Association of p300 and CBP with Simian Virus 40 Large T Antigen

Transcription

1 MOLECULAR AND CELLULAR BIOLOGY, July 1996, p Vol. 16, No /96/$ Copyright 1996, American Society for Microbiology Association of p300 and CBP with Simian Virus 40 Large T Antigen RICHARD ECKNER, JOHN W. LUDLOW, NANCY L. LILL, ELIZABETH OLDREAD, ZOLTAN ARANY, NAZANINE MODJTAHEDI, JAMES A. DECAPRIO, DAVID M. LIVINGSTON,* AND JEFFERY A. MORGAN Dana-Farber Cancer Institute and Harvard Medical School, Boston, Massachusetts Received 29 November 1995/Returned for modification 19 January 1996/Accepted 5 April 1996 p300 and the CREB-binding protein CBP are two large nuclear phosphoproteins that are structurally highly related. Both function, in part, as transcriptional adapters and are targeted by the adenovirus E1A oncoprotein. We show here that p300 and CBP interact with another transforming protein, the simian virus 40 large T antigen (T). This interaction depends on the integrity of a region of T which is critical for its transforming and mitogenic properties and includes its LXCXE Rb-binding motif. T interferes with normal p300 and CBP function on at least two different levels. The presence of T alters the phosphorylation states of both proteins and inhibits their transcriptional activities on certain promoters. Although E1A and T show little sequence similarity, they interact with the same domain of p300 and CBP, suggesting that this region exhibits considerable flexibility in accommodating diverse protein ligands. Small DNA tumor viruses such as simian virus 40 (SV40) and polyomavirus rely on host cell proteins for the replication of their genomes. Since these viruses normally infect resting or differentiated cells, they have evolved means of stimulating host cell DNA synthesis. In the case of SV40, large T antigen (T) is the only viral protein required for this task (reviewed in references 39 and 46). The stimulation of host cell DNA replication depends on the ability of T to interact physically with a set of cellular proteins negatively regulating S-phase entry. The binding of T to these proteins appears to reduce their potential for preventing DNA synthesis and cell growth. Of these cellular targets, the tumor suppressor protein p53 and the retinoblastoma (Rb) family of proteins, comprising the tumor suppressor protein Rb itself, p107, and p130, play key roles in controlling the cell cycle. The latter three proteins interact with an amino-terminal domain of T antigen which encompasses amino acid residues 102 to 114. Included in this region is a motif, LXCXE, that also exists in several analogous viral proteins which perform functions similar to those of T, such as polyomavirus large T antigen, adenovirus E1A, and papillomavirus E7. These viral polypeptides can disrupt complexes between Rb family members and the E2F family of transcription factors (44). Once released from negative regulation by Rb and related proteins, E2F members can activate a diverse set of genes, ultimately leading to cellular DNA synthesis and an intracellular environment permissive for full replication of the viral genome. Extensive analyses of T mutants revealed the existence of two additional regions which contribute to its mitogenic function (12, 40, 41, 43, 47, 49, 50, 62). The first is located in the N-terminal 82 residues and has been postulated to bind p300. This proposal was mainly based on the results of transformation assays in which a T mutant unable to bind to Rb family * Corresponding author. Phone: (617) Fax: (617) Present address: Division of Tumor Biology, University of Rochester Cancer Center, Rochester, NY Present address: Institut Gustave Roussy, Villejuif, France. Present address: Division of Hematology/Oncology, Brigham and Women s Hospital, Boston, MA members complemented in trans an E1A protein defective for interaction with p300 (57). Deletion of T residues 17 to 27 abolished complementation in this assay. The second region has been localized to a discrete segment of T which binds DNA and the transcription factor TEF1 (12, 20). T molecules bearing mutations in the first 82 amino-terminal residues often display several defects. In addition to their impaired mitogenic potential, they are also defective in transforming cultured cells (40, 49, 62), despite their residual ability to interact with Rb family members and with p53. In transgenic mice, such a T mutant induced tumors in a subset of tissues in which it was expressed and it did so at a reduced frequency compared with wild-type (wt) T (50). Several cellular proteins have been reported to bind to the N terminus of T. One of these proteins has a molecular mass of 185 kda (34). This protein may be related to the insulin receptor substrate 1 protein, which also has a molecular mass of 185 kda and requires sequences in the N-terminal half of T for binding (60). Because insulin receptor substrate 1 protein is cytoplasmic, it can bind only to the small fraction of T which is cytosolic (48). A nuclear target of the N terminus of T may be the TATA-binding protein TBP (20). p300 is a nuclear phosphoprotein, first described as one of several cellular proteins coprecipitating with E1A (27, 59). The binding of p300 to E1A is essential for the oncogenic and growth-stimulatory activities of E1A and for its ability to repress several tissue-specific, transcriptional control elements (reviewed in references 6 and 45). Molecular cloning of p300 revealed it to contain two signature motifs for transcriptional coactivator proteins, namely, a bromodomain and a region with homology to the yeast ADA-2 transcriptional adapter protein (14, 15). The presence of these two motifs suggested a similar role for p300. Indeed, when fused to a DNA-binding domain, p300 efficiently stimulated transcription (1). p300 displays striking homology (2) to another large nuclear protein, CBP, which was cloned as part of a search for proteins capable of binding to the transcription factor CREB after its phosphorylation by protein kinase A (9, 35). Most notably, both p300 and CBP share the two transcriptional adapter motifs noted above as well as three discrete regions rich in cysteine and histidine residues, the third of which mediates the 3454

