| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
J. Biol. Chem., Vol. 275, Issue 47, 37194-37201, November 24, 2000
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
From the Department of Biochemistry and Molecular Biology,
University of Kansas Medical Center,
Kansas City, Kansas 66160-7421
Received for publication, May 5, 2000, and in revised form, August 15, 2000
Metal response element-binding transcription
factor-1 (MTF-1) is a unique, zinc-inducible transcription factor that
binds to metal response elements in the metallothionein promoter and activates transcription in response to metals and oxidative stress. MTF-1 contains six zinc fingers of the
Cys2-His2 type. It was previously shown
that MTF-1 is reversibly activated to bind DNA in response to changes
in zinc status, unlike other zinc finger transcription factors, which
do not appear to be reversibly activated by zinc in the cellular
environment. Here we show that zinc fingers 2-4 constitute the core
DNA-binding domain, whereas fingers 5 and 6 appear to be unnecessary
for DNA binding in vitro. Deletion of finger 1 resulted in
a protein that bound DNA constitutively in vitro.
Furthermore, transfer of MTF-1 finger 1 to a position immediately
preceding the three zinc fingers of Sp1 resulted in a chimeric protein
that required exogenous zinc to activate DNA binding in
vitro, unlike native Sp1, which binds DNA constitutively. Transient transfection experiments demonstrated that intact MTF-1 activated a reporter 2.5-4-fold above basal levels after metal treatment in mouse MTF-1 knockout cells, Drosophila SL2
cells, and yeast. However, the metal response was lost in all three
systems when finger 1 was deleted, but was unaffected by deletion of
fingers 5 and 6. These data suggest that finger 1 of MTF-1 constitutes a unique metal-sensing domain that, in cooperation with the
transactivation domains, produces a zinc-sensing metalloregulatory
transcription factor.
Metallothioneins (MTs)1
constitute a conserved family of cysteine-rich heavy metal-binding
proteins (1). In the mouse, MT-I and MT-II display a wide tissue
distribution and have been demonstrated to participate in zinc
homeostasis (2), detoxification of cadmium (3, 4), and protection
against oxidative stress (5). MT gene transcription is induced
dramatically by heavy metals (especially zinc and cadmium) (6). Metal
response elements (MREs) are essential for metal induction, and there
are multiple copies of the MREs in the proximal promoters of MT genes.
MREs were initially shown to mediate the transcriptional response of MT
genes to metals (7) and, more recently, to participate in the
transcriptional response to oxidative stress (8, 9).
MRE-binding transcription factor-1 (MTF-1) was cloned (10, 11) and
found to be a six-zinc finger transcription factor in the
Cys2-His2 family. Targeted disruption of both
MTF-1 alleles in mouse embryonic stem cells demonstrated its essential
role for basal as well as heavy metal-induced MT gene expression (12). Unlike other zinc finger transcription factors, which appear to be
constitutively active to bind DNA under normal physiological conditions, MTF-1 is reversibly activated to bind DNA in response to
changes in zinc availability (9, 10, 13-16). This reversible activation involves direct interaction between zinc and the zinc finger
domain of MTF-1. Activation of MTF-1 in response to zinc is reminiscent
of copper activation of Ace1 in yeast. Ace1 binds to DNA in response to
excess copper and activates transcription of the yeast MT
(CRS5 and CUP) and superoxide dismutase
(SOD1) genes (17, 18). Copper activation of transcription in
yeast involves reversible binding of copper to the DNA-binding domain of Ace1.
Based on the observation that a single zinc finger interacts with 3 or
4 bases in DNA (19), it seems likely that the zinc fingers of MTF-1 are
not all required for binding to the 12-base pair consensus MRE sequence
(7). Chen et al. (20) reported that there is structural
heterogeneity in the zinc-binding sites in the isolated MTF-1 zinc
finger domain, and they detected at least two classes of zinc-binding
sites. Their observations indicated that there are three or four high
affinity zinc-binding fingers and that the remainder of the fingers
have a much lower affinity for zinc. However, in their experimental
system, no change in the DNA binding of the purified MTF-1 zinc finger
domain was detectable when three zinc molecules were bound compared
with six.
Multiple zinc fingers are often arranged in close proximity to each
other, suggesting the potential for cooperative binding, which might,
in turn, produce a metalloregulatory function inherent in zinc finger
domains. Krizek et al. (21) examined the metal-binding characteristics of a peptide with two consensus zinc finger domains and
found no evidence for cooperativity. They concluded that cooperative metal binding is not an inherent property of tandem zinc finger domains, but that specially evolved zinc fingers might still act as
mechanisms sensing changes in free zinc concentration. Several lines of
evidence suggest that MTF-1 may function as a metalloregulatory protein
serving as an intracellular sensor of free zinc. First, treatment of
mammalian cells with zinc in vivo has been reported to cause
a rapid, dramatic increase in the DNA-binding activity of MTF-1
measured in vitro (14, 16). Second, zinc causes nuclear translocation of MTF-1 (22) and the concomitant occupancy of MREs in
the MT promoter in vivo (9). Third, MTF-1 is more sensitive to metal chelators than are other zinc finger transcription
factors (10, 14). Fourth, the DNA-binding activity of native and
recombinant MTF-1 can be reversibly modulated by zinc (12, 14, 23); and
fifth, mouse MTF-1 can function as a zinc sensor in
yeast.2 The studies presented
herein examined the roles that individual zinc fingers play in
zinc-sensing, DNA-binding, and gene activation. The data indicate that
three of the zinc fingers constitute the core DNA-binding domain, that
two fingers have an unknown function, and that a single finger controls
zinc-activated DNA binding.
Cell Culture and Treatment--
Mouse dko7
(MTF-1 Preparation of Whole Cell Extracts--
Whole cell extracts were
prepared as described previously (26).
Nuclear and Cytosolic Extracts--
Nuclear and cytosolic
extracts were prepared using NE-PER nuclear and cytoplasmic
extraction reagent (Pierce) according to the manufacturer's suggestions.
Immunoblotting--
Cell extracts (50-100 µg of protein) were
separated by 10 or 12% SDS-polyacrylamide gel electrophoresis (27)
under reduced conditions and transferred to nitrocellulose membranes.
