 2F) and showed that the binding of TAF1 required the C-terminal domain of p53 (Figure 2D). To determine if the interaction is direct, we showed that affinity-purified TAF1 (Figure 3H) interacts with purified p53 (Figure 3H) and this interaction can be completed by an addition of the excess C-terminal peptide of p53 (Figure 2E). To determine if wild-type p53 conformation is required for the interaction, we assayed the ability of TAF1 to interact

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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.

with wild-type p53 and p53 mutants that exhibit wildtype conformation (T55A and R248W) and with p53 mutants that exhibit structure mutant conformation (R175H and R273H; Bullock and Fersht, 2001; H.-H.L. and X.L., unpublished data). Our results showed that wild-type p53 conformation is required for the TAF1-p53 interaction (Figure 2G). Together, data derived from these experiments suggest that TAF1 directly interacts with the C-terminal domain of p53 in a conformation-dependent manner. TAF1 Phosphorylates p53 at Thr-55 Since TAF1 could form physical complexes with p53, we proceeded to test whether TAF1 could control p53

by phosphorylation. We first performed in vitro phosphorylation assays using baculovirus-expressed and purified TAF1 and bacterial-expressed GST-p53. As a measure of the specific kinase activity of TAF1, we assayed its effects on phosphorylation of RAP74 under identical conditions (Figure 3A, lane 5). Our results showed that TAF1 phosphorylated p53 in vitro (Figure 3A, lane 3). In order to better mimic in vivo conditions, we next used affinity-purified TFIID to substitute TAF1 and vaccinia virus-expressed and purified p53 to substitute GST-p53 (Figure 3B). As shown in Figure 3A (lanes 6–10), the purified TFIID complex also phosphorylated p53 (Figure 3A, lane 8). These results suggest that p53 could serve as a substrate for TAF1.

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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.

We note that, under our assay conditions, a very low level of phosphorylation of affinity-purified p53 was also observed without exogenously added TFIID (Figure 3A, lane 7). We suspect that this may be due to low levels of TAF1 copurified with vaccinia virus-expressed p53 because TAF1 associates p53 in vivo (Figure 2A). To demonstrate this directly, we performed Western blot analysis using a newly generated anti-TAF1 polyclonal antibody (Ab1230). As shown in Figure 3C, this antibody specifically detects a band at TAF1 position in affinitypurified p53 preparation. Presumably due to the limited amounts of endogenous TAF1 associated with overexpressed p53, the detected band, however, is very weak. To confirm this result, we performed immunodepletion experiments using the anti-TAF1 antibody. A significant decrease in phosphorylation but not overall level of p53 was observed upon removal of associated TAF1, while a control IgG had no effect on p53 phosphorylation (Figure 3D). In light of the previously demonstrated association of TAF1 and p53 in vivo, our results indicate that TAF1 is responsible for the low level of phosphorylation of affinity-purified p53.

To study the effect of phosphorylation on TAF1-mediated cell G1 progression, we first determined the phosphorylation sites of p53 by TAF1. Using a biochemical analysis, we have previously identified the residue of Thr-55 as the major phosphorylation site of p53 by a copurified kinase (Gatti et al., 2000). Our observation that TAF1 is associated with p53 in vivo and that it phosphorylates p53 in vitro led us to hypothesize that TAF1 might be the copurified kinase responsible for the phosphorylation at Thr-55. To test this hypothesis, a phosphospecific antibody for Thr-55 (Ab202) was generated; the specificity of this antibody had been validated previously (Gatti et al., 2000). Using this antibody, we have confirmed that Thr-55 indeed is phosphorylated by exogenously added TFIID in vitro (Figure 3E). To test whether Thr-55 is the major phosphorylation site for TAF1, we generated a recombinant vaccinia virus expressing a p53 phosphorylation mutant (alteration of Thr to Ala at position 55, T55A) and showed that phosphorylation of this mutant by TAF1 and TFIID was abolished (Figure 3F). To confirm that the intrinsic kinase activity of TAF1 is

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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 ␮l). (B) Silver staining of the purified TFIID, p53, and partially purified RAP74 and TAF1. (C) TAF1 is copurified with p53. A total of 2 ␮g 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 ATP␥S. 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 denaturationrenaturation. The phosphorylation reaction was carried out in the presence of 1 ␮g 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).

