Originally published In Press as doi:10.1074/jbc.M202937200 on April 23, 2002
J. Biol. Chem., Vol. 277, Issue 26, 23888-23897, June 28, 2002
Myotrophin/V-1, a Protein Up-regulated in the Failing Human Heart
and in Postnatal Cerebellum, Converts NF
B p50-p65 Heterodimers to
p50-p50 and p65-p65 Homodimers*
Pascal
Knuefermann
,
Peter
Chen§,
Arunima
Misra,
Shu-Ping
Shi,
Maha
Abdellatif¶, and
Natarajan
Sivasubramanian
From the Winters Center For Heart Failure Research,
Molecular Cardiology Unit, Cardiology Section of Department of
Medicine, Baylor College of Medicine, Veterans Affairs Medical Center,
Houston, Texas 77030
Received for publication, March 26, 2002, and in revised form, April 22, 2002
 |
ABSTRACT |
Myotrophin/V-1 is a cytosolic
protein found at elevated levels in failing human hearts and in
postnatal cerebellum. We have previously shown that it disrupts nuclear
factor of 
B (NFB)-DNA complexes in vitro. In this
study, we demonstrated that in HeLa cells native myotrophin/V-1 is
predominantly present in the cytoplasm and translocates to the nucleus
during sustained NFB activation. Three-dimensional alignment studies
indicate that myotrophin/V-1 resembles a truncated IB
without the
signal response domain (SRD) and PEST domains. Co-immunoprecipitation
studies reveal that myotrophin/V-1 interacts with NFB proteins
in vitro; however, it remains physically associated only
with p65 and c-Rel proteins in vivo during NFB
activation. In vitro studies indicate that myotrophin/V-1
can promote the formation of p50-p50 homodimers from monomeric p50
proteins and can convert the preformed p50-p65 heterodimers into
p50-p50 and p65-p65 homodimers. Furthermore, adenovirus-mediated
overexpression of myotrophin/V-1 resulted in elevated levels of both
p50-p50 and p65-p65 homodimers exceeding the levels of p50-p65
heterodimers compared with Ad
gal-infected cells, where the levels of
p50-p65 heterodimers exceeded the levels of p50-p50 and p65-p65
homodimers. Thus, overexpression of myotrophin/V-1 during NFB
activation resulted in a qualitative shift by quantitatively reducing
the level of transactivating heterodimers while elevating the levels of
repressive p50-p50 homodimers. Correspondingly, overexpression of
myotrophin/V-1 resulted in significantly reduced B-luciferase
reporter activity. Because myotrophin/V-1 is found at elevated levels
during NFB activation in postnatal cerebellum and in failing human
hearts, this study cumulatively suggests that myotrophin/V-1 is
a regulatory protein for modulating the levels of activated NFB
dimers during this period.
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INTRODUCTION |
Myotrophin/V-1 (Myo/V1)1
protein was initially characterized in the mammalian heart, where it
was called myotrophin (1), and in the rat cerebellum, where it was
called V-1 (2). It was later found to be ubiquitously expressed in all
mammalian tissues (3, 4). Myo/V1 is a 12-kDa ankyrin repeat-containing intracellular protein that has been found at elevated levels in failing
human hearts (5) as well as in the hearts of spontaneously hypertensive
rats (6). Although Myo/V1 was originally described as a trophic protein
(myotrophin) exhibiting growth properties exogenously on rat neonatal
myocytes (1), other studies showed that this protein was only present
in intracellular space (2, 7-9) and its trophic growth properties on
neonatal myocytes were not confirmed (10). Moreover, this protein was
originally identified and isolated only from an intracellular location
(1), and a transcriptional regulatory function has been proposed (3,
9).
Since its discovery, investigators have proposed various functions for
Myo/V1 protein (1-3). In the postnatal rat cerebellum, the cellular
level of soluble Myo/V1 was found to be transiently up-regulated
immediately after birth and later declined displaying a unique pattern
of expression among 120 soluble proteins, implicating its role during
postnatal cerebellum development (2). Because of its aberrant
expression in genetically defective cerebellar granular cells, this
protein was proposed to play a role in granular cell differentiation
process (Unigene Mm.4123) (7). To date, however, the molecular function
of Myo/V1 protein is still lacking. We recently reported that Myo/V1
protein exhibits significant homology to IB protein and that
Myo/V1 can disrupt the NFB-DNA complexes in vitro (3).
Utilizing our recombinant Myo/V1 protein, the NMR structure of Myo/V1
was determined (11, 12), and the ankyrin repeats of Myo/V1 exhibited
structural features similar to those of IB (13, 14) at the
three-dimensional level.
In response to a variety of pathophysiological and developmental
signals, the NFB/Rel family of transcription factors are activated
and form different types of hetero- and homodimers among themselves to
regulate the expression of target genes containing B-specific
binding sites (15, 16). Among the activated NFB dimers, the p50-p65
heterodimers are known to be involved in enhancing the transcription of
target genes and the p50-p50 homodimers in transcriptional repression
(17-22). However, the p65-p65 homodimers are known for both
transcriptional activation and repressive activity against target genes
(23-31). NFB activation is regulated at multiple levels. The
dynamic shuttling of the inactive NFB dimers between the cytoplasm
and nucleus by IB proteins (32-35) and its termination by
phosphorylation and proteasomal degradation (36, 37), direct phosphorylation (38), acetylation of NFB factors (39), and dynamic
reorganization of NFB subunits among the activated NFB dimers
(40-42) have all been identified as key regulatory steps in
NFB-mediated transcription process. B DNA binding sites with varied affinities to different NFB dimers (43) have been discovered in the promoters of several eukaryotic genes (16, 20, 22, 44, 45), and
the balance between activated NFB homo- and heterodimers ultimately
determines the nature and level of gene expression within the cell (18,
22). However, thus far the underlying molecular mechanism for the
generation and dynamic reorganization of NFB dimers during chronic
activation is unknown. Here, for the first time, we show that Myo/V1
acts as a "zipper chaperone" protein to generate NFB homodimers
from monomeric p50 proteins and with its "unzipping" function
converts the transcriptionally active p50-p65 heterodimers to
transcriptionally repressive homodimers in HeLa cells, thus attenuating
NFB-mediated transcription.