The preparation and development of the chemiluminescent signal were as
described previously (22). Relative band intensities were quantitated using Biomax 1D image analysis software (Kodak Scientific Imaging Systems). Equal protein loading and transfer were verified visually by
staining membranes with Ponceau solution.
In Vitro Transcription/Translation--
The cDNA clones
encoding mouse MTF-1 and human Sp1 were previously described (14, 15).
Recombinant and chimeric proteins were synthesized in vitro
using a TnT coupled reticulocyte lysate system (Promega, Madison, WI)
containing 1 µg of the plasmid and Sp6 RNA polymerase according to
the manufacturer's instructions (15).
Electrophoretic Mobility Shift Assays (EMSAs)--
EMSA, using 1 µl of a TnT lysate and MRE and/or Sp1 oligonucleotide, was
carried out as described in detail previously (14, 15).
Plasmid Preparation--
DNA sequences coding for chimeric
proteins were constructed by polymerase chain reaction amplification of
individual domains and ligating domains together using endogenous
restriction sites or sites introduced by altering nucleotides at
redundant codon positions. The resulting chimeric proteins (Fig.
1A) retained the exact amino
acid sequences of the original domains.
MTF-1 deletion constructs were prepared by amplifying MTF-1 in two
components containing the 5' and 3' termini of the coding sequence.
Fingers were deleted from the first cysteine of the deleted finger to
the amino acid preceding the first cysteine of the next finger. The two
amplified fragments were ligated using an endogenous KpnI
site for deletion of finger 1, fingers 1 and 2, and fingers 1-3 and a
BspHI site for deletion of finger 6, fingers 5 and 6, and
fingers 4-6. This allowed the amino acid sequence to be maintained
exactly as that of full-length MTF-1 with the exception of the
indicated deletion. All constructs were verified by sequencing at the
Biotechnology Support Facility of the University of Kansas Medical Center.
F1Sp1 (Fig. 1B) was constructed by polymerase chain reaction
amplification of the Sp1 5' terminus with the addition of a
KpnI site preceding the zinc finger coding sequence. The
KpnI site was added by modifying the redundant nucleotides
without altering the resulting amino acid sequence. The 3' terminus of
Sp1, including the zinc finger region, was amplified with the addition
of an EcoRI site to the 5'-end. MTF-1 finger 1 was amplified
by polymerase chain reaction and included an endogenous
KpnI site on the 5'-side and an added
EcoRI site on the 3'-side. To introduce the EcoRI site, a phenylalanine codon was added between MTF-1 finger 1 and the
zinc fingers of Sp1. This resulted in seven amino acids between MTF-1
finger 1 and the first zinc finger of Sp1. The first three amino acids
following MTF-1 finger 1 were from MTF-1, followed by phenylalanine and
then the three amino acids preceding Sp1 finger 1. This is the same
spacing relationship as that between fingers 1 and 2 in MTF-1 and in
Sp1. The three DNA fragments were ligated into pGem7 and expressed from
the Sp6 promoter in a TnT lysate reaction. Sequences expressed
in Drosophila SL2 cells were inserted into the multicloning
site of the pAC5/V5-His vector (Invitrogen, Carlsbad, CA), which
utilizes the Drosophila actin promoter to drive expression.
The yeast expression vector pVT101u:MTF-1 was engineered for episomal
expression of mouse MTF-1 cDNA and deletions of MTF-1 under the
control of the yeast ADH promoter and for selection for
URA3 auxotrophy. The plasmid pVT101u was as described (28). The yeast reporter gene vector pYEp363:MREd5- Transfection--
LipofectAMINE (Life Technologies,
Inc.)-mediated transfections were performed according to the
manufacturer's suggestions. Briefly, dko7
(MTF-1
SL2 cells were transfected using the calcium phosphate transfection
system kit (Life Technologies, Inc.) according to the manufacturer's
suggestions. Briefly, cells were plated at a density of 1 × 106 cells/ml in 6-well plates. The following morning, the
DNA/calcium phosphate suspension was prepared as recommended in a
volume equivalent to 200 µl/well. Each 200-µl aliquot contained 5 ng of pAC-MTF-1, 5 µg of MREd5-luciferase, and 5 µg of
CMV-
Yeast transfections were as described elsewhere.2 ZHy6
cells were cotransformed using the lithium acetate procedure (30) and
selected for growth in uracil and leucine dropout Complete Medium. Transformed colonies were selected and grown overnight in
dropout medium, and glycerol stocks were prepared and frozen at
Comparison of the Concentrations of Exogenous Zinc Required to
Activate DNA Binding by MTF-1 and Sp1--
MTF-1 in whole cell
extracts from control cells and recombinant MTF-1 synthesized in
vitro require micromolar concentrations of exogenous zinc to
activate DNA binding (14, 15, 23, 31). To make a more direct comparison
between the zinc concentrations required to activate DNA binding by
MTF-1 and Sp1, both constructs were simultaneously transcribed and
translated in the same TnT reaction, and their individual DNA-binding
activities were detected by EMSA. Most of the DNA-binding activity of
Sp1 (~60%) was activated in the TnT lysate and did not require the
addition of exogenous zinc to the EMSA reaction (Fig.
2, lanes 2, 4, and
5). In contrast, without exogenous zinc, there was little
detectable (<5%) MTF-1 DNA-binding activity (Fig. 2, lanes
4 and 5). Furthermore, if zinc was added to the
reaction, but the reaction was kept on ice, very little activation of
MTF-1 occurred, whereas there was a significant Sp1 complex formed
(Fig. 2, lane 8). Thus, MTF-1 requires exogenous zinc and an
increase in temperature for activation of DNA binding to occur (Fig. 2,
lanes 1 and 3).
In Vivo and in Vitro Analysis of Chimeric Proteins--
Previous
studies support the concept that zinc induction of DNA binding is
contained within the zinc fingers of MTF-1 (14, 15, 20). To further
examine the roles of the zinc fingers in zinc activation, the zinc
fingers of Sp1 and MTF-1 were exchanged to produce chimeric proteins
(Fig. 1A). The Sp1 peptide backbone with the MTF-1 zinc
fingers is termed SMS, whereas the MTF-1 peptide backbone with the Sp1
zinc fingers is called MSM. The chimeric DNA constructs were prepared
for transcription and translation in TnT lysates and for expression in
dko7 (MTF-1
The intramolecular and intermolecular interactions that combine to
induce transcription in a nucleus cannot be assessed merely by EMSA.