responsible for Thr-55 phosphorylation, we performed a modified version of an in-gel phosphorylation assay (Siegert and Robbins, 1999). In this assay, baculovirusexpressed and purified TAF1 was subjected to SDSPAGE and transferred to nitrocellulose membrane. The membrane-bound TAF1 was renatured and incubated with purified p53 or T55A. As controls, mock-infected insect cells were subjected to identical purifications and renaturation. As illustrated in Figure 3G, the renatured TAF1 exhibited a significant level of phosphorylation of p53. The phosphorylation was not observed with T55A,

suggesting that TAF1 directly phosphorylates p53 at Thr-55. Next, we determined whether endogenous p53 could be phosphorylated at Thr-55 in U2OS cells. As illustrated in Figure 4A, endogenous p53 is indeed phosphorylated at Thr-55 (lane 3, upper panel) under cell growth conditions. To test whether TAF1 is responsible for this phosphorylation, we overexpressed TAF1 in U2OS cells and observed a 2-fold increase in the phosphorylation (lanes 3 and 4, upper panel). Since overexpression of TAF1 affects p53 protein levels (see later results), these exper-

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Figure 4. TAF1 Phosphorylates p53 at Thr55 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 antiTAF1, 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 ␮M apigenin. (C) U2OS cells were treated with 40 ␮M 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, antiMdm2, 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.

iments were performed in the presence of the proteasome inhibitor MG132. Because p53 is already phosphorylated at Thr-55 in vivo, it is not surprising that the increased Thr-55 phosphorylation under TAF1 overexpression is very subtle. To ensure that the kinase activity of TAF1 is responsible for the phosphorylation, we introduced into the U2OS cells a pCMV-A2/N7Ala vector that expresses a TAF1 deletion mutant lacking the C-terminal

kinase and carrying kinase-inactive mutations on the N-terminal kinase domain (O’Brien and Tjian, 1998). Our results showed that this mutant inhibited the Thr-55 phosphorylation in a dose-dependent manner (lanes 12 and 14, upper panel), indicating that A2/N7Ala functions as a dominant-negative mutant when highly overexpressed. Collectively, our data suggested that the kinase activity of the introduced TAF1 was responsible

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for the increase in Thr-55 phosphorylation of endogenous p53. We recently found that a naturally occurring flavonoid, apigenin, can inhibit the kinase activity of TAF1 in vitro (Figure 4B). We therefore tested Thr-55 phosphorylation by treating cells with apigenin. As illustrated in Figure 4C, the Thr-55 phosphorylation level is significantly reduced in apigenin-treated U2OS cells. To provide specificity, we showed that Ser-15, Ser-20, and Ser-46 phosphorylations were not affected by apigenin treatment (Figure 4C). As a control, we examined Ser-392 phosphorylation as apigenin has been shown to inhibit casein kinase II (CKII; Critchfield et al., 1997; Tsai and Seto, 2002) specifically, which can phosphorylate p53 at Ser392 (Fiscella et al., 1994; Figure 4C). To provide further evidence that TAF1 is responsible for phosphorylation of p53 at Thr-55, we transfected U2OS cells with two TAF1-specific RNA oligonucleotides (TAF1-RNAi). As shown in Figure 4D, endogenous TAF1 was significantly reduced after transfection, while the levels of TBP and vinculin remained the same. The levels of Mdm2 protein were also decreased in our TAF1RNAi experiments. This result is consistent with a previous report that the expression of the Mdm2 gene in tsBN462 cells (which contains an identical G690E mutation in TAF1 as in ts13 cells) was reduced at the nonpermissive temperature (Wasylyk and Wasylyk, 2000). Strikingly, inhibition of endogenous TAF1 by TAF1-RNAi significantly reduced p53 phosphorylation at Thr-55. Overexpression of A2/N7Ala further reduced the phosphorylation (Figure 4E), suggesting that A2/N7Ala functions as a dominant-negative mutant under RNAi-mediated TAF1 reduction condition. These results together with our previous findings that TAF1 forms a complex with p53 (Figure 2) and phosphorylates p53 at Thr-55 in vitro (Figure 3) suggest that TAF1 directly phosphorylates p53 at Thr-55 in vivo. Notably, overexpression of TAF1 resulted in a decrease in p53 protein levels (Figure 4A), whereas inhibition of TAF1 led to an increase in the levels (Figures 4A, 4C, and 4D), indicating that Thr-55 phosphorylation might lead to p53 degradation. Phosphorylation at Thr-55 Leads to Mdm2-Mediated p53 Degradation To further explore the possibility that phosphorylation at Thr-55 leads to p53 degradation, the protein levels of wild-type p53 and the phosphorylation mutant T55A were determined in Saos-2 cells following transfection. Our results showed a moderate increase in the protein levels when cells were transfected with T55A (Figure 5A). Significantly, when cells were cotransfected with Mdm2, wild-type p53 protein but not T55A levels were markedly reduced. Consistent with these results, the inhibition of p53-dependent transcription by Mdm2 was observed with wild-type p53 but to a much-reduced extent with T55A (Figure 5F). Furthermore, we found that the proteasome inhibitor MG132 blocked wild-type p53 degradation but had little effect on T55A (Figure 5B). We next measured the half-life of p53 and T55A. As expected, the half-life of T55A was significantly longer than that of p53 (Figure 5C), and the overexpression of TAF1 leads to a decrease in the half-life of wild-type p53 but not that of T55A. These results support the