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EXPERIMENTAL PROCEDURES |
Recombinant Expression Plasmids and
Adenoviruses--
Recombinant Myo/V1 protein was expressed in
Escherichia coli in two different forms (~12-kDa
full-length and histidine-tagged ~14-kDa fusion protein) using pET
expression vectors as described before (3). The following mammalian
expression plasmids were constructed by recombinant DNA methods. For
pcDNA-AM1.1-Myo/V1, because of the poor translation initiation
Kozak site in the Myo/V1 mRNA, we engineered a heterologous highly
efficient Kozak site into 5'-untranslated region of Myo/V1 so that
Myo/V1 is expressed at high levels in mammalian cells. In
vitro transcription and translation with pcDNA3-AM1.1-Myo/V1
template DNA confirmed the synthesis of 12-kDa Myo/V1 protein at higher
levels than the native Myo/V1 mRNA (data not shown). For
pB-tk-luc, the parent chloramphenicol acetyltransferase reporter
plasmid containing a minimal thymidine kinase promoter and two B
enhancer sites was obtained and replaced with the coding region of
luciferase enzyme. pRSV-RelA vector expressing p65 was obtained through
the AIDS Research and Reference Reagent Program, Division of AIDS,
NIAID, National Institutes of Health. The expression plasmid is from
Dr. Gary Nabel and Dr. Neil Perkins. The recombinant adenoviruses
expressing Myo/V1 and -galactosidase were constructed as follows.
For AdMyo/V1/Adgal, the respective expression plasmids
pcDNA3-AM1.1-Myo/V1 and pcDNA3-gal were incorporated into
Ad5 adenovirus through allelic recombination. Recombinant adenoviruses
were propagated, purified, and titered as previously reported (46).
Cell Biology Techniques--
HeLa cells (ATCC-CCL2) were
maintained in minimal essential medium. Cell fixation and indirect
immunofluorescence studies were performed as previously described (47).
Cytoplasmic and nuclear extracts were prepared using NE-PERTM nuclear
and cytoplasmic extraction reagents (Pierce). Plasmid DNA transient
transfection experiments were performed using FuGENE6 reagent (Roche).
Luciferase assays were conducted with reagents from Promega Inc. The
protein concentration was determined by BCA method using Pierce reagents.
HeLa cells were treated for 2 h with TNF (50 ng/ml) to induce
NFB. For superinduction of NFB, cells were further treated with
cycloheximide (10 µg/ml). After 2 h of treatment, cells were harvested and subcellular fractionation for cytoplasmic and nuclear extracts were carried out as previously described (48). Briefly, HeLa
cells were harvested at 150 × g and Dounce-homogenized
in a hypotonic lysis buffer (buffer A; Ref. 49), and nuclei were collected at 4300 × g. The nuclei were extracted with
buffer C (49) and were used for GSA and Western blot analysis. After the removal of the nuclei, the supernatant was further centrifuged at
20,000 × g for isolating mitochondrial fractions.
The remaining cytosolic supernatant was further concentrated by
acetone precipitation. Identical amounts of cytosolic (40 µg),
nuclear extract (30 µg), and mitochondrial proteins (35 µg) were
fractionated on a 10% Tris-Tricine SDS-PAGE and processed for Myo/V1
ECL immunoblotting.
In Vitro Myo/V1-NFB Interaction
Experiments--
Pure recombinant p50 protein was obtained from
Promega Inc., and pure truncated recombinant p65
(p65
RHRs) protein was obtained from Dr. Gaurishankar Ghosh
(50). The p65 contains only the Rel DNA binding domain (~31 kDa;
aa 19-291) of p65, and the carboxyl terminus containing the
transactivating domain of p65 has been removed (50). Monomeric p50 (2.5 ng in 50 µl; 2 nM) and p65 (0.5 µg in 50 µl; 7 µM) proteins were separately incubated with increasing
concentrations of Myo/V1 (0-100 ng) and the resulting B-DNA
complexes from these reactions were analyzed by GSA (3, 46). Because
p50 and p65 proteins exhibit high affinity to each other, equimolar
concentrations of p50 (16 pmol; 0.8 µg) and p65 (16 pmol; 0.5 µg) proteins were mixed and incubated in a binding buffer at 4 °C
to preform the p50-p65 heterodimers. Later, increasing concentrations of 12-kDa Myo/V1 protein (0-56 pmol; 0-800 ng) were
added and incubated for an additional 30 min at 4 °C. Finally, the
radiolabeled B oligonucleotides (25,000 cpm) were added and incubated for another 10 min at 4 °C. The resulting B-DNA
complexes from these reactions were analyzed by GSA (3, 46). The
mobility patterns of the p50-p65, p50-p50, and p65-p65 dimers
on GSA exactly resemble the previously published patterns for these
dimers (43, 51, 52). To identify the nature of Myo/V1-generated NFB
dimers, antibodies to p50 (#sc114x; Santa Cruz Biotechnology, Santa
Cruz, CA) and p65 (#ab243, Abcam, Cambridge, UK) were added to
preformed heterodimers (equimolar mixture of p50 (80 ng) and p65 (50 ng) proteins) with increasing concentrations of Myo/V1 (0, 100, 400 ng), and its effects were studied on GSA. This experiment was also
repeated with preformed p50-p65 heterodimers with 100-fold excess
p65 protein (5 µg).
Quantification of Individual NFB Dimers in HeLa Cell Nuclear
Extracts--
We developed a more accurate method to quantify NFB
dimers in mammalian cell nuclear extracts. We chose three B sites
from native genes, which have been previously well characterized to exhibit high affinity to individual NFB dimers. The rationale behind
this approach is with varied amounts of activated NFB dimers in the
mammalian cell, using a single B oligo, which exhibits high affinity
to one dimer, and not to others and using it to quantify the various
NFB dimers will yield erroneous results. Moreover, the ideal B
oligo, which exhibits equal affinity toward all NFB dimers with
varied mobilities on GSA to distinguish them, does not exist.
Therefore, the conventional B-Ig/HIV oligonucleotide (5'-AGTTGAGGGGACTTTCCCAGGC-3' from Santa Cruz Biotechnology, Inc.) was used to quantify only p50-p65 heterodimers because it exhibited high affinity only toward p50-p65 heterodimers. Because this
B site exhibited lower affinities toward p50-p50 and p65-p65 homodimers (5- and 15-fold, respectively) (53), we chose two other B
oligonucleotides B#SeqB (44) and B#u-iNOS (45), which were
previously shown to exhibit high affinity and exclusive specificity
toward p50-p50 and p65-p65 homodimers (44, 45). The B#SeqB
(5'-GTAGGGGGCCTCCCCGGCTCGAGATCCTATG-3') and B#u-iNOS
(5'-GTACCGGAAATTCCGGGCTCGAGATCCTATG-3') oligonucleotides were custom synthesized (Synthetic Genetics, CA) and used for quantifying respective NFB homodimers. To determine the relative levels of various NFB dimers, nuclear extracts (~20 µg) from AdMyo/V1- and Adgal-infected cells were incubated with radiolabeled individual B oligonucleotides (B-Ig, #SeqB; #u-iNOS; 25,000 cpm with similar specific activities), and the resulting DNA-protein complexes were fractionated on the same 4% PAGE. Radiographic images
of the gels were captured using a STORM 860 imager (Molecular Dynamics,
CA) and quantified using ImageQuant 4.2 software. NFB antibody
supershifts were done as described previously; however, p65 antibody
from Geneka Biotechnology (Montreal, Canada) was used in these
experiments. Additionally, heterologous chase experiments were done to
further confirm the nature of the NFB dimers.
