TAFII 250 Phosphorylates Human Transcription Factor IIA on Serine Residues Important for TBP Binding and Transcription Activity*

Steven SolowDagger, Moreh Salunek, Robert Ryan, and Paul M. Lieberman§

From the Wistar Institute, Philadelphia, Pennsylvania 19104

Received for publication, October 13, 2000, and in revised form, February 14, 2001


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Transcription factor IIA (TFIIA) is a positive acting general factor that contacts the TATA-binding protein (TBP) and mediates an activator-induced conformational change in the transcription factor IID (TFIID) complex. Previously, we have found that phosphorylation of yeast TFIIA stimulates TFIIA·TBP·TATA complex formation and transcription activation in vivo. We now show that human TFIIA is phosphorylated in vivo on serine residues that are partially conserved between yeast and human TFIIA large subunits. Alanine substitution mutation of serine residues 316 and 321 in TFIIA alpha beta reduced TFIIA phosphorylation significantly in vivo. Additional alanine substitutions at serines 280 and 281 reduced phosphorylation to undetectable levels. Mutation of all four serine residues reduced the ability of TFIIA to stimulate transcription in transient transfection assays with various activators and promoters, indicating that TFIIA phosphorylation is required globally for optimal function. In vitro, holo-TFIID and TBP-associated factor 250 (TAFII250) phosphorylated TFIIA on the beta  subunit. Mutation of the four serines required for in vivo phosphorylation eliminated TFIID and TAFII250 phosphorylation in vitro. The NH2-terminal kinase domain of TAFII250 was sufficient for TFIIA phosphorylation, and this activity was inhibited by full-length retinoblastoma protein but not by a retinoblastoma protein mutant defective for TAFII250 interaction or tumor suppressor activity. TFIIA phosphorylation had little effect on the TFIIA·TBP·TATA complex in electrophoretic mobility shift assay. However, phosphorylation of TFIIA containing a gamma  subunit Y65A mutation strongly stimulated TFIIA·TBP·TATA complex formation. TFIIA-gamma Y65A is defective for binding to the beta -sheet domain of TBP identified in the crystal structure. These results suggest that TFIIA phosphorylation is important for strengthening the TFIIA·TBP contact or creating a second contact between TFIIA and TBP that was not visible in the crystal structure.


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

TFIIA1 was identified originally as an activity required for optimal in vitro transcription with HeLa cell nuclear extracts and various viral and cellular templates (1) (reviewed in Refs. 2-5). TFIIA was isolated as three polypeptides from human and Drosophila embryo extracts, and the two largest subunits are proteolytic cleavage products of a single open reading frame (6-13). TFIIA could be purified as a TATA-binding protein (TBP)-associated factor, although it could also be found as a chromatographically separate complex from TBP (7). In addition to binding directly to TBP, TFIIA stabilized the binding of TBP with TATA DNA in electrophoretic mobility shift assays (EMSA) (14, 15). In transcription reactions reconstituted with partially purified general factors and coactivators, TFIIA is essential for activator-mediated transcription and has a weak stimulatory effect on basal transcription lacking exogenous activators. In transcription reactions reconstituted with recombinant and affinity-purified general factors and coactivators, TFIIA is dispensable (16, 17). Based on these findings, TFIIA is thought to be a transcription accessory factor required for complex regulation in the presence of positive and negative cofactors in vivo.

The two genes encoding the polypeptides of TFIIA share significant sequence similarity between human and yeast Saccharomyces cerevisiae (18). The yeast TFIIA genes, TOA1 and TOA2, are both essential for viability (19). Reduction of TOA1 expression results in a general 2-fold decrease in RNA polymerase II transcription, although some genes are more dramatically affected and others are elevated in gene expression (20, 21). Depletion of TOA1 led to a cell cycle arrest at the G2/M border but did not have a dramatic affect on transcription activation by several activators. TOA2 mutants compromised for TBP binding were defective for transcription activation with several activators and promoters and also accumulated at the G2/M border (22). In this respect, TFIIA behaves similar to several TAFs that when depleted have limited effect on global gene expression but result in cell cycle arrest.

Cross-talk between TFIIA and the largest TAF (human TAFII250 and yeast TAFII145) has been described both biochemically and genetically (23-25). The amino-terminal domain of TAFII250 contains a negative regulatory element that binds TBP competitively with TFIIA (23, 26, 27). TAFII250 can inhibit TBP-TATA binding, and TFIIA can reverse this inhibition in a competitive manner (26). Deletion of the amino-terminal domain of yeast TAFII145 results in temperature-sensitive growth defects that can be suppressed by high copy expression of both subunits of TFIIA, supporting a genetic interaction of these two proteins in vivo (23). TFIIA interacts with several other positive and negative transcriptional cofactors. TFIIA binds to the positive cofactor PC4 and is required in PC4-mediated transcription reactions in vitro (28). TFIIA can stimulate transcription repressed by several negative cofactors, including NC2 (DR1/DRAP1), Mot1, and NC1 (TopoI) (29-32). The interaction between TFIIA and NC2 has been clearly established by yeast genetic experiments where a point mutation in TFIIA relieves the requirement for the otherwise essential DR1(Ydr1) and DRAP1(Bur6) genes (33). This point mutation in TFIIA suppresses a slow growth phenotype induced by a reduced expression of DR1. Thus, TFIIA plays an important role in the balance between positive and negative acting cofactors of transcription.