hypothesis that the phosphorylation at Thr-55 promotes Mdm2-mediated p53 degradation. To determine the cause of p53 degradation when phosphorylated at Thr-55, we tested whether T55A interaction with Mdm2 is different from wild-type p53 interaction. Cell lysates from p53 or T55A and Mdm2-transfected Saos-2 cells were immunoprecipitated with anti-p53 antibody (DO-1) and Western blotted with either an antiMdm2 (N-20) or an anti-p53 polyclonal antibody (FL393), respectively. As shown in Figure 5D, a significantly lower level of Mdm2 interaction was observed with T55A compared with wild-type p53. These data indicate that phosphorylation of p53 at Thr-55 leads to a higher level of interaction with Mdm2, thus promoting p53 protein degradation. To further analyze the contribution of the phosphorylation to the interaction, we tested the p53-mdm2 interaction under a TAF1 overexpression condition or in the presence of apigenin. As expected, the overexpression of TAF1 leads to a subtle increase in the interaction, whereas apigenin treatment completely abolishes the interaction (Figure 5E). These results indicate that phosphorylation on Thr-55 promotes the interaction of p53 with Mdm2 and p53 protein degradation. If this result is correct, we reasoned that introduction of the degradation-resistant mutant T55A into cells should result in an increase in G1 arrest because high p53 protein levels are known to lead to cell G1 arrest. In addition, introducing the T55A mutant in HCT116⫺/⫺ cells should not have any significant effect on the recovery of the TAF1-mediated G1 progression. We first compared the cell cycle profiles of Saos-2 cells transfected with either wild-type p53 or T55A mutant. Our results indeed revealed an increase (34%–52%) in T55A-transfected cells in the G1 phase (Figure 5G), whereas a moderate increase (from 34%–42%) in wild-type p53 cells. We again used the G1/S ratio to represent the ability of p53 to induce G1 arrest, and a higher G1/S ratio (2.32) was clearly observed with T55A as compared with wild-type p53 (1.35). Next, we introduced the T55A mutant into HCT116⫺/⫺ cells and found that it, unlike the wild-type p53, failed to show a significant effect on the recovery of the TAF1-mediated G1 progression (Figure 1B). Taken together, our data suggest a model for the regulation of p53 by TAF1, in which TAF1 phosphorylates p53 at Thr-55, leading to a subsequent degradation of p53 and cell cycle progression. Reduction of Thr-55 Phosphorylation in Response to DNA Damage The observations that TAF1 phosphorylates p53 at Thr-55 in vivo thereby affecting p53 protein stability raised the question of whether this phosphorylation is involved in the DNA-damage response, because DNA damage is known to stabilize p53. To test this, Thr-55 phosphorylation levels following DNA damage were determined using the phosphoThr-55-specific antibody, Ab202. Our results revealed a marked decrease in Thr55 phosphorylation after UV and IR irradiation in U2OS cells (Figure 6A). Because the amount of p53 increases in response to DNA damage, these experiments were performed in the presence of the proteasome inhibitor MG132. To exclude the possibility that this inhibitor may have an effect on the Thr-55 phosphorylation, CEM cells,