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RESULTS |
Myo/V1 Is Localized in the Cytoplasm and
Nucleus--
Indirect immunofluorescence studies were conducted to
locate the native Myo/V1 protein in HeLa cells. Under basal conditions (Fig. 1A), Myo/V1 was
predominantly observed in a wide area of the cytoplasm surrounding the
nucleus. Further analysis with confocal microscopy revealed that native
Myo/V1 was also present in the nucleus to a lesser extent
(spotted green fluorescence in Fig. 1C). Upon treatment with TNF for 1 h, Myo/V1 was found
to cluster around the perinuclear region in the cytoplasm.
Additionally, Myo/V1 was found to increase slightly within the nucleus
(green FITC masking the blue DAPI nuclear
staining, resulting in pale blue
nucleus) suggesting migration of Myo/V1 to the nucleus (Fig. 1B). Because TNF is known to reorganize the cytoskeleton
(54-56), the present observation of increased perinuclear clustering
and nuclear migration of Myo/V1 in TNF-treated HeLa cells (Fig.
1B) suggests that Myo/V1 might be associated with the
cytoskeleton and its associated organelles and might participate in the
signal transduction process from these locations.
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Fig. 1.
Native Myo/V1 is localized in the cytoplasm
and nucleus of HeLa cells. A, detection of
Myo/V1 by indirect immunofluorescence light microscopy. HeLa cells
under basal conditions were fixed and stained with anti-Myo/V1 antibody
and a FITC-conjugated secondary antibody. Nuclei were stained with
DAPI, and images of FITC and DAPI staining (60×; A and
B) were captured using a phase contrast Nikon Eclipse E800
microscope. A nonfluorescent blue color was assigned for
DAPI image during image capture and superimposed with FITC image using
Metaview software. B, HeLa cells treated with TNF for 2 h. C, image of untreated HeLa cells was acquired using a
laser-scanning microscope. White arrows denote the
cytoplasmic and nuclear localization of Myo/V1.
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Myo/V1 Translocates to Nucleus during Sustained
Induction of NFB--
Cellular partitioning studies were performed
to determine the location of Myo/V1 under the NFB inducing
conditions. Cells were treated with TNF or TNF plus CHX for 2 h.
Cytoplasmic, mitochondrial, and nuclear extracts were prepared and
immunoblotted for Myo/V1 protein (Fig.
2). Under basal conditions the majority
of Myo/V1 was present in the cytoplasmic fraction (lane 5 in
Fig. 2B) and only a small quantity in the nuclear fraction
(lane 3 in Fig. 2B). After TNF stimulation for
2 h, Myo/V1 levels in the cytoplasmic fraction did not change
significantly (data not shown). However, with TNF plus CHX stimulation,
the levels of Myo/V1 reduced significantly in the cytoplasmic fraction
compared with control (lanes 5 and 6 in Fig.
2B). Additionally, a simultaneous increase in the levels of
Myo/V1 was observed in the nucleus (lane 4 in Fig.
2B), indicating that the translocated Myo/V1 migrated from
cytoplasm. We did not observe any Myo/V1 in mitochondrial fractions
(lanes 1 and 2 in Fig. 2B). These data
suggest that, under acute stress conditions, a major relocalization of
Myo/V1 first occurs within the cytoplasm (Fig. 1B),
following which Myo/V1 is translocated to the nucleus.
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Fig. 2.
Myo/V1 is translocated to nucleus during
superinduction of NFB. A, GSA
showing the B DNA binding activity of HeLa cell nuclear extracts.
Cells were treated for 2 h with TNF (50 ng/ml) (lanes 2 and 3) or TNF plus CHX (10 µg/ml) (lanes 4 and
5) to superinduce NFB. B DNA binding activity was
performed with 20 µg of nuclear extracts. B, Western blot
analysis of native Myo/V1. HeLa cells were treated with TNF (50 ng/ml)
plus CHX (10 µg/ml) for 2 h and cytoplasmic (CYT; 40 µg), mitochondrial (MC; 35 µg), and nuclear
(NE; 30 µg) extracts were prepared, fractionated on a 10%
Tris-Tricine SDS-PAGE, and processed for ECL immunoblotting.
CON, control.
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Myo/V1 Physically Interacts with NFB
Proteins--
To confirm that Myo/V1 physically interacts with NFB
proteins, co-immunoprecipitation studies were conducted both in
vitro and in vivo. Fig.
3A shows that, in
vitro, p50, p65, and c-Rel proteins strongly interacted with
Myo/V1 protein (lanes 2-4). To further confirm these
physical interactions in vivo, HeLa cells were treated with
diluent (lanes 8-10 in Fig. 3B), TNF plus CHX (lanes 5-7 in Fig. 3B), or phorbol ester
(lanes 11-13 in Fig. 3C) for 2 h or
infected with AdMyo/V1 for 12 h (lanes 14 and 15 in Fig. 3C), cellular extracts were
prepared, and co-immunoprecipitation experiments were conducted. In
control diluent-treated cells, Myo/V1 did not associate with any of the
NFB proteins (lanes 8-10 in Fig. 3B).
However, in TNF plus CHX- or AdMyo/V1-treated cells, Myo/V1
predominantly associated with p65 protein (lane 7 in Fig.
3B and lane 15 in Fig. 3C) but not
with p50 protein (lane 6 in Fig. 3B and
lane 14 in Fig. 3C). In phorbol ester-treated HeLa cells (Fig. 3C), Myo/V1 co-immunoprecipitated with
c-Rel and p65 protein (lanes 11 and
12 in Fig. 3C) compared with p50 protein
(lane 13 in Fig. 3C).
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Fig. 3.