Therefore, to test the in vivo function of the chimeric protein, the SMS construct was transfected into dko7
(MTF-1 In Vitro Analysis of Zinc Finger Deletions--
To examine the
contribution of the individual MTF-1 zinc fingers to DNA binding and
zinc response, finger deletion constructs were made. Surprisingly,
deletion of finger 1 (D1) led to the constitutive DNA-binding activity
of MTF-1 (Fig. 4A, lanes
2 and 3). In the absence of finger 1, most of the DNA
binding (~60%) was present without the addition of exogenous
zinc.
Deletion of fingers 1 and 2 (D1,2) produced a peptide that bound MREs
poorly (Fig. 4A, lanes 5 and
6). Deletion of fingers 1-3 (D1,2,3) resulted in a peptide
with no detectable DNA-binding activity (Fig. 4A,
lanes 7 and 8). Similarly, deletion of
fingers 4-6 (D4,5,6) resulted in the drastic reduction of DNA binding, although a small zinc-inducible MTF-1·MRE complex was detectable by
EMSA (Fig. 4A, lanes 9 and
10). The losses of DNA binding were not the result of poor
translation of the deletion constructs, as each TnT lysate reaction
produced similar amounts of immunoreactive peptide (Fig.
4B).
Deletion of finger 6 (D6) or of both fingers 5 and 6 (D5,6) had little
detectable effect on DNA-binding activity in an EMSA reaction (Fig.
4A, lanes 11-14). The resulting
peptide required concentrations of exogenous zinc to activate DNA
binding that were similar to concentrations required to activate the
native protein. The intensities of the bands detected by immunoblotting of lysates containing native MTF-1, D5,6, or D6 were similar (Fig. 4B), as were the intensities of the respective MTF-1·MRE
complex in EMSA reactions.
Expression of MTF-1, D1, and D5,6 in Mouse dko7
(MTF-1
MTF-1, D1, and D5,6 were transfected into dko7 cells using conditions
that were optimal for detecting changes in expression of the MREd5
reporter in response to zinc treatment (22). In cells transfected with
MTF-1 or D5,6, expression from the reporter increased ~2-fold when
the medium was adjusted to 100 µM zinc or 10 µM cadmium (Fig. 5C). In contrast, in cells
transfected with D1, there was a 3-fold increase in basal level
reporter activity compared with cells transfected with reporter alone,
but no change in reporter activity after zinc or cadmium treatment of
the cells (Fig. 5C).
Expression of MTF-1, D1, and D5,6 in Drosophila SL2 Cells--
We
examined the behavior of MTF-1 in Drosophila SL2 cells to
determine if MTF-1 could function as a zinc-activated transcription factor in a heterologous system and to further examine the effects of
finger deletions. SL2 cells transfected with pAC-MTF-1 produced a
protein recognized by the mouse anti-MTF-1 antibody that was of the
expected size (Fig. 6A).
Drosophila expression vectors encoding MTF-1, D5,6, or D1
were cotransfected with the MREd5 reporter into Drosophila
SL2 cells. This resulted in an increase in reporter activity compared
with that found in cells transfected with the reporter alone. Zinc
treatment (1 mM) of SL2 cells transfected with MTF-1 or
D5,6 resulted in an ~3.5- or 2.5-fold increase in reporter activity,
respectively (Fig. 6B). In contrast, luciferase activity was
unaffected after zinc treatment of cells transfected with D1 (Fig.
6B), in agreement with the results from dko7 transfections (Fig. 5C).
Expression of MTF-1, D1, and D5,6 in Yeast--
The function of
the MTF-1 finger deletion mutants was further examined in S. cerevisiae. We have shown that mouse MTF-1 can function as a zinc
sensor in yeast.2 Immunoblotting demonstrated that D1 and
D5,6 were produced in three independently selected colonies of yeast
transformed with pVT-D1 or pVT-D5,6 and the MREd5 expression vector
(Fig. 7A). Cells expressing D1
consistently contained lower levels of immunoreactive protein
than did cells expressing D5,6 (Fig. 7A) or MTF-1. There was
a rapid increase in
Six independently selected colonies expressing either D5,6 or D1 were
examined for their response to zinc. The MTF-1 Finger 1 Transferred in Front of the Sp1 Zinc Fingers Confers
Zinc-inducible DNA Binding in Vitro--
The experiments described
above indicated that finger 1 was responsible for the zinc-activated
DNA binding of MTF-1. Whether this function was transferable to a
constitutive DNA-binding zinc finger domain, such as that in Sp1, was
examined. The coding sequence for finger 1 was transferred from MTF-1
to a position immediately preceding finger 1 of Sp1 (F1Sp1) (Fig.
1B). The chimeric protein was expressed in the TnT reaction
and analyzed by EMSA (Fig.
8A). In contrast to the
constitutive DNA binding of Sp1, F1Sp1 had little detectable
DNA-binding activity when zinc was not added to the EMSA reaction (Fig.
8A). However, the addition of 1 µM zinc and
raising the temperature to 37 °C caused a dramatic increase in F1Sp1
DNA-binding activity. MTF-1 behaved similarly to F1Sp1 under these
experimental conditions (Fig. 8A). Immunoblotting using an
anti-Sp1 antibody demonstrated that similar amounts of protein were
made in the Sp1 and F1Sp1 reactions (Fig. 8B). A protein
extract from mouse Hepa cells was included as a reference (Fig.
8B, Hepa).
To examine the in vivo function of F1Sp1, a
Drosophila expression construct encoding F1Sp1 was
transfected into Drosophila SL2 cells, which are devoid of
Sp1-like DNA-binding activity. Although a 5-10-fold increase in the
basal activity of the Sp1 reporter was detected when either F1Sp1 or
Sp1 was transfected into these cells, zinc (1 mM) had no
detectable effect on Sp1 reporter expression (data not shown). Neither
pVT-F1Sp1 nor pVT-SMS were zinc-responsive in SL2 cells, even though
both demonstrated zinc-activated DNA binding in vitro.
