RelB Cellular Regulation and Transcriptional Activity Are
Regulated by p100*
Nancie J.
Solan
,
Hiroko
Miyoshi,
Eva M.
Carmona,
Gary D.
Bren, and
Carlos V.
Paya§¶
From the Departments of Immunology and
§ Experimental Pathology and Laboratory Medicine and the
¶ Division of Infectious Diseases, Mayo Clinic,
Rochester, Minnesota 55905
Received for publication, October 4, 2001
 |
ABSTRACT |
RelB mediates the constitutive nuclear pool of
NF-
B transcriptional activity in myeloid and lymphoid cells, which
is believed to be secondary to its weak interaction with the classical
NF-B inhibitor proteins, the IBs. In other cell types, RelB is
located in the cytosol, thus suggesting that RelB is also regulated by an inhibitory protein(s). In this study, it is demonstrated that RelB
is associated in the cytosol with p100 but not with IB
, IB
,
IB
, nor p105. Its cytosolic control is not affected by stimuli
that lead to RelA nuclear translocation, and RelB nuclear localization
is prevented by p100, but not by p105 or IB. Structure function
analysis p100-RelB interactions indicates that p100 amino acids
623-900 are required for effective interaction and repression of
nuclear translocation and RelB driven NF-B-dependent
transcription. Moreover, this carboxyl-portion of p100 contains
a nuclear export signal(s), which is required for effective retrieval
of RelB from the nucleus. Finally, overexpression of NF-B-inducing
kinase, a kinase that has recently been shown to induce p100
processing, possibly through IKK activation, causes nuclear
translocation of RelB protein. Thus, these studies indicate that p100
is a bone fide inhibitor of RelB and that this transcription factor may be regulated by NF-B-inducing kinase and/or IKK.
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INTRODUCTION |
NF-B is a family of ubiquitously expressed transcription
factors involved in the regulation of activation, proliferation, differentiation, and death of numerous cell types (1-4). The NF-B
family is composed of proteins that form homodimers or heterodimers with each other, and includes NF-B1 (p50), NF-B2 (p52), RelA, RelB, and c-Rel. The NF-B dimers remain transcriptionally inactive when anchored in the cytosol through association with one of the ankyrin-containing NF-B inhibitors, the IBs: IB, IB,
and IB (5), or with p100 or p105 (atypical ankyrin-containing inhibitors that share a gene transcript with p52 and p50, respectively) (6-10). Phosphorylation of the NF-B inhibitor protein at specific serine residues by the IKK complex targets it for ubiquitination and
degradation by the proteasome, thus enabling the NF-B dimer to
translocate into the nucleus where it can bind to the promoter regions
of numerous genes (1, 11-15). The phosphorylation leading to
ubiquitination and degradation of NF-B inhibitor molecules has been
well characterized for the typical IB molecules, such as IB,
-, and - (1, 13-17). p105 has recently been shown to contain
analogous serine sites which are also susceptible to phosphorylation by
the IKK complex, and stimuli that target the activation of the IKK
complex, such as TNF1
(18), do lead to the phosphorylation of p105 on these serine residues
(19). No such TNF-sensitive IKK sites have yet been identified on
p100 (19, 20).
Recent investigations have given NF-B-inducing kinase (NIK) and
IKK (a component of the IKK complex) important roles in the
regulation of p100 protein (21, 22). When NIK was first characterized
as a mitogen activated protein kinase kinase kinase, it was
shown to be involved in IKK activation; however, subsequent studies
clarified that NIK may not be crucial for IKK activation by
inflammatory cytokines (23-25). Recent work has identified IKK, upon activation by NIK, as a specific inducer of p100 processing, causing site-specific phosphorylation, leading to its ubiquitination and subsequent processing to p52 (21, 22).
IB inhibitor proteins also exert their control of NF-B by
entering the nucleus and retrieving the NF-B heterodimer, thereby escorting it out of the nucleus and into the cytoplasm. This function is mediated by nuclear import sequences located within the ankyrin repeats (26) and by nuclear export signals (NES) (27-31). Mutation of
the NES sequence in IB, or inhibition of nuclear export of IB with the pharmacological inhibitor leptomycin B, which targets the nuclear export receptor CRM1, prevents IB from exiting the nuclear compartment of the cell (32-34). No CRM1-binding export sequences have been identified in the other IB molecules (31).
RelB was originally described as an inhibitor of NF-B activity, as
it cannot form homodimers nor directly bind to DNA (35). Yet, when
coupled with either p50 or p52, RelB can trigger potent transcriptional
activation (36, 37). RelB contains an amino-terminal activating domain,
not present in RelA or c-Rel, which is required, along with the
carboxyl-terminal domain, for effective gene transactivation (38). RelB
expression is mainly restricted to lymphoid tissues including the
thymic medulla, the periarterial lymphatic sheaths of the spleen, and
the deep cortex of the lymph nodes (39). These areas all correspond to
regions containing antigen-presenting interdigitating dendritic cells.
In fact, in mature B cells, differentiated dendritic cells, and
macrophages, RelB has a strong and predominant nuclear localization
(39-43). RelB
/ mice survive in utero, but die as early
as 10 days after birth from extensive multiorgan mixed
lymphoproliferation, myeloid hyperplasia (excess macrophages and
granulocytes), and splenomegaly due to extramedullary hematopoiesis (44). These mice lack mature antigen-presenting dendritic cells and a
thymic medulla (45). The studies performed in RelB-deficient mice
highlight the fact that RelB is essential to the development and
regulation of certain cell types, and that its absence cannot be
compensated by the presence of the other NF-B proteins.