Co-immunoprecipitation of
NFB with Myo/V1 protein. A,
recombinant NFB proteins were incubated separately with immobilized
histidine-tagged Myo/V1 protein (bound to nickel resin) and were washed
several times with 1 M NaCl. The bound proteins were eluted
and immunoblotted with respective NFB antibodies. Lane
1, empty nickel resin beads incubated with p65 protein;
lane 2, Myo/V1-bound nickel resin beads incubated with
p65 protein; lane 3, Myo/V1-bound nickel resin incubated
with c-Rel (GST-c-Rel; ~75 kDa); lane 4, Myo/V1-bound
nickel resin incubated with the p52 protein (homologue of p50; GST-p52;
~80 kDa). B, cellular extracts from HeLa cells treated
with TNF plus CHX (lanes 5-7) and diluent (lanes
8-10) were immunoprecipitated with IgG, p50, and p65 antibodies,
and the bound proteins were eluted and immunoblotted for Myo/V1.
C, cellular extracts from HeLa cells treated with phorbol
ester (lanes 11-13) and AdMyo/V1 (lanes
14 and 15) were immunoprecipitated with p50, p65,
and c-Rel antibodies, and the bound proteins were eluted and
immunoblotted for Myo/V1.
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Myo/V1 Promotes the Formation of p50-p50
Homodimers from Monomeric p50 Proteins in Vitro--
Because our
earlier studies indicated that Myo/V1 disrupts and induces the
formation of new NFB-DNA complexes (3) and can physically interact
with NFB proteins in vitro and in vivo (Fig.
3), we designed a detailed study to identify the role of Myo/V1 in
altering the specific NFB dimers in vitro. For this purpose, we initially studied the effect of Myo/V1 on monomeric NFB
proteins. NFB proteins, p50, and p65 have low affinity to form
homodimers among themselves, and a majority of the proteins remain
monomeric without binding to B DNA. However, when incubated together, they exhibit high affinity to its heterologous partner and
they form heterodimers very easily. Purified recombinant p50 protein
and truncated p65 protein (from Dr. Gaurishankar Ghosh) was used for
these studies. First, monomeric p50 proteins were mixed at very low
concentrations (2.5 ng in 50 µl; 2 nM) with Myo/V1
(0-100 ng) and the formation of p50-p50 homodimers were studied using
gel-shift assay (lanes 1-5 in Fig.
4A). Similarly, monomeric
p65 proteins were mixed at very low concentrations (0.5 µg in 50 µl; 7 µM) with Myo/V1 (0-100 ng), the formation of
p65-p65 homodimers were studied, and the effect of Myo/V1 was
studied (lanes 6-10 in Fig. 4A). Although the
addition of Myo/V1 (0-100 ng) led to an increased formation of p50-p50
homodimers (lanes 1-5 in Fig. 4A), there was no
enhanced formation of p65-p65 homodimers (lanes 6-10
in Fig. 4A). We additionally tested the monomeric p65
proteins with higher concentrations of Myo/V1 (0-1500 ng), which also
did not show any formation of additional p65-p65 homodimers (data
not shown). These results indicate that Myo/V1 actively promotes the
formation of p50-p50 homodimers by dynamically interacting with the p50
subunit of NFB. Although Myo/V1 was found to be physically
associated with p65 and its homologues in NFB-activated HeLa cells
(Fig. 3), we do not observe the active promotion of p65-p65 homodimers
by Myo/V1 in vitro.
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Fig. 4.
Myo/V1 promotes the formation of p50-p50
homodimers from monomeric p50 proteins and converts
NFB p50-p65 heterodimers to p50-p50 and
p65-p65 homodimers in vitro. A, GSA
showing the effect of Myo/V1 on monomeric NFB proteins. Increasing
concentrations of Myo/V1 (0-100 ng in 25-ng increments) were added to
p50 (lanes 1-5; 2.5 ng in 50 µl) and p65 proteins
(lanes 6-10; 0.5 µg in 50 µl) separately, and B-DNA
binding activity was measured. Lanes 1 and 6 consist of p50-p50 and p65-p65 homodimers, respectively, without
Myo/V1. In lanes 2-5 and in lanes 7-10,
increasing amounts of Myo/V1 (0-100 ng) were added. B, GSA
showing the effect of Myo/V1 on preformed NFB p50-p65
heterodimers. Increasing concentrations of Myo/V1 were added to
preformed p50-p65 heterodimers, and B-DNA binding activity was
measured. p50 and truncated p65 protein were incubated to form
p50-p65 heterodimers. To serve as a reference, preformed p50-p50
homodimers, p50-p65 heterodimers, and p65-p65 homodimers are
shown in lanes 1, 2, and 12.
Increasing concentrations of Myo/V1 protein were added to preformed
heterodimers in lanes 3-11. The subtle upward shift of the
protein-DNA complex in lanes 3-5 is indicated by
parallel arrows. Arrows in lanes
9-11 denote the freed p65 subunits forming p65-p65
homodimers. C, GSA showing the effect of NFB antibodies
on Myo/V1-generated NFB dimers. Increasing concentrations of Myo/V1
were added to preformed p50-p65 heterodimers (lanes
1-3), and p50 (lanes 4-6) and p65 antibodies
(lanes 7-9) were added. ss, supershifted
complex. D, GSA showing the effect of NFB antibodies on
Myo/V1-generated NFB dimers with excess p65 protein. Increasing
concentrations of Myo/V1 were added to preformed p50-p65
heterodimers with 100-fold excess p65 protein (lanes
1-3), and p50 (lanes 5-7) and p65 antibodies
(lanes 9-11) were added. ss, supershifted
complex. These results are representative of at least three different
experiments.
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Myo/V1 Converts NFB p50-p65 Heterodimers to
p50-p50 and p65-p65 Homodimers in Vitro--
Because Myo/V1 promoted
the formation of p50-p50 homodimers from monomeric p50 proteins, we
tested its effect on p50-p65 heterodimers. Three different
experiments were conducted to study the effect of Myo/V1 (Fig. 4,
B-D).
First, an equimolar mixture of p50 and p65 proteins (16 pmol each)
were allowed to preform p50-p65 heterodimers, and the effect of
Myo/V1 (4-56 pmol) on these dimers was studied by GSA. The results of
this experiment is shown in Fig. 4B, and the effects of
NFB antibodies on Myo/V1-generated dimers is shown in Fig. 4C. Because p50 has higher affinity with p65 than to itself,
the formation of p50-p65 heterodimers under basal conditions (Fig. 4B, lane 2) was expected. The preformed
p50-p65 heterodimers migrated at a location (lane 2 in
Fig 4B) in between the p50-p50 (lane 1 in Fig.