Furthermore, it was noted that the F1Sp1 zinc finger cassette in the
context of the MTF-1 protein backbone (F1MSM) (Fig. 1C) was
also not zinc-responsive in SL2 cells (data not shown).
In mammals, zinc homeostasis appears to be maintained, in part, by
zinc-dependent activation of the transcription factor
MTF-1, which, in turn, modulates gene expression. The MT genes are
activated by MTF-1, and MTs chelate zinc and provide a labile pool of
zinc during periods of zinc deficiency (2, 32). MTF-1 also regulates the expression of other homeostatic genes such as ZnT1
(zinc transporter 1),
which functions to remove excess zinc from the
cell.3
The mechanisms by which MTF-1 senses zinc and subsequently activates
gene expression are not well understood. The zinc finger domain of
MTF-1 appears to be essential for zinc activation of DNA binding, and
this likely reflects heterogeneity in the zinc-binding properties of
the zinc fingers. Chen et al. (20) have described at least
two classes of zinc-binding sites using the purified zinc finger domain
of MTF-1. Three or four high affinity binding sites were detected, as
were two or three low affinity zinc-binding sites. The evidence
presented here suggests that three fingers (fingers 2-4) are essential
for DNA binding and that these fingers display constitutive DNA-binding
activity in the absence of finger 1. This suggests that these fingers
represent the high affinity zinc-binding sites in MTF-1 observed by
Chen et al. (20). This, in turn, is consistent with the fact
that MTF-1 interacts specifically with a 12-base pair MRE-binding site,
which would be expected to require three zinc fingers.
Remarkably, zinc finger 1 of MTF-1 was found to mediate zinc activation
of the DNA-binding activity of adjacent zinc fingers of MTF-1 or Sp1
and to be essential for zinc activation of gene expression by MTF-1.
This suggests that finger 1 represents a low affinity zinc-binding site
that, in the absence of zinc, can actually mask the constitutive
DNA-binding activity of adjacent zinc fingers. The mechanisms by which
finger 1 senses zinc and masks DNA binding are unclear. The length of
finger 1 and the placement of the cysteine and histidine residues are
the same as those of each of the other zinc fingers in MTF-1. However, finger 1 is significantly different in primary amino acid sequence relative to the other fingers in MTF-1. In addition, four of the seven
residues in the linker between fingers 1 and 2 are unique compared with
the residues in those positions in the other linking sequences (the
linker region between zinc fingers has been shown to modulate the
folding of the adjacent finger (33)). Presumably, these differences in
sequence influence the affinity of finger 1 for zinc, resulting in a
finger with unique zinc-binding characteristics. Alternatively, these
residues may influence the interaction of finger 1 with the other
fingers, thus affecting the zinc response.
The majority of MTF-1 in control cells is found in the cytoplasm, but
it is re-localized to the nucleus after zinc treatment (22). Although
deletion of finger 1 could have potentially inhibited accumulation of
MTF-1 in the nucleus, thus attenuating the zinc response, transient
transfection experiments demonstrated that D1 was recovered primarily
from the nuclear fraction of transfected cells and was competent to
bind to DNA. Therefore, deletion of finger 1 does not interfere with
nuclear localization.
In the course of these experiments, it was noted that fingers 5 and 6 of MTF-1 are dispensable for DNA binding in vitro and for
zinc activation of gene expression in vivo. These findings contradict a recent report (34) that suggested that fingers 5 and 6 are
low affinity zinc-binding sites that are essential for the DNA-binding
affinity and specificity of MTF-1. The reason for this discrepancy is
unclear. However, in those studies, the zinc finger domain, in
isolation from surrounding amino acids, was analyzed for in
vitro activity. Thus, it is conceivable that the functions
(in vitro and in vivo) of these zinc fingers are not accurately reflected when the isolated finger domain is studied. Nevertheless, the functions of fingers 5 and 6 remain unclear. The high
degree of conservation of the amino acid sequence between fingers 5 and
6 of mouse and human (10, 35) and fish (Fugu rubripes) (36) suggests that these fingers have an important function. Thus, it seems likely that transfection assays and in vitro DNA binding assays do not measure all functions of MTF-1. In
addition to protein-DNA interactions, zinc fingers have been shown to
participate in protein-protein interactions (37). Perhaps, fingers 5 and 6 are important for intermolecular interactions between MTF-1 and coactivators.
Cadmium is a potent inducer of MT, and MTF-1 is required for metal
activation of MT gene transcription (38). However, cadmium does not
cause measurable changes in the DNA binding of MTF-1 in
vitro (15, 16) and does so only at high concentration in vivo (22). Mutational analysis demonstrated that loss of zinc activation in vitro and in vivo was accompanied
by a loss of cadmium induction in vivo. This finding is
consistent with the concept that cadmium might displace zinc in
vivo, making it available for activation of MTF-1 (39). Cadmium
activation of MT gene expression may require only a small change in the
amount of activated MTF-1 in the nucleus in combination with other
components, e.g. protein modification or a coactivator.
These questions remain to be resolved.
Intramolecular interactions are clearly important for MTF-1 function
(24). Substitution of the Sp1 zinc fingers or transactivation domains
for their counterparts in MTF-1 resulted in a chimeric protein that was
not zinc-responsive in vivo. Therefore, both the zinc
fingers and the transactivation domains of MTF-1 are necessary for
efficient zinc response in vivo. Activation of DNA binding
by MTF-1 is not sufficient for zinc activation of MT transcription. MTF-1 is a complex protein with multiple domains that may have an
equally complicated folded structure. The chimeric proteins used in
these studies may not be able to achieve a structure capable of
responding to zinc treatment in vivo.