RelB regulation differs significantly from the other two NF-B
transactivators, RelA and c-Rel. It is not apparently regulated by
IB or IB (46). In fact, RelB binds IB only weakly
(47), and IB not at all (48). RelB's weak interaction with these IBs, coupled with its constitutive nuclear presence in some cell types, such as the antigen-presenting dendritic cells mentioned above,
has led RelB to be classified as the member of the NF-B family that
contributes to the constitutive pool of NF-B within a cell (47, 49).
In mature B cells, RelB is mainly located in the cytosol, and
translocates to the nucleus only within several hours following CD40
activation (50-52) or treatment with platelet-derived growth factors
or serum in fibroblasts (53). Because these stimuli also result in an
increase in RelB transcription and protein synthesis, it is infered
that its nuclear translocation may be secondary to an excess of RelB
levels over those of a putative cytosolic inhibitor. What controls
RelB's cytosolic retention in certain cell types and not others
remains unknown.
In this study we have investigated whether IB molecules, other than
IB or IB, control the cellular localization of RelB. We
report here that p100 is the only IB molecule able to associate with
RelB in vivo and inhibit both RelB nuclear localization and RelB-dependent transcriptional activation. Moreover, it is
demonstrated that NIK triggers the nuclear translocation of RelB
secondary to the degradation of p100.
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EXPERIMENTAL PROCEDURES |
Plasmids--
DNA encoding human RelB or RelA was cloned into
the pcDNA3 vector already containing an HA-tag immediately upstream
of the transcription start site for the inserted DNA. p100 cDNA
encoding amino acids 407-582, 407-623, 407-744, 407-900; amino
acids 401-920 of p105; or full-length IB cDNA were cloned
into pcDNA3 vector containing a Flag-tag immediately upstream of
the transcription start site for the inserted DNA. cDNA encoding
amino acids 407-582, 407-623, 407-744, or 407-900 of p100 were
cloned into pcDNA3 vector containing a cDNA sequence for green
fluorescent protein (GFP) immediately upstream of the transcription
start site for the inserted DNA. cDNA encoding human RelB or RelA
were cloned into pcDNA3 vector containing sequence for red
fluorescent protein (RFP) immediately upstream of the transcription
start site for the inserted DNA. The B-luciferase reporter plasmid
consists of three NF-B concatamers from the human immunodeficiency
virus-long terminal repeat cloned upstream of a concavalin-A
minimal promoter driving the expression of luciferase. The
TK-renilla-luciferase plasmid contains cDNA encoding
Renilla luciferase under the control of an upstream herpes simplex virus-thymidine kinase promoter (Promega, Madison, WI). cDNA encoding p52, RelB, p100, p105, IB, IKK, or
kinase-dead IKK were cloned into pcDNA3 vector. myc-NIK plasmid
was obtained from Tularik (South San Francisco, CA). The p105
expression vector was obtained from the National Institutes of Health
AIDS Research and Reference Reagent Program, Division of AIDS, NIAID,
National Institutes of Health, from Gary Nabel.
Cell Culture--
HeLa and 293T cells were cultured in
Dulbecco's modified Eagle's medium (Invitrogen, Rockville, MD)
supplemented with 10% heat-inactivated fetal bovine serum (Intergen,
Purchase, NY), penicillin (50 units/ml), streptomycin (50 µg/ml), and
glutamine (2 mM). In one experiment, cells were treated in
culture with 10 ng/ml tumor necrosis factor (TNF)- (R&D Systems,
Minneapolis, MN) for the times indicated.
Gene Expression and Cell Extract Preparation--
Transfections
were performed with FuGENE6 (Roche Molecular Biochemicals,
Indianapolis, IN) or LipofectAMINE Plus (Invitrogen) according to the
manufacturer's protocol. Cytosolic proteins used in
immunoprecipitation experiments were obtained from the supernatant of
cells first washed with cold phosphate-buffered saline and then lysed
with buffer containing 50 mmol/liter Tris, 150 mmol/liter NaCl, and
0.1% Triton X-100 plus 300 µM sodium orthovanadate, 1 mM dithiothreitol, 0.5 mM
3,4-dichloroisocoumarin, 2 µM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 1 µg/ml leupeptin, and 1 µg/ml
pepstatin. Lysates were either subjected to immunoprecipitation, as
described below, or to SDS-PAGE followed by electrophoretic transfer to polyvinylidene difluoride membrane and analysis for immunoreactivity with the appropriate antibody. Nuclear and cytosolic proteins were
prepared using a modification of the method of Dignam (54). Briefly,
cells were washed with cold buffer A (10 mM Hepes, pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 10 µg/ml
aprotinin, 2 µg/ml leupeptin, 1 µg/ml pepstatin, 1 mM
dithiothreitol, and 2 µM phenylmethylsulfonyl fluoride)
and lysed in buffer A plus 0.1% Nonidet P-40 for 10 min on ice. The
cytosolic fraction was harvested by centrifuging the lysate at
4500 × g for 3 min and collecting the
supernatant. The nuclear pellet was washed twice with buffer A
(centrifuging at 4500 × g for 3 min each time). The
nuclear proteins were then extracted by resuspending the pellet in 20 mM Hepes, pH 7.9, 0.42 M NaCl, 1.5 mM MgCl2, 0.2 mM EDTA,
dithiothreitol, 3,4-dichloroisocoumarin, and protease inhibitors as in
buffer A, plus 25% glycerol for 30 min on ice.
