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Advances in Brief |
Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, and Southwest Cancer Center at Texas Tech University Medical Center, Lubbock, Texas 79430
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ABSTRACT |
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Introduction |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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JunD) are expressed in all
cell types and tissues thus far tested and are expressed at
approximately the same stoichiometry.
MEN-1 is an autosomal dominant disease characterized by a variety of
tumors in endocrine tissues including the anterior pituitary,
parathyroid, and pancreatic islets (8)
. The gene was
identified by positional cloning in 1997 and is located at 11q13
(9, 10, 11, 12)
. The encoded protein, named Menin, contains 610
amino acids and bears no discernable similarity with known sequences.
Most of the identified sporadic and germ-line mutations result in
truncations that likely inactivate the Menin protein; thus, Menin is
considered a tumor suppressor protein. Loss or mutation of both MEN-1
alleles is thought to be etiological for familial MEN-1 tumor formation
(12
, 13) . Using the yeast two-hybrid interaction assay
Menin was found to interact specifically with the JunD transcription
factor, although the significance of this interaction is presently
unknown (14)
. Agarwal et al.
(14)
demonstrated that JunD residues 8–70 were
required for JunD-Menin interaction. This identified Menin-binding
sequence overlaps the junction between JunD-FL and JunD; thus, any
possible difference in Menin binding between the two JunD isoforms was
not revealed. Here we report that Menin binds only the JunD-FL isoform
and that the binding site for Menin is entirely contained within the
first 48 amino acids. Menin strongly suppresses the transcriptional
transactivation activity of JunD-FL, whereas it does not affect the
activity of the shorter JunD isoform (JunD). We have tested the
effects of two point mutations that commonly occur in familial MEN-1
tumors (A176P and A242V; Ref. 13
). Either of these
mutations resulted in lower binding of the mutant Menin protein to
JunD-FL and less suppression of JunD-FL transcriptional activity.
An important regulatory mechanism for c-Jun and JunD function involves phosphorylation of their NH2-terminal transactivation domains by the JNKs (15) . Because the docking domain within the JunD protein is immediately adjacent to the Menin interaction domain, we tested whether phosphorylation by JNK had any influence on Menin binding. We demonstrate that phosphorylation of the JNK target residues has no impact on Menin interaction.
These results suggest that JunD-FL is a functional target of the Menin tumor suppressor protein and that disruption of proper JunD-FL/Menin interaction is a component of the mechanism of tumorigenesis in MEN-1 disease.
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Materials and Methods |
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Expression Vector Construction.
GST fusion proteins were prepared using the pGEX4T-3 vector
(Pharmacia). Insert DNA encoding the
NH2-terminal fragments of c-Jun (amino acids
1–123), JunD (amino acids 1–149), JunD (amino acids 1–115)
and JunD (amino acids 49–149) with BamHI and
EcoRI restriction sites at their 5' and 3' ends,
respectively, were generated using PCR from cDNA templates. The inserts
were gel-purified, digested, and ligated into pGEX4T-3 cut with the
same enzymes. Expression vectors for the GAL4-fusion proteins were
constructed using the pGal4–0 plasmid (16)
. Insert DNA
encoding the same NH2-terminal fragments
indicated above were prepared by PCR with BamHI and
XbaI restriction sites at the 5' and 3' ends, respectively.
The inserts were gel-purified, digested, and ligated into pGAL4–0 cut
with the same enzymes. Plasmid DNA stocks used for transfections were
prepared using a standard cesium chloride density gradient procedure.
The myc-tagged Menin expression vector (pcDNA3.1-Menin-Myc) was kindly
provided Dr. S. Chandrasekharappa (Bethesda, MD). The A176P and A242V
point mutations in Menin were introduced using a PCR-based
site-directed mutagenesis protocol (Quick Change, Stratagene). All of
the constructs were verified by direct sequencing using an ABI
automated sequencer.
Binding and Kinase Assays.