In summary, our studies suggest that within the context of an intact
protein, finger 1 is necessary for the zinc activation of DNA binding
by MTF-1 as measured in vitro and for an efficient response
to zinc and cadmium in vivo. Furthermore, fingers 2-4 appear to be the core DNA-binding fingers. Our studies indicate that
there is an intricate interplay between the zinc fingers and other
domains within MTF-1 that is required for an efficient response to
increased zinc concentrations in vivo. The function of
fingers 5 and 6 is unknown. These two fingers apparently behave differently in their interaction with zinc compared with the other fingers, and our data indicate that they do not play a significant role
in the zinc or cadmium response, either in vitro or in
vivo, at least under these experimental conditions. Studies
examining protein-protein interactions and modification of MTF-1 in
response to inducers may help resolve these questions.
We are indebted to Jim Geiser and Steve
Eklund for excellent technical assistance.
*
This work was supported in part by National Institutes of
Health Grant ES05704 (to G. K. A.).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.
Published, JBC Papers in Press, August 24, 2000, DOI 10.1074/jbc.M003863200
2
G. K. Andrews, D. Bittel, I. Smirnova, R. Ravindra, and D. Winge, submitted for publication.
3
Langmade, J., Ravindra, R., and Andrews, G. K. J. Biol. Chem. 275, in press.
The abbreviations used are:
MT, metallothionein;
MRE, metal response element;
MREd, metal response element "d" from
the mouse MT-I promoter;
MTF-1, MRE-binding transcription factor-1;
EMSA, electrophoretic mobility shift assay;
CMV, cytomegalovirus.
Functional Heterogeneity in the Zinc Fingers of
Metalloregulatory Protein Metal Response Element-binding
Transcription Factor-1*
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/) cells were maintained in high
glucose Dulbecco's modified Eagle's medium supplemented with 10%
fetal bovine serum. The mouse dko7 cell line is a simian virus 40 large
T-antigen-immortalized fibroblast line derived from embryonic stem
cells lacking MTF-1 (MTF-1 double knockout) and was a generous gift of
Dr. Walter Schaffner (University of Zurich, Zurich, Switzerland) (24). Drosophila SL2 cells were maintained in Schneider's
Drosophila medium supplemented with 10% fetal bovine serum.
The culture of Saccharomyces cerevisiae was as described
elsewhere.2 S. cerevisiae strain ZHy6 (25) was
obtained from Dr. David Eide (University of Missouri at Columbia).
ZnSO4 and CdCl2 were dissolved in acidified
H2O as 1000-fold concentrated stock solutions.
View larger version (26K):
[in a new window]
Fig. 1.
Diagram of chimeric proteins.
A, the zinc fingers of MTF-1 and Sp1 were switched without
gain or loss of amino acids (AA) in the backbone of each.
The domains that were exchanged encompass the first cysteine of finger
1 to the last histidine of the final finger. The Sp1 peptide backbone
with the MTF-1 finger cassette is termed SMS, whereas the MTF-1 peptide
backbone with the Sp1 finger cassette is called MSM. B, the
F1Sp1 fusion construct contains the Sp1 amino acid sequence to lysine
632, followed by 29 residues encompassing finger 1 of MTF-1, including
the three amino acids immediately preceding the first cysteine and the
three amino acids following the last histidine. C, the Sp1
zinc fingers were inserted following finger 1 of MTF-1, replacing MTF-1
zinc fingers 2-6. The amino acid sequence at the fusion of finger 1 and Sp1 finger 1 was the same as in F1Sp1. Sp1 finger 3 ended with the
last histidine of the finger and was followed by aspartate, which is
the first amino acid following the final histidine of finger 6 of
MTF-1.
Gal was engineered to
express -galactosidase under the control of five tandem copies of
mouse MREd, as described (8), and for LEU2 auxotrophy.
/) cells were transfected and assayed
for reporter gene expression as described (22, 29).
-galactosidase. The DNA mixture was incubated for 20 min at room
temperature and added to the well. The following morning, the medium
was replaced, and the cells were treated with metal in the afternoon.
The next morning, the cells were harvested, and luciferase and
-galactosidase activities were quantified.
80 °C. Experiments were initiated using colonies of freshly plated
cells from the glycerol stocks.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (58K):
[in a new window]
Fig. 2.
Comparison of the zinc requirement for
in vitro activation of DNA-binding by MTF-1 and
Sp1. A, MTF-1 and Sp1 were cotranslated in a coupled
transcription/translation (TnT) reaction. EMSA reactions using 1 µl
of TnT lysate were carried out at different zinc concentrations and
temperatures as described under "Experimental Procedures."
Lanes 1 and 2 contained only the MRE-
or Sp1-binding sites, respectively. Lanes 3-8 (labeled
M/S) contained both binding sites. Arrows
indicate the specific complex. B, the band intensities of
the EMSA in A were quantified using a phosphorimaging system
as described under "Experimental Procedures." Each bar
represents the mean ± S.E. of three EMSA bands.
/) cells. When expressed in a
TnT lysate, there was a low, but detectable level of MRE binding by SMS
(Fig. 1) without exogenous zinc being added to the EMSA (Fig.
3, lane 3). However, there was
a dramatic increase in binding after zinc was added (Fig. 3,
lane 4). As expected, this chimeric protein did
not bind to the Sp1-binding site (Fig. 3, lanes 5 and 6). In contrast, MSM (Fig. 1) bound to the Sp1-binding
site without the addition of exogenous zinc (Fig. 3, lane
9), and only a small increase in DNA binding occurred after
exogenous zinc was added (lane 10). The Sp1
oligonucleotide contains two Sp1-binding sites, which likely accounts
for the second EMSA band detected after the addition of exogenous zinc
(Fig. 3, lane 10).
View larger version (47K):
[in a new window]
Fig. 3.
In vitro and in vivo
behavior of chimeric proteins. A, full-length
MTF-1 and Sp1 and chimeric SMS and MSM (Fig. 1) constructs were used to
program TnT lysates. EMSA reactions containing 1 µl of the lysate
treated with 30 µM zinc were warmed to 15 °C for 15 min and returned to ice as described under "Experimental
Procedures." Reactions not treated with zinc were kept on ice.
Arrows indicate the specific complex. Oligo,
oligonucleotide. B, MTF-1 and chimeric SMS constructs were
cotransfected into dko7 (MTF-1 /) cells with
the MREd5 reporter and a CMV--galactosidase vector to normalize for
transfection efficiency. Transfected cells were treated with 60 µM zinc or 6 µM cadmium overnight,
harvested, and stored at 80 °C until luciferase and
-galactosidase were quantified. The -fold change is relative to the
control, which was transfected with the reporter construct, but not the
expression vector. Each bar represents the mean ± S.E.
of normalized luciferase counts from at least three transfection
experiments.