Post-translational modifications of transcription factors and chromatin components have been shown to be important in transcription regulation. TAFII250 possesses histone acetyltransferase activity and protein kinase activity (34, 35). The kinase domain of TAFII250 has been mapped to two distinct regions spanning the amino-terminal and carboxyl-terminal domains (35, 36). The amino-terminal kinase domain of TAFII250 has been shown to be capable of phosphorylating the 74-kDa subunit of RAP74 and, to a lesser extent, the large subunit of TFIIA (35, 36). The retinoblastoma (Rb) tumor suppressor protein can bind to the amino-terminal domain of TAFII250 and inhibit kinase activity (37, 38). TAFII250 plays an important role in cell cycle progression in mammalian cells, since a point mutation in the central domain is responsible for the G1/S arrest in ts13 hamster cells (39, 40). Whether Rb binding to TAFII250 or the kinase activity of TAFII250 contributes to cell cycle function is unclear.

In previous work, we demonstrated that yeast TFIIA was phosphorylated on the C-terminal domain of the large subunit (TOA1) in vivo (41). Substitution of three serine residues in TOA1 resulted in the complete loss of TFIIA phosphorylation. TFIIA mutants incapable of being phosphorylated were reduced for T-A binding in vitro and incapable of high level transcription activation in vivo at some promoters. While phosphorylation-defective TFIIA did not lead to an apparent growth defect, the serine to alanine mutations were lethal when combined with a single alanine substitution in a second position known to contact TBP. The crystal structure of the yeast TFIIA·TBP·TATA ternary complex shows that TFIIA residues Tyr69 and Trp76 on TOA2 and Trp285 on TOA1 make direct contact with TBP residues surrounding the S2 beta -sheet domain residues Arg105, Ili106, and Arg107 (42, 43). Mutagenesis of these residues reduced T-A complex formation and had growth defects in vivo (22, 44). However, mutagenesis experiments suggest that additional residues in the basic repeat region of helix H2 in TBP were important for T-A complex formation (45, 46). The crystal structure did not detect a clear contact between TFIIA and these residues, although in one structure a nonresolvable electron density appeared near TBP helix H2 (42). A substantial region of TOA1 was not structured in the ternary complex and therefore could not be assigned a fixed position. Recently, two NMR studies suggest that the unstructured region of TFIIA can contact helix H2 of TBP (27, 47). We present evidence here that phosphorylation of TFIIA on serine residues in the unstructured domain of TOA1 (human TFIIA beta ) stabilizes the T-A complex when the primary contact is disrupted. Phosphorylation competent TFIIA stimulated transcription in transient transfection assays, suggesting that phosphorylation is important for high level transcription in vivo. Finally, we present evidence that TAFII250 amino-terminal kinase domain can phosphorylate TFIIA on serine residues known to be phosphorylated in vivo.

    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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Plasmid Constructs and Yeast Strains-- The FLAG-tagged alpha beta subunit of human TFIIA was expressed in transient transfection assays using the plasmid pFLAGhIIAalpha beta (gift of J. Zhang, University of Pennsylvania, Philadelphia, PA and M. Lazar, University of Medicine and Dentistry of New Jersey, Piscataway, NJ), and the gamma  subunit of TFIIA was expressed using pCMV-hIIAgamma (gift of D. Reinberg) (8). Some or all of the codons for the four serine residues at sites 280, 281, 316, and 321 of the alpha beta gene of pFLAGhIIAalpha beta were changed to those for alanine using the QuikChange site-directed mutagenesis kit (Stratagene), according to the manufacturer's instructions. This yielded plasmids pSPS762 (S280/281A), pSPS806 (S316/321A), and pSPS825 (S280/281/316/321A), all confirmed by sequencing. Expression plasmids expressing GAL4 DNA binding domains fused to the activation domains of E2F, EWS, VP16, or Myc were gifts of (F. Rauscher III and S. McMahon). The control plasmid pFLAG-CMV-2 was purchased from Sigma. The pG5-TK-Luc plasmid contains five GAL4 binding sites cloned upstream of the herpes simplex virus thymidine kinase core promoter regulating luciferase (gift of M. Lazar). The pCD1-76Luc promoter contains the cyclin D1 core promoter sequence -76 to +45 regulating luciferase expression (gift of R. Pestell, Albert Einstein College of Medicine, New York). Experiments involving the Zta activator used pZtaSRalpha to express the activator (48) and pZ7E4TCAT as the reporter gene (49). The eukaryotic expression vector for hTAFII250 was obtained from E. Wang and R. Tjian (University of California, Berkley, CA).

