From the Wistar Institute, Philadelphia, Pennsylvania 19104
Received for publication, October 13, 2000, and in revised form, February 14, 2001
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ABSTRACT |
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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 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
Plasmid Constructs and Yeast Strains--
The FLAG-tagged 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 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%
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).
The amino-terminal ( 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
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
subunit Y65A
mutation strongly stimulated TFIIA·TBP·TATA complex formation.
TFIIA-
Y65A is defective for binding to the
-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
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-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
) 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
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
subunit of human TFIIA was expressed in transient transfection assays
using the plasmid pFLAGhIIA
(gift of J. Zhang, University of
Pennsylvania, Philadelphia, PA and M. Lazar, University of Medicine and
Dentistry of New Jersey, Piscataway, NJ), and the
subunit of
TFIIA was expressed using pCMV-hIIA
(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
gene of pFLAGhIIA
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 pZtaSR
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).
protein was expressed in
E. coli using the pQEhIIA
plasmid (11, 44) and
isolated as described above. The hexahistidine-tagged human TFIIA
protein or
protein with tyrosine 65 mutated to alanine (
Y65A)
was expressed using pQEhIIA
(11, 44) and isolated as described
above. To make recombinant
+
or
+
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-Rb
22, and the
control GST proteins were expressed using
pGEX-(GST)-Rb379-928, pGEX(GST)-Rb379-928
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.
-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
-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.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
) and carboxyl-terminal (
) domains of
the large subunit of TFIIA (
) 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
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
was transfected into 293 cells and assayed for
incorporation of phosphate by metabolically labeling transfected cells
with [32P]orthophosphate. We found that TFIIA
was
efficiently labeled under these conditions, while the tightly
associated heterodimeric partner TFIIA
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
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
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
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
subunits aligned with yeast TOA1. B, sequence alignment of
human TFIIA
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.
View larger version (35K):
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Fig. 2.
Phosphorylation of TFIIA
in human cells. Human 293 cells
were transfected with FLAG-tagged TFIIA
expression vectors
containing wild type and alanine substitution mutants of TFIIA
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
in vitro (Fig. 3A, lanes 2 and 4). To determine if TFIIA
was phosphorylated on
the carboxyl-terminal domain in vitro, we tested the ability
of TAFII250 and hIID to phosphorylate the
subunit of
TFIIA when presented as the trimeric form of TFIIA (referred to as
+
+
). Both hIID and TAFII250 preferentially
phosphorylated the
subunit of TFIIA in the trimeric complex,
although some background phosphorylation occurred on
and
(Fig.
3A, lanes 6 and 8). The
phosphorylation of TFIIA
by TAFII250 and hIID is likely
to be specific, since the
subunit has six serine and five threonine
residues, while the
subunit has 11 serines and 17 threonines, and
the
subunit has five serines and nine threonines.
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To determine if the and
subunit of TFIIA were important for the
TAFII250 phosphorylation of TFIIA
, we expressed TFIIA
as a GST fusion protein and assayed its ability to be
phosphorylated in vitro by TAFII250 (Fig.
3B). We found that GST-
-(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-
-(252-376) to two
amino-terminal truncation mutants GST-
-(310-376) and
GST-
-(329-376). We found that TAFII250 efficiently
phosphorylated
-(310-376) but failed to phosphorylate
-(329-376), suggesting that important phosphoacceptor sites reside between amino acids 310 and 329 (Fig. 3B). Phosphorylated
TFIIA
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
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
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 abrogated phosphorylation in
vivo (Fig. 2). To determine if the same serine residues in TFIIA
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
in vivo than is casein kinase II, since mutant IIA is
poorly phosphorylated in vivo.
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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 (Rbex22) 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-Rb
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 Rb
ex22 could inhibit TFIIA
phosphorylation. Concentrations of
GST, GST-Rb, and GST-Rb
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 was
cotransfected with equimolar amounts of TFIIA
and assayed for the
stimulation of reporter activity. Western blots were used to confirm
that both
and m
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
stimulated transcription between 2- and
3-fold. Wild type TFIIA stimulated Zta activation of the
Z5E4TCAT promoter almost 4-fold, while mutant
had no
detectable activation (Fig. 5B). Similarly, wild type
(wt-
) stimulated the cyclin D1-Luc reporter, while mutant
(m-
) had no effect (Fig.
5C). These results indicate that transfection of TFIIA
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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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 -Tyr65.
TFIIA
-Tyr65 is the primary contact in the
crystal-determined structure, and mutagenesis of this residue severely
compromises T-A formation in EMSA (44). IIA-
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-
Y65A was
strongly stimulated in forming the T-A complex (Fig. 6A,
lanes 6 and 8). To determine if the
serine residues in TFIIA
were required for this stimulation of
T-A complex formation, we compared wild type
(IIA-Y65A) with
lacking all four phosphoserine residues (mIIA-Y65A) in EMSA reactions (Fig. 6B).
We found that phosphorylation stimulated the T-A complex with wild type
(Fig. 6B, lanes 1 and
2) but did not stimulate complex formation with mutant
(Fig. 6B, lanes 3 and
4), indicating that stimulation by ATP and CKII was
dependent upon the phosphoacceptor serine residues in TFIIA
.
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
-Tyr65.
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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 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 Rb
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
-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 (
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.
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ACKNOWLEDGEMENTS |
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We thank D. Reinberg, R. Tjian, X. Liu, M. Lazar, P. Robbins, S. McMahon, and F. Rauscher III for plasmid reagents.
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FOOTNOTES |
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* 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.
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
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ABBREVIATIONS |
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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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