| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
J. Biol. Chem., Vol. 275, Issue 21, 15912-15916, May 26, 2000
From the Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, Texas 77030
Received for publication, January 20, 2000, and in revised form, March 21, 2000
 |
ABSTRACT |
|---|
 TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
|
|---|
Activation domains are functional modules that
enable DNA-binding proteins to stimulate transcription.
Characterization of these essential modules in transcription factors
has been hampered by their low sequence homology. Here we delineate the
peptide sequences that are required for transactivation and interaction with hTAFII31, a classical target of the acidic class
of activation domains. Our analyses indicate that hTAFII31
recognizes a diverse set of sequences for transactivation. This
information enabled the identification of hTAFII31-binding
sequences that are critical for the activity of the activation domains
of five human transcription factors: NFAT1, ALL1, NF-IL6, ESX, and
HSF-1. The interaction surfaces are localized in short peptide segments
of activation domains. The brevity and heterogeneity of the motifs may
explain the low sequence homology among acidic activation domains.
Transcription factors typically have distinct domains for binding
specific DNA sequences and for activating transcription through
protein-protein interactions (1-3). Although a large number of
activation domains are known, these functional modules share little
sequence homology and have only loosely been classified by the
preponderance of amino acid residues such as acidic residues, glutamine, and proline (1, 4). This low homology of activation domains
has made it difficult to characterize these essential modules in
transcription factors. Identification of functional sequence motifs
that are hidden in activation domains would dissect the functions of
activation domains and help to understand their composite regulations.
Multiple target proteins for each class of activation domains have been
proposed, including the basal transcriptional factors, mediators, and
chromatin-remodeling factors. One such direct target for acidic
activators is hTAFII31 (a human TFIID TATA box-binding protein-associated factor) (5-9). Functional inactivation of its yeast
homolog, yTAFII17, results in the loss of transcription for
approximately 67% of the actively expressed yeast genes (10-13). Moreover, hTAFII31 has been found in a human
histone-acetylase complex in addition to TFIID (14, 15). These previous
results collectively suggest a general role of hTAFII31 and
its homologs in the regulation of eukaryotic gene transcription both at
the level of chromatin modification and RNA polymerase recruitment (16).
It has been reported that hTAFII31 makes direct contacts
with the activation domains of VP16, p53, and NF- Here we delineate the peptide sequences that are required for
transactivation and for interaction with hTAFII31. Our
analyses indicate that hTAFII31 recognizes a more divergent
set of peptide sequences than FXX Mutant Library Screening--
We constructed four small
libraries of a mammalian expression vector, each encoding a 17-amino
acid peptide from the VP16 activation domain (VP16-(469-485)) fused
with the GAL4 DNA-binding domain. Each of the four libraries consists
of random point mutants at one of the four positions within the
FXX In Vitro Protein-Protein Interaction Assay--
The protein
hTAFII31-(1-140) was purified as described (18).
Glutathione S-transferase fusion proteins of activation
domains were expressed in BL21(DE3)pLysS and purified by affinity
chromatography using glutathione-agarose beads. The beads binding
either glutathione S-transferase (GST) fusion protein or GST
only were incubated with hTAFII31-(1-140) in 200 µl of
binding buffer containing 25 mM NaCl, 0.005% Nonidet P-40,
10% glycerol, 20 mM Tris-HCl, pH 7.5, 10 mM
MgCl2, and 2 mM dithiothreitol for 1 h at
4 °C. After extensive washing with the same buffer, the bound
hTAFII31-(1-140) was analyzed by SDS-polyacrylamide gel electrophoresis.
Transcription Assay--
The DNA encoding the GAL4 DNA-binding
domain (residues 1-94) was subcloned into the
HindIII/KpnI site of pcDNA3 (Invitrogen) and
the resultant pcDNAGAL4 plasmid was used to construct mammalian expression vectors for GAL4 fusions of ALL1-(2829-2883),
NFIL6-(24-124), ESX-(129-159), HSF-1-(371-430) and NFAT1-(1-96).
Jurkat Tag cells (~2 × 106) were transfected with
500 ng of each GAL4 fusion construct along with 2 µg of pG5IL2SX.
After a 48-h incubation, an aliquot of the culture was removed and
assayed for SEAP activity as described (19). The expression levels of
the GAL4 fusion proteins were comparable, as judged by Western blot
analyses using an antibody against the GAL4 DNA-binding domain.