Immunoprecipitation and Western Blotting--
100-300 µg of
cytosolic protein were incubated with one of the following antibodies:
rabbit polyclonal anti-human RelB (raised against amino-terminal
peptide PSRRVARPPAAPE), rabbit polyclonal anti-human IB sc-371,
rabbit polyclonal anti-human IB sc-945, and rabbit polyclonal
anti-human IB sc-7156 (Santa Cruz, Santa Cruz, CA), rabbit
anti-human p100 and rabbit anti-human p105, generated against peptide
epitopes within the COOH termini of p100 and p105 (generous gifts from
Dr. Nancy Rice) (10), mouse monoclonal anti-HA, clone 12CA5 (Roche
Molecular Biochemicals), or anti-Flag (Sigma) for 1-2 h at 4 °C,
after which protein A-agarose beads (Invitrogen) were added for 1 h at 4 °C. The beads were then washed three times with lysis buffer
and heat denatured to separate proteins from beads. Immunoprecipitated
proteins were subject to SDS-PAGE and electrophoretically transferred
to polyvinylidene difluoride membrane. Immunoblotting was performed
with specific antibodies and visualized by using the ECL Western
blotting detection kit (Amersham Bioscience, Inc., Buckinghamshire,
UK). Antibodies used for immunoblotting include those listed above in
addition to rabbit polyclonal anti-RelB sc-226, rabbit polyclonal
anti-RelA sc-109, rabbit polyclonal anti-p50 sc-7178, rabbit polyclonal anti-p52 sc-298 (Santa Cruz, Santa Cruz, CA), and mouse monoclonal anti-histone (Qiagen, Valencia, CA).
Immunofluorescence--
HeLa cells were plated onto washed glass
coverslips in six-well culture plate chambers. 24 h later they
were transfected by FuGENE6 (Roche Molecular Biochemicals) according to
the manufacturer's protocol. Immunofluorescence was performed 18 h after transfection as follows. Cells were washed 3 times with
phosphate-buffered saline at 37 °C, fixed in phosphate-buffered
saline plus 2% paraformaldehyde for 10 min at 37 °C, washed again,
and quenched in phosphate-buffered saline containing 50 mM
glycine for 5 min at room temperature. Cells were washed again,
permeabilized with methanol for 2 min at room temperature, washed, and
blocked with phosphate-buffered saline containing 5% goat serum and
5% glycerol for 30 min at room temperature. Blocking was done only for
those experiments using anti-HA antibody. After blocking, cells were
again washed and incubated with anti-HA antibody (2 µg/ml, Roche
Molecular Biochemicals) in phosphate-buffered saline containing 0.09%
sodium azide, 1% bovine serum albumin, 0.1% Tween 20, and 10 µg/ml
DAPI (4',6-diamidino-2-phenylindole) (Sigma) for 1-2 h at room
temperature, followed by washing and further incubation with a
fluorescein isothiocyanate-conjugated goat antibody to rat IgG (ICN,
Costa Mesa, CA). Incubation mixtures for experiments visualizing GFP or
RFP constructs did not include the anti-HA antibody. Following antibody
and/or DAPI incubation, cells were washed, inverted, and mounted onto
microscope slides. Slides were viewed by confocal microscopy at ×400
magnification with UV, HeNe, and argon lasers for excitation.
Inhibition of Nuclear Export--
Cells studied for inhibition
of nuclear export were incubated with 200 ng/ml leptomycin B (generous
gift from Dr. Minoru Yoshida, Tokyo, Japan) for 6 h before
preparation for immunofluorescence.
Gene Transfection and Reporter Assays--
HeLa
cells were plated 24 h before transfection at 50% confluency into
24-well plates, and then transiently transfected by FuGENE6 (Roche
Molecular Biochemicals) according to the manufacturer's protocol.
Transfected HeLa cells were lysed in Passive Lysis Buffer supplied in
the Dual-LuciferaseTM Reporter Assay System (Promega)
according to instructions in the accompanying technical manual.
Briefly, 10-µl lysates were mixed with 100 µl of luciferase Assay
Reagent II (Promega) and luminescence was measured with a Berthold
Lumat to analyze firefly luciferase levels. Then 100 µl of Stop & GloTM Reagent (Promega) was added and luminescence was
again measured to analyze Renilla luciferase levels.
Relative luciferase units are equivalent to firefly luciferase
normalized to Renilla luciferase. All transfection
experiments were performed in duplicate.
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RESULTS |
RelB Physically Associates with p100--
To determine the ability
of RelB to physically interact with previously identified NF-B
inhibitors, RelB and the various IB molecules were
immunoprecipitated from HeLa cell lysates, and the immunoprecipitates
were analyzed by immunoblot using either anti-RelB antibodies or
antibodies recognizing the corresponding IB molecules. In the case
of p100 and p105, these molecules were immunoprecipitated using
antibodies directed against the COOH terminus of each molecule, to
avoid immunoprecipitating p52 or p50, respectively. As shown in Fig.
1A, RelB is present in the p100 immunoprecipitate (lane 3) but not in the
immunoprecipitates of the other IB molecules. Moreover, p100 is
present in the RelB precipitate (Fig. 1A, lane
1), further stressing the native and specific association between
RelB and p100. RelB does not physically associate with p105, IB,
IB, and IB (Fig. 1A, lanes 2 and 4-6). Similar results were obtained in other cell types,
including the monocytic cell line U937 and the B lymphocyte cell line
BJAB (data not shown).
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Fig. 1.
RelB is associated with p100, and
TNF- does not target p100 for
degradation. A, association between RelB and p100. 100 µg of HeLa cellular lysates were immunoprecipitated with antibodies
reactive against RelB, a carboxyl-terminal epitope of p105, a
carboxyl-terminal epitope of p100, IB, IB, or IB.