Preparation of Proteins-- Recombinant human TBP was isolated as an amino-terminal tagged hexahistidine fusion protein (gift of T. Curran, St. Judes Children's Research Hospital, Memphis, TN). The tagged TBP was expressed in Escherichia coli, purified on a Ni2+-nitrilotriacetic acid-agarose column (Qiagen), and renatured as described previously (26). Recombinant hexahistidine-tagged human TFIIA alpha beta protein was expressed in E. coli using the pQEhIIAalpha beta plasmid (11, 44) and isolated as described above. The hexahistidine-tagged human TFIIA gamma  protein or gamma  protein with tyrosine 65 mutated to alanine (gamma Y65A) was expressed using pQEhIIAgamma (11, 44) and isolated as described above. To make recombinant alpha beta  + gamma  or alpha beta  gamma Y65A for either in vitro phosphorylation or EMSA, the proteins were renatured in equimolar amounts, as referenced above. Casein kinase II from rat liver was acquired from Sigma. GST-Rb, GST-RbDelta 22, and the control GST proteins were expressed using pGEX-(GST)-Rb379-928, pGEX(GST)-Rb379-928Delta ex22 (gifts of P. Robbins (37)), and pGEX-2T (Amersham Pharmacia Biotech), respectively. Purification of these GST-fused proteins was described previously (11). Hemagglutinin epitope-tagged TAFII250 and GST-TAFII250-NTK were expressed as baculovirus proteins in Sf9 cells (gifts of R. Tjian) and isolated from lysate by immunoprecipitating with 12CA5 monoclonal antibody as described previously (50) or glutathione-Sepharose beads. Human holo-TFIID was isolated from LTR3 cell lysate using the 12CA5 antibody as described (50). Recombinant RAP74, a subunit of TFIIF, was made using expression plasmids obtained from D. Reinberg.

DNA Binding Reactions-- Electrophoretic mobility shift assays were regularly performed in 12.5 mM Hepes, pH 7.9, 12.5% glycerol, 0.4 mg/ml bovine serum albumin, 6 mM MgCl2, 16 µg/ml poly(dGdC:dGdC), 0.4% beta -mercaptoethanol (12.5 µl final volume) using the adenovirus E1B TATA box as a probe (51). Binding reactions were done for 50 min at 30 °C, separated on a 0.5× TBE, 5% acrylamide gel, and visualized by autoradiography. When recombinant TFIIA was incubated with a kinase prior to EMSA, the treatment was performed in the same concentrations of Hepes, pH 7.9, bovine serum albumin, MgCl2, dGdC:dGdC oligonucleotide, glycerol, and beta -mercaptoethanol, as described above. A final concentration of 2 units/ml CKII (Sigma) was used or the indicated amount of TAFII250 NH2-terminal kinase and 100 µM ATP, if required, in a 30-min incubation at 30 °C. The DNA probe and other components of the binding reaction were then added, and the assay continued.

Transient Transfections-- Transient transfections were performed using a modified CaPO4 transfection protocol (52). BHK21 or 293 cells were used, as indicated, and always grown at 37 °C in 5% CO2. Cells were harvested 48 h after transfection and assayed using the Luciferase Assay System from Promega or a chloramphenicol acetyl transferase assay (48).

In Vivo Phospholabeling-- Metabolic labeling of phosphorylated proteins was performed on 293 cells, essentially as described previously (53). Briefly, 24 h after transfection, the cells were washed with phosphate-free Dulbecco's modified Eagle's medium containing 10% dialyzed fetal bovine serum and 1× penicillin-streptomycin-glutamine (all from Cellgro) and then refed with this same medium. 2 h later, [32P]orthophosphate (200 µCi per ml of medium; PerkinElmer Life Sciences) was added, and growth continued for an additional 2 h. The plates were then set on ice and washed twice with ice-cold phosphate-buffered saline and then lysed with radioimmune precipitation buffer (1% Nonidet P-40, 1% deoxycholic acid, 0.1% SDS, 150 mM NaCl, 10 mM Tris-HCl, pH 7.4) with phosphatase and proteinase inhibitors (1 mM phenylmethylsulfonyl fluoride, 1 mM sodium orthovanadate, 2 µg/ml pepstatin A, and 2 µg/ml leupeptin) in an amount one-tenth of the volume of the medium used. After incubating for 15 min on ice, the lysate was removed to an Eppendorf tube and spun at 16,000 rpm for 2 min, and the supernatant was frozen on dry ice.