NMR Studies--
Peptides were synthesized using Rink Amide MBHA
resin, purified by high performance liquid chromatography, and
characterized by NMR, amino acid analyses, and electrospray ionization
mass spectroscopy. The peptide was dissolved in 95% H2O
plus 5% 2H2O containing 130 mM
KCl, 5 mM perdeuterated dithiothreitol, 20 mM
perdeuterated Tris-AcOH (pH 6.2), and 10 µM EDTA, and
then the pH of the solution was adjusted to ~6.2 by adding dilute
KOH. The final concentrations of peptides were determined by UV
absorption or amino acid analyses to be ~4 mM. NMR
experiments were performed in the absence or presence of
hTAFII31-(1-140) (240 µM) on a Bruker AMX600
spectrometer. The sequential assignment of the peptide signals was
obtained by using a combination of total correlation spectroscopy,
DQF-COSY, and nuclear Overhauser effect spectroscopy (NOESY) data sets
of a free peptide sample. Sequential
d Divergent Motif for hTAFII31--
To delineate the
peptide sequences required for transactivation through interaction with
hTAFII31, we first constructed four small libraries of a
mammalian expression vector, each encoding a 17-amino acid peptide from
the VP16 activation domain (VP16-(469-485)) fused with the GAL4
DNA-binding domain. VP16-(469-485) was chosen because it is the
minimal VP16 peptide that binds a fragment of hTAFII31
(hTAFII31-(1-140)) and activates transcription (18). Each
of the four libraries consists of random point mutants at one of the
four positions within the FXX
The DNA sequence analyses of positive clones (>50% SEAP activity of
the wild type) revealed that the ability of VP16-(469-485) to activate
transcription can endure a variety of amino acid substitutions (Fig.
1). The screen identified Trp, Ile, and
Leu at the conserved position of Phe479; substitution of
Phe479 with any one of the three residues exhibited no
substantial loss in its transcriptional activity. The mutation of
Phe479 with Val abrogated much of its activation potential,
thus validating our screen and indicating the presence of a clear
boundary between Leu and Val for the activity. At the positions of
Thr480 and Ala482, we obtained many clones that
activate transcription more than 50% of the wild type. Although
hydrophobic residues are favored at these two positions, clones with
some of hydrophilic residues such as Tyr also had significant activity.
The identity of Leu483 is more tightly controlled, since we
isolated only Trp, Phe, and Leu as positive clones. Clones with the
other bulky hydrophobic residues, i.e. Ile and Val, at this
position exhibited less than 20% activity of the wild type. However,
the simultaneous substitution of Ala482 with a bulky
hydrophobic residue (Trp, Phe, Ile, or Leu) restored the
transcriptional level of the Ile and Val mutants to >50% of the wild
type, indicative of the complementarity between these two adjacent
positions.
The peptide sequences examined above were then fused with GST and
tested for the ability to bind hTAFII31-(1-140). As shown in Fig. 1, the activation-positive peptides bound
hTAFII31-(1-140) to the same extent as the wild type,
whereas the binding of activation-deficient peptides was significantly
impaired. Thus the strength of the interaction with
hTAFII31-(1-140) in vitro correlates with the ability to activate transcription in transfected cells.
Search for hTAFII31-binding Sequences--
Guided by
the information obtained from the screen, we searched for potential
hTAFII31-binding sequences in human activation domains. A
series of selection steps was carried out on the activation domains of
65 distinct human transcription factors. The amino acid sequences of
these 65 activation domains and their original references are available
on the World Wide Web and in the supplemental materials. In an initial
step, we selected for any activation domains that contain a signature
P1-P2-X-P3-P4 sequence (where P1
represents Phe, Trp, Ile, or Leu; P2 represents Ile, Leu, Tyr, Trp,
Met, Asn, Ala, Thr, Val, Ser, Glu, or Gln; X represents any
amino acid; and P3-P4 represents Trp/Phe/Ile/Leu-Trp/Phe/Leu/Ile/Val or
Ala/Tyr/Val/Cys/Met-Trp/Phe/Leu) and found 26 that did so. Those
activation domains whose signature sequences were not conserved among
species and subtypes were eliminated in a second step, and a third
elimination step was then run on the remaining candidates by
calculating the probability of
The full-length activation domains of these factors were further
analyzed. NFAT1 (nuclear factor of
activated T cells 1) belongs to the
NFAT family of transcription factors and plays a central role in
inducible gene transcription during the immune response. Whereas the
full-length activation domain of NFAT1 (amino acids 1-96) bound
hTAFII31-(1-140) to the same extent as its peptide version, mutation of Phe30 and Phe34 with Ala
impaired its interaction (Fig.