2 VOL. 16, 1996 p300 AND CBP INTERACT WITH SV40 LARGE T ANTIGEN 3455 binding of p300 and CBP to E1A (15). CBP also binds to E1A and requires for this interaction the same regions of E1A as does p300. The binding of E1A to p300 and CBP inhibited the transcriptional activation function of both proteins on some promoters (1, 38). We have investigated whether p300 and CBP interact in vivo with SV40 T. Using antibodies directed against either T, p300, or CBP, we have detected and characterized complexes containing these proteins. A mutation removing the Rb-binding motif of T strongly affected its ability to bind to p300 and CBP. Unlike wt T, this mutant also failed to alter the phosphorylation state or to inhibit the transcriptional activity of either target protein. These results support the view that p300 and CBP are targets of multiple oncogenic events, leading to deregulated cell growth. MATERIALS AND METHODS Cell culture. Mouse embryo fibroblast (MEF) cells stably expressing wt, K1, PVU-1, or T50L7 T (7, 30, 51) were grown in Dulbecco s modified Eagle s medium containing 10% iron-supplemented calf serum. Two different cell lines synthesizing wt T were used. The first, PVU-0, expresses small t and large T. The second, dl2005 (51), synthesizes only large T. No differences between these two cell lines were found for the binding of p300 or CBP to T or the phosphorylation state of p300 or CBP. The spontaneously transformed B6S c17 (referred to subsequently as B6) cell line (51) and U-2 OS human osteosarcoma cells were cultured in Dulbecco s modified Eagle s medium plus 10% Fetalclone I serum (HyClone). For the determination of growth rates and saturation densities, 10 5 MEF cells were seeded on eight duplicate 60-mm-diameter dishes. Every 24 h, cells on duplicate dishes were trypsinized and counted with a hemocytometer. The culture medium was changed daily. Immunoprecipitations and Western blotting (immunoblotting). To assay for the presence of p300 and CBP in anti-t immunoprecipitates, MEF cells were labeled at 80% confluency with 1 mci of [ 35 S]methionine per 10-cm-diameter dish for 4 h. Cells were lysed in 1 ml of EBC-150 buffer (50 mm Tris-HCl [ph 8.0], 150 mm NaCl, 0.5% Nonidet P-40, 10 g each of aprotinin and leupeptin per ml, 1 mm phenylmethylsulfonyl fluoride, 50 mm NaF, 10 mm Na 2 - -glycerophosphate) for 30 min, cell debris was removed by centrifugation at 14,000 g, and 90% of the clarified extract from a 10-cm-diameter dish was incubated for 1hat4 C with a T monoclonal antibody (24). PAb419 was used for most experiments, but PAb416 and PAb423 worked as well. The remaining 10% of the lysate was mixed with an extract prepared from unlabeled 293 cells and subsequently incubated with the E1A monoclonal antibody M73 (25). Immune complexes were collected by adding 30 l (packed volume) of protein A-Sepharose beads and rocking samples for 30 min, with three subsequent washes with NET-N (20 mm Tris-HCl [ph 7.5], 100 mm NaCl, 1 mm EDTA, 0.5% Nonidet P-40). Proteolytic mapping experiments were performed as previously described (10) with Staphylococcus aureus V8 protease (Boehringer Mannheim). For reprecipitations, sodium dodecyl sulfate (SDS) and dithiothreitol were added to the washed beads to give a final concentration of 1% and 5 mm, respectively. Samples were boiled for 5 min, and the supernatant ( 50 l) was transferred to a new tube and diluted with 1 ml of cold NET-N. Samples were then incubated overnight with the second antibody. To probe for the presence of T in p300 and CBP immunoprecipitates, cells were labeled and lysed as described above. Typically, the extract was then divided into three parts; 45% of it was precipitated with AC 238, 45% was precipitated with a control antibody raised against rat myogenin, F5D (56) (gift of W. Wright), and the remaining 10% was precipitated with an excess of PAb419. Incubations with the primary antibodies were carried outfor1hat4 C and were followed by a 30-min incubation with a rabbit anti-mouse immunoglobulin G secondary antibody. Fifteen microliters of packed protein A-Sepharose beads was added to capture immune complexes. Beads were washed five times with NET-N containing 250 mm NaCl, except in the case of immunoprecipitations performed with extracts from T50L7 cells. In the latter case, four washes with standard NET-N were performed. Washing with NET-N containing higher salt concentrations did not lead to the reproducible recovery of T50L7 in AC 238-containing immune complexes. The treatment of p300 and CBP with phosphatase (New England Biolabs) was carried out on purified immune complexes bound to protein A-Sepharose by using 400 U of phosphatase per sample. The reaction conditions were those recommended by the manufacturer. For Western blots, proteins separated on SDS-protein gels were transferred to BA85 membranes (Schleicher & Schuell) by standard procedures (26). Membranes were either blocked in 5% bovine serum albumin in Tris-buffered saline for nitroblue tetrazolium-bcip (5-bromo-4-chloro-3-indolylphosphate toluidinium) colorimetric detection or in 5% nonfat dry milk powder in Tris-buffered saline for detection with enhanced chemiluminescence reagents (Amersham). The presence of T was monitored with PAb419. GST binding assays. Glutathione S-transferase (GST) fusion proteins were isolated from 10-ml bacterial cultures as previously described (29). Cell lysates from MEF cells expressing wt T or a mutant were prepared with EBC-150 buffer as outlined above. Of each lysate, 4/10 was incubated (1 h at 4 C) with glutathione beads loaded with one of the GST proteins (see Fig. 6). Beads were washed four times with EBC-150 buffer and resuspended in SDS-sample buffer, and the eluted proteins were separated on SDS-protein gels. The binding of T to various GST proteins was analyzed by Western blotting with PAb419. GST protein production was monitored by separating duplicate GST protein sample preparations on SDS-protein gels with subsequent Coomassie blue staining. Sucrose gradient sedimentation. Cells on 10-cm-diameter dishes were extracted with 400 l of EBC-120 buffer. Two hundred microliters of the lysate was layered on top of a sucrose gradient (5 to 20%) which was prepared in EBC-120 buffer. Sedimentation was for 4hat48,000 rpm at 4 C in a Beckman SW50.1 rotor. Gradient fractions ( 180 l) were collected from the top. Even-numbered fractions were immunoprecipitated with PAb419. Molecular size markers were sedimented on parallel gradients and consisted of ovalbumin (3.6S), catalase (11.2S), apoferritin (17.6S), and thyroglobulin (26.5S). Monoclonal