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Figure 5. Thr-55 Phosphorylation Leads to p53 Degradation Saos-2 cells were transfected with pcDNAp53 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 ␮M 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 ␮g of p53 reporter (pRGCE4Luc), and increasing amounts of Mdm2 and luciferase activity were assayed. (G) Saos-2 cells were transfected with 0.5 ␮g of GFP and 1.5 ␮g of pcDNA-p53 or pcDNAT55A for 42 hr. Cell cycle profile of the GFPpositive 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.

which express a high-level mutant p53 that interacts with TAF1 (Figure 6D), were exposed to UV light without the inhibitor and showed a decrease in the Thr-55 phosphorylation (Figure 6C). As a control, a corresponding increase in p53 protein levels following DNA damage in U2OS cells (Figure 6B) was observed. Thus, DNA damage leads to a decrease in p53 phosphorylation at Thr-55, which in turn alleviates Mdm2-mediated degradation of p53. Notably, the reduced phosphorylation of Thr-55 in response to DNA damage is transient, suggesting that Thr-55 phosphorylation may coordinate with other p53 modifications to stabilize p53 after DNA damage. To explore the molecular mechanism by which DNA damage leads to the decrease in p53 phosphorylation at Thr-55, we tested whether DNA damage will directly affect the interaction of p53 with TAF1. As shown in Figure 6E, under normal growth conditions, p53 is associated with TAF1. Strikingly, however, after DNA damage, p53 was dissociated from TAF1. To exclude the possibility that Thr-55 phosphorylation may have an effect on the interaction, we showed that TAF1 interacts with wildtype p53 and T55A to a similar extent both in vitro (Figure 2G) and in vivo (Supplemental Figure S1 at http://www. molecule.org/cgi/content/full/13/6/867/DC1). These results indicate that DNA damage leads to a dissociation

of p53 and TAF1 thus resulting in a reduction in Thr-55 phosphorylation. Discussion TAF1 is the largest component of the TFIID complex and appears to function as a major scaffold by which TBP and other TAFs interact in the assembly of TFIID. TAF1 plays a critical role in the regulation of cell growth in ts13 cells (Sekiguchi et al., 1991). TAF1 possesses intrinsic protein kinase activity (Dikstein et al., 1996). Despite the lack of definitive substrates, it is clear that the kinase activity is required in vivo, because a recombinant TAF1 protein that lacks the NTK domain is unable to rescue ts13 G1 arrest phenotypes (O’Brien and Tjian, 1998). In the present work, we report that TAF1 forms a complex with p53 and phosphorylates p53 at Thr-55 in vivo. This phosphorylation, unlike many other reported p53 phosphorylations, occurs under cell growth conditions and leads to p53 degradation. Thus, phosphorylation and the resultant destabilization of p53 provide a mechanism for regulation of the cell cycle by TAF1. Because the phosphorylation of p53 by TAF1 results in p53 degradation, it is not surprising that it plays a role in the TAF1-mediated G1 progression. Indeed, we

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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 ␥ 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.

show that TAF1 induces G1 progression in p53⫹/⫹ but not p53⫺/⫺ cell lines. Introducing wild-type p53 but not T55A back into ⫺/⫺ cells rescues the TAF1-induced G1 progression phenotype. With these observations in mind, we hypothesized that TAF1 promotes cells to progress through G1, at least in part, via phosphorylation of p53 at Thr-55. This hypothesis, however, could not be confirmed directly using the model hamster cell line ts13, as Thr-55 is not present in hamster p53. It remains unclear how TAF1 rescues the hamster ts13 cells at 39.5⬚C. It is possible, however, that in hamster cells an amino acid functionally equivalent to Thr-55 in human p53 may be involved in p53 protein degradation and progression through G1. Several lines of evidence have lent support to this hypothesis. Rescue of the ham-