3A), indicative of direct involvement of the signature sequence in the interaction with hTAFII31. ALL1 (acute lymphoblastic
leukemia gene product; also referred as HRX or MILL) is a
human transcription factor that is involved in acute lymphoblastic
leukemia (20, 21), and its transcriptional activity is considered to be
responsible for malignant transformation (22). We identified the
Ile2849-Met2850-Asp2851-Phe2852-Val2853
sequence in its activation domain as a hTAFII31-binding
motif. This was in good agreement with the previous mutational studies showing the importance of these residues in transactivation (22). As
shown in Fig. 3A, the full-length activation domain of ALL1 (amino acids 2829-2883) bound hTAFII31-(1-140) as tightly
as its peptide version. Substitution of Ile2849 and
Val2853 by Ala greatly impaired the interaction, indicative
of the involvement of the IMDFV sequence in the interaction. NF-IL6
(nuclear factor interleukin-6; also referred as C/EBP
To analyze the ability to activate transcription, each activator was
fused with the GAL4 DNA-binding domain, and its expression plasmid was
transfected into human Jurkat T cells along with the reporter plasmid
driven by five copies of the GAL4-binding element. As shown in Fig.
3B, the interaction-deficient mutants of the activation
domains were correspondingly unable to activate transcription of the
reporter gene, whereas the wild type proteins activated transcription,
similar to the VP16 activation domain (VP16452-490). This
functional reduction of the mutants is not the result of differences in
their expression levels as judged by Western analyses. Thus, the
hTAFII31-binding sequences of NFAT1, ALL1, NF-IL6, HSF-1, and ESX are critical for their ability to activate transcription; perhaps hTAFII31 directly mediates the transcriptional
activation by these human factors. However, it is not impossible to
imagine that the same amino acids involved in the interaction with
hTAFII31 may also interact with surfaces in some other
co-activators.
Comparison of hTAFII31-binding
Sequences--
Including p53 and p65, we have now obtained a total of
seven human activation domains whose activities are critically
dependent on their hTAFII31-binding sequences. Their amino
acid sequences are compared in Fig. 2A. Apart from the
COOH-terminal signature sequences, the hTAFII31-binding
peptides have no sequence similarity among themselves. Nonetheless, the
NH2-terminal nonhomology region is necessary for binding
because truncation of the NH2-terminal five residues in
ALL1-(2839-2855) and VP16-(469-485) abolish hTAFII31 binding (18) (Fig. 4A).
To compare the conformations of the hTAFII31-binding
peptides upon binding to hTAFII31, each peptide was
chemically synthesized and analyzed by transferred nuclear Overhauser
effects (TRNOE), an NMR technique that provides conformational
information of a small ligand interacting weakly with its
macromolecular receptor (18, 32). Only ALL12839-2855 and
NF-IL6108-124 showed good physical properties under NMR
conditions and were amenable to TRNOE analyses. NOESY spectra of the
free peptides exhibited few NOEs, as expected from low molecular weight
peptides tumbling freely in solution. In the presence of
hTAFII31-(1-140), however, numerous TRNOE peaks newly
appeared, including those between successive amide protons in the main
chain (Fig. 4, B and C). The pattern of these
cross-peaks and the presence of long range
d
In ALL1-(2839-2855) and NF-IL6-(108-124), the
NH2-terminal halves appear to be in an extended
conformation, and we failed to detect any NOEs that suggest the
formation of a folded structure in this region. These nonhomology
segments thus may make variable contributions to the association,
possibly by lowering the energetic barrier for helix formation or by
making additional contacts with the surface of hTAFII31,
perhaps including those between main chain amide groups in the peptides
and chemically complementary functional groups in hTAFII31.
Our analyses indicate that hTAFII31 recognizes a
diverse set of peptide sequences in activation domains. There are two
advantages for the cells in using such a promiscuous interaction for
transactivation. One is the weakness of the interaction; the
dissociation constant of the interaction between hTAFII31
and the VP16 activation domain is in the high micromolar range, and the
weakness of the interactions is often translated to the diversity of
binding sequences. Synergism of such weak interactions between
activators and co-activators makes transactivation signals diverse and
steep enough to emulate a binary switch (3, 33, 34). Low affinity
interactions also permit dynamic modulation in response to the
alteration of signals that high affinity interactions would be unable
to generate (35). Therefore, the coupling of weak interactions with
transcriptional activation may be ideal for eukaryotic cells that
respond to various signals in a highly tuned manner.
Another advantage is the fact that each one of the binding sequences
can be unique enough to be recognized specifically by its regulatory
proteins. This permits specific modulation of activity of particular
transcription factors in response to the alteration of signals. For
example, the MDM2 protein, a cellular attenuator of p53, specifically
recognizes and masks the hTAFII31-binding motif in the p53
activation domain while exhibiting no detectable affinity to any other
activation domains that bind hTAFII31 (17). This
differential recognition is evidently enabled by the heterogeneity of
hTAFII31-binding sequences. Many phosphorylation sites can also be arranged in the hTAFII31-binding peptides for
specific regulation (Fig. 2A). Some of the Ser residues are
indeed known to be phosphorylated upon particular stimuli.