Immunoprecipitation was performed as described below. To the labeled lysate, 300 µl of Net Gel buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl 0.1% Nonidet P-40, 1 mM EDTA, 0.25% gelatin, 0.02% sodium azide) (53) was added with the phosphatase and proteinase inhibitors in the concentrations listed above. The lysate was first precleared using 100 µl of a 10% solution of fixed Staphylococcus aureus protein A-positive cells (Roche Molecular Biochemicals) in Net Gel buffer for 1 h while rolling at 4 °C. The lysate was then spun down twice to eliminate all solid material. 5 mg of anti-FLAG M5 monoclonal antibody from Sigma was added to immunoprecipitate the protein of interest for 16 h at 4 °C. The antibody was brought down using 50 µl (packed volume) of protein A-Sepharose Fast Flow (Amersham Pharmacia Biotech) that had previously been washed with the Net Gel buffer containing proteinase and phosphatase inhibitor mix described above. This incubation was performed for 1 h at 4 °C before being washed with 130 µl of radioimmune precipitation buffer containing proteinase and phosphatase inhibitor mix three times for 30 min each. The proteins were eluted using Laemmli buffer at 95 °C for 10 min. One-half the volume of eluted material was run on a Tris-glycine-SDS-10% polyacrylamide gel and visualized by autoradiography.

Western Blot Analysis-- Antibodies against FLAG (anti-FLAG M5 monoclonal antibody from Sigma) or hemagglutinin (12CA5 from (Roche Molecular Biochemicals)) were used at concentrations recommended by the manufacturer.

In Vitro Kinase Reactions and Phosphoamino Acid Analysis-- Recombinant TFIIA (100 ng) was incubated with ~10 ng of affinity-purified HA-tagged TAFII250 or HA-tagged TFIID in a reaction volume of 15 µl containing 12.5 mM Hepes, pH 7.9, 12.5% glycerol, 0.4 mg/ml bovine serum albumin, 6 mM MgCl2, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, and phosphatase inhibitor mix described above. Reactions were incubated at 30 °C for 30 min. Phosphorylated proteins were subject to phosphoamino acid analysis essentially as described (54).

    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The amino-terminal (alpha ) and carboxyl-terminal (beta ) domains of the large subunit of TFIIA (alpha beta ) are highly conserved between human and yeast (Fig. 1A). In previously published work, we had shown that yeast TOA1 subunit of TFIIA was phosphorylated in vivo, and alanine substitution of serines 220, 225, and 231 abrogated TFIIA phosphorylation. Alignment of the carboxyl-terminal domain of TOA1 with the alpha beta subunit of TFIIA shows that yeast serine 225 and 230 can be partially aligned with human serine residues 316 and 321 (Fig. 1B). To determine if human TFIIA was similarly phosphorylated in human cells, FLAG-tagged TFIIA alpha beta was transfected into 293 cells and assayed for incorporation of phosphate by metabolically labeling transfected cells with [32P]orthophosphate. We found that TFIIA alpha beta was efficiently labeled under these conditions, while the tightly associated heterodimeric partner TFIIA gamma  was not labeled in these experiments (Fig. 2 and data not shown). To determine if the serine residues conserved between yeast and human TFIIA large subunits were important for phosphorylation in vivo, we mutated two sets of serine residues in TFIIA beta  domain, Ser280/Ser281 and Ser316/Ser321 to alanines. FLAG-tagged TFIIA wild type and serine to alanine mutants were transfected into 293 cells, metabolically labeled with [32P]orthophosphate, and assayed by immunoprecipitation (Fig. 2, upper panel). We found that alanine substitutions at serines 316 and 321 reduced phosphorylation of TFIIA considerably, while a mutation in serines 280 and 281 had only a small effect on in vivo phosphorylation. Alanine substitution at all four positions (S280A/S281A/S316A/S321A) produced the most dramatic reduction of TFIIA phosphorylation. All of the TFIIA alpha beta proteins were expressed to similar levels in transfections and after immunoprecipitations as determined by Western blot analysis (Fig. 2B, lower panel, and data not shown). These results indicate that human TFIIA alpha beta can be phosphorylated in vivo and that serine residues 316 and 321 are important for this phosphorylation.


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Fig. 1.   A, schematic of human TFIIA alpha beta subunits aligned with yeast TOA1. B, sequence alignment of human TFIIA alpha beta amino acids 307-376 with yeast TOA1 amino acids 216-287. Serine residues in TOA1 shown to be phosphorylated in yeast are indicated by an asterisk above amino acids 220, 225, and 232. The circles indicate two of the four serine residues shown in this manuscript to be essential for TFIIA phosphorylation of TFIIA.


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Fig. 2.   Phosphorylation of TFIIA alpha beta in human cells. Human 293 cells were transfected with FLAG-tagged TFIIA alpha beta expression vectors containing wild type and alanine substitution mutants of TFIIA alpha beta at residues Ser280/Ser281, Ser316/Ser321, or Ser280/Ser281/Ser316/Ser321. Transfected cells metabolically labeled with [32P]orthophosphate were subject to immunoprecipitation with anti-FLAG antibody and analyzed by autoradiography of SDS-PAGE gels (top panel). The same immunoprecipitates were analyzed by Western blotting with antibody specific for the FLAG epitope (lower panel).