GST-fusion proteins were inducibly expressed in
Escherichia coli and purified by a standard protocol using
glutathione-coated agarose beads (Sigma). GST-fusion proteins were
eluted from the beads with excess glutathione and dialyzed in 50
mM Tris (pH 7.5) and 20 mM
MgCl2. Menin protein was prepared using a coupled
in vitro transcription/translation system (TNT, Promega),
that was supplemented with [35S]methionine and
[35S]cysteine (Trans-label,
ICN). Binding assays were performed using approximately 4 µg
of GST-fusion proteins, 5 µl of Menin TNT-lysate, and 20 µl of 50%
glutathione-agarose beads in binding buffer [50
mM Tris (pH 8.0); 0.01% NP40; 5
mM MgCl2; 10% glycerol; 10
mM NaCl; and 4 µg/ml each aprotonin, leupeptin,
and pepstatin A] at 4°C for 3 h. After centrifugation, the
beads were washed three times in binding buffer and boiled in
SDS-sample buffer, and the complexed proteins were resolved on 12%
SDS-polyacrylamide gels. The proteins were then transferred to
nitrocellulose (Schleicher & Schull, Dassel, Germany) by
electroblotting and the membranes dried down. For the
kinase/GST-pull-down dual assay, the kinase reaction was performed
first in a total volume of 40 µl using 4 µg of each fusion protein,
1 µl of TNT-lysate programmed with a cDNA for JNK1ß2 (kindly
provided by Dr. R. Davis) in the presence of 50
µM [
32P]ATP (10
Ci/mmol) in kinase buffer [50 mM Tris (pH 7.5),
20 mM MgCl2, 20
mM ß-glycerophospate, 2
mM DTT, and 0.1 mM sodium
orthovanadate] at room temperature for 1 h.
35S-labeled Menin protein and 20 µl of 50%
glutathione-agarose in binding buffer were then added and the
mixture further incubated for 4 h at room temperature. The beads
were then harvested by centrifugation. The protein complexes were
analyzed as described above. The 35S and
32P signals were detected using a PhosphorImager
instrument (Molecular Dynamics).
Transfection Assays.
CHO cells were plated at a density of 45,000 cells/well in 24-well
plates, 24 h before transfection. The reporter plasmid
G5-E1b-Luc (0.3 µg) along with 2 ng of the
indicated expression vector (pGAL4-Jun), encoding each of the GAL4-Jun
fusion proteins, and the indicated amount (0, 5, or 10 ng) of
expression vector (pcDNA3.1-Menin-Myc) for the wild-type or mutant
Menin proteins were transfected into the cells using Transfast reagent
(Promega) in serum-free media according to the manufacturer’s
instructions. At 24 h posttransfection, cells were harvested by
incubation in Passive Lysis Buffer (Promega) for 30 min at room
temperature with agitation. The luciferase activity in the extracts was
measured in a Turner Designs luminometer using the Luciferase Assay
System (Promega).
Western Blot Analysis.
Transfected CHO cells were rinsed with cold PBS, harvested, and boiled
in SDS-sample buffer for 5 min. Equal amounts of total protein from
whole-cell extracts were fractionated on a 12% denaturing
SDS-polyacrylamide gel and transferred to nitrocellulose membranes.
Nonspecific interactions were blocked by preincubation of the membranes
with 5% nonfat milk powder in PBS supplemented with 0.1% Tween 20 for
1 h. GAL4-Jun fusion proteins were detected using an 
-GAL4
mouse monoclonal antibody directed against the GAL4 DNA binding domain
(SC-510, Santa Cruz Biotechnology). Myc-tagged Menin proteins were
detected using an -myc mouse monoclonal antibody (Invitrogen).
Horseradish peroxidase-conjugated donkey antimouse antibody (Jackson
Labs) was used for the secondary antibody. The blots were developed
using an enhanced chemiluminescence system (Pierce).
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Results |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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JunD) is truncated by 48
NH2-terminal amino acids in the rodent and 53
residues in human. JunD is ubiquitously expressed as determined by
immunoblotting analyses from a wide range of cell lines and animal
tissues.3
A representative JunD Western blot using extract
from mouse thymus is shown in Fig. 1A
. No functional differences between JunD-FL and JunD have
yet been reported.
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JunD, or c-Jun (Fig. 1B)
. These fusion proteins
were expressed in bacteria, purified, and used in pull-down assays.
GST-Jun fusion proteins were incubated with Menin protein that was
produced by in vitro transcription/translation. Menin
protein was radiolabeled by incorporation of
[35S]methionine and
[35S]cysteine. Fig. 2A
illustrates a typical binding assay. Menin efficiently
binds to GST-JunD-FL and copellets with GST-JunD-FL when harvested
using glutathione-agarose beads (Fig. 2A
, Lanes 4
and 5). However, JunD displays no measurable binding to
Menin above the background detected when GST alone was used as a
control (Fig. 2A
, Lanes 6 and 8).
Furthermore, GST-cJun also exhibits no measurable binding to Menin in
this assay. A GST fusion containing the first 48
NH2-terminal amino acids of JunD-FL (the residues
truncated in JunD) also bound Menin strongly (Fig. 2A
,
Lane 1). As can be seen comparing Lanes 1 and
Lane 4 in Fig. 2
, most, if not all, of the Menin binding
activity within the JunD molecule is fully contained within the first
48 amino acids.