antibodies of the AC series. Mice were injected four times with 50 g of GST-CBP (residues 720 to 1676) protein [GST-CBP ( )]. Serum samples from immunized animals were checked for antibody titers by assaying the efficiency with which they immunoprecipitated in vitro-translated CBP protein. In vitro translation reactions were performed with the TNT system (Promega). Spleen cells from the mouse displaying the best response were fused to NS-1 cells (26). Positive hybridoma clones were identified by testing the ability of the hybridoma supernatants to immunoprecipitate in vitro-translated CBP. The epitopes recognized by the antibodies were mapped by using a series of CBP in vitro translation products containing progressive carboxy-terminal deletions. AC 26, AC 235, AC 238 and AC 240 all recognize an epitope located between mouse CBP amino acid residues 781 and 909. The epitope recognized by AC 217 is localized between CBP residues 1098 and Plasmids. In vitro-translated CBP was generated from pbluescript mcbp, which contains an 8-kb mouse CBP cdna fragment (35) (gift of R. Goodman) inserted into the BamHI site of pbluescript SK (Stratagene). pgem-p53 (gift of M. Oren) served as the template for in vitro transcription and translation of murine p53. Both CBP and p53 were transcribed by T7 polymerase. To produce GST fusion proteins of the third CH-rich region of p300, two p300 fragments were cloned into pgex2tk (Pharmacia). The first fragment was used to generate GST-p300 ( ) and comprised nucleotides 6326 to 6942, and the second fragment gave rise to GST-p300 ( ) and contained p300 nucleotides 6653 to The numbers in parentheses refer to the amino acid residues of p300 fused to the GST moiety. The GST-CBP ( ) fusion protein (see Fig. 6) has been previously described (1). The GST-CBP ( ) protein used to immunize mice encompassed nucleotides 2160 to 5037 of murine CBP. To facilitate fusion of the bovine papillomavirus E2 protein DNA-binding domain to CBP, a KpnI site was first introduced by site-directed mutagenesis near the 3 end of the CBP cdna. The mutagenesis primer had the sequence 5 -CCACGG GAGACACACTAGAAATGGTACCAAGTTTGTGGAGGGTTTGTAG-3, leading to the insertion of a KpnI site just after the codon encoding residue 2336 of CBP. A KpnI-HindIII fragment encompassing the E2 DNA-binding region was ligated to the newly generated KpnI site in the CBP cdna, and the fusion construct was transferred to the CMV expression vector (15). The p300-e2 plasmids used in this study have been described previously (1). Transient-transfection assays. U-2 OS cells were transfected overnight by the CaPO 4 method (3). To measure the transcriptional activities of E2 fusion proteins, 5 g of 6xE2-tk-CAT and 1 g of CMV-lacZ (internal standard) along with either 3 g of p300-e2 or 8 g of CBP-E2 were cotransfected into U-2 OS cells. Six micrograms of psg5-t, psg5-k1, psg5-t50l7, or psg5 (vector) was added to the transfection mixture to determine the effect of wt or mutant T on p300 and CBP transactivation activities. The best repression was observed when U-2 OS cells growing exponentially were harvested. As indicated, 2 g of CMV-12S E1A was included in the transfection mixture. Chloramphenicol acetyltransferase assays were performed as previously described (3), and the conversion of chloramphenicol to its acetylated forms was quantitated with a Betascope. RESULTS T antigen antibodies coprecipitate a 300 kda protein. MEF cell lines expressing three different SV40 T species were analyzed in an immunoprecipitation experiment. These three MEF cell lines synthesize wt T and two T mutants, K1 and T50L7. Both K1 and T50L7 fail to bind Rb and its family members (Fig. 1A). Cells were labeled with [ 35 S]methionine, and extracts were immunoprecipitated with a monoclonal antibody (PAb419) directed against T. In parallel reactions, a 1/10 volume of each extract was first mixed with a lysate from unlabeled 293 cells (containing E1A) and subsequently immunoprecipitated with an E1A antibody (M73) to monitor the presence and migration

3 3456 ECKNER ET AL. MOL. CELL. BIOL. FIG. 1. A T antigen antibody coprecipitates a p300-related protein. (A) Schematic diagrams of the four T species used in this study. The site of interaction of Rb family members with T is represented as a solid box, whereas the two segments required for p53 binding (31) are displayed as crosshatched boxes. The K1 mutant has a lysine instead of a glutamic acid residue at position 107; in the PVU-1 mutant (Pvu 1), residues 106 to 113 are replaced by a tyrosine residue; and in T50L7, amino acids 98 to 126 are replaced by the residues Arg-Asn-Ser-Ala. (B) Protein gel displaying immunoprecipitates from K1-, T50L7-, and wt-t-expressing cells. 35 S-labeled extracts from these three different MEF cell lines were immunoprecipitated with the indicated antibodies. In lanes 1, 4, and 7, 90% of each extract was precipitated with the T antibody PAb419. The remaining 10% was first mixed with a 293 cell lysate and subsequently precipitated with the E1A antibody M73 (M73/293; lanes 2, 5, and 8). Lanes 3, 6, and 9, control precipitates with M73 [ctrl (M73)], for which lysates, each from a complete dish, were employed in the absence of added E1A. The positions of p300 and the 180-kDa prestained protein marker are shown on the left. (C) Protease V8 mapping to compare the proteolytic products of the E1A-associated 300-kDa doublet (E1A/300kD; lanes 1 to 3) with those of the 300-kDa doublet coprecipitated by PAb419 (T/300kD; lanes 4 to 6). position of labeled p300 protein from these three MEF cell lines. Figure 1B shows than an 300-kDa protein doublet was immunoprecipitated with PAb419 from both K1- and wt-texpressing cells (lanes 1 and 7). In both cases, this doublet comigrated with a 300-kDa doublet coprecipitated by the E1A antibody from the mixed MEF-293 lysates (Fig. 1B, lanes 2 and 8). In contrast, no protein in the 300-kDa range was apparent in anti-t precipitates of a T50L7 cell extract and the 300-kDa protein coprecipitating with E1A migrated more slowly than did the doublet observed in the wt-t immunoprecipitate (Fig. 1B, lanes 4 and 5). Control precipitations performed with M73 on MEF lysates not mixed with 293 extracts did not reveal detectable 300-kDa bands (Fig. 1B, lanes 3, 6, and 9). V8 mapping was performed to investigate the degree of identity of the two doublets immunoprecipitated with either