TAFII250 has been reported to phosphorylate TFIIA in vitro (35). We compared the ability of affinity-purified holo-TFIID and baculovirus-expressed TAFII250 to phosphorylate TFIIA in vitro (Fig. 3). Both holo-TFIID (hIID) and TAFII250 were capable of phosphorylating TFIIA alpha beta in vitro (Fig. 3A, lanes 2 and 4). To determine if TFIIA alpha beta was phosphorylated on the carboxyl-terminal domain in vitro, we tested the ability of TAFII250 and hIID to phosphorylate the beta  subunit of TFIIA when presented as the trimeric form of TFIIA (referred to as alpha  + beta  + gamma ). Both hIID and TAFII250 preferentially phosphorylated the beta  subunit of TFIIA in the trimeric complex, although some background phosphorylation occurred on alpha  and gamma  (Fig. 3A, lanes 6 and 8). The phosphorylation of TFIIA beta  by TAFII250 and hIID is likely to be specific, since the beta  subunit has six serine and five threonine residues, while the alpha  subunit has 11 serines and 17 threonines, and the gamma  subunit has five serines and nine threonines.


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Fig. 3.   TFIID and TAFII250 phosphorylation of TFIIA in vitro. Affinity-purified hIID (lanes 1, 2, 5, and 6) or baculovirus HA-tagged TAFII250 (lanes 3, 4, 7, and 8) were incubated with TFIIA alpha beta  + gamma  (lanes 2 and 4) or with TFIIA alpha  + beta  + gamma  (lanes 6 and 8) in the presence of [gamma -32P]ATP. Reaction products were visualized by autoradiography of SDS-PAGE gels. B, TAFII250 was tested for its ability to phosphorylate GST (lane 1), GST-beta -(329-382) (lane 2), GST-beta -(310-382) (lane 3), or GST-beta -(252-382) (lane 4). C, TAFII250-phosphorylated TFIIA beta  was assayed by two-dimensional phosphoamino acid analysis. The arrows indicate migration of major phosphoamino acid species. D, hIID phosphorylates yeast TOA1 in vitro. Human TFIIA (alpha beta  + gamma ) or increasing concentrations of recombinant yeast TOA1 were assayed for their ability to serve as kinase substrates for affinity-purified hIID.

To determine if the alpha  and gamma  subunit of TFIIA were important for the TAFII250 phosphorylation of TFIIA beta , we expressed TFIIA beta  as a GST fusion protein and assayed its ability to be phosphorylated in vitro by TAFII250 (Fig. 3B). We found that GST-beta -(252-375) was efficiently phosphorylated by TAFII250 in vitro, while GST was not phosphorylated. To partially map the phosphorylation sites in vitro, we compared GST-beta -(252-376) to two amino-terminal truncation mutants GST-beta -(310-376) and GST-beta -(329-376). We found that TAFII250 efficiently phosphorylated beta -(310-376) but failed to phosphorylate beta -(329-376), suggesting that important phosphoacceptor sites reside between amino acids 310 and 329 (Fig. 3B). Phosphorylated TFIIA beta  subunit was subjected to phosphoamino acid analysis to determine the predominant amino acid species phosphorylated (Fig. 3C). We found that the majority of phosphorylation occurred on serine residues, although a trace of threonine phosphorylation was detected. Tyrosine phosphorylation was not detected. Identical observations were made when TFIIA alpha beta was subject to phosphoamino acid analysis, indicating that serine is the predominant species phosphorylated throughout the entire polypeptide. To determine if the phosphorylation of TFIIA by TFIID-associated kinase was conserved between human and yeast, we compared the ability of hIID to phosphorylate human TFIIA alpha beta with yeast TOA1 (Fig. 3D). We found the human TFIID was similarly capable of phosphorylating yeast TOA1, suggesting that the conserved serine residues in TFIIA were important for phosphorylation.

The phosphorylation of TFIIA by TAFII250 and hIID was further characterized in vitro by comparing the relative specificity of phosphorylation for the wild type and mutant TFIIA. We have previously shown that alanine substitution of serine residues 280/281/316/321 in TFIIA alpha beta abrogated phosphorylation in vivo (Fig. 2). To determine if the same serine residues in TFIIA alpha beta were required for phosphorylation by TFIID and TAFII250 in vitro, we compared TFIIA wild type (wt IIA) and S280A/S281A/S316A/S321A mutant (m-IIA) in the in vitro kinase reaction (Fig. 4). TAFII250 phosphorylated wild type TFIIA efficiently but had little activity on mutant IIA (Fig. 4A, lanes 3 and 4). Similarly, holo-TFIID phosphorylated wild type TFIIA efficiently but had no detectable kinase activity with mutant IIA (Fig. 4A, lanes 5 and 6). In contrast, commercial preparations of casein kinase II phosphorylated wild type TFIIA and mutant IIA to similar levels (Fig. 4A, lanes 7 and 8), suggesting that sequence specificity is different for TAFII250 kinase and casein kinase II. Equal amounts of wild type TFIIA and mutant IIA were included in these reactions as shown by Coomassie staining of SDS-PAGE gels (Fig. 4B). These results indicate that TAFII250 is a better candidate for phosphorylation of TFIIA alpha beta in vivo than is casein kinase II, since mutant IIA is poorly phosphorylated in vivo.