Immunoprecipitates were subjected to SDS-PAGE and membranes were
blotted with antibodies reactive against RelB, p105, p100, IB,
IB, and IB. B, TNF- stimulation causes
degradation of IB and p105, but not p100, and nuclear
translocation of RelA, but not RelB. HeLa cells were treated with 10 ng/ml TNF- for various time points. Nuclear and cytosolic
compartments were isolated and 20 µg of each lysate were subjected to
SDS-PAGE. Membranes were blotted with antibodies reactive against RelB,
RelA, IB, p100, and p105.
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TNF Targets IB and p105, but Not p100, and Results in the
Nuclear Translocation of RelA, but Not RelB--
Having demonstrated
that p100 is physically associated with RelB, we next investigated
whether stimuli, such as TNF-, that induce the nuclear translocation
of RelA by triggering the degradation of IB molecules like IB,
result in the nuclear translocation of RelB. HeLa cells were treated
with TNF- for up to 100 min, followed by the isolation of nuclear
extracts from the cytosolic compartment. Nuclear and cytosolic extracts
were analyzed in SDS-PAGE and subjected to immunoblot using antibodies
against RelB, RelA, IB, p100, and p105. While TNF- triggers a
rapid nuclear translocation of RelA, it does not trigger the nuclear
translocation of RelB (Fig. 1B). Previously identified
degradation of both IB and p105 are observed to temporarily
coincide with RelA nuclear translocation, while p100 levels and
migration remain unchanged. Similar results were obtained in the
promonocytic cell line U937 (data not shown). This implies that
TNF-mediated activity on the IKK complex, known to trigger
IB and p105 degradation, does not target p100 degradation; this
is in direct correlation with absent nuclear translocation of RelB.
Altogether, this data further establishes the connection between
p100/RelB association in vivo and lack of the
TNF--induced nuclear translocation of RelB in the absence of p100
modification and degradation.
p100 Associates with RelB--
The significant homology between
p100 and p105 suggests that RelB may also interact with p105 in a
similar manner as that observed with p100. However, results shown in
Fig. 1A indicate that native RelB is constitutively
associated in the cytosol with p100, and not p105. To further confirm
this, we investigated the interaction between ectopic expression of
tagged RelB with the inhibitor proteins p100
N and p105N. p100N
is the carboxyl-terminal portion of p100, containing its entire
inhibitory ankyrin region, amino acids 407-900; p105N is the
carboxyl-terminal of p105, containing its entire inhibitory ankyrin
region, amino acids 401-920. We chose to examine the interaction of
RelB with only the inhibitory ankyrin containing regions of p100 and
p105, to dissect it from the potential interaction of RelB with the Rel
homology domain present in the amino-terminal portions of these
atypical inhibitor proteins, i.e. p52 and p50, respectively.
Co-expression of HA-RelB with Flag-p100N, followed by
immunoprecipitation of RelB with anti-HA antibodies and immunoblotting
with anti-Flag antibodies, demonstrates that p100N is associated
with RelB (Fig. 2A, lane 5). However, co-expression of HA-RelB with Flag-p105N, followed by HA-RelB immunoprecipitation, indicates that RelB does not associate with p105N (Fig. 2A, lane 4). When the same
experiment was performed expressing HA-RelA, instead of HA-RelB, it is
demonstrated that HA-RelA can associate with either Flag-p100N or
Flag-p105N (Fig. 2A, lanes 2 and 1,
respectively). This indicates that the lack of association between
p105N and RelB is not due to an abnormal configuration of the
expressed p105 transgene, as it is shown that p105N effectively
interacts with RelA.
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Fig. 2.
RelB associates with
p100N, but not p105
78 or IB.
A, HeLa cells were transfected with 500 ng of HA-RelB (or
HA-RelA) cDNA and 500 ng of Flag-100N, 3 µg of Flag-p105N,
or both, as indicated above the figure. 300 µg of
cytosolic lysates were immunoprecipitated with anti-HA antibodies and
subjected to SDS-PAGE. Membranes were immunoblotted with anti-Flag
antibodies (upper panel). Expression of transfected proteins
was confirmed by Western blot of 10 µg of cytosolic lysates with
anti-Flag antibodies followed by a second immunoblot with anti-HA
antibodies (lower panel). B, HeLa cells were
transfected with 500 ng of HA-RelB (or HA-RelA) and 500 ng of
Flag-p100N, 500 ng of Flag-IB cDNA, or both, as indicated
above the figure. 300 µg of cytosolic lysates were
immunoprecipitated with anti-HA antibodies and subjected to SDS-PAGE.
Membranes were blotted with anti-Flag antibodies (upper
panel). Expression of transfected proteins was confirmed by
Western blot of 10 µg of cytosolic lysates with anti-Flag antibodies
followed by a second immunoblot with anti-HA antibodies (lower
panel).
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The observation that RelA, but not RelB, binds with equal affinity to
p105N or p100N was surprising and prompted us to investigate whether, in the presence of both p105N and p100N, RelA or RelB would preferentially interact with one of the IB molecules over the
other. This was achieved by co-expressing both Flag-p100N and
Flag-p105N together with either HA-RelA or HA-RelB. Cells were then
lysed, and cytosolic extracts were subjected to immunoprecipitation by
antiHA antibodies, followed by immunoblotting with anti-Flag antibodies, to determine which of the two IB molecules, p100N or
p105N, preferentially interact with either RelA or RelB. Results from these experiments indicate that RelA preferentially associates with p105N (Fig. 2A, lane 3), while RelB
preferentially associates with p100N (Fig. 2A, lane
6) when both inhibitor molecules are expressed at equivalent
levels (Fig. 2A, lower panel). Surprisingly, RelB
levels in the cytosol are significantly decreased in the presence of
co-expressed p105N, but not p100N (Fig. 2A,
lower panel, lane 4 versus 5 or 6),
suggesting that the inability of p105N to maintain RelB in the
cytosol may allow the overexpressed RelB to localize in the nucleus
when it is co-expressed with p105N. This potential explanation is
supported by results discussed in Fig. 4. Low cytosolic RelB levels
prevent an absolute comparison of RelB affinity to p100N or
p105N. However, the competitive co-immunoprecipitation indicates a
preferential association of RelB with p100N, (Fig. 2A,
lane 6). Furthermore, functional experiments to follow (Fig.