Ser15 and Ser20 in the p53 activation domain
are phosphorylated upon DNA damage, and Ser15 is the site
of the phosphorylation by the ATM kinase (36-38) and the DNA-activated
protein kinase (39). Phosphorylation of Ser536 in the
activation domain of NF- The physical association between activation domains and
hTAFII31 requires a small surface comprising divergent
peptide motifs in activation domains. The brevity and heterogeneity of
the motifs obscure their existence in transactivators but add
flexibility to the functions. It is now clear that hTAFII31
is just one of the many targets of activation domains, and other
targets may also recognize short, divergent peptide sequences. The
combinatorial presence of such motifs in a single activation domain
would render a highly mosaic and cryptic nature to the activation domain.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B p65 and that the
strength of the interactions correlates with the ability to activate
transcription (5-9, 17, 18). NMR and biochemical studies have shown
that the activation domains of VP16 and p53 undergo an induced
transition from random coil to 
-helix upon interaction with
hTAFII31, with key hydrophobic residues along one face of
the nascent helix (17, 18). The pattern of such hydrophobic residues,
FXX
(where X represents any residue and represents any hydrophobic residue) is conserved among the activation domains of VP16, p53, and NF-B p65, suggesting that this sequence represents a recognition element for hTAFII31.
for the transmission
of activation signals. This sequence characterization enabled the
identification of hTAFII31-binding sequences hidden in the
activation domains of NFAT1, ALL1, NF-IL6, ESX, and HSF-1. A
combination of mutational studies and NMR analyses indicated that the
interaction surfaces comprise short peptide regions containing
signature -helical motifs. Furthermore, the strength of the
interactions between these activators and hTAFII31 correlates with the ability to activate transcription in human cells,
supporting the notion that hTAFII31 and its homologs are important targets of eukaryotic transactivators.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
sequence of VP16-(469-485) (Phe479-Thr480-Asp481-Ala482-Leu483).
192 clones from each library were miniprepped and individually transfected into human Jurkat Tag cells in two 96-well plates (100 ng/well of expression plasmid and ~105 cells/well). The
reporter construct we used (120 ng/well) was pG5IL2SX in which the
secreted alkaline phosphatase
(SEAP)1 gene is controlled by
five GAL4-binding sites. After a 48-h incubation, each well was assayed
for SEAP activity through fluorescence change of 4-methylumbeliferyl
phosphate as described (19). Fluorescence measurements were carried out
by a microplate reader, Fluoroskan II (Labsystems). Positive clones
were characterized by DNA sequencing, and their activities were
quantitatively estimated through repeated transfection experiments in a
larger volume.
N(i, i + 1) NOEs,
although weak, were observed in the NOESY spectra of the free peptide,
which served as a basis for the complete sequential assignment. In the
NOESY spectra, 512 free induction decays were recorded at 290 or 300 K
with mixing times of 350 ms. The data were processed with the Felix
98.0 software (Biosym Technologies) with appropriate apodization and
zero-filling.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
sequence of
VP16-(469-485) (Phe479-Thr480-Asp481-Ala482-Leu483).
192 clones from each library were miniprepped and individually transfected into human Jurkat T cells in 96-well plates. We
cotransfected the cells with a reporter containing the SEAP gene under
the control of five GAL4-binding sites. This permits convenient
detection of transcriptional activation through fluorescence change of
a phosphatase substrate.
View larger version (26K):
[in a new window]
Fig. 1.
Divergent hTAFII31-binding
motifs. We constructed four small mutant libraries of a mammalian
expression vector of GAL4-VP16-(469-485), each of which consisted of
point mutants at one of the four positions within the
FXX sequence of VP16-(469-485)
(Phe479-Thr480-Asp481-Ala482-Leu483).
192 clones were miniprepped for each library and individually
transfected into human Jurkat T cells in a 96-well format. Each well
was assayed for reporter gene activity (SEAP) through fluorescence
change. Positive clones were characterized by DNA sequencing, and their
activities were quantitatively estimated through repeated transfection
experiments in a larger volume (upper). The mutants of Val
and Glu at Phe479; Pro at Thr480; Ser at
Ala482; and Ile, Val, and Pro at Leu483 are
shown as negative clones. The transcriptional level of mutants of Val
and Ile at Leu483 is restored to >50% of the wild type
when Ala482 is simultaneously replaced by a bulky
hydrophobic residue as shown at the bottom for
Ala482-Leu483. The DNAs encoding these mutants
were then subcloned into pGEX3X vectors, which provided GST fusions of
the mutants. It is evident that activation-positive mutants were able
to bind hTAFII31-(1-140), whereas activation-deficient
mutants had substantially lower affinity (lower).