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Fig. 4.   Substrate specificity of TAFII250 and holo-TFIID kinase. A, TFIIA proteins generated with wild type alpha beta (wt IIA) or alpha beta S280A/S281A/S316A/S321A (m-IIA) were compared for their ability to be phosphorylated by TAFII250 (lanes 3 and 4), holo-TFIID (lanes 5 and 6), or casein kinase II (CKII) (lanes 7 and 8). B, the amino-terminal kinase domain of TAFII250 (NTK) was incubated with RAP74 (lanes 1-3) and either m-IIA (lane 2) or wild type TFIIA (lane 3). C, TFIIA alpha beta subunits from wild type or mutant TFIIA were analyzed by Coomassie staining of SDS-PAGE gels. D, Rb inhibits TAFII250 phosphorylation of TFIIA in vitro. TAFII250 phosphorylation of TFIIA alpha beta was assayed in the presence of GST (lane 1), GST-Rb-(379-928) (lane 2), or GST-RbDelta ex22 (lane 3). Phosphorylated TFIIA alpha beta is indicated. Coomassie staining of GST (lane 4), GST-Rb-(379-928) (lane 5), and GST-RbDelta ex22 (lane 6) was analyzed for relative protein abundance. The arrow indicates the position of full-length Rb and RbDelta ex22.

TAFII250 consists of two functional and distinct kinase domains. We tested the ability of the NH2-terminal kinase (NTK) domain to phosphorylate TFIIA in vitro (Fig. 4C). TAFII250 NTK was expressed and purified as a GST fusion protein from baculovirus-infected cells. NTK activity was demonstrated by phosphorylation of the TFIIF subunit RAP74 (Fig. 4C, lane 1). We then compared the ability of TAFII250 NTK to phosphorylate mutant IIA or wild type TFIIA in the presence of RAP74 (Fig. 4C, lanes 2 and 3). We found that TAFII250 NTK preferentially phosphorylated wild type TFIIA relative to mutant IIA, although the activity was significantly less than found in full-length TAFII250. These results suggest that the NH2-terminal kinase domain of TAFII250 can phosphorylate TFIIA with specificity similar to that observed for full-length TAFII250 and hIID.

The Rb tumor suppressor has been shown to inhibit TAFII250 kinase activity directed toward RAP74 in vitro (37). A mutation in Rb (RbDelta ex22) that abrogates tumor suppressor activity was shown to be incapable of inhibiting TAFII250 kinase activity. We tested the ability of GST, GST-Rb, and GST-RbDelta ex22 to inhibit the phosphorylation of TFIIA by TAFII250 (Fig. 4D, lanes 1-3). Similar to what was found for the phosphorylation of RAP74, we found that Rb but not RbDelta ex22 could inhibit TFIIA alpha beta phosphorylation. Concentrations of GST, GST-Rb, and GST-RbDelta ex22 were shown to be equivalent by Coomassie staining of SDS-PAGE gels (Fig. 4D, lanes 4-6). The inhibition of TFIIA phosphorylation by Rb further suggests that TFIIA is phosphorylated by TAFII250 and not by a contaminating activity in the affinity-purified baculovirus preparation of TAFII250.

TFIIA Phosphorylation Correlates with Transcription Stimulation-- To determine if TFIIA phosphorylation was important for transcription function, we employed a transient transfection assay (Fig. 5). Wild type or mutant alpha beta was cotransfected with equimolar amounts of TFIIA gamma  and assayed for the stimulation of reporter activity. Western blots were used to confirm that both alpha beta and malpha beta were expressed in vivo at equal levels (data not shown). TFIIA subunits were tested for their ability to affect transcription activated by GAL4-E2F, GAL4-EWS, GAL4-VP16, or GAL4-MYC on the G5-TK-Luc promoter construct (Fig. 5A). In all cases, wild type alpha beta stimulated transcription between 2- and 3-fold. Wild type TFIIA stimulated Zta activation of the Z5E4TCAT promoter almost 4-fold, while mutant alpha beta had no detectable activation (Fig. 5B). Similarly, wild type alpha beta (wt-alpha beta ) stimulated the cyclin D1-Luc reporter, while mutant alpha beta (m-alpha beta ) had no effect (Fig. 5C). These results indicate that transfection of TFIIA alpha beta stimulates various activators and promoters, and this activation is dependent upon serine residues shown to be essential for phosphorylation in vivo.