PAb419 or M73 (after mixing with unlabeled 293 cell lysate) from wt-t-containing cell extracts. Both bands of the doublet were excised in one slice and partially digested with three different quantities of V8 protease. The resulting digestion patterns of the two sets of doublets were nearly identical (Fig. 1C), suggesting that E1A and T bind to the same 300-kDa protein(s). Other T antibodies, such as PAb416 and PAb423, also precipitated a 300-kDa doublet from wt-t-containing extracts (data not shown). From the results of several experiments, we estimate that the recovery of the 300-kDa protein doublet from T immunoprecipitates is 10- to 20-fold less efficient than that from E1A immunoprecipitates. Both p300 and CBP interact with T. The identity of the protein doublet coprecipitated with PAb419 was further investigated in reimmunoprecipitation experiments. We have previously described the RW series of monoclonal antibodies raised against p300 (15). A new set of monoclonal antibodies (termed the AC series for anti CBP) was generated by using a fragment of CBP as the immunogen. Both sets of antibodies were analyzed for reactivity with p300 and CBP (see below). Immunoprecipitates of lysates prepared from MEF cells expressing wt or K1 T were boiled in SDS-containing buffer, and the released proteins were exposed to a control antibody or to three different p300 and/or CBP antibodies. Only the lower band of the doublet was reprecipitated by a p300-specific antibody, whereas the upper band alone was brought down with a CBP-specific antibody (Fig. 2, lanes 2 and 6 and lanes 4 and 8, respectively). An antibody reacting with both p300 and CBP again yielded a doublet (Fig. 2, lanes 3 and 7). No signal was detected with the control antibody. These data indicate that T binds both p300 and CBP. The abilities of these two sets of monoclonal antibodies to react with p300 and CBP were further examined (Fig. 3A). In vitro translates of full-length p300 and CBP were immunoprecipitated with various antibodies. Most antibodies recognized both p300 and CBP. Antibodies AC 235 and AC 238 preferentially bound CBP, and RW 128 preferentially bound p300. All antibodies of the AC series efficiently coprecipitated E1A from 293 cell extracts (Fig. 3B). Notably, the longest visible CBP in vitro translation product migrated like an 180-kDa protein. This was faster than would

4 VOL. 16, 1996 p300 AND CBP INTERACT WITH SV40 LARGE T ANTIGEN 3457 FIG. 2. Both p300 and CBP can be recovered in T-containing immunoprecipitates. Immunoprecipitates (i.p.) from wt-t- (lanes 1 to 4) and K1 T (lanes 5 to 8)-containing cell lysates were boiled and reprecipitated (re - i.p.) with a control antibody (F5D; anti-myogenin) or with antibodies reacting specifically with either p300 (RW 128) or CBP (AC 238) only or both (p300/cbp; RW 105). RW 105 exhibits a higher affinity for p300 than for CBP, explaining the more intense p300 signal compared with that of CBP. be expected from knowledge of the molecular weight of CBP and the migration of the longest p300 translation product. This shortened CBP species appeared reproducibly after in vitro transcription and translation of more than one CBP cdna clone. On the other hand, after prolonged exposures, a faint CBP species comigrating with the longest p300 in vitro translation product was detected. The reason for failure to generate full-length CBP efficiently is not known. A CBP antibody coprecipitates T. The monoclonal antibodies characterized above were tested for the ability to coprecipitate T. Of the 10 antibodies investigated, only one, AC 238, efficiently coprecipitated T (data not shown). AC 238 was subsequently used for more detailed experiments. Extracts of MEF cells expressing either wt T or a mutant species, K1, PVU-1, or T50L7, were subjected to immunoprecipitation with three different antibodies. The spontaneously transformed MEF cell clone, B6, which does not synthesize T was used as a negative control. In these experiments, lysates were split into three parts; 45% of each extract was precipitated with AC 238, 45% was exposed to a control antibody of the same isotype as AC 238, and the remaining 10% was incubated with an excess of the T antibody PAb419 to obtain an estimate of the T protein levels in extracts. Figure 4A shows a Western blot of such immunoprecipitates. The CBP-specific antibody AC 238 coprecipitated approximately 10 to 20% of the available wt T (compare lanes 4 and 5), whereas only 1% of the two mutants K1 and PVU-1 was recovered with the same antibody (lanes 7, 8, 10, and 11). The T protein levels in all three MEF cell lines were comparable, and, as expected, no T signal was detected in the control lanes or in any of the precipitates of B6 cell extracts. The Western blot in Fig. 4A was developed with color development reagents. With this technique, no T50L7 T signal was observed when the relevant lysate was incubated with AC 238. However, when a more sensitive development technique based on chemiluminescence was used, a small amount of T50L7 protein was detected in an AC 238 immunoprecipitate (Fig. 4B, lane 5). On the basis of several experiments of this type, we estimated that AC 238 coprecipitated less than 0.25% of the total cellular T50L7 protein pool. Since only one of the p300 and CBP monoclonal antibodies, AC 238, coprecipitated T, it was necessary to determine whether AC 238 reacted directly either with T itself or with Downloaded from on January 14, 2019 by guest FIG. 3. Specificities of monoclonal antibodies against p300 and CBP. (A) In vitro translation products (IVT) encoded by full-length murine CBP cdna or full-length human p300 cdna were incubated with the antibodies indicated. Lanes 1 and 8, 10% of the amounts of CBP and p300 translates (input), respectively, employed for immunoprecipitation reactions. The control antibody used in lanes 7 and 14 was F5D (anti-myogenin). The positions of molecular mass standards (in kilodaltons) are marked on the left. (B) Western blot to monitor the presence of E1A in CBP immunoprecipitates from 293 cells. Lysates prepared from 293 cells were incubated either with a CBP antibody (lanes 2, 3, and 5 to 7), with the E1A antibody M73 (lane 1), or with an irrelevant antibody (control; lane 4). The membrane containing the transferred immunoprecipitates was incubated with M73. Ig H marks the migration position of the immunoglobulin heavy chain.