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JunD via a JNK docking
domain positioned between residues 49 and 73 (numbered with respect to
JunD-FL). Both JunD-FL and JunD are phosphorylated by JNK on three
residues adjacent to the docking domain (serine-90, serine-100, and
threonine-117). Phosphorylation of JunD-FL and JunD by JNK
significantly increases their transcriptional activity in transfection
reporter assays, which suggests that JNKs are important regulators of
JunD
function4
. Therefore, we tested whether phosphorylation by JNK affected the
interaction between JunD and Menin. GST-binding assays were performed
after incubation of GST-Jun in the absence or presence of in
vitro transcribed/translated JNK along with
[-32P]ATP. Under these reaction
conditions, phosphorylation of the GST-JunD protein is nearly complete
(Fig. 2B
and data not shown). Again, JunD-FL bound
35S-labeled Menin (Fig. 2B
, Lane
1), but this binding was unaffected by phosphorylation of the
GST-JunD-FL (Fig. 2B
, Lane 3). Although JunD
was similarly phosphorylated under these conditions, it showed no
measurable binding to Menin (Fig. 2B, Lane 7).
Menin Suppresses Transcriptional Activity of the JunD-FL Isoform.
To test the effect of Menin on JunD transcriptional activity, transient
transfections were performed using a GAL4 transcription reporter
system. GAL4 fusions were prepared between the DNA binding domain of
GAL4 and NH2-terminal fragments of JunD or c-Jun
and placed in a mammalian expression vector (Fig. 1C)
. These
vectors were tested by Western blot analysis for their ability to
produce the correct size fusion proteins after transfection and
expression in CHO cells as shown in Fig. 3B
. These expression vectors were cotransfected into CHO cells
along with a GAL4-luciferase reporter vector
(G5-E1b-Luc). Gal4-JunD-FL displayed consistently
higher transactivation capacity than either GAL4-JunD or GAL4-c-Jun
(Fig. 3A)
. When cotransfected with an expression vector for
wild-type Menin, transactivation of JunD-FL was suppressed in a
dose-responsive manner, with activity decreased 5-fold with 10 ng of
Menin expression vector. Transactivation by either JunD or c-Jun was
unaffected by coexpression of Menin. Menin protein was monitored by
Western blot analysis using an anti-myc antibody to detect the myc
epitope-tagged Menin protein (Fig. 3B)
.
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. Both of these mutations (A176P and
A242V) are found in both sporadic and familial tumors
(13)
. As shown in Fig. 2A
, Lanes 2
and 3, these mutations resulted in lower levels of Menin
binding to JunD in GST-pull-down assays. In the case of the A242V
mutation, the amount of Menin that copelleted with GST-JunD was similar
to that in the GST control, indicating that this residue is critical
for Menin binding (Fig. 2A
, Lanes 3 and 8). These
mutant isoforms of Menin were also tested in cotransfection
transcriptional assays. Both mutations had a similar impact. Each
mutant Menin protein resulted in a lower level of inhibition of JunD-FL
transcriptional activity than that seen with the wild-type Menin (Fig. 3A)
. The mutants exhibited 
50% of the inhibitory
activity of the wild-type Menin (Fig. 3A
, top bar
graph). No effect of either mutation was detected on the
transcriptional activity of JunD or c-Jun. |
Discussion |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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JunD in their ability to slow fibroblast growth or to antagonize
ras-induced transformation.
The positional cloning of the gene responsible for multiple endocrine
neoplasia (MEN-1) and its characterization as a classical
tumor suppressor has focused attention on the encoded protein
(10)
. The Menin protein is 610 amino acids in length and
has no identified similarities with database sequences except for two
putative nuclear localization signals (Fig. 1D
; Ref.
24
). Using the yeast two-hybrid interaction system with
Menin as the bait, JunD was identified as a Menin-interacting protein
(14)
. Here we have demonstrated that Menin binds
directly to the full-length isoform of JunD (JunD-FL) but not to the
truncated JunD isoform (JunD). Furthermore, the Menin binding domain
of JunD is contained within the first 48 amino acids, precisely the
same residues that are truncated in JunD. We have demonstrated that
this differential binding has pronounced functional effects. Menin
suppresses transcriptional activity mediated by a GAL4-JunD-FL fusion
molecule in a dose-dependent manner, but activity of GAL4-JunD is
unaffected. Supporting the hypothesis that JunD-Menin interaction is
functionally relevant, two naturally occurring Menin mutations that
were engineered into our Menin expression vector resulted in lower
suppression of JunD-FL transcriptional activity, although having no
measurable effect on JunD activity. Consistent with these
observations, these same Menin mutations resulted in lower levels of
binding to GST-JunD-FL fusion proteins in our in vitro
assays.