4) support the increased affinity of RelB for p100N over
p105N.
We next investigated the ability of the classical IB molecule,
IB, to associate with RelB, for previous data have indicated its
weak association with RelB (47). Moreover, co-immunoprecipitation data
from Fig. 1A demonstrated that native RelB does not interact with native IB, nor any of the other IB molecules, IB
and IB. Co-expression of HA-RelB with Flag-p100N, followed by
immunoprecipitation of RelB with anti-HA antibodies and immunoblotting
with anti-Flag antibodies, demonstrates once more that p100N is
associated with RelB (Fig. 2B, lane 5). However,
co-expression of HA-RelB with Flag-IB, followed by HA-RelB
immunoprecipitation and anti-Flag immunoblot, indicates that RelB does
not associate with IB (Fig. 2B, lane 4).
When the same experiment was performed expressing HA-RelA, instead of
HA-RelB, it is demonstrated that HA-RelA can equally associate with
Flag-p100N and Flag-IB (Fig. 2B, lanes 2 and 1, respectively), similarly to what was observed for
p105. Again, this indicates that the lack of association between
IB and RelB is not due to an abnormal configuration of the
expressed IB transgene, for we show that IB effectively
interacts with RelA. This observation was further investigated by
co-expressing, within the same cell, both Flag-p100N and
Flag-IB together with either HA-RelA or HA-RelB. Cells were then
lysed, and cytosolic extracts were subjected to immunoprecipitation by
antiHA antibodies to determine which of the two IB molecules,
p100N or IB, preferentially interact with either RelA or RelB.
Results from these experiments indicate that RelA preferentially
associates with IB (Fig. 2B, lane 3), while
RelB preferentially associates with p100N (Fig. 2B,
lane 6) when both inhibitor molecules are present at
equivalent levels (Fig. 2B, lower panel). Again, as seen
when co-expressed with p105N (Fig. 2A, lane
4), RelB levels in the cytosol are significantly decreased in the
presence of co-expressed IB, but not of p100N (Fig. 2B,
lower panel, lane 4 versus 5 or
6). This could be explained by the inability of IB to
maintain RelB in the cytosol, allowing it to localize in the nucleus.
This hypothesis is supported by the next set of results summarized in
Fig. 4. Thus, RelA preferentially interacts with IB (Fig.
2B, lane 3), while RelB preferentially interacts
with p100N (Fig. 2B, lane 6).
RelB does not form homodimers, and only heterodimerizes with either p50
or p52 (36, 37, 55). Since the true interaction between the RelB
heterodimer and the ankyrin-containing portion of the inhibitor
molecule may require the presence of a dimer partner, or at least the
amino-terminal portion of the inhibitor, we wished to confirm the
results we obtained in Fig. 2 by repeating the competitive
co-immunoprecipitations in the presence of overexpressed p52 dimer
partner. p52 was chosen as dimer partner in this experiment, for it is
the amino-terminal portion of the p100 protein, which has thus far been
shown to preferentially associate with RelB. Nevertheless, similar
experiments were also performed using p50. HeLa cells were transfected
with p52, either HA-RelA or HA-RelB, and either both Flag-p100N and
Flag-IB together, or both Flag-p100N and Flag-p105N
together. Cells were then lysed, and cytosolic extracts were subjected
to immunoprecipitation by anti-HA antibodies to determine which of the
IB molecules preferentially interact with either RelA or RelB when
p52 is co-expressed. Results from these experiments confirm the
previous findings, and indicate that RelA preferentially associates
with IB or p105N, while RelB preferentially associates with
p100N (data not shown).
We next addressed the ability of full-length tagged p100 or p105 to
bind to RelA or RelB. Co-expression of HA-tagged RelA or HA-tagged RelB
with Flag-tagged p100 and Flag-tagged p105, followed by
immunoprecipitation with anti-HA antibodies and immunoblotting with
anti-Flag antibodies, demonstrates that both RelA and RelB can
associate with full-length p100 or full-length p105 (Fig. 3A). Because of the difficulty
in separating full-length p100 from full-length p105 by gel
electrophoresis, additional immunoprecipitations were blotted with
antibodies specific for the N terminus of p100 (Fig. 3A,
lanes 5 and 6) or p105 (Fig. 3A,
lanes 7 and 8). Results from these experiments
indicate that RelB preferentially associates with p100 (Fig.
3A, lanes 5 and 6) over p105 (Fig.
3A, lanes 7 and 8), similar to that
observed with the N p100 truncated protein.
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Fig. 3.
RelB associates with full-length p100 and
p105 but not IKB. 293 T cells were
transfected with expression vectors coding for HA-RelA, HA-RelB,
Flag-IB, Flag-p100, and Flag-p105 as indicated. Anti-HA immuno-
precipitations were performed and run on polyacrylamide gels along with
10 µg of cytosolic extracts. The proteins were transferred to
polyvinylidene difluoride membranes and blotted with antibodies
specific for the HA or Flag tags (A and B) as
well as antibodies to the NH2 terminus of p100 or p105
(A).