/) cells along with a luciferase
reporter construct driven by five copies of MREd. Transfection with SMS
caused an increase in basal luciferase activity with increasing DNA
concentration. However, there was no significant increase in luciferase
activity in response to added zinc or cadmium (Fig. 3B). In
contrast, cells transfected with MTF-1 displayed an increased basal
luciferase activity compared with cells transfected only with reporter.
The luciferase activity in extracts from cells transfected with MTF-1
was increased up to 2.5-fold in response to zinc or cadmium (Fig.
3B).
View larger version (70K):
[in a new window]
Fig. 4.
Analysis of the DNA binding of MTF-1 zinc
finger deletion constructs. A, EMSA reactions
containing 1 µl of the indicated TnT lysate were brought to 30 µM zinc, and the reactions were carried out as indicated
under "Experimental Procedures." The arrow indicates the
specific complex. B, MTF-1 was detected with the anti-MTF-1
polyclonal antibody as described under "Experimental Procedures." 1 µl of the TnT lysate used for EMSA in A was loaded onto
each lane of a 10% SDS-polyacrylamide gel.
/) Cells--
The effects of finger deletions on
the in vivo function of MTF-1 were examined by transient
transfection of mouse cells as well as insect cells and yeast (see
below). D1 and D5,6 in a CMV expression vector were transfected into
dko7 (MTF-1/) cells. A putative nuclear
localization sequence just before finger 1 could have been affected by
the deletion of finger 1, disturbing correct nuclear trafficking.
Therefore, proteins were isolated from nuclear and cytoplasmic
fractions of dko7 cells after transfection with MTF-1 or D1 to verify
that the proteins could be transported to the nucleus. Both MTF-1 and
D1 were detected in the nuclear fraction before and after zinc
treatment (Fig. 5A). However,
before zinc treatment, the majority of MTF-1 was found in the
cytoplasmic fraction, as previously reported (22). In contrast, the
majority of D1 was present in the nucleus in control cells as well as
in zinc-treated cells (Fig. 5A). The nuclear fractions from
these transfected cells were also analyzed by EMSA (Fig.
5B). Extracts from non-transfected dko7 cells contain no
detectable MTF-1 (14). In contrast, nuclear extracts from dko7 cells
transfected with CMV-MTF-1 or CMV-D1 contained a protein that could
bind MREs (Fig. 5B).
View larger version (41K):
[in a new window]
Fig. 5.
Function of MTF-1, D1, and D5,6 in
transiently transfected dko7
(MTF-1 /)
cells. A, shown is an immunoblot of nuclear (nuc)
and cytosolic (cyto) extracts made from dko7 cells
transfected with MTF-1 or D1 and treated with 100 µM zinc
for 1 h as described under "Experimental Procedures." 100 µg
of protein was separated by 10% SDS-polyacrylamide gel electrophoresis
and detected with anti-MTF-1 antibody. The arrow indicates
the position of the 98-kDa molecular mass marker. B, EMSA
was carried out as described under "Experimental Procedures" using
20 µg of protein from nuclear extracts from dko7 cells transfected
with MTF-1 or D1 and treated with 100 µM zinc for 1 h. The arrow indicates the specific complex. C,
shown is the luciferase activity in extracts from dko7 cells
transfected with MTF-1, D1, or D5,6 as described under "Experimental
Procedures." Cells were treated with 60 µM zinc or 6 µM cadmium as indicated, harvested the next morning, and
stored at 80 °C until luciferase and -galactosidase activities
were determined. Luciferase activity was normalized to
-galactosidase activity to account for variations in transfection
efficiency. Each bar represents the mean ± S.E. of
three experiments.
View larger version (17K):
[in a new window]
Fig. 6.
Function of MTF-1, D1, and D5,6 in
transiently transfected Drosophila SL2 cells.
A, immunoblot using 100 µg of protein from extracts from
Drosophila SL2 cells transfected with MTF-1 as described
under "Experimental Procedures." Each well received 250 ng
(Low) or 2 µg (High) of MTF-1 as indicated. The
zinc-treated cells were exposed to 100 µM zinc for 1 h prior to harvest. The Mock lane was loaded with protein
from SL2 cells not transfected with MTF-1, and TnT lane
contained recombinant MTF-1 produced in a TnT lysate. The
arrow indicates the position of the 98-kDa molecular mass
marker. B, luciferase activity in SL2 cells transfected with
MREd5-luciferase, CMV- -galactosidase, and pAC-MTF-1, D1, or D5,6 in
6-well plates as described under "Experimental Procedures." Cells
were treated with 1 mM zinc overnight, as indicated, prior
to harvest. Luciferase activity was normalized to -galactosidase
activity to account for variations in transfection efficiency. Each
bar represents the mean ± S.E. of at least three
transfection experiments.
-galactosidase mRNA after the addition of
zinc in cells expressing MTF-1 or D5,6 (4- and 3-fold, respectively) (Fig. 7B). The change in mRNA was observed 1 h
after the addition of zinc, indicating a rapid transcriptional response
(Fig. 7B). In contrast, there was no change in the level of
-galactosidase mRNA after the addition of zinc in cells
expressing D1.
View larger version (58K):
[in a new window]
Fig. 7.
Function of MTF-1, D1, or D5,6 in yeast.
A, proteins extracted from three independent colonies
of yeast strain ZHy6 transformed with D1 or D5,6 were separated by 10%
SDS-polyacrylamide gel electrophoresis. Specific bands were detected by
anti-MTF-1 antibody. Recombinant MTF-1 synthesized in a TnT lysate was
used as a positive control in the first lane. The ZHy6
lane contained protein from the non-transfected parental yeast
strain. The arrow indicates the position of the
98-kDa molecular mass marker. B, shown is a Northern blot
probed with an antisense -galactosidase riboprobe (as described
under "Experimental Procedures.") using 3 µg of total RNA
extracted from yeast strains cotransfected with MTF-1, D1, or D5,6 plus
an MREd5--galactosidase reporter. Cells were grown overnight in
medium containing 2 µM zinc and brought to 60 µM zinc as indicated for 1 h. Arrows
indicate the presence of two -galactosidase
(-gal) transcripts. C, independent
colonies transfected with D1 or D5,6 were grown overnight in 2 µM zinc and then brought to 60 µM zinc for
8 h before harvest. -Galactosidase is expressed as
units/min/µg of protein. Note that the axes are different.