-helix formation of their signature sequences. 15 candidates were eliminated by the second and third steps.
For example, the activation domain of cell cycle regulator E2F1 was
eliminated because its signature sequence, FSGLL, was not conserved in its chicken homolog
(FPGFL) and because these sequences had
little -helix probability. The activation domain of MSG1 was also
eliminated because its signature sequence, LMSLV,
was not conserved in a subtype of its mouse homolog
(LTSLE). As expected, the activation domains of E2F1 and MSG1 had no detectable affinity to
hTAFII31, validating our elimination steps (data not
shown). 11 activation domains survived all three elimination steps.
These are the activation domains of ALL1, NF-IL6, NFAT1, Sox-4, MyoD,
c-Jun, HIF-1, TEF-1, HSF-1, TREB5, and ESX (Fig.
2A). The peptide segments that
correspond to VP16-(469-485) were fused to GST and assayed for the
ability to bind hTAFII31. Only the peptides of ALL1,
NF-IL6, ESX, HSF-1, and NFAT1 bound hTAFII31 as tightly as
VP16-(469-485) (Fig. 2B).
View larger version (41K):
[in a new window]
Fig. 2.
Identification of short
hTAFII31-binding peptides in the activation domains.
A, sequence alignment of relevant regions in the activation
domains of p53, p65, and the 11 activators that survived all three
elimination steps. The signature sequences are highlighted.
B, in vitro binding assay. It is evident that GST
fusion proteins of the peptide segments of ALL1 (lane
3), NF-IL6 (lane 4), ESX
(lane 5), HSF-1 (lane 6),
and NFAT1 (lane 7) bind
hTAFII31-(1-140), whereas those of TEF-1 (lane
8), HIF-1 (lane 9), TREB5
(lane 10), Sox-4 (lane 11),
MyoD (lane 12), c-Jun (lanes
13 and 14), and GST alone (lane
15) have no detectable affinity to
hTAFII31-(1-140).
or
LAP) induces cytokine genes and has been implicated as a master
regulator of the acute-phase response (23). Its full-length activation
domain (amino acids 24-124) (24) bound hTAFII31-(1-140)
to the same extent as its peptide version. This interaction appears to
be mediated by the LSDLF sequence, since substitution of
Leu118 and Phe122 by Ala impairs the
interaction. The (L/F)(S/A)DLF sequence is conserved among the
activation domains of C/EBPs, and amino acid substitutions in the
conserved region in rC/EBP adversely affect its transactivation
potential (25, 26). HSF-1 (heat shock factor 1) responds a multitude of
stress conditions and plays an important role in the molecular response
to nonnative proteins (27), and its activation domain is known to be
highly potent (28). We found a hTAFII31-binding motif in
its COOH-terminal half, which was consistent with the previous
truncation studies (29). Whereas the full-length activation domain of
HSF-1 (amino acids 371-430) bound hTAFII31-(1-140) to the
same extent as its peptide version, mutation of Leu414 and
Phe418 with Ala impaired its interaction (Fig.
3A), indicative of direct involvement of the signature
sequence in the interaction with hTAFII31. Last, we
identified a hTAFII31-binding sequence in the activation
domain of ESX (an epithelium-restricted Ets factor) that regulates the
expression of the HER2/neu (c-erbB2) oncogene in
human breast cancer (30) and has been found to be overexpressed at an
early stage of human breast cancer development (31). Once again, the
ESX activation domain (amino acids 129-159) bound
hTAFII31-(1-140) as tightly as its peptide version.
Substitution of Ile139 and Leu143 by Ala
compromised this interaction, indicating that the interaction is
mediated at least in part by the IIELL sequence.
View larger version (43K):
[in a new window]
Fig. 3.
A, in vitro binding assay. It
is evident that GST fusions of the activation domains of ALL1
(lane 3), NF-IL6 (lane 5),
ESX (lane 7), HSF-1 (lane
9), and NFAT1 (lane 11) bind
hTAFII31-(1-140), whereas the glutamine-rich activation
domain of Sp1 (lane 13) and GST alone
(lane 14) have no affinity to
hTAFII31-(1-140). Substitutions of key hydrophobic
residues in the signature motifs greatly impair interactions
(lanes 4, 6, 8,
10, and 12). The position of
hTAFII31-(1-140) is indicated. B, activities of
the activation domains of ALL1, NF-IL6, ESX, HSF-1, NFAT1, and their
mutants in human cells when fused with the DNA-binding domain of GAL4.
The expression construct of each GAL4 fusion was transiently
transfected into human Jurkat T cells along with a reporter gene that
expresses SEAP under the control of five GAL4- binding sites. SEAP
activities were monitored by fluorescence.