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Co-expression of HA-RelB with Flag-p100 and Flag-IB, followed by
immunoprecipitation of RelB with anti-HA antibodies and immunoblotting
with anti-Flag antibodies, demonstrates that RelB preferentially
associates with full-length p100 over IB (Fig. 3B,
lane 4). However, when the same experiment was performed
expressing HA-RelA, instead of HA-RelB, it is demonstrated that HA-RelA
associates with either Flag-IB or Flag-p100 (Fig. 3B,
lane 3). These results demonstrate that in an overexpression
system, RelA will associate with IB, p100, or p105 while RelB
will associate preferentially with p100 or p105. The association of
RelB to full-length p105 is likely due to affinity for the Rel homology
domain in the NH2 terminus since no association of RelB was
found with the COOH terminus of p105 (Fig. 2A, lanes
4 and 6). Altogether, these data confirm the findings
with endogenous proteins (Fig. 1A) indicating that RelB
preferentially associates with p100 over the other IB proteins.
p100, but Not p105 nor IB, Inhibits RelB Nuclear
Localization--
Based on the above, we hypothesized that p100 is a
bone fide cytosolic inhibitor of RelB as compared with p105 or
IB. To confirm this, we analyzed the ability of RelB to localize
in the nuclear compartment when overexpressed in the presence of p100, p105, or IB. HeLa cells were co-transfected with HA-RelB or HA-RelA and either full-length p100, p105, or IB cDNA.
Transfected cells were fixed and incubated with anti-HA antibodies
followed by a fluorescein-conjugated secondary antibody, stained with
DAPI to identify the nuclear compartment, and analyzed by confocal microscopy. Only a fraction of the cells are transfected, yet all cells
stain blue for DAPI. As shown in Fig. 4,
HA-RelB is found in the nuclear compartment when expressed in the
absence of other inhibitor molecules (box 1). Co-expression
of p100, but not p105 or IB, blocks RelB presence in the nucleus
(Fig. 4, boxes 2, 3, and 4,
respectively). Rather, co-expression with p105 or IB allows
HA-RelB to undergo a nuclear localization. HA-RelA likewise localizes
to the nucleus when expressed alone (Fig. 4, box 5), yet it
is retained in the cytosol when co-expressed with p100, p105, or
IB (Fig. 4, boxes 6, 7, and 8,
respectively). Thus, while RelA nuclear localization is inhibited by
p100, p105, or IB RelB nuclear presence is only inhibited by
p100. The inability of p105 and IB to retain RelB in the
cytosolic compartment (Fig. 4, boxes 3 and 4) as
seen by immunofluorescence, confirms that the low expression of HA-RelB
in the cytosol seen in Fig. 2 is indeed due to the inability of both
p105N and IB to prevent the nuclear localization of HA-RelB.
This experiment was also performed using p100N or p105N instead
of full-length protein, and similar results were observed (data not
shown). Altogether, this experiment indicates that RelB nuclear
localization is preferentially inhibited by p100.
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Fig. 4.
RelB nuclear localization is inhibited by
p100, but not by p105 or
IB.
Top panels, HeLa cells were transfected with 500 ng of
HA-RelB cDNA, either alone (box 1), with 500 ng of p100
cDNA (box 2), with 500 ng of p105 cDNA (box
3), or with 500 ng of IB cDNA (box 4).
Bottom panels, HeLa cells were transfected with 500 ng of
HA-RelA cDNA, either alone (box 5), with 500 ng of p100
(box 6), with 500 ng of p105 cDNA (box 7), or
with 500 ng of IB cDNA (box 8). Cells were fixed
and probed with anti-HA antibodies followed by fluorescein-conjugated
secondary antibodies. Nuclei are stained with DAPI.
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The p100 Carboxyl-terminal Domains Are Required for p100
Interaction with RelB and Inhibition of RelB-dependent
Transcriptional Activity--
To identify which domain(s) of p100
is/are responsible for its association with RelB, a series of 3' to 5'
deletions were introduced into either the Flag-p100N or GFP-p100N
(Fig. 5A). The various
Flag-tagged expression vectors were then co-transfected with HA-RelB
into HeLa cells. Cells were lysed and cytosolic extracts were
immunoprecipitated for HA-RelB using anti-HA antibodies. The
immunoprecipitates and the cytosolic extracts were subsequently analyzed by immunoblot using anti-FLAG antibodies. While the four p100
constructs (407-582, 407-623, 407-744, and 407-900) were expressed
at similar levels (Fig. 5B, left panel), only
those constructs containing ankyrin region 4 (407-623, 407-744, or
407-900) were able to physically associate with RelB (Fig.
5B, right panel, lanes 2-4),
suggesting that such ankyrin contained in p100 (407), but not in
p100 (407), is necessary for its association with RelB.
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Fig. 5.
Domains of the COOH terminus of p100 required
for its association with RelB. A, representation of the
carboxyl-terminal region of p100 with the six ankyrin domains (numbered
1-6) and two incomplete ankyrin regions (one before and one
after the first and sixth ankyrin domain). Carboxyl-terminal deletions
were introduced into p100N expression vectors. B,
Flag-p100N deletion constructs 407-623, 407-744, and 407-900, but
not 407-582 can associate with HA-RelB. HeLa cells were transfected
with 500 ng of HA-RelB and 500 ng of Flag-tagged 407-582, 407-623,
407-744, or 407-900 cDNA. 300 µg of cell lysates were
immunoprecipitated with anti-HA antibodies and subjected to SDS-PAGE.