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Fig. 5.   Phosphorylation-defective TFIIA fails to coactivate transcription. A, expression vectors for wild type TFIIA alpha beta or mutant TFIIA alpha beta were cotransfected with TFIIA gamma  and the GAL4-TK-Luc reporter plasmid with GAL4-E2F, -EWS, -VP16, or -MYC activators as indicated for each graph. B, the Zta reporter plasmid was the Z7E4TCAT promoter, and chloramphenicol acetyltransferase units were measured in counts/min (cpm). C, cyclin D1 promoter-Luc construct was cotransfected with wild type alpha beta  + gamma  (wt-alpha beta  + gamma ; black bars) or mutant alpha beta  + gamma  (m-alpha beta  + gamma ; stippled bars). No activators were transfected for the cyclin D1 promoter. All transfections were performed in triplicate, with the S.E. shown on the graph.

TFIIA Phosphorylation Strengthens the TBP·TFIIA Complex-- Previous work with yeast TFIIA indicated that phosphorylation of TOA1 stimulated T-A complex formation in EMSA. We tested the effect of human TFIIA phosphorylation on the T-A complex. We found that phosphorylation had no detectable effect on wild type TFIIA (Fig. 6A, lanes 3 and 4). However, the increase in stability induced by phosphorylation may not be detectable unless the primary contact between TFIIA and TBP is compromised. To test this possibility, we assayed the effect of phosphorylation on a TFIIA derivative containing an alanine substitution at gamma -Tyr65. TFIIA gamma -Tyr65 is the primary contact in the crystal-determined structure, and mutagenesis of this residue severely compromises T-A formation in EMSA (44). IIA-gamma Y65A was significantly reduced for T-A complex formation in the absence of phosphorylation (Fig. 6A, lanes 5 and 7). However, in the presence of ATP and casein kinase II, IIA-gamma Y65A was strongly stimulated in forming the T-A complex (Fig. 6A, lanes 6 and 8). To determine if the serine residues in TFIIA alpha beta were required for this stimulation of T-A complex formation, we compared wild type alpha beta (IIA-Y65A) with alpha beta lacking all four phosphoserine residues (mIIA-Y65A) in EMSA reactions (Fig. 6B). We found that phosphorylation stimulated the T-A complex with wild type alpha beta (Fig. 6B, lanes 1 and 2) but did not stimulate complex formation with mutant alpha beta (Fig. 6B, lanes 3 and 4), indicating that stimulation by ATP and CKII was dependent upon the phosphoacceptor serine residues in TFIIA alpha beta . These results were found to be essentially identical when TAFII250 was used as the kinase (Fig. 6C), but the efficiency of TAFII250 phosphorylation is significantly less than that of CKII in these reactions. The reduced complex formation in the presence of TAFII250 results partly from the inhibitory activity of TAFII250 on TBP-DNA and TBP-TFIIA interactions (26, 55). Together, these results suggest that TFIIA phosphorylation by CKII or TAFII250 stabilizes T-A complex when TFIIA is compromised for interacting with TBP through its primary contact residue gamma -Tyr65.


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Fig. 6.   Phosphorylation of TFIIA gamma Y65A stimulates complex formation with TBP in EMSA. A, wild type TFIIA (IIAwt) or TFIIA gamma Y65A (IIA-Y65A) was incubated with CKII (lanes 1-8) in the presence (+) or absence (-) of ATP and then assayed for the ability to form a stable complex with TBP. TBP·DNA complex (T) and TBP·TFIIA·DNA complex (T-A) are indicated. B, TFIIA gamma Y65A (lanes 1 and 2) or mutant TFIIA gamma Y65A (lanes 3 and 4) were compared for T-A complex formation with TBP in the presence (+) or absence (-) of ATP with CKII as the kinase. C, TAFII250 was incubated with TFIIA gamma Y65A with (+) or without (-) ATP and then assayed for complex formation with TBP.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

TFIIA Phosphorylation Is Conserved between Yeast and Humans-- Human and yeast TFIIA share significant sequence similarity in the amino- and carboxyl-terminal domains of the large subunits. The intervening spacer region has no known function but is important for normal growth in yeast. The region of yeast TFIIA that we have found to be phosphorylated (TOA1 amino acids 220-232) is essential for viability but is only partially conserved with human TFIIA (19). Only one of the serine residues can be aligned with human TFIIA, although several other serines are similarly positioned in acidic patches. This domain of TFIIA could not be visualized in the crystal structure, presumably because this domain lacks ordered structure in the ternary complex. Our results suggest that phosphorylation of TFIIA in this domain is a conserved post-translation modification that promotes additional contacts with TBP that are important for transcription functions.