5 3458 ECKNER ET AL. MOL. CELL. BIOL. FIG. 4. A CBP antibody coprecipitates T. (A) Extracts from cell lines expressing wt T and the K1 and PVU-1 (Pvu 1) mutants and from the spontaneously transformed B6 cell line were immunoprecipitated with AC 238 for CBP, PAb419 for T antigen (T ag), and F5D as a control. Forty-five percent each of extracts was used for precipitation with either AC 238 or F5D, and 10% was used for immunoprecipitation with an excess of PAb419 to monitor T protein levels. Immunoprecipitates were separated on protein gels and transferred to a membrane which was probed with PAb419. The blot was developed with color development reagents. (B) Western blot showing inefficient coprecipitation of the T50L7 mutant. The experimental details were the same as described for panel A, except that only 2.5% of the total extract was subjected to immunoprecipitation with the T antibody PAb419. The blot was developed with enhanced chemiluminescence reagents. ctrl, control antibody (F5D) precipitations. The positions of the T ag and immunoglobulin heavy chain (Ig H) are shown on the right. (C) The CBP antibody AC 238 does not react with T or p53. In vitro-translated (IVT) T (lanes 3 to 5), CBP (lanes 6 to 8), and p53 (lanes 10 to 12) were incubated with AC 238, an irrelevant antibody (F5D) serving as a negative control, or an antibody recognizing the respective in vitro translation products. Lanes 1, 2, and 9, inputs of the in vitro translates. i.p., immunoprecipitation. p53, the major cellular protein binding to all of the T species studied. AC 238 did not precipitate in vitro-translated T or murine p53 (Fig. 4C, lanes 3 and 12, respectively), but it did efficiently recognize in vitro-translated CBP (Fig. 4C, lane 6). Thus, the recovery of T in AC 238 immunoprecipitates does not appear to be due to unexpected cross-reactivity between this antibody and either T or p53. Taken together, these results suggest that the region of T encompassing the Rb-binding motif contributes to the interaction of T with p300 and CBP. This was most obvious in the case of the T50L7 mutant, which lacks residues 98 to 126 and barely bound to CBP. Compared with wt T, the recovery of the K1 mutant, which like T50L7 is defective in Rb binding, was less efficient when exposed to AC 238. In contrast, the T antibody PAb419 coprecipitated p300 and CBP from wt and K1 T- expressing cells with comparable, albeit relatively low, efficiency (see Discussion). p300 and CBP exist in a ternary complex with T and p S-labeled cellular extracts derived from T-expressing MEF cells were sedimented through sucrose gradients to learn whether p300 and/or CBP exist in a stable complex with T, p53, and/or other associated proteins. After sedimentation, gradient fractions were immunoprecipitated with PAb419. Figure 5A illustrates that the bottom fractions 22 to 26 contained p53, T, p107, and p300 or CBP. To clarify whether all four of these proteins exist in one complex, immunoprecipitation with AC 238 was performed with extracts from MEF cells. As can be seen in Fig. 5B, AC 238 coprecipitated several proteins from lysates of wt-t-expressing cells, two of which comigrated with T and p53, respectively (lanes 4 to 6). The protein comigrating with p53 in the CBP immunoprecipitate (Fig. 5B, lane 6) was indeed p53, since it was reimmunoprecipitated by a p53-specific antibody (lane 7). Moreover, neither T nor p53 coprecipitated with CBP when lysates of B6 cells (lacking T) were used (Fig. 5B, lanes 1 to 3). No p107 was detected in these AC 238 immunoprecipitates. We conclude that in T-containing cells, CBP exists in a ternary complex with T antigen and p53. The third cysteine/histidine-rich region of p300 and CBP interacts with T. To map the region of p300 and CBP directing complex formation with T, various segments of p300 or CBP were fused to GST (Fig. 6C) and the recombinant proteins were incubated with extracts containing different T species. As shown in the Western blot (Fig. 6A), wt, K1, and PVU-1 T bound almost equally to GST-p300 ( ), which encompasses the third CH-rich region of p300, but not at all to GST-p300 ( ), which lacks most of the third CH-rich region (lanes 1 to 6). One-fifth of the input extract was monitored for T protein levels, which were found to be comparable (Fig. 6A, lanes 7 to 9). The analogous region of CBP was similarly capable of interacting with both wt T and the K1 mutant, whereas GST by itself did not bind to these two T versions (Fig. 6B, lanes 3 to 6). Relative to wt T, only a small proportion of the T50L7 mutant was recovered with the p300- GST and CBP-GST fusion proteins (data not shown). These results suggest that T, similar to E1A, binds to the third CHrich region of p300 and CBP. SV40 T influences the phosphorylation state of p300. After long periods of electrophoresis, p300 isolated from cells synthesizing wt or K1 T migrated faster than did p300 from cells not containing T (Fig. 7A; compare lanes 1 and 7 with lane 4). In contrast, p300 isolated from cells containing the T50L7 mutant displayed a more retarded mobility than did p300 from wt-t- or K1-expressing cells (Fig. 7B; compare lane 5 with lanes 1 and 9). In fact, p300 from T50L7 cells comigrated with p300 from cells devoid of any T (data not shown). To determine whether these variations in p300 migration were due to differential phosphorylation, p300 and CBP immunoprecipitates were treated with phosphatase. As shown in Fig. 7B, the