Membranes were blotted with anti-Flag antibodies (right
panel). Expression of the transfected Flag-p100N deletion
constructs was confirmed by Western blot of 10 µg cytosolic lysates
with anti-Flag antibodies (left panel).
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To investigate whether p100N constructs 407-582,
407-623, 407-744, and 407-900 lead to the cytoplasmic retention of
RelB, HeLa cells were transfected with the RFP-tagged RelB and its
dimer partner p52, with or without GFP alone, or GFP-tagged p100
deletion mutants: 407-582, 407-623, 407-744, or 407-900.
Transfected cells were then subjected to immunofluorescence studies.
Only a fraction of cells were transfected with the RFP and GFP
constructs, yet all nuclei stained blue with DAPI. As shown in Fig.
6, RFP-RelB, in the absence of p100N,
localizes to the nucleus (Fig. 6, row 1). RFP-RelB
co-expressed with GFP (row 2), GFP-p100N 407-582 (row 3), GFP-p100N 407-623 (row 4), or
GFP-p100N 407-744 (row 5) was still located in the
nucleus, although there appears to be some weak cytosolic presence of
RFP-RelB when GFP-p100N 407-744 is co-expressed. However, only the
GFP-p100N construct 407-900 retains RFP-RelB in the cytoplasm (Fig.
6, row 6).
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Fig. 6.
p100N 407-900
prevents RelB nuclear translocation. HeLa cells plated onto
coverslips were transfected with 5 µg of RFP-RelB cDNA either
alone (row 1), with 500 ng GFP vector (row 2), or
with 500 ng of GFP-tagged p100N deletion constructs containing amino
acids 407-582 (row 3), 407-623 (row 4),
407-744 (row 5), or 407-900 cDNA (row 6).
Nuclei are stained with DAPI. Cellular location of RFP- and GFP-tagged
proteins was determined by immunofluorescence. Column 1,
location of RFP-RelB; column 2, location of GFP-p100N
deletion constructs; column 3, nuclei stained with DAPI;
column 4, overlay of RFP, GFP, and DAPI.
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The functional relevance of the inhibition of RelB nuclear localization
by the p100N construct 407-900 was further analyzed by measuring
the transcriptional activity of RelB in the presence or absence of the
p100N constructs. HeLa cells were co-transfected with an NF-B
concatemer driving firefly luciferase, a minimal TK promoter driving
renilla luciferase, and RelB and p52 expression vectors in the presence
or absence of the different p100N deletion constructs (407-582,
407-623, 407-744, or 407-900). Cells were lysed and measured for
normalized firefly luciferase activity. As shown in Fig. 6A,
p100N constructs 407-900, and to a lesser degree 407-744,
inhibited the transcriptional activity driven by RelB/p52 in a
dose-dependent manner. However, p100N constructs 407-582 and 407-623 were unable to prevent
RelB/p52-dependent transcriptional activity. Similar
results were seen with RelB expressed without its dimer partner p52
(Fig. 7B). Moreover,
full-length p100 was also seen to inhibit the transcriptional activity
driven by RelB or RelB/p52 in a dose-dependent manner (Fig.
7C).
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Fig. 7.
Both p100 and p100N
inhibit RelB-dependent transcriptional activity.
A, HeLa cells were transfected with 100 ng of a B-firefly
luciferase reporter construct, 25 ng of a Renilla luciferase
reporter construct under the control of a thymidine kinase promoter,
100 ng of RelB, 100 ng of p52, and increasing amounts (100-300 ng) of
Flag-p100N deletion constructs 407-582, 407-623, 407-744, or
407-900. Cells were lysed and luciferase activity was determined by
luminescence. B, HeLa cells were transfected with 100 ng of
a B-firefly luciferase reporter construct, 25 ng of a
Renilla luciferase reporter construct under the control of a
TK promoter, 100 ng of RelB, and increasing amounts (100-300 ng) of
Flag-p100N deletion constructs 407-582, 407-623, 407-744, or
407-900. Cells were lysed and luciferase activity was determined by
luminescence. C, HeLa cells were transfected with 100 ng of
a B-firefly luciferase reporter construct, 25 ng of a
Renilla luciferase reporter construct under the control of a
TK promoter, 100 ng of RelB with or without 100 ng of p52, and
increasing amounts (50-300 ng) of full-length p100 cDNA. Cells
were lysed and luciferase activity was determined by luminescence. All
readings are expressed in relative luciferase units (RLU)
equivalent to expression of firefly luciferase normalized to the
constitutively expressed Renilla luciferase.
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Cytosolic Localization of RelB by p100N Is in Part Dependent on
Its Nuclear Export--
To explain the inability of p100N construct
407-623, and to a lesser extent 407-744, to inhibit neither RelB
nuclear translocation (Fig. 6) nor RelB transcriptional activity (Fig.
7), despite being able to physically associate with RelB (Fig.
5B), we next studied the ability of the various p100N
constructs to effectively shuttle between the nuclear and cytosolic
compartments. To do so, we first determined the cellular location of
each GFP-p100N protein. HeLa cells were transfected with
GFP-p100N constructs containing amino acids 407-623, 407-744, or
407-900. Cells were then fixed and visualized by confocal microscopy
for the subcellular localization of the GFP-p100N deletion
constructs. Deletion constructs truncating at amino acid 623 or 744 were located within the nuclear compartment of HeLa cells (Fig.
8, rows 1 and 2),
but a construct containing the entire carboxyl-terminal,
ankyrin-containing region, i.e. amino acids 407-900, was
located entirely in the cytosolic compartment of the cell (Fig. 8,
row 3).
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Fig. 8.