View larger version (20K):
[in a new window]
Fig. 4.
A, truncation study. Deletion of the
NH2-terminal five residues from ALL1-(2839-2855) impaired
the interaction with hTAFII31-(1-140) (compare
lanes 2 and 3). B and
C, transferred NOE NMR experiments. Amide regions of 350-ms
NOESY spectra of ALL1-(2839-2855) (B) and NF-IL6-(108-124)
(~4 mM) (C) in the presence of
hTAFII31-(1-140) (240 µM). The identities of
residues that exhibit NOE cross-peaks are indicated. Sequential NOEs
characteristic of a helix formation are summarized in the
lower panels. For the NMR analysis of ALL1, the
peptide in which Cys2841 was substituted with Ser was used
for technical convenience. The GST pull-down experiment independently
verified that this substitution had no effect on the interaction with
hTAFII31-(1-140).
N(i, i + 3) and
d
(i, i + 3) NOEs
suggest the formation of short -helices encompassing the signature
motifs in ALL1-(2839-2855) and NF-IL6-(108-124). No TRNOEs were
observed with a control 17-amino acid peptide that has a similar
acidicity/hydrophobicity profile but no affinity to
hTAFII31, indicating that the interaction with
hTAFII31-(1-140) under the NMR condition is specific (data not shown). These results support the notion that the interactions between activation domains and hTAFII31 are mediated
generally by short -helices in the activators.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B p65 has been detected in TNF--induced cells (40). It is also known that the activation potential of rat
NF-IL6/LAP is directly enhanced by phosphorylation of the Ser residue
that is 13 amino acids away from the hTAFII31 motif (41).
Phosphorylation of nonconserved Ser residues within or adjacent to
hTAFII31-binding motifs may be a general strategy for
building dynamic and specific characters into the regulation of
transcription factors in higher eukaryotes.
| |
ACKNOWLEDGEMENTS |
|---|
We thank Xiaolian Gao for assistance in using the NMR instrument at the University of Houston, Florante A. Quiocho for generous instrumental support, Donghai Yu for technical assistance, and Jun Qin for mass analyses. We are also grateful to Tadamitsu Kishimoto, Gretchen Darlington, Eli Canaani, Anjana Rao, Akira Nakai, Toshi Shioda, and Wade Harper for plasmid constructs, Sheldon Park for helpful comments on the manuscript, and the members of the Wakil laboratory for encouragement and discussion.
| |
FOOTNOTES |
|---|
* This work was supported in part by research funds from Yoshitomi Pharmaceutical Industries and the Leukemia Society of America. The 600-MHz NMR spectrometer at the University of Houston is funded by the W. M. Keck Foundation.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.
The on-line version of this article (available at
http://www.jbc.org) contains one table.
To whom correspondence should be addressed. Fax: 713-798-1625;
E-mail: muesugi@bcm.tmc.edu.
| |
ABBREVIATIONS |
|---|
The abbreviations used are: SEAP, secreted alkaline phosphatase; GST, glutathione S-transferase; NOE, nuclear Overhauser effect; NOESY, NOE spectroscopy.
| |
REFERENCES |
|---|
|
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
|
|---|
| 1. | Tjian, R., and Maniatis, T. (1994) Cell 77, 5-8[CrossRef][Medline] [Order article via Infotrieve] |
| 2. | Radhakrishnan, I., Perez-Alvarado, G. C., Parker, D., Dyson, H. J., Montminy, M. R., and Wright, P. E. (1997) Cell 91, 741-752[CrossRef][Medline] [Order article via Infotrieve] |
| 3. | Ptashne, M., and Gann, A. (1997) Nature 386, 569-577[CrossRef][Medline] [Order article via Infotrieve] |
| 4. | Triezenberg, S. J. (1995) Curr. Opin. Genet. Dev. 5, 190-196[CrossRef][Medline] [Order article via Infotrieve] |
| 5. | Goodrich, J. A., Hoey, T., Thut, C. J., Admon, A., and Tjian, R. (1993) Cell 75, 519-530[CrossRef][Medline] [Order article via Infotrieve] |
| 6. |
Klemm, R. D.,
Goodrich, J. A.,
Zhou, S.,
and Tjian, R.
(1995)
Proc. Natl. Acad. Sci. U. S. A.
92,
5788-5792 |
| 7. | Burley, S. K., and Roeder, R. G. (1996) Annu. Rev. Biochem. 65, 769-799[CrossRef][Medline] [Order article via Infotrieve] |
| 8. |
Thut, C. J.,
Chen, J. L.,
Klemm, R.,
and Tjian, R.
(1995)
Science
267,
100-104 |
| 9. |
Lu, H.,
and Levine, A. J.