6 VOL. 16, 1996 p300 AND CBP INTERACT WITH SV40 LARGE T ANTIGEN 3459 FIG. 5. CBP exists in a ternary complex with T antigen and p53. (A) A sucrose gradient containing 5 to 20% sucrose was loaded with a 35 S-labeled extract of MEF cells expressing wt T. After a 4-h run, gradient fractions were collected and precipitated with PAb419. The migration positions of sedimentation standards are given above the lanes and those of T antigen (T ag) and associated proteins are indicated on the right. The positions of molecular mass markers (in kilodaltons) are shown on the left. (B) p53 coprecipitates with CBP in T-containing cells. 35 S-labeled lysates prepared from cells either devoid of T (B6) or expressing wt T were immunoprecipitated with the indicated antibodies (lanes 1 to 6). The antibodies were AC 238 for CBP, PAb419 for T ag, and PAb421 for p53. Lanes 3 and 6, 5-h exposure; other lanes, 24-h exposure. In lane 5, CBP and p300 are visible as a protein doublet coprecipitating with T. In lanes 7 to 9, extracts of wt-t-containing MEF cells were first immunoprecipitated with antibodies against CBP (AC 238), T ag (PAb419), or an irrelevant protein (control; F5D). These three precipitates were boiled and reprecipitated (re-i.p.) with the p53 antibody ( -p53) PAb421. The positions of CBP, T ag, and p53 are marked. treatment of p300 from wt-t-, T50L7-, or K1-synthesizing cells led to the collapse of the broad p300 band, resulting in the appearance of a focused p300 band that comigrated in all three cases (compare lanes 1, 5, and 9 with lanes 3, 7, and 11). Hence, differential phosphorylation is responsible for differential migration. The same phenomenon was observed for CBP, although the differences in migration were less pronounced. The results presented in Fig. 7B provide an explanation for the appearance of a 300-kDa doublet in immunoprecipitates of wt-t- and K1 T-containing extracts and that of a single, more slowly migrating 300-kDa band in precipitates of T50L7-containing lysates (Fig. 1B). Unlike in cells synthesizing wt or K1 T, there is no interference with the phosphorylation of p300 and CBP in cells expressing T50L7 or lacking T. Accordingly, when extracted from these cells, p300 and CBP comigrate on protein gels. It has been reported that E1A inhibits the phosphorylation of p300 in vitro (4). To investigate whether E1A can interfere with p300 phosphorylation as does T, E1A-associated 300-kDa proteins, as well as p300 and CBP, were immunoprecipitated from 35 S-labeled 293 cell extracts with specific antibodies. The migrations of these proteins were compared with that of p300 and CBP isolated from human U-2 OS cells. Figure 7C shows that the E1A-associated 300-kDa proteins as well as p300 and CBP, all isolated from 293 cells, virtually comigrated with each other (lanes 1 to 3). In U-2 OS cells, the majority of p300 migrated faster than did CBP (lanes 7 and 8); it also migrated faster than did p300 from 293 cells. Phosphatase treatment of E1A-bound 300-kDa proteins resulted in a visible downshift of the initial band and led to the appearance of a doublet, with the bottom band comigrating with phosphatase-treated p300 and the top band comigrating with dephosphorylated CBP (Fig. 7C, lanes 4 to 6). In U-2 OS cells, p300 and CBP protein bands underwent a less dramatic shift in mobility and became more focused upon incubation with phosphatase (Fig. 7C, lanes 9 and 10). We conclude that p300 from 293 cells is hyperphosphorylated relative to that from U-2 OS cells and, therefore, that E1A does not appear to markedly inhibit p300 and CBP phosphorylation in vivo. In addition, the observation that E1A-associated 300-kDa proteins resolved in just two discrete species after phosphatase treatment suggests that p300 and CBP are the only two components of the 300-kDa band in E1A immunoprecipitates. T represses the transcriptional activities of p300 and CBP. Transient transfections into U-2 OS cells were performed to investigate the influence of T on the transcriptional activation potential of p300 and CBP. For these assays, both proteins were fused to the DNA-binding domain of the bovine papillomavirus E2 protein. The transactivation function of the fusion proteins was measured with a chloramphenicol acetyltransferase reporter construct containing six E2 binding sites upstream of a thymidine kinase promoter. wt T and the K1 mutant repressed the transcriptional activities of p300-e2 and CBP-E2 as efficiently as E1A did (Fig. 8A). Efficient repression depended on the integrity of the third CH-rich region of p300 since p300-e2 fusion proteins lacking part of this domain (p300

7 3460 ECKNER ET AL. MOL. CELL. BIOL. FIG. 6. The third CH-rich region of p300 and CBP mediates binding to T. (A) Western blot showing the binding of three T species to GST-p300 ( ) but not to GST-p300 ( ). The numbers in parentheses indicate the p300 residues fused to the GST moiety. MEF cell extracts (4/10 each) containing the T version indicated were incubated with either GST-p300 ( ) (lanes 1 to 3) or GST-p300 ( ) (lanes 4 to 6). The remaining 2/10 were immunoprecipitated with an excess of PAb419 to monitor T protein levels (lanes 7 to 9). The Western blot was developed with PAb419. The positions of T and immunoglobulin heavy chain (Ig H) are shown on the left and right, respectively. (B) The same experimental procedure as described for panel A was used to assess the binding of GST-CBP to wt or K1 T. The GST-CBP protein contains residues 1746 to The position of T is shown on the left. (C) Schematic diagrams of the three GST fusion proteins employed in panels A and B. Downloaded from del 33-E2 or p300 del 47-E2) were only marginally repressed, as was a VP16-E2 fusion protein (Fig. 8C and B, respectively). Interestingly, as described previously (1), p300 del 47-E2 was actually activated by E1A rather than inhibited. These results indicate that a physical interaction between T and p300 and/or CBP is important for repression. This view is supported by the results for the T50L7 mutant, which elicited only low-level repression of p300-e2 and no repression of CBP-E2 (Fig. 8D). In these experiments, comparable levels of wt T, K1, and T50L7 proteins were expressed (Fig. 8E). Reduced growth rate and saturation density of cells expressing T50L7. Immortalized MEF cells expressing comparable amounts of wt, K1, and T50L7 T were seeded on 60-mmdiameter dishes to determine their growth rates and saturation densities in medium containing 10% calf serum. Cells from duplicate dishes were trypsinized and counted daily over 8 days. MEF cells harboring wt T sloughed and clumped after 6 days and could no longer be counted. Figure 9 illustrates that wt-t- and K1 T-expressing cells exhibited comparable growth rates and saturation densities. In contrast, cells containing the T50L7 mutant grew markedly more slowly and attained a saturation density of