4B) and p65-p65 homodimers (lane 12 in Fig. 4B) in the GSA, and this observed subtle difference in the
mobility between these dimers is similar to that previously described
(43, 51, 52). Because of the deletion of the carboxyl terminus of p65
protein, the truncated p65-p65 homodimers (~31-kDa dimers) migrate faster (lane 12 in Fig. 4B) than p50-p50
homodimers (lane 1 in Fig. 4B). With increasing
concentrations of Myo/V1 (lanes 4-11 in Fig.
4B), we observed a subtle upward shift in the mobility of
the NFB dimers, reaching a position occupied by the p50-p50 homodimers. This gradual upward shift suggests that Myo/V1 converted p50-p65 heterodimers to p50-p50 homodimers (lanes 4-11
in Fig. 4B) in a dose-dependent fashion. 100%
conversion (lane 6 in Fig. 4B) occurred when the
ratio of Myo/V1 to p50-p65 heterodimers reached 1 (16 pmol). At a
lower ratio, the unconverted p50-p65 heterodimers were still present
(lane 3 in Fig. 4B) in the reaction. Furthermore,
at high concentrations of Myo/V1 (32-56 pmol), p65-p65 homodimers started to appear (arrows in lanes
9-11 in Fig. 4B) in these reactions. Because Myo/V1
does not actively promote the formation of p65-p65 homodimers
(lanes 6-10 in Fig. 4A), it is possible the
p65 subunits, freed from the splitting of the p50-p65
heterodimers, accumulated to reach high concentrations and may have
self-associated to form p65-p65 homodimers. This is not
surprising, because p65 exhibits lowest affinity to itself among NFB
subunits; hence, the p65-p65 homodimers are formed only when they
accumulate at high concentrations (43).
Later, to further confirm the nature of Myo/V1-generated dimers, the
effect of NFB antibodies on these complexes was studied (Fig.
4C). The p50-p65 heterodimers were pre-assembled at
equimolar ratio as previously described, and Myo/V1 was added at two
(100 and 400 ng) concentrations (lanes 2 and
3 in Fig. 4C). These reactions were performed in
triplicate for p50 and p65 antibody supershifts (lanes 5,
6, 8, and 9 in Fig 4C). As
expected, we observed both an upward shift in the mobility of
Myo/V1-shifted dimers occupying the position of p50-p50 homodimers
(lanes 2 and 3 in Fig. 4C) as well as
an increased formation of p50-p50 homodimers with increasing amounts of
Myo/V1 (lanes 2 and 3 in Fig. 4C) in
these reactions. Lanes 4-6 confirm that these dimers have
p50 proteins as revealed by p50 antibody supershifted complexes.
Lanes 7-9 exhibit the inhibitory effects of p65 antibody on
Myo/V1-generated dimers. Because the p65 antibody exhibits inhibitory
activity on p65 proteins (Rel DNA binding domain antibodies are not
supershiftable), the formation of heterodimers as well as the
conversion to homodimers was also affected (lanes 7-9).
Hence, the subtle upward shift observed in lanes 2 and
3 was not observed in lanes 8 and 9. Lanes 8 and 9 contain a mixture of
p50-p50 homodimers and p50-p65 heterodimers. Close examination of
the migratory patterns and the levels of these dimers (lanes
8 and 9 in Fig. 4C) reveals that, in
the presence of p65 antibody, Myo/V1 promoted the formation of p50-p50
homodimers from the residual free monomeric p50 proteins, although at a
reduced level. The splitting of the heterodimers as observed in
lanes 2 and 3 did not occur in these
reactions (lanes 8 and 9) because of
the presence of p65 antibody. The NFB dimers that appear in lanes 7-9 are predominantly p50-p50 homodimers,
as evidenced by its location.
To further confirm the chaperone functions of Myo/V1, we conducted
another experiment (Fig. 4D), where the amount of p65 was
kept excess by 100-fold to that of p50 so that, in addition to the
preformed p50-p65 heterodimers, free p65 proteins will be present
in the reaction. The effect of Myo/V1 (lanes 1-3 in Fig.
4D), and the effect of NFB antibodies on these
Myo/V1-shifted complexes were studied (lanes 6-8 and
10-12 in Fig. 4D). The rationale for this
experiment is that, when the p50-p65 heterodimers are split by Myo/V1,
there will be a competition among the released monomeric proteins to
form either homodimers or heterodimers. With excess p65 protein in the
reaction and because of its high affinity toward p50 protein to form
heterodimers (43), there will be a continuous formation of new p50-p65
heterodimers as the old heterodimers are being converted by Myo/V1.
Thus, in this experiment Myo/V1-generated "intermediate products"
can be identified. The p50-p65 heterodimers were pre-assembled at
1:100 ratios, and Myo/V1 was added at two (100 and 400 ng)
concentrations (lanes 2 and 3 in Fig.
4D). These reactions were performed in triplicate for p50
and p65 antibody supershifts (lanes 5-7 and
9-11 in Fig. 4D). As expected (shown in Fig.
4D), both heterodimers and homodimers were formed when
Myo/V1 was present at high concentrations with excess p65 (lane
3). The complete subtle upward shift observed in lane 3 of Fig. 4C was not similarly observed in lane 3 of Fig. 4D, strongly indicating the presence of both
heterodimers and homodimers. The presence of both of these dimers is
more clearly seen in lanes 7 and 11.
Additionally, we also observed a simultaneous reduction in
p65-p65 homodimers (lanes 1-3) with the addition of
Myo/V1. Because Myo/V1 does not have any direct effect on p65 per
se (lanes 6-10 in Fig. 4A), the reduction
in p65-p65 homodimers in lanes 1-3 is a result of its
incorporation into p50 protein in the reaction to form p50-p65
heterodimers. Furthermore, the absence of p50-p65 heterodimers in
lanes 6 and 10 and the presence of these dimers
in lanes 7 and 11 correlate with the reduction of
p65-p65 homodimers in lanes 7 and 11. Lanes 5-7 confirm that the Myo/V1-generated
dimers have p50 proteins, as revealed by supershifted complexes.
Because of excess p65, p50 antibody supershifts did not occur
completely. Because the p65 antibody used in lanes 9-11
exhibits inhibitory activity on p65 proteins, the complete conversion
of heterodimers to homodimers is affected. Thus, these results further
confirm the chaperone functions of Myo/V1 in promoting NFB
homodimers and the formation of p50-p65 heterodimers is an indirect
event promoted by the chaperone functions of Myo/V1 on p50.