(1995)
Proc. Natl. Acad. Sci. U. S. A.
92,
5154-5158 |
| 10. | Holstege, F. C., Jennings, E. G., Wyrick, J. J., Lee, T. I., Hengartner, C. J., Green, M. R., Golub, T. R., Lander, E. S., and Young, R. A. (1998) Cell 95, 717-728[CrossRef][Medline] [Order article via Infotrieve] |
| 11. | Moqtaderi, Z., Keaveney, M., and Struhl, K. (1998) Mol. Cell 2, 675-682[CrossRef][Medline] [Order article via Infotrieve] |
| 12. | Michel, B., Komarnitsky, P., and Buratowski, S. (1998) Mol. Cell 2, 663-673[CrossRef][Medline] [Order article via Infotrieve] |
| 13. | Apone, L. M., Virbasius, C. A., Holstege, F. C., Wang, J., Young, R. A., and Green, M. R. (1998) Mol. Cell 2, 653-661[CrossRef][Medline] [Order article via Infotrieve] |
| 14. | Ogryzko, V. V., Kotani, T., Zhang, X., Schlitz, R. L., Howard, T., Yang, X. J., Howard, B. H., Qin, J., and Nakatani, Y. (1998) Cell 94, 35-44[CrossRef][Medline] [Order article via Infotrieve] |
| 15. | Grant, P. A., Schieltz, D., Pray-Grant, M. G., Steger, D. J., Reese, J. C., Yates, J. R., III, and Workman, J. L. (1998) Cell 94, 45-53[CrossRef][Medline] [Order article via Infotrieve] |
| 16. | Hahn, S. (1998) Cell 95, 579-582[CrossRef][Medline] [Order article via Infotrieve] |
| 17. |
Uesugi, M.,
and Verdine, G. L.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
14801-14806 |
| 18. |
Uesugi, M.,
Nyanguile, O.,
Lu, H.,
Levine, A. J.,
and Verdine, G. L.
(1997)
Science
277,
1310-1313 |
| 19. |
Belshaw, P. J.,
Ho, S. N.,
Crabtree, G. R.,
and Schreiber, S. L.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
4604-4607 |
| 20. | Tkachuk, D. C., Kohler, S., and Cleary, M. L. (1992) Cell 71, 691-700[CrossRef][Medline] [Order article via Infotrieve] |
| 21. | Gu, Y., Nakamura, T., Alder, H., Prasad, R., Canaani, O., Cimino, G., Croce, C. M., and Canaani, E. (1992) Cell 71, 701-708[CrossRef][Medline] [Order article via Infotrieve] |
| 22. |
Prasad, R.,
Yano, T.,
Sorio, C.,
Nakamura, T.,
Rallapalli, R.,
Gu, Y.,
Leshkowitz, D.,
Croce, C. M.,
and Canaani, E.
(1995)
Proc. Natl. Acad. Sci. U. S. A.
92,
12160-12164 |
| 23. | Akira, S., Isshiki, H., Sugita, T., Tanabe, O., Kinoshita, S., Nishio, Y., Nakajima, T., Hirano, T., and Kishimoto, T. (1990) EMBO J. 9, 1897-1906[Medline] [Order article via Infotrieve] |
| 24. |
Trautwein, C.,
Walker, D. L.,
Plumpe, J.,
and Manns, M. P.
(1995)
J. Biol. Chem.
270,
15130-15136 |
| 25. | Nerlov, C., and Ziff, E. B. (1995) EMBO J. 14, 4318-4328[Medline] [Order article via Infotrieve] |
| 26. | Williams, S. C., Baer, M., Dillner, A. J., and Johnson, P. F. (1995) EMBO J. 14, 3170-3183[Medline] [Order article via Infotrieve] |
| 27. |
Morimoto, R. I.
(1998)
Genes Dev.
12,
3788-3796 |
| 28. | Molinari, E., Gilman, M., and Natesan, S. (1999) EMBO J. 18, 6439-6447[CrossRef][Medline] [Order article via Infotrieve] |
| 29. | Newton, E. M., Knauf, U., Green, M., and Kingston, R. E. (1996) Mol. Cell. Biol. 16, 839-846[Abstract] |
| 30. |
Neve, R.,
Chang, C. H.,
Scott, G. K.,
Wong, A.,
Friis, R. R.,
Hynes, N. E.,
and Benz, C. C.
(1998)
FASEB J.