only cells per dish relative to the cells observed with wt or K1 T. A similar growth disadvantage was seen with two other T50L7 MEF clones, and the differences in growth rates were even more pronounced when the three cell lines were grown in 1% calf serum (data not shown). Thus, a correlation exists between the impaired ability of T50L7 to interact with p300 and CBP and the reduced mitogenic activity of this mutant. This correlation suggests that the binding of p300 and CBP by T contributes to its growth-stimulating activity. DISCUSSION In this report, we have shown that both p300 and CBP bind specifically to SV40 T. This interaction leads to at least two different consequences for p300 and CBP. It antagonizes their transactivation function on certain promoters, and it perturbs their proper phosphorylation. The failure of a specific T mutant, T50L7, to bind efficiently to p300 and CBP correlated with its inability to interfere with both the transcriptional activity and phosphorylation state of both proteins. This T mutant also exhibited severely reduced mitogenic activity. Thus, there is a link between the efficient binding of T to p300 and CBP and expression of its mitogenic functions. Three independent biochemical approaches were employed to analyze whether a particular T species could interact with p300 or CBP. These approaches were immunoprecipitation withatorcbpantibody and in vitro binding of T to GSTp300 and GST-CBP fusion proteins. A fourth and more indirect measure of the ability of a particular T mutant to interact with p300 and CBP was provided by transcriptional repression assays performed with p300-e2 and CBP-E2 fusion proteins. The results of these four assays are summarized in Table 1. wt T and the K1 mutant scored positively in all four assays. For the K1 and T50L7 mutants, there were quantitative differences in binding efficiency observed with the three biochemical approaches. This may be related to the different natures and on January 14, 2019 by guest

8 VOL. 16, 1996 p300 AND CBP INTERACT WITH SV40 LARGE T ANTIGEN 3461 FIG. 7. Altered phosphorylation state of p300 in cells expressing wt or K1 T. (A) Two cell lines lacking T (B6 and 10T1/2) and MEF cells synthesizing the K1 mutant were labeled with [ 35 S]methionine. Extracts were prepared and immunoprecipitated with the antibodies indicated. The antibodies were the same as those described in the legend to Fig. 2. The immunoprecipitates were resolved on 5% protein gels which were electrophoresed until the 116-kDa marker had left the gels. (B) Extracts from 35 S-labeled cells were immunoprecipitated in duplicate with either a p300- or CBP-specific antibody. The first set of immunoprecipitates was left untreated ( ). The second set was incubated with phosphatase ( -phosph.; ). When the phosphatase treatment was carried out in the presence of phosphatase inhibitors, no change in p300 mobility was observed (data not shown). (C) Duplicate sets of 35 S-labeled lysates of 293 and U-2 OS cells were immunoprecipitated with the antibodies indicated and either left untreated ( ) or exposed to phosphatase ( -phos.). The antibodies used were M73 for E1A, RW 128 for p300, and AC 235 for CBP. Downloaded from sensitivities of the affinity binding reagents employed. In general, none of the T species studied here appeared to be completely defective in p300 and CBP binding. Even the T mutant with the most severe impairment in p300 and CBP binding, T50L7, displayed at least some interaction in two independent assays. Our difficulties in detecting T in p300 and CBP immunoprecipitates may reflect an exquisite sensitivity of these complexes to distortions in protein folding induced by antibody binding. Of the 10 p300 and CBP antibodies tested, only 1 (AC 238) was capable of precipitating intact T-containing complexes. In contrast, 9 of the 10 antibodies efficiently coprecipitated E1A (Fig. 3A and data not shown). Thus, the failure to recover T in these experiments is not due to a general inability of these antibodies to precipitate intact protein complexes. Rather, it may be linked to different mechanisms or affinities of E1A and T binding to p300 and CBP. We also noted that CBP-T antigen complexes precipitated by AC 238 were relatively stable, since they could be extracted from cells in the presence of 400 mm salt and resisted up to seven washes with buffer containing 300 mm NaCl (data not shown). In contrast, complexes containing T50L7 were less stable and did not resist such conditions (see Materials and Methods), implying that there is a correlation between the strength of binding by the oncoprotein and subsequent T- dependent perturbation of p300 and CBP function. The impaired interaction of the T50L7 mutant with T suggests that the integrity of the relatively small region deleted from this mutant (residues 98 to 126) is important not only for the ability of T to associate with Rb and related proteins (11, 13, 19, 23) but also for high-affinity binding to p300 and CBP. The importance of this region in influencing the quality of interaction with p300 and CBP is further emphasized by the failure of T50L7 to alter the phosphorylation of p300 and CBP and to repress transactivation by these two proteins. Further studies to determine which regions of T are necessary and sufficient for directing interaction with p300 and CBP are in progress. Preliminary results indicate that the T sequences governing p300 binding are complex. Consistent with the ability of T50L7 to bind p300 and CBP, T sequences outside of residues 98 to 126 also appear to be critical for complex formation (36). Although the T sequences required for interaction with p300, CBP, and Rb family members may partially overlap, the binding of these two protein families clearly depends on different elements of T structure. This was best exemplified by the K1 and PVU-1 mutants, which bound demonstrably to p300 and CBP but failed to interact with Rb-related proteins. Compared with E1A, T interacts with a lower efficiency with members of the Rb family (13, 37). This may be of physiological importance if the binding of Rb and the binding of p300 and CBP by T are mutually exclusive events. If this is true, balancing the affinities with which T interacts with these two groups of proteins could be critical for the ability of T to interfere with the cellular growth control pathways governed both by Rb family members and by p300 and CBP. In that case, replacement of the suboptimal Rb-binding motif of T with a more optimal one (e.g., from E1A) would reduce the likelihood of the recombinant T protein binding efficiently to p300 and CBP, thereby impairing the mitogenic functions of T. In the case of on January 14, 2019 by guest