The basal -galactosidase activities in cells expressing D1 and D5,6
were ~3 and 10 times that of the background -galactosidase
activity in cells transfected with reporter only. Each bar
represents the mean ± S.E. of three experiments.
-galactosidase activity in
cells expressing D5,6 increased significantly (up to 3-fold) by 8 h after the addition of zinc to the medium (Fig. 7C),
similar to cells expressing MTF-1. In contrast, the -galactosidase activity in cells expressing D1 was unchanged or even slightly reduced
after the addition of zinc to the medium (Fig. 7C).
View larger version (66K):
[in a new window]
Fig. 8.
Comparison of zinc activation of the DNA
binding of MTF-1, Sp1, and chimeric F1Sp1. A, TnT
lysates were programmed with MTF-1, Sp1, or chimeric F1Sp1. EMSA
reactions contained 1 µl of the indicated TnT lysate and were treated
with the indicated amounts of zinc at the indicated temperatures for 15 min as described under "Experimental Procedures." Arrows
indicate specific bands. B, shown is an immunoblot
containing 1 µl of TnT lysates programmed with Sp1 or F1Sp1 or a
whole cell extract from Hepa cells detected with anti-Sp1 antibody.
The positions of the 64- and 98-kDa molecular mass markers are
indicated. Oligo, oligonucleotide.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
ACKNOWLEDGEMENTS
FOOTNOTES
To whom correspondence should be addressed: Dept. of Biochemistry
and Molecular Biology, University of Kansas Medical Center, 3901 Rainbow Blvd., Kansas City, KS 66160-7421. Tel.: 913-588-6935; Fax:
913-588-7350; E-mail: gandrews@kumc.edu.
ABBREVIATIONS
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1.
Kagi, J. H. R.
(1991)
Methods Enzymol.
205,
613-626
2.
Dalton, T. P.,
Fu, K.,
Palmiter, R. D.,
and Andrews, G. K.
(1996)
J. Nutr.
126,
825-833
3.
Masters, B. A.,
Kelly, E. J.,
Quaife, C. J.,
Brinster, R. L.,
and Palmiter, R. D.
(1994)
Proc. Natl. Acad. Sci. U. S. A.
91,
584-588
4.
Michalska, A. E.,
and Choo, K. H. A.
(1993)
Proc. Natl. Acad. Sci. U. S. A.
90,
8088-8092
5.
Lazo, J. S.,
Kondo, Y.,
Dellapiazza, D.,
Michalska, A. E.,
Choo, K. H. A.,
and Pitt, B. R.
(1995)
J. Biol. Chem.
270,
5506-5510
6.
Andrews, G. K.
(1990)
Prog. Food Nutr. Sci.
14,
193-258
7.
Stuart, G. W.,
Searle, P. F.,
Chen, H. Y.,
Brinster, R. L.,
and Palmiter, R. D.
(1984)
Proc. Natl. Acad. Sci. U. S. A.
81,
7318-7322
8.
Dalton, T. P.,
Palmiter, R. D.,
and Andrews, G. K.
(1994)
Nucleic Acids Res.
22,
5016-5023
9.
Dalton, T. P.,
Li, Q. W.,
Bittel, D.,
Liang, L. C.,
and Andrews, G. K.
(1996)
J. Biol. Chem.
271,
26233-26241
10.
Radtke, F.,
Heuchel, R.,
Georgiev, O.,
Hergersberg, M.,
Gariglio, M.,
Dembic, Z.,
and Schaffner, W.
(1993)
EMBO J.
12,
1355-1362
11.
Brugnera, E.,
Georgiev, O.,
Radtke, F.,
Heuchel, R.,
Baker, E.,
Sutherland, G. R.,
and Schaffner, W.
(1994)
Nucleic Acids Res.
22,
3167-3173
12.
Heuchel, R.,
Radtke, F.,
Georgiev, O.,
Stark, G.,
Aguet, M.,
and Schaffner, W.
(1994)
EMBO J.
13,
2870-2875
13.
Westin, G.,
and Schaffner, W.
(1988)
EMBO J.
7,
3763-3770
14.
Dalton, T. D.,
Bittel, D.,
and Andrews, G. K.
(1997)
Mol. Cell. Biol.
17,
2781-2789
15.
Bittel, D.,
Dalton, T.,
Samson, S.,
Gedamu, L.,
and Andrews, G. K.
(1998)
J. Biol. Chem.
273,
7127-7133
16.
Koizumi, S.,
Suzuki, K.,
Ogra, Y.,
Yamada, H.,
and Otsuka, F.
(1999)
Eur. J. Biochem.
259,
635-642
17.
Gralla, E. B.,
Thiele, D. J.,
Silar, P.,
and Valentine, J. S.
(1991)
Proc. Natl. Acad. Sci. U. S. A.
88,
8558-8562
18.
Labbé, S.,
and Thiele, D. J.
(1999)
Methods Enzymol.
306,
145-153
19.
Rebar, E. J.,
Greisman, H. A.,
and Pabo, C. O.
(1996)
Methods Enzymol.
267,
129-149
20.
Chen, X. H.,
Agarwal, A.,
and Giedroc, D. P.
(1998)
Biochemistry
37,
11152-11161
21.
Krizek, B. A.,
Zawadzke, L. E.,
and Berg, J. M.
(1993)
Protein Sci.
2,
1313-1319
22.
Smirnova, I. V.,
Bittel, D. C.,
Ravindra, R.,
Jiang, H.,
and Andrews, G. K.
(2000)
J. Biol. Chem.
275,
9377-9384
23.
Otsuka, F.,
Iwamatsu, A.,
Suzuki, K.,
Ohsawa, M.,
Hamer, D. H.,
and Koizumi, S.