Overexpression of Myo/V1 Changes the Ratio of
Activated NFB Dimers in Vivo in Favor of p50-p50 Homodimers--
To
further confirm our in vitro results, in vivo
studies were conducted to identify the role of Myo/V1 in NFB
homodimer generation. For this purpose, a strategy of
adenovirus-mediated overexpression of Myo/V1 was chosen to exploit the
inherent activation of NFB heterodimers by recombinant adenoviral
vectors (57-59). Thus, Myo/V1 overexpressing recombinant adenovirus is
expected to alter the composition of activated NFB dimers induced by
its viral vector backbone (57-59). HeLa cells were infected with
recombinant adenoviruses expressing Myo/V1 (AdMyo/V1) and
-galactosidase (Adgal), and nuclear extracts were prepared
12 h after infection. B DNA binding reactions (Fig.
5A) were conducted with three
high affinity NFB dimer-specific oligos, and the levels of
individual NFB dimers were quantified (Fig. 5, B and
C). Identical amounts of B oligos (25,000 cpm) with
similar specific activities were incubated with identical amounts of
nuclear extracts, and the resulting NFB-DNA complexes were
fractionated on the same gel before autoradiography.
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Fig. 5.
Myo/V1 changes the ratio of
NFB dimers in vivo
in favor of p50-p50 homodimers. A, GSA showing
the effect of Myo/V1 on in vivo generated NFB
dimers. HeLa cells were infected with AdMyo/V1 or Adgal recombinant
adenoviruses at a multiplicity of infection of 10. Twelve hours after
infection, nuclear extracts were prepared and GSAs were conducted with
three high affinity NFB dimer-specific oligonucleotides (B#seqB
for p50-p50 homodimers; B#u-iNOS for p65-p65 homodimers; IgGB
oligonucleotide for p50-p65 heterodimers) (lanes 1-6).
Supershift experiments with NFB p50 and p65 antibodies were
conducted to confirm the nature of the NFB dimers (lanes
7-15). p50 supershift complexes are indicated by *,
and p65 supershift complexes are indicated by arrows.
B, quantitative comparison of NFB dimers between Adgal
and AdMyo/V1. The bar graph shows the -fold
change in the levels of individual NFB dimers in relation to
Adgal-infected HeLa cells. C, relative levels of NFB
dimers in Adgal- and AdMyo/V1-infected cells. The bar
graph shows the NFB dimer ratio in relation to p50-p65
heterodimers in Adgal- and AdMyo/V1-infected HeLa cells. These
results are representative of six different experiments, conducted four
times at a multiplicity of infection of 10 and twice at a multiplicity
of infection of 50.
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Results show that overexpression of AdMyo/V1 enhanced the generation of
p50-p50 (lane 2 in Fig. 5A) and p65-p65
(lane 4 in Fig. 5A) homodimers compared with the
control Adgal (lanes 1 and 3 in Fig.
5A). Interestingly, p50-p65 heterodimers are less abundant
in AdMyo/V1 compared with the Adgal-overexpressing cells (lanes 5 and 6 in Fig. 5A). Thus, the
observation of a simultaneous decrease in the levels of p50-p65
heterodimers and an increase in p50-p50 and p65-p65 homodimers suggests
a conversion from heterodimers to homodimers (Fig. 5B).
Moreover, an unequal quantitative shift (Fig. 5B) occurred
between p50-p50 homodimers and p50-p65 heterodimers in
Myo/V1-overexpressing cells. Although the levels of p50-p50 homodimers
were elevated by 70%, the p50-p65 heterodimers declined by 26% (Fig.
5B). This observation suggests that Myo/V1, in addition to
generating the p50-p50 homodimers from the p50-p65 heterodimeric substrates, might directly generate nascent p50-p50 homodimers from
other in vivo substrates, further confirming our previous in vitro results (Fig. 4B). Furthermore, analysis
of the relative levels of NFB dimers (Fig. 5C) in
AdMyo/V1-infected cells (p50-p65:p50-p50:p65-p65; 1.0:1.3:0.5) revealed
that the levels of p50-p50 homodimers exceeded the levels of p50-p65
heterodimers compared with Adgal (1.0:0.55:0.2). Because p50-p50
homodimers possess transcriptionally repressive activity (17-22), the
observed shifts in the NFB dimer ratio in Myo/V1-overexpressing
cells might have repressive effects on the NFB-mediated
transcription process.
Antibody supershift experiments and heterologous B oligo chase
experiments (data not shown) confirmed the nature of NFB dimers
(lanes 7-15 in Fig. 5A) binding to the B
oligos B#SeqB, B-Ig, and B#u-iNOS. However, the p50
antibody supershifting the NFB complexes bound to B#u-iNOS oligo
(lane 11 in Fig. 5A) was unexpected. We believe
this is because of "antibody-induced effect" rather than residual
p50-p65 heterodimers binding to these B oligos. The p50 antibody
after binding to a p50-p65 heterodimer could change the affinity of the
"supershifted heterodimeric ternary complex" now to recognize and
bound to a otherwise p65-p65 homodimer specific oligo. Although
antibodies used in gel-shift assays are generally described as
"supershifting antibodies," these antibodies change the affinity of
homo- and heterodimers and thereby affect their binding to B sites.
Evidence of these artifacts is seen in lanes 12 and 15 where increased (lane 12) and
decreased (lane 15) binding is observed after the
antibody was added. Therefore, as described previously (44, 45) without
the NFB antibodies, B#seqB and B#u-iNOS oligos exhibit high
affinity and exclusive specificity toward p50-p50 and p65-p65
homodimers. Additionally, we conducted heterologous B oligo chase
experiments (B#u-iNOS-bound complexes chased with 100× cold
B#SeqB and B-Ig oligos), and the results revealed that
Myo/V1-overexpressing HeLa cells still produced higher levels of
p65-p65 homodimers compared with Adgal (data not shown). Additional
chase experiments (B#SeqB complexes chased with 100× cold
B#u-iNOS and B-Ig oligos; B-Ig complexes chased with
100× cold B#50-2 and B#u-iNOS oligos) were done, and the
results were exactly the same as in Fig. 5A (data not shown).
Myo/V1 Represses NFB-mediated Transcription
Process--
To identify the functional relevance of Myo/V1 with
respect to NFB dimers, we studied the role of Myo/V1 on
NFB-mediated transcription. HeLa cells were cotransfected with
pB-tk-luc, p65 pRSV-RelA, and pcDNA-AM1.1-Myo/V1 expression
vector. Forty-eight hours after transfection, HeLa cells were harvested
and lysed and luciferase enzyme activity was measured. The results
(Fig. 6) show that overexpression of
Myo/V1 significantly reduced the NFB-mediated transcription by 42%
(p 
0.05) on p65-mediated luciferase reporter
activity. Because p50-p65 heterodimers are not available for Myo/V1 to
act, and because Myo/V1 does not actively promote p65-p65 homodimers
per se, the observed inhibition is probably the result of
the initial generation of abundant p50-p50 homodimers (Myo/V1 acting on
p50-p105 complexes) that are well known for transcriptional repression
of their target genes (17-22).