9 3462 ECKNER ET AL. MOL. CELL. BIOL. FIG. 8. T can inhibit transactivation by p300 or CBP. (A) The transcriptional activities of p300 and CBP, fused to the DNA-binding domain of the bovine papillomavirus E2 protein, were measured in U-2 OS cells with the 6xE2-tk-CAT reporter plasmid (1). Chloramphenicol acetyltransferase assays were performed with extract quantities normalized to -galactosidase activity. The first three bars show that the basal transcriptional activity of the reporter itself ( ) was not altered by the presence of either wt or K1 T. In contrast, expression of these T species or 12S E1A diminished the transcriptional activities of p300-e2 and CBP-E2. Data are the averages standard errors of four independent transfection assays. (B) The activation domain of VP16, fused to the E2 DNA-binding domain, is marginally inhibited by either wt or K1 T. The reporter plasmid was 6xE2-tk-CAT. Data are the averages standard errors of three independent experiments. (C) Efficient repression of p300-e2 depends on the integrity of the third CH-rich region. p300 del 33-E2 and p300 del 47-E2 lack residues 1737 to 1836 and 1676 to 1750, respectively. These residues lie within the third CH-rich region of p300. Data from three independent experiments are shown. (D) The T50L7 mutant fails to inhibit p300-e2 and CBP-E2 efficiently. Assays were performed as described for panel A. (E) Western blot of cell lysates isolated from one of the transfection experiments performed in panel D. Extracts from 1/10 of the transfected U-2 OS cells were monitored for T protein levels with PAb419. Lane, extract from cells transfected with only the reporter plasmid and p300-e2 (no T expression plasmid). This blot also shows the expression level of the K1 mutant. The data for this mutant have not been included in panel D. direct contacts between T and p300 and/or CBP. T exhibits little sequence similarity with the segments of E1A responsible for binding to p300 and CBP. Thus, the third CH-rich region of p300 and CBP may prove to be flexible in its seeming ability to recognize proteins unrelated in primary sequence. In keeping with this notion, the C terminus of c-fos has recently been shown to interact with this domain of CBP (5). On the basis of computer comparisons, the protein sequence of the c-fos C terminus does not show any similarities to T or E1A. Although T and E1A contact the same region of p300 and CBP, the effects of viral oncoprotein binding on p300 and CBP phosphorylation are, at first glance, disparate. From the results of our in vivo experiments, T suppressed the state of phosphorylation of p300 and CBP, while E1A induced hyperphosphorylation of these two proteins. It is conceivable that the strong kinase activity associated with E1A (28, 33) is in part responsible for this hyperphosphorylation. The binding of T to p300 and CBP could sterically interfere with the access of kinases, the presence of T might downregulate the activities of kinases which phosphorylate p300 and CBP, or it might lead to the activation of a putative p300 phosphatase. In theory, E1A might also be able to interfere with the phosphorylation of p300 and CBP in a way similar to that of T. However, this inhibition may be masked by its associated kinase activity. Consistent with the view that E1A may also inhibit the phosphorylation of certain sites on p300, the results of in vitro kinase assays have shown that the incorporation of phosphate groups into p300 proceeded more slowly when E1A was present (4). Our work provides an example of a protein negatively regulating cellular signal transduction to p300 and CBP. To fully understand the functional consequences of this negative regulation on the activities of p300, it will be necessary to characterize p300 phosphorylation in greater detail. Previous studies have shown that p300 phosphorylation is subject to regulation during the cell cycle (58) and during cellular differentiation (32, 42). For example, p300 isolated from undifferentiated F9 cells is essentially unphosphorylated. Upon differentiation induced by either retinoic acid or the expression of E1A, p300 is phosphorylated and competent to activate transcription (32). There is an important question concerning the mechanism by which complexes between T and p300 and/or CBP exert E1A, the domains directing interactions with pocket proteins, p300, and CBP can be more readily separated (54), and unlike T, even a large deletion in conserved region 2 of E1A does not diminish the binding of p300 and CBP (2, 17, 53, 55). Previous work indicated that E1A contacts the third CH-rich region of p300 and CBP (1, 15, 37). Two lines of evidence suggest that T also interacts with this region. First, GST fusion proteins consisting of this region directed stable T binding. Second, efficient inhibition of the transcriptional activity of p300-e2 by T required the integrity of the third CH-rich region. Since T purified from a baculovirus expression system is capable of binding to a GST-p300 protein comprising the third CH-rich region (16), it is likely that there are at least some FIG. 9. Reduced growth rate and saturation density of MEF cells harboring T50L7. MEF cells (10 5 ) expressing wt, K1, and T50L7 T were seeded on eight duplicate 60-mm-diameter dishes on day 0. Every 24 h, cells from duplicate dishes were trypsinized and counted. The growth of MEF cells synthesizing wt T could be followed for only 6 days, at which time they sloughed and clumped, preventing any further counting. Data are the average cell numbers from duplicate dishes. Note the logarithmic scale for the y axis (cell number).