TAFII250 NTK Is a Candidate TFIIA Kinase-- We have shown that TAFII250 can phosphorylate TFIIA in vitro (Figs. 3 and 4). Phosphorylation was mapped to serine residues on the beta  subunit between amino acid residues 310 and 329. Alanine substitution of serine residues 316 and 321 strongly reduced TFIIA phosphorylation and, in combination with alanine substitutions at serines 280 and 281, completely eliminated detectable phosphorylation by affinity-purified TFIID or TAFII250. The specificity of serine phosphorylation by TAFII250 in vitro corresponds well to the serine phosphorylation specificity found in vivo (Fig. 2). Interestingly, we found that Rb, but not the tumor suppressor-defective mutant RbDelta ex22, could inhibit TAFII250 phosphorylation of TFIIA in vitro. These experiments support earlier studies demonstrating that Rb can inhibit TAFII250 phosphorylation of RAP74 (37). Considering the fact that TFIIA has been implicated in regulating cell cycle progression in yeast, it is possible that TAFII250 phosphorylation of TFIIA may be a transcription regulatory signal required for stimulation of cell cycle control genes (22). Consistent with this hypothesis is the observation that TFIIA phosphorylation-defective mutants were incapable of stimulating transcription from the cyclin D1 promoter (Fig. 5). Transfection of cyclin D1 bypasses the TAFII250-dependent cell cycle defect found in ts13 cells, suggesting that cyclin D1 is a target of TAFII250 activation (56). Our results are consistent with these findings and further suggest that TFIIA contributes to the regulation of cyclin D1 presumably by TAFII250 phosphorylation of TFIIA.

A second line of evidence that TAFII250 may positively regulate TFIIA comes from genetic studies in yeast (23). Deletion of the amino-terminal domain of yeast TAFII145 (the homologue of human TAFII250) results in temperature-sensitive growth. This deletion is predicted to abolish both the TBP interaction domain and the NH2-terminal kinase domain, although no kinase activity in yeast TAFII145 has been reported. This deletion mutant can be suppressed by high copy expression of both subunits of TFIIA (23). Increased expression of TFIIA may be predicted to bypass the need for phosphorylation by increasing the effective concentration of TFIIA and therefore driving the TFIIA-TBP association forward by mass action. These observations from yeast genetics are consistent with our model that the amino-terminal domain of TAFII250 promotes TFIIA·TBP complex formation. While we and others have also found that TAFII250 can inhibit TBP-TATA binding and TFIIA-TBP binding, these processes must be dynamic in vivo (25, 26). It is possible that TAFII250 inhibits unphosphorylated TFIIA from binding TBP but that phosphorylation of TFIIA is sufficient to reverse this inhibition. In this way, TAFII250 can function both to inhibit and promote TFIIA·TBP complex formation depending on the phosphorylation status of TFIIA.

Regulation of Transcription by TFIIA Phosphorylation-- We show that a phosphorylation-defective mutant of TFIIA can not stimulate transcription in transient transfection assays, while wild-type TFIIA can stimulate most activators and promoters 2-4-fold (Fig. 5). While these activation levels are modest, they are similar to the levels of activation typically observed for cotransfection of coactivators like CBP/p300. Phosphorylation of TFIIA stabilized the T-A complex when TFIIA mutants compromised in TBP binding were used (Fig. 6). This suggests that TFIIA phosphorylation stabilizes T-A complex formation either by increasing affinity of TFIIA for TBP through the gamma -Tyr65 contact or by enhancing the interaction between the unstructured domain of TFIIA with TBP. Since this increase in stability is only detectable in the yeast ternary complex or in mutated human TFIIA (gamma Y65A), we suggest that stabilization of the ternary complex is not the major function of phosphorylation. Rather, we suggest that interaction of phosphorylated TFIIA with TBP may alter the interaction between coactivators and repressors and TBP. This could be a method of regulating preinitiation complex assembly and disassembly as well as promoter clearance and transcription reinitiation cycles. Future studies will be required to determine how TFIIA phosphorylation regulates the transcription process.

    ACKNOWLEDGEMENTS

We thank D. Reinberg, R. Tjian, X. Liu, M. Lazar, P. Robbins, S. McMahon, and F. Rauscher III for plasmid reagents.

    FOOTNOTES

* This work was supported by National Institutes of Health Grant GM 54687-04, the W. W. Smith Charitable Trust, and the Leukemia/Lymphoma Society (to P. M. L.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Supported by post-doctoral National Institutes of Health training grant CA 09171 to the Wistar Institute.

§ To whom correspondence should be addressed. Tel.: 215-898-9491; Fax: 215-898-0663; E-mail: lieberman@wistar.upenn.edu.

Published, JBC Papers in Press, February 20, 2001, DOI 10.1074/jbc.M009385200

    ABBREVIATIONS

The abbreviations used are: TFIIA, transcription factor IIA; TBP, TATA-binding protein; EMSA, electrophoretic mobility shift assay(s); TAF, TBP-associated factor; Rb, retinoblastoma; T-A, TFIIA·TBP·TATA; GST, glutathione S-transferase; CKII, casein kinase II; HA, hemagglutinin; hIID, holo-TFIID; PAGE, polyacrylamide gel electrophoresis; NTK, NH2-terminal kinase.

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