ster ts13 cells at 39.5⬚C was less efficient when an N-terminal kinase-deficient TAF1 was expressed (O’Brien and Tjian, 1998), suggesting that the kinase activity is important for G1 arrest and apoptosis. Furthermore, we have observed a decrease in Thr-55 phosphorylation at 39.5⬚C when ts13 cells were transfected with human p53 (Supplemental Figure S2 on Molecular Cell’s website). This is in agreement with the finding that the expression of p53 target genes (e.g., p21 and GADD45) was increased at 39.5⬚C (Sekiguchi et al., 1996; O’Brien and Tjian, 2000). Finally, several viral proteins including SV40 large T antigen (Damania and Alwine, 1996) and hepatitis B virus pX (Haviv et al., 1998), which can rescue the hamster ts13 phenotypes, are known to bind and inactivate p53. In light of these data and our results, it seems likely that the kinase activity of TAF1 is involved in p53 degradation. In theory, phosphorylation and the resultant degradation of p53 could facilitate cells to progress through G1. Therefore, our data are in agreement with previous observations suggesting that TAF1 is involved in cell cycle progression and thereby provide a direct link between the kinase activity of TAF1 and cell cycle progression. One other noteworthy mechanism by which TAF1 has been implicated in p53 stability is a faulty expression of Mdm2 gene in tsBN462 cells at the nonpermissive temperature (Wasylyk and Wasylyk, 2000). Exogenous Mdm2 expression rescues the temperature-sensitive phenotype of tsBN462 cells, as shown by activation of cell cycle-regulated gene promoters. Mdm2 with point mutations that inhibit p53 binding had reduced effects on the cell cycle (Wasylyk and Wasylyk, 2000). These results support our notion that p53 may play an important role in the response to inactivation of TAF1. In the present study, we also observed a reduction on the Mdm2 protein levels under the TAF1 inhibition condition (Figure 4D). Together, these results suggest that TAF1 may function to downregulate p53 via two mechanisms: (1) by directly phosphorylating Thr-55, which leads to p53 protein degradation, and (2) by enhancing the expression of Mdm2, which results in decreased levels of p53. Since we have not observed a significant effect of TAF1 overexpression on Mdm2 expression in U2OS cells (data not shown), TAF1 can probably downregulate p53 without affecting the expression of Mdm2. Although our studies indicate that the dissociation of p53 and TAF1 leads to a reduction in Thr-55 phosphorylation in response to DNA damage, we can not exclude the possibility that Thr-55 phosphorylation is also regulated by other protein kinase(s) and phosphatase(s). In fact, Thr-55 has been implicated as a phosphorylation site for ERK2 in vitro, and this phosphorylation appears to increase with use of ERK2 immunoprecipitated from Doxorubicin-treated cells (Yeh et al., 2001). However, we failed to detect increased Thr-55 phosphorylation in Doxorubicin-treated U2OS cells using our phosphoThr-55-specific antibody (data not shown). Moreover, an increased ERK2 activity has been reported in response to UV irradiation (Li et al., 2002), while a significant decrease in Thr-55 phosphorylation after UV irradiation was observed in our experiments. Thus, it is unlikely that ERK2 contributes to the decreased Thr-55 phosphorylation in response to UV irradiation. However, the kinase activity of ERK-2 might play a role in Thr-55