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Fig. 6.
Myo/V1 represses
NFB-mediated transcription process. HeLa
cells were cotransfected with constant amounts of
tk-B2-luciferase reporter (500 ng) and pRSV-RelA (500 ng). Myo/V1 or -gal control expressing plasmid (200 ng) was
cotransfected, and, 48 h later, cells were harvested and lysed and
luciferase enzyme activity was measured. Luciferase activity was
normalized for protein concentration and transfection efficiency.
Transfection experiments were performed four times, and the data are
shown as -fold change over control cells. Comparison of two groups was
done by unpaired Student's t test. All statistical analysis
was performed with SigmaStat (SPSS Inc.). Significance was accepted at
p < 0.05.
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Three-dimensional Structural Alignment of Myo/V1
with Rel-interacting Ankyrin Repeats of IB Reveals the Potential
Chaperone Functions of Myo/V1 on NFB--
To
correlate the observed chaperone functions of Myo/V1 with its
structural domains, we conducted the three-dimensional superimposition analysis (60) using the three-dimensional NMR structure of Myo/V1 (Fig.
7A) (11, 12) and the
three-dimensional structure of IB (Fig. 7B) (13, 14),
where NFB-interacting domains were well characterized. The
superimposed C traces of Myo/V1 with IB (Fig. 7, C
and D) show that entire Myo/V1 aligned with ankyrin repeats
3-6 of IB protein, further confirming our earlier primary structural homologies with IB (3). This finding is highly significant because these specific ankyrin repeats of IB interact with the Rel domain of p50 and p65 subunits (Fig. 7, C and
D) that are known for dimerization and B DNA binding.
Moreover, the ankyrin repeats 1 and 2 of IB (aa 67-142), which
mask the NLS signal of p65, the amino-terminal SRD (aa 1-66 of
IB), and the carboxyl-terminal acidic PEST domain (aa 281-317)
that is responsible for degradation of IB, are all absent in the
Myo/V1 protein. Thus, the fact that Myo/V1 resembles only the
Rel-interacting ankyrin repeat domains of IB (AR 3-6) strongly
suggests that Myo/V1 might similarly interact with the Rel DNA binding
domain of NFB but, unlike IB, Myo/V1 might mediate its
chaperone functions in a zipper-like fashion using the terminal
residues of its three finger loops (Fig. 7, C and
D). One of the overlooked functions of IB is its use
of these ankyrin repeats (AR 3-6) to assemble the inactive
NFB dimers in the cytoplasm. Similar to Myo/V1, IB might also
possess zipper chaperone function; however, it is only used to assemble
the inactive NFB dimers in the cytoplasm. Because of its other NLS
masking ankyrin repeats (repeats 1 and 2) and SRD domain, it behaves as
an inhibitor or a carrier of NFB. Because of the observed similarity
in Rel-interacting ankyrin repeats, one could envision that IB and
Myo/V1 proteins could behave as lock and key of NFB dimers in the
mammalian cell.
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Fig. 7.
Three-dimensional structural homologies
between Myo/V1 and IB. The
three-dimensional superimposition analysis (60) was done on the Dali
server (www2.ebi.ac.uk/dali/fssp/). The C traces of Myo/V1
(A) and IB (B) were superimposed
(C), viewed by Rasmol plug-in software, and captured.
D, primary sequence alignment of Myo/V1 with IB using
the three-dimensional superimposition analysis data. Purple
and dark blue colored residues in IB are known to
interact with p50 and p65 proteins, respectively (13, 14). Red
AR1-6 denote ankyrin repeats of IB, and purple
AR1-3 denote ankyrin repeats of Myo/V1. PEST is the
degradation domain of IB.
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DISCUSSION |
Myo/V1 was first identified as an up-regulated intracellular
protein in pathophysiological conditions (1, 4-6) such as human heart
failure, as well as during early organ developmental processes such as
in postnatal mammalian cerebellum (2, 4, 8, 10). However, later it was
identified to express in every mammalian organ and cell type. Moreover,
Myo/V1 is an evolutionarily conserved protein from multicellular
organisms to mammals (Fig. 8). Among
mammals it shows 97% homology, and with invertebrates it exhibits 67%
homology. Putative nuclear import and export signals as well as
potential phosphorylation sites were mostly conserved between
invertebrates and vertebrates (Fig. 8). Our three-dimensional structural alignment studies (Fig. 7C) further revealed that
Myo/V1 possessed only Rel domain-interacting ankyrin repeats and
resembles a truncated form of IB protein without the SRD, NLS
masking, and PEST degradation domains (Fig. 7D).
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Fig. 8.
Evolutionary conservation of Myo/V1.
Sequences of Myo/V1 from various species were identified in the
GenBankTM database (rat, U21661; human, AC015987; mouse, AAA86719;
chicken, BAA05379; Drosophila, AAF48915;
Caenorhabditis elegans, AAA96086; marine sponge, CAC38782)
and aligned using CLUSTAL W alignment program. Potential
phosphorylation and nuclear import (aa 36-51) and export (aa 52-60)
signals are highlighted. Nuc. Imp. Signal,
nuclear import signal; Nuc. Exp. Sig., nuclear export
signal; GSK3/CK-I, glycogen synthase kinase 3/casein kinase
I; PKC, protein kinase C; CK-II, casein kinase
II.
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Results presented in this study indicate that, under basal conditions,
cellular Myo/V1 predominantly present in the cytoplasm and probably
associates with cytoskeletal structures (Fig. 1) similar to IB
(61, 62). Additionally, low levels of Myo/V1 are also present in the
nucleus (Fig. 1C) of unstimulated HeLa cells and in other
cell types such as Jurkat T cells and rat neonatal cardiac
myocytes.2 Our studies also
demonstrated that, upon stimulation with TNF, Myo/V1 relocates within
the cytoplasm (Fig. 1B) and further translocates to the
nucleus during sustained induction of NFB (Figs. 1B and 2B). Because Myo/V1 possesses both nuclear import and export
signals (Fig. 8), it is expected that Myo/V1 might shuttle between
cytoplasm and nucleus similar to IB family of proteins (32-36).
Co-immunoprecipitation studies showed that, although Myo/V1 interacts
with all NFB proteins in vitro, it remains physically
associated only with p65 and c-Rel proteins in vivo (Fig.