12,
1541-50 |
| 31. | Chang, C. H., Scott, G. K., Kuo, W. L., Xiong, X., Suzdaltseva, Y., Park, J. W., Sayre, P., Erny, K., Collins, C., Gray, J. W., and Benz, C. C. (1997) Oncogene 14, 1617-1622[CrossRef][Medline] [Order article via Infotrieve] |
| 32. | Campbell, A. P., and Sykes, B. D. (1993) Annu. Rev. Biophys. Biomol. Struct. 22, 99-122[Medline] [Order article via Infotrieve] |
| 33. | Frankel, A. D., and Kim, P. S. (1991) Cell 65, 717-719[CrossRef][Medline] [Order article via Infotrieve] |
| 34. |
Sauer, F.,
Hansen, S. K.,
and Tjian, R.
(1995)
Science
270,
1783-1788 |
| 35. |
Nguyen, J. T.,
Turck, C. W.,
Cohen, F. E.,
Zuckermann, R. N.,
and Lim, W. A.
(1998)
Science
282,
2088-2092 |
| 36. |
Chehab, N. H.,
Malikzay, A.,
Stavridi, E. S.,
and Halazonetis, T. D.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
13777-13782 |
| 37. |
Banin, S.,
Moyal, L.,
Shieh, S.,
Taya, Y.,
Anderson, C. W.,
Chessa, L.,
Smorodinsky, N. I.,
Prives, C.,
Reiss, Y.,
Shiloh, Y.,
and Ziv, Y.
(1998)
Science
281,
1674-1677 |
| 38. |
Canman, C. E.,
Lim, D. S.,
Cimprich, K. A.,
Taya, Y.,
Tamai, K.,
Sakaguchi, K.,
Appella, E.,
Kastan, M. B.,
and Siliciano, J. D.
(1998)
Science
281,
1677-1679 |
| 39. | Woo, R. A., McLure, K. G., Lees-Miller, S. P., Rancourt, D. E., and Lee, P. W. (1998) Nature 394, 700-704[CrossRef][Medline] [Order article via Infotrieve] |
| 40. |
Sakurai, H.,
Chiba, H.,
Miyoshi, H.,
Sugita, T.,
and Toriumi, W.
(1999)
J. Biol. Chem.
274,
30353-30356 |
| 41. | Trautwein, C., Caelles, C., van der Geer, P., Hunter, T., Karin, M., and Chojkier, M. (1993) Nature 364, 544-547[CrossRef][Medline] [Order article via Infotrieve] |
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
This article has been cited by other articles:
|
|
 |
|
  J. Rovnak and S. L. Quackenbush Walleye Dermal Sarcoma Virus Retroviral Cyclin Directly Contacts TAF9 J. Virol., December 15, 2006; 80(24): 12041 - 12048. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. H. Kim, C. K. Yang, and M. R. Stallcup Downstream signaling mechanism of the C-terminal activation domain of transcriptional coactivator CoCoA. Nucleic Acids Res., January 1, 2006; 34(9): 2736 - 2750. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Buss, A. Dorrie, M. L. Schmitz, E. Hoffmann, K. Resch, and M. Kracht Constitutive and Interleukin-1-inducible Phosphorylation of p65 NF-{kappa}B at Serine 536 Is Mediated by Multiple Protein Kinases Including I{kappa}B Kinase (IKK)-{alpha}, IKK{beta}, IKK{epsilon}, TRAF Family Member-associated (TANK)-binding Kinase 1 (TBK1), and an Unknown Kinase and Couples p65 to TATA-binding Protein-associated Factor II31-mediated Interleukin-8 Transcription J. Biol. Chem., December 31, 2004; 279(53): 55633 - 55643. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. D. Prescott, K. S. N. Koto, M. Singh, and A. Gutierrez-Hartmann The ETS Transcription Factor ESE-1 Transforms MCF-12A Human Mammary Epithelial Cells via a Novel Cytoplasmic Mechanism Mol. Cell. Biol., June 15, 2004; 24(12): 5548 - 5564. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. B. Hock and M. A. Brown Nuclear Factor of Activated T Cells 2 Transactivation in Mast Cells: A NOVEL ISOFORM-SPECIFIC TRANSACTIVATION DOMAIN CONFERS UNIQUE Fc{epsilon}RI RESPONSIVENESS J. Biol. Chem., July 11, 2003; 278(29): 26695 - 26703. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P.-A. Defossez and E. Gilson The vertebrate protein CTCF functions as an insulator in Saccharomyces cerevisiae Nucleic Acids Res., December 1, 2002; 30(23): 5136 - 5141. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Asada, Y. Choi, M. Yamada, S.-C. Wang, M.-C. Hung, J. Qin, and M. Uesugi External control of Her2 expression and cancer cell growth by targeting a Ras-linked coactivator PNAS, October 1, 2002; 99(20): 12747 - 12752. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| All ASBMB Journals | Molecular and Cellular Proteomics |
| Journal of Lipid Research | ASBMB Today |