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phosphorylation at later time points after UV irradiation. Different molecular pathways have been attributed to signal DNA damage and activate p53 via the phosphorylation of various amino acids within p53 (Hirao et al., 2000; Chehab et al., 2000; Shieh et al., 2000; Waterman et al., 1998; Banin et al., 1998; Canman et al., 1998; Ashcroft et al., 2000; Keller et al., 2001). Our data presented here revealed the existence of a p53 regulatory pathway that is controlled by TAF1. How p53 dissociates from TAF1 in response to DNA damage remains to be elucidated. Nevertheless, phosphorylation on Thr-55 and other residues likely cooperatively regulates the stability of p53 and thereby more effectively activates p53 in response to DNA damage. Finally, apigenin, a plant flavonoid, was reported to inhibit activity of casein kinase II (CKII) that can phosphorylate p53 at Ser-392. Therefore, the high p53 levels might be due to inhibition of CKII or even other protein kinases that phosphorylate p53 by apigenin. We do not consider that this is the case because inhibition of Ser392 phosphorylation appears after p53 stabilization, and phosphorylation by CKII is reported to activate p53 (Keller et al., 2001). Furthermore, several other p53 phosphorylation sites, the phosphorylation of which are known to stabilize p53, were not affected after apigenin treatment (Figure 4C). Interestingly, apigenin has been shown to induce p21 expression and cell G1 arrest in a p53-dependent manner (McVean et al., 2000; Figure 4C). Our results show that apigenin inhibits the kinase activity of TAF1 and subsequently stabilizes p53 (Figure 4C), suggesting a molecular mechanism by which the chemopreventive agent apigenin activates p53 and possibly providing an avenue for targeted therapy for cancer. Experimental Procedures Vaccinia Virus Construction and Protein Expression and Purification pTM-T55A was constructed by PCR amplification of T55A from the pcDNA-T55A plasmid (Gatti et al., 2000) and cloned between the NcoI and BamHI sites of the pTM.1 vector. Recombinant vaccinia virus expressing T55A was constructed by the procedure of ElroyStein et al. (1989). Vaccinia virus-expressed p53 was purified as described previously (Nie et al., 2000). To purify TAF1, Sf21 cells were infected with recombinant baculoviruses expressing HA-tagged TAF1 (a gift from Dr. R. Tjian) and TAF1 was purified with antiHA antibody, 12CA5, as described (Ruppert and Tjian, 1995). TFIID complex was purified from LTR␣3 cell nuclear extract as described (Liu et al., 1993). Recombinant GST-p53 and His-tagged RAP74 were purified with either glutathione-Sepharose (Amersham) or NiNTA agarose (Qiagen) according to the manufacturer’s protocols. Transient Transfection and Inhibition of Endogenous TAF1 by RNAi U2OS cells were transfected using LipofectAMINE with 1 ␮g TAF1expressing vector (pCMV-HAhTAF1; Ruppert and Tjian, 1995) or A2/ N7Ala-expressing vector (p-LXSN-MT-TAF1N1398 A2/N7Ala; O’Brien and Tjian, 1998) that expresses a CTK-deleted TAF1 protein with deficient NTK activity. To obtain overexpression of A2/N7Ala, U2OS cells were cotransfected with a CD4-expressing vector and transfected cells were selected using Dynabeads M-450 CD4 according to manufacturer’s protocol (Dynal). Saos-2 cells were transfected using the calcium phosphate method with 0.2 ␮g pcDNA-p53, or pcDNA-T55A, in the presence or absence of 0.5 ␮g of the Mdm2expressing vector pCHDM1B (a gift from Dr. A.J. Levine). For the luciferase assays, p53 was cotransfected with 0.5 ␮g p53 reporter (pRGCE4Luc; Nie et al., 2000) and increasing amounts of Mdm2. For inhibition of the kinase activity of TAF1, apigenin (Sigma), dis-