(1994)
J. Biol. Chem.
269,
23700-23707
24.
Radtke, F.,
Georgiev, O.,
Müller, H.-P.,
Brugnera, E.,
and Schaffner, W.
(1995)
Nucleic Acids Res.
23,
2277-2286
25.
Zhao, H.,
and Eide, D. J.
(1997)
Mol. Cell. Biol.
17,
5044-5052
26.
Zimarino, V.,
and Wu, C.
(1987)
Nature
327,
727-730
27.
Laemmli, U. K.
(1970)
Nature
227,
680-685
28.
Donahue, T. F.,
and Cigan, A. M.
(1988)
Mol. Cell. Biol.
8,
2955-2963
29.
Chu, W. A.,
Moehlenkamp, J. D.,
Bittel, D.,
Andrews, G. K.,
and Johnson, J. A.
(1999)
J. Biol. Chem.
274,
5279-5284
30.
Lundblad, V.
(1997)
in
Current Protocols in Molecular Biology
(Ausubel, F. M.
, Brent, R.
, Kingston, R. E.
, Moore, D. D.
, Seidman, J. G.
, Smith, J. A.
, and Struhl, K., eds)
, pp. 13.0.1-13.13.7, John Wiley & Sons, Inc., New York
31.
Welsh, N.,
and Sandler, S.
(1994)
Mol. Cell. Endocrinol.
103,
109-114
32.
Andrews, G. K.,
and Geiser, J.
(1999)
J. Nutr.
129,
1643-1648
33.
Laity, J. H.,
Dyson, H. J.,
and Wright, P. E.
(2000)
J. Mol. Biol.
295,
719-727
34.
Chen, X. H.,
Chu, M. H.,
and Giedroc, D. P.
(1999)
Biochemistry
38,
12915-12925
35.
Müller, H.-P.,
Brugnera, E.,
Georgiev, O.,
Badzong, M.,
Müller, K. H.,
and Schaffner, W.
(1995)
Somatic Cell Mol. Genet.
21,
289-297
36.
Auf der Maur, A.,
Belser, T.,
Elgar, G.,
Georgiev, O.,
and Schaffner, W.
(1999)
Biol. Chem.
380,
175-185
37.
Tsai, R. Y.,
and Reed, R. R.
(1998)
Mol. Cell. Biol.
18,
6447-6456
38.
Sapin, V.,
Ward, S. J.,
Bronner, S.,
Chambon, P.,
and Dollé, P.
(1997)
Dev. Dyn.
208,
199-210
39.
Palmiter, R. D.
(1994)
Proc. Natl. Acad. Sci. U. S. A.
91,
1219-1223
Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
This article has been cited by other articles:
|
|
 |
|
  Y. Li, T. Kimura, J. H. Laity, and G. K. Andrews The Zinc-Sensing Mechanism of Mouse MTF-1 Involves Linker Peptides between the Zinc Fingers Mol. Cell. Biol., August 1, 2006; 26(15): 5580 - 5587. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. M. Potter, L. S. Feng, P. Parasuram, V. A. Matskevich, J. A. Wilson, G. K. Andrews, and J. H. Laity The Six Zinc Fingers of Metal-responsive Element Binding Transcription Factor-1 Form Stable and Quasi-ordered Structures with Relatively Small Differences in Zinc Affinities J. Biol. Chem., August 5, 2005; 280(31): 28529 - 28540. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
X. Chen, B. Zhang, P. M. Harmon, W. Schaffner, D. O. Peterson, and D. P. Giedroc A Novel Cysteine Cluster in Human Metal-responsive Transcription Factor 1 Is Required for Heavy Metal-induced Transcriptional Activation in Vivo J. Biol. Chem., February 6, 2004; 279(6): 4515 - 4522. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. C. Rutherford and A. J. Bird Metal-Responsive Transcription Factors That Regulate Iron, Zinc, and Copper Homeostasis in Eukaryotic Cells Eukaryot. Cell, February 1, 2004; 3(1): 1 - 13. [Full Text] [PDF]
|
|
|
|
 |
|
 P. Meerarani, G. Reiterer, M. Toborek, and B. Hennig Zinc Modulates PPAR{gamma} Signaling and Activation of Porcine Endothelial Cells J. Nutr., October 1, 2003; 133(10): 3058 - 3064. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Jiang, P. J. Daniels, and G. K. Andrews Putative Zinc-sensing Zinc Fingers of Metal-response Element-binding Transcription Factor-1 Stabilize a Metal-dependent Chromatin Complex on the Endogenous Metallothionein-I Promoter J. Biol. Chem., August 8, 2003; 278(32): 30394 - 30402. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Tacahashi, S. Helmling, and C. L. Moore Functional dissection of the zinc finger and flanking domains of the Yth1 cleavage/polyadenylation factor Nucleic Acids Res., March 15, 2003; 31(6): 1744 - 1752. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. J. Daniels, D. Bittel, I. V. Smirnova, D. R. Winge, and G. K. Andrews Mammalian metal response element-binding transcription factor-1 functions as a zinc sensor in yeast, but not as a sensor of cadmium or oxidative stress Nucleic Acids Res., July 15, 2002; 30(14): 3130 - 3140. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Saydam, T. K. Adams, F. Steiner, W. Schaffner, and J. H. Freedman Regulation of Metallothionein Transcription by the Metal-responsive Transcription Factor MTF-1. IDENTIFICATION OF SIGNAL TRANSDUCTION CASCADES THAT CONTROL METAL-INDUCIBLE TRANSCRIPTION J. Biol. Chem., May 31, 2002; 277(23): 20438 - 20445. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
O. LaRochelle, V. Gagne, J. Charron, J.-W. Soh, and C. Seguin Phosphorylation Is Involved in the Activation of Metal-regulatory Transcription Factor 1 in Response to Metal Ions J. Biol. Chem., November 2, 2001; 276(45): 41879 - 41888. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. P. Giedroc, X. Chen, M. A. Pennella, and A. C. LiWang Conformational Heterogeneity in the C-terminal Zinc Fingers of Human MTF-1. AN NMR AND ZINC-BINDING STUDY J. Biol. Chem., November 2, 2001; 276(45): 42322 - 42332. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