3). The differences between in vitro and in vivo
results might also reflect the potential function of Myo/V1 protein.
For example, Myo/V1 may interact dynamically with p50 proteins in
vivo in a transient fashion to mediate its zipper chaperone
function; however, it remains physically associated with p65 or its
homologue c-Rel protein to prevent them from reassociating with p50.
In vitro NFB interaction studies indicated that Myo/V1 can generate p50-p50 homodimers from monomeric p50 proteins and can
convert the preformed p50-p65 heterodimers into p50-p50 and p65-p65
homodimers (Figs. 4 and 9A).
Compared with control Adgal, overexpression of AdMyo/V1 resulted in
an increase in both p50-p50 and p65-p65 homodimers exceeding the levels
of p50-p65 heterodimers (Fig. 5). A simultaneous decrease in the levels
of p50-p65 heterodimers was also observed, suggesting a conversion from
transactivating heterodimers to transcriptionally repressive homodimers
(17-25). However, the observation of disproportionate increase in the
levels of p50-p50 homodimers compared with the decreased levels of
heterodimers (70% increase versus 26% decrease) raises the
possibility that Myo/V1 might also generate p50-p50 homodimers from
other inactive NFB substrates. Because a majority of the Myo/V1 is
localized in the cytoplasm, it could very well be involved in the
activation of p50-p50 homodimers by acting on p50-p105 substrates (63) in vivo (Fig. 9B). Alternatively, Myo/V1 could
assemble the p50-p50 homodimers from free monomeric p50 proteins in the
cytoplasm similar to the conditions mentioned in Fig. 4A.
Additionally, Myo/V1 could act on any p50 subunit containing complexes
(p50-p65, p50-c-Rel, p50-RelB, and "p50-non-NFB" dimers) in the
nucleus and further generate the transcriptionally repressive p50-p50
homodimers (Fig. 9B). In accordance with these observations,
overexpression of Myo/V1 also resulted in significantly reduced
NFB-mediated luciferase reporter gene expression (Fig. 6), which is
probably a result of the generation of p50-p50 homodimers.
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Fig. 9.
Model for zipper chaperone function of
Myo/V1 (A) and potential in
vivo NFB substrates for
Myo/V1 protein (B). Model shows potential
involvement of Myo/V1 in multiple NFB activation pathways in HeLa
cells. B, 1, potential involvement of Myo/V1 in
generating p50-p50 homodimers from p50-p105 complexes, and
2, conversion of heterodimers to homodimers by Myo/V1 during
sustained activation of NFB. HDAC, histone
deacetylase.
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Although we observed an increased level of p65-p65 homodimers during
AdMyo/V1 overexpression (Fig. 5A), and Myo/V1 being
physically associated with p65 and its homologues (Fig. 3), in
vitro interaction experiments (Fig. 4A) do not suggest
an active role for Myo/V1 in the generation of p65-p65 homodimers.
Moreover, in overexpression of Myo/V1 in HeLa cells through transient
transfection methods (Fig. 6), where "active p50-p65 heterodimers"
are not available for Myo/V1 to act upon, we only observed an
inhibition of B driven luciferase reporter activity. If Myo/V1 would
have actively promoted the generation of p65-p65 homodimers in these
cells, we should have observed an increased B-luciferase reporter
activity during Myo/V1 overexpression. Because Myo/V1 does not retain
or sequester p65 in the cytoplasm2 under basal conditions
(Fig. 3B), the observed inhibition of B-luciferase
reporter activity is the result of the active generation of p50-p50
homodimers by Myo/V1, as observed in our in vitro and in vivo experiments (Figs. 4 and 5). Therefore, we believe
the increased generation of p65-p65 homodimers during AdMyo/V1
expression is an indirect effect of the conversion of p50-p65
heterodimers by Myo/V1 and the physical association of Myo/V1 with p65
is probably to prevent the p65 from reassociating with p50 (Fig.
9A).
Even though our studies indicate that Myo/V1 could favor the active
generation of NFB homodimers in HeLa cells, it is possible that,
under certain scenarios (similar to conditions mentioned in Fig.
4D), Myo/V1 could indirectly induce the formation of NFB heterodimers in other cell types. For example, Myo/V1 could potentially act on inactive p50-p65-IB substrate complexes in the cytoplasm and generate active NFB heterodimers in immune cells. Because Myo/V1
has been identified as a "severe combined immunodeficiency complementing gene" (GenBankTM accession no. D78188), and because this protein is also found in the nuclear extracts of phorbol ester-treated Jurkat T cells,2 the potential role of Myo/V1
in the activation of other NFB dimers is highly likely.
The data presented in this study were further strengthened when, from
previously published independent studies (2, 64-66), we identified a
strong temporal relationship between the intracellular levels of Myo/V1
and NFB activation in vivo in mammalian organs. Myo/V1
was found to occur at elevated levels in failing human hearts (1, 4-6)
and found to be transiently elevated in postnatal mammalian cerebellum
(2, 7). Interestingly, NFB dimers were also found to be activated in
failing human hearts2 (67) as well as in developing
postnatal cerebellum (64-66) during this period. Specifically, in
postnatal cerebellum, the intracellular level of Myo/V1 protein was
found to rise at the 2nd day, peak at the 7th day, and rapidly decrease
by the 12th day (2). Similarly, independent studies have reported the
transient activation of NFB dimers begin to occur around postnatal
day 2, peaking at the 7th day, and declining by 12th day in rat (64,
65) and mouse cerebellum (66). This strong temporal observation that the intracellular levels of Myo/V1 being elevated exactly at the same
time during NFB dimer activation and its decline during the
disappearance of NFB dimers in postnatal cerebellum further highlights the important potential role of Myo/V1 even in initial NFB activation. The earlier observation of disproportionate increase in the levels of p50-p50 homodimers compared with the decreased levels
of heterodimers in AdMyo/V1-infected cells (Fig. 5) also raises the
possibility that Myo/V1 might be involved in the initial activation of
p50-p50 homodimers in vivo (Fig. 9B). Therefore, the results presented in this study strongly suggest that not only
Myo/V1 is involved in converting heterodimers into homodimers during
sustained NFB activation, it could potentially be involved in the
initial activation of NFB dimers. The recent identification that
entire NFB machinery (NIK, IKKs, IB, ubiquitination,
and proteasome) is shuttling (68, 69) between cytoplasm and the nucleus
of resting mammalian cells (32-35) and the observation of NFB
activation without IB degradation (70, 71) changes the central tenet
of NF
TOP
ABSTRACT
INTRODUCTION