solved in DMSO, was added to the cell culture at a final concentration of 40 ␮M. For RNAi-mediated TAF1 reduction, two 21 nucleotide siRNA duplexes with 3⬘dTdT overhangs corresponding to TAF1 mRNA (5⬘-AAGACCCAAACAACCCCGCAT-3⬘ and 5⬘-AACTACGAC TACGCTCCACCA-3⬘) were synthesized. RNAi transfections were performed using Lipofectamine 2000 Reagent according to manufacturer’s protocol (Invitrogen). Western Blot Analysis and Immunoprecipitation Whole-cell extract was prepared by lysing the cells in a buffer containing 50 mM Tris-Cl (pH 8.0), 120 mM NaCl, 0.5% NP-40, 1 mM DTT, 2 ␮g/ml aprotinin, and 2 ␮g/ml leupeptin. Cell lysates were subjected to SDS-PAGE and then Western blotting using anti-p53 (DO-1, Santa Cruz), monoclonal (6B3, Santa Cruz), or polyclonal anti-TAF1 (Ab1230), anti-TAF5 (a gift from Dr. E. Martinez), antiMdm2 (N-20, Santa Cruz), anti-Vinculin (Sigma), and anti-Actin (Sigma) antibodies. For half-life experiments, 1 ␮g of pCEP4-p53 or T55A plasmids were transfected into Saos-2 cells in the 60 mm plates. Transfected cells were treated with 50 ␮g/ml cyclohexamide (Calbiochem) at 26 hr after transfection. Cells were lysed at indicated time points and analyzed by immunoblotting. The phosphorylation level of endogenous p53 at Thr-55 was determined by immunoprecipitation with the phosphospecific antibody for Thr-55 Ab202 (Gatti et al., 2000). The amount of p53 in the immunoprecipitates was determined by DO-1 antibody. Protein-Protein Interaction For p53 and TAF1 interaction in vivo, U2OS cell lysate was immunoprecipitated with 1 ␮g of anti-p53, anti-GST (B-14, Santa Cruz), anti-TAF1 antibody, or mouse IgG (Santa Cruz), and the resulting immunocomplexes were analyzed by immunoblotting with either anti-TAF1 or anti-TAF5 antibody. For p53 and Mdm2 interaction in vivo, 0.5 ␮g pCEP4-p53 or pCEP4-T55A was cotransfected into Saos-2 cells with 1 ␮g pCHDM1B. Cells were treated with MG132 for 4 hr before harvesting. Immunoprecipitation was performed using anti-p53 polyclonal antibody (FL-393, Santa Cruz). The amounts of Mdm2 and p53 in the immunoprecipitates were determined by Western blot analysis. For protein interaction in vitro, far-Western blotting was performed as described (Liu et al., 1993). In brief, purified TFIID complex (60 ␮l, approximately 60 ng TBP) or partially purified recombinant TBP (150 ng) was separated by SDS-PAGE, renatured, and incubated with 35S-labeled wild-type p53, ⌬N92, ⌬N160, or ⌬C292 proteins. For mapping the interaction domain on p53, 35S-labeled p53 protein was incubated with baculovirus-expressed TAF1, and immunoprecipitation was performed with anti-TAF1 antibody. For peptide competition assays, a 100-fold excess of the p53 C-terminal peptide (364-AHSSHLKSKKGQSTSRHKKLMFKTEG-389) was included in the reaction. Kinase Assay and Immunodepletion For TAF1 phosphorylation assay, 100–200 ng of bacterial-expressed GST-p53 or RAP74 was incubated with ⵑ50 ng of affinity-purified TAF1. For TFIID phosphorylation assay, 100 ng of vaccinia virusexpressed p53 (or T55A) was incubated with 1 to 2 ␮l of purified TFIID complex (approximately 10 ng of TAF1). Reactions were carried out in 15 ␮l phosphorylation buffer (25 mM HEPES [pH 7.9], 12.5 mM MgCl2, 100 mM KCl, 0.1 mM EDTA, 100 ␮M ATP, and 1 mg/ml bovine serum albumin) at 30⬚C for 30 min. Phosphorylated proteins were subjected to SDS-PAGE and autoradiography. For inhibition of the kinase activity of TAF1, apigenin was added to the reactions at a final concentration of 20 ␮M. To deplete the associated TAF1 from purified p53, 2 ␮g purified p53 was immunodepleted with 0.2 ␮g anti-TAF1 antibody in 120 ␮l incubation buffer containing 50 mM Tris-Cl (pH 8.0), 120 mM NaCl, 0.5% NP-40, 1 mM DTT, 2 ␮g/ml aprotinin, and 2 ␮g/ml leupeptin. Half of the supernatant was collected as the primary immunodepletes. The rest of the supernatant was subjected to another run of immunodepletion. The supernatant was collected as the secondary immunodepletes. “In-gel” kinase assays were performed as described (Siegert and Robbins, 1999). In brief, baculovirus-expressed TAF1 was immunopurified, separated by SDS-PAGE, and transferred to nitrocellulose