The Jun proteins have a highly modular structure with the dimerization,
DNA-binding, and nuclear localization signal domains located within the
COOH-terminus, and the transactivation domain located within the
NH2-terminal one-third of the molecule
(1)
. In addition, a discrete JNK docking domain is present
in both JunD isoforms (Fig. 1A)
. JNK binds both JunD-FL and
JunD and activates transcription of both isoforms by phosphorylating
target residues located adjacent to the docking domain. Phosphorylation
of these sites did not affect the binding of Menin to JunD-FL in our
binding assays, suggesting that JNK-mediated phosphorylation does not
modulate Menin binding. However, these assays used in vitro
translated and transcribed JNK and Menin with the GST-JunD substrate in
large excess. Thus, whereas the kinase reactions were allowed to run to
completion, thereby phosphorylating most of the available target
residues, the possibility of direct steric interference between Menin
and JNK was not tested. Because Menin has a molecular mass of 68 kDa
and JNK isoforms have masses of 45 or 54 kDa, the possibility of steric
interference certainly exists, considering that their respective
binding domains within JunD are immediately adjacent.
In addition to Menin, several other nuclear proteins have been shown to interact with the NH2-terminal region of JunD, including JAB-1 (25) and CBP (26) . The precise binding domains for these proteins have not yet been mapped, nor have any complexes composed of JunD and several of these binding proteins been described. Thus, although the manner in which Menin suppresses the transcriptional activity of JunD-FL is presently unknown, a reasonable conjecture is that Menin functions by regulating the binding of JunD-interacting proteins or by participating in protein complexes positioned on the NH2-terminal of JunD.
These data together suggest that the full-length isoform of JunD is a
relevant physiological target for the Menin tumor suppressor protein.
Moreover, disruption of proper JunD-FL-Menin interaction by mutational
inactivation or deletion of Menin may underlie at least part of the
mechanism leading to tumor development in MEN-1 disease. Interestingly,
the JunD-FL isoform has higher basal transcriptional activity than the
JunD isoform, which suggests that JunD-FL activity must be more
tightly modulated for growth regulation. Because we have previously
shown that constitutive expression of both isoforms of JunD had
growth-inhibitory effects in mouse fibroblasts, an important
experimental question is whether this effect is predominantly
attributable to the JunD isoform.
A working model for tumorigenesis resulting from loss of Menin activity
is presented in Fig. 4
. Although it is not known whether JunD-FL and JunD regulate unique
subsets of target genes, there is presently no evidence to suggest that
they possess different dimerization properties. Therefore, both
isoforms would be expected to form dimers with other Jun and Fos
proteins, thereby contributing to the total AP-1-dependent regulation
of target genes. Some of these genes are likely to be critical for
proper regulation of cell proliferation (Fig. 4A)
. Our data
suggest that JunD-FL would be capable of exerting a stronger
transcriptional activation response than would JunD. Although
wild-type Menin would inhibit JunD-FL, the strength of this inhibition
in any given cell type or tissue is unknown; i.e., important
parameters such as the stoichiometry of Menin and JunD-FL or the
regulation of Menin-JunD binding within the nucleus have not yet been
determined. When Menin activity is absent or attenuated, as is the case
for MEN-1 disease, the transcriptional inhibition of JunD-FL would be
lifted resulting in an altered AP-1 activity. This in turn would lead
to an alteration of AP-1 target gene expression leading, through
unknown mechanisms, to tumorigenesis. Although JunD is the only
Menin-interacting protein identified to date, undoubtedly additional
interactions will be uncovered that also play a role in MEN-1 disease.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 To whom requests for reprints should be
addressed, at Department of Cell Biology and Biochemistry, Texas Tech
University Health Sciences Center, 3601 4th Street, Lubbock, Texas
79430. Phone: (806) 743-1559; Fax: (806) 743-2990; E-mail: curt.pfarr{at}ttmc.ttuhsc.edu 
2 The abbreviations used are: JunD-FL; full-length
JunD; JNK, Jun-NH2-terminal kinase; CHO, Chinese hamster
ovary; GST, glutathione-S-transferase.
3 J. D. Short and C. M. Pfarr. Translational
regulation of the JunD mRNA, manuscript in preparation.
4 O. Yazgan and C. M. Pfarr. Interaction of
Jun-N-terminal kinases with JunD, manuscript in preparation.
Received 9/12/00. Accepted 12/ 4/00.
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