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membrane. Following Ponceau-S staining, the region of the membrane containing TAF1 was excited and subjected to a renature protocol as described (Dikstein et al., 1996). The phosphorylation reaction was carried out in the presence of 1 ␮g of p53 or T55A in 200 ␮l of phosphorylation buffer. Cell Cycle Profile and Cell Growth Analysis The amounts of plasmid used for transfection in a 100 mm plate were as follows: 1 ␮g pCMV-EGFP, 2 ␮g pcDNA-p53, 2 ␮g pcDNAT55A, 6 ␮g pCMV-HAhTAF1. Cells were harvested 42 hr after transfection, fixed in 0.4% paraformaldehyde, and resuspended in PBS plus 0.1% Tween-20 and 2% fetal bovine serum. Propidium iodide (PI) and RNase A were added to final concentrations of 50 ␮g/ml and 100 ␮g/ml, respectively, and incubated at 37⬚C for 1 hr. Cell cycle phase distribution of GFP-positive cells was determined by FACScan flow cytometry and analyzed by CellQuest software (Becton Dickinson). The cell cycle profile was created using the ModFit software (Verity). For measuring cell division and growth, HCT116 cells were transfected with either the empty CMV vector or pCMVHAhTAF1 and labeled with 5 ␮M of 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE, Molecular Probes) 5 hr prior to transfection. Distribution of parental and later cell generations was analyzed by flow cytometry at day 0, 1, 2, 3, and 4 (Lyons and Parish, 1994) and analyzed by ModFit software. Acknowledgments We are grateful to A.J. Berk, R. Tjian, and A.J. Levine for providing TAF1, RAP74, and Mdm2 expression vectors; to B. Vogelstein for providing HCT116 cell lines; to E. Martinez for providing anti-TAF5 antibody; to B. Walter and L. Owen-Shaub for assistance with FACS analysis; to T. Maile and F. Sauer for assistance with in-gel phosphorylation; and to G. Wei and G. Shouse for technical assistance. We thank R. Liu, E. Martinez, J.A. Traugh, D.S. Straus, and all members of our laboratory for many helpful discussions and valuable comments on the manuscript. We also thank an anonymous reviewer for an incisive suggestion. This work was supported by NIH grant CA75180 from the National Institute of Cancer. Received: July 29, 2003 Revised: January 30, 2004 Accepted: February 2, 2004 Published: March 25, 2004 References Appella, E., and Anderson, C.W. (2001). Post-translational modifications and activation of p53 by genotoxic stresses. Eur. J. Biochem. 268, 2764–2772. Ashcroft, M., Taya, Y., and Vousden, K.H. (2000). Stress signals utilize multiple pathways to stabilize p53. Mol. Cell. Biol. 20, 3224– 3233. Banin, S., Moyal, L., Shieh, S., Taya, Y., Anderson, C.W., Chessa, L., Smorodinsky, N.I., Prives, C., Reiss, Y., Shiloh, Y., et al. (1998). Enhanced phosphorylation of p53 by ATM in response to DNA damage. Science 281, 1674–1677. Bullock, A.N., and Fersht, A.R. (2001). Rescuing the function of mutant p53. Nat. Rev. Cancer 1, 68–76. Bunz, F., Dutriaux, A., Lengauer, C., Waldman, T., Zhou, S., Brown, J.P., Sedivy, J.M., Kinzler, K.W., and Vogelstein, B. (1998). Requirement for p53 and p21 to sustain G2 arrest after DNA damage. Science 282, 1497–1501. Canman, C.E., Lim, D.S., Cimprich, K.A., Taya, Y., Tamai, K., Sakaguchi, K., Appella, E., Kastan, M.B., and Siliciano, J.D. (1998). Activation of the ATM kinase by ionizing radiation and phosphorylation of p53. Science 281, 1677–1679. Chehab, N.H., Malikzay, A., Appel, M., and Halazonetis, T.D. (2000). Chk2/hCds1 functions as a DNA damage checkpoint in G(1) by stabilizing p53. Genes Dev. 14, 278–288. Critchfield, J.W., Coligan, J.E., Folks, T.M., and Butera, S.T. (1997). Casein kinase II is a selective target of HIV-1 transcriptional inhibitors. Proc. Natl. Acad. Sci. USA 94, 6110–6115.

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