Cytosolic localization of
p100N relies on a putative NES located between
amino acids 744 and 900. HeLa cells plated onto coverslips were
transfected with 500 ng of GFP-fused p100N deletion construct
cDNA containing amino acids 407-623 (row 1), 407-744
(row 2), or 407-900 (rows 3 and 4).
Row 4, cells were treated for 6 h with 200 ng/ml
leptomycin B (LMB). Cells were fixed and visualized by
confocal microscopy for the subcellular localization of the
GFP-p100N deletion constructs. Nuclei are stained with DAPI.
Column 1, location of GFP-p100N deletion constructs;
column 2, nuclei stained with DAPI; column 3,
overlay of GFP and DAPI.
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Based on the above result, we asked whether the cytosolic localization
of p100N 407-900 was due to the presence of putative NES that are
not present in p100N constructs truncated prior to amino acid 744. For this, HeLa cells transfected with GFP-p100N 407-900 were
treated or not with a pharmacological inhibitor of the CRM1-mediated
nuclear export, leptomycin B. Treatment with LMB resulted in
GFP-p100N 407-900 being localized to the nuclear compartment (Fig.
8, row 4, as compared with Fig. 8, row 3).
Moreover, HeLa cells transfected with GFP-p100N 407-623 or
GFP-p100N 407-744, and treated with leptomycin B, resulted in no
discernable change in the nuclear presence of these constructs (data
not shown). Thus, the cytosolic localization of p100N, and
therefore, the ability of p100 to inhibit RelB transcriptional
activity, appears to rely upon the ability of p100 to shuttle between
the nuclear and cytoplasmic compartments of the cell secondary to one
or more NES located between amino acids 744 and 900 of the p100 protein.
NIK Causes p100 Degradation and RelB Nuclear Localization--
To
confirm the functional relevance of p100 control over RelB subcellular
localization, we reasoned that inducing the degradation of p100 should
result in the nuclear translocation of RelB. For this, we exploited the
recent finding that p100 degradation can be induced by NIK (21). HeLa
cells were co-transfected with either control vector, RelB alone, or
RelB plus p100, alone or in the presence of NIK, IKK, or kinase-dead
IKK. Nuclear and cytosolic compartments were separated, analyzed by
SDS-PAGE, and subjected to immunoblotting using antibodies against
RelB, p100, histone, and -actin (Fig.
9A). HeLa cells transfected
with RelB alone had detectable RelB in the nuclear compartment (Fig.
9A, lane 2), which was decreased when p100 was
co-expressed (Fig. 9A, lane 3). However,
co-expression of RelB and p100 with NIK resulted in significant
degradation of p100, and this was accompanied by movement of RelB to
the nucleus (Fig. 9A, lane 4). Co-expression of
RelB, p100, and IKK demonstrates that IKK has no effect on p100
processing or RelB nuclear localization (Fig. 9A, lane
6). Moreover, when kinase-dead IKK is co-transfected with NIK,
p100, and RelB, this mutant kinase does not interfere with NIK-induced p100 degradation and RelB nuclear translocation (Fig. 9A,
lane 5), confirming the lack of involvement of IKK in
targeting p100 for degradation. These results correlate with and
provide an explanation for the inability of TNF to either degrade
p100 or translocate RelB into the nucleus, as seen in Fig.
1B, for TNF is targeting IKK activity and, apparently,
not NIK. In addition, this set of results confirms that full-length
p100 is an effective inhibitor of RelB and that it is susceptible to
regulation by NIK. Expression of IKK does not result in lower p100
levels in the cytosol, nor in RelB nuclear translocation (Fig.
9A, lane 6).
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Fig. 9.
NIK overexpression causes p100 degradation
and RelB nuclear localization. A, HeLa cells were
transfected with control vector (lane 1), with RelB cDNA
alone (lane 2), or with RelB and p100 cDNA (lanes
3-6) with NIK (lane 4), NIK plus
kinase-dead IKK (lane 5), or IKK (lane 6).
Nuclear and cytosolic compartments were isolated and 10 µg of each
lysate were subjected to SDS-PAGE. Membranes were blotted with
antibodies reactive against RelB, histone, p100, and -actin.
B, HeLa cells plated onto coverslips were transfected with 3 µg of RFP-RelB cDNA, 500 ng of p52 cDNA, and 500 ng of
GFP-tagged p100N deletion constructs containing amino acids 407-900
cDNA with (row 2) or without (row 1) 500 ng
of Myc-NIK. Nuclei are stained with DAPI. Cellular location of RFP- and
GFP-tagged proteins was determined by immunofluorescence. Column
1, location of RFP-RelB; column 2, location of
GFP-p100N deletion constructs; column 3, nuclei stained
with DAPI; column 4, overlay of RFP, GFP, and DAPI.
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To extend the findings shown in Fig. 8A, we decided to
confirm the influence of NIK on p100 degradation and RelB cellular localization using immunofluorescence. HeLa cells were transfected with
the RFP-tagged RelB, its dimer partner p52, and green
fluorescent protein-tagged p100 deletion mutant 407-900 with or
without NIK. Transfected cells were then subjected to
immunofluorescence studies. Only a fraction of cells were transfected
with the RFP and GFP constructs, yet all nuclei stained blue with DAPI.
As shown in Fig. 9B, and as seen before, RFP-RelB
co-expressed with GFP-p100N 407-900 (row 1) was retained in the
cytoplasm (Fig. 9B, row 1), whereas the addition
of NIK prevented RFP-RelB's cytosolic retention and allowed RFP-RelB
to localize in the nucleus (row 2). Furthermore, in these
panels, and in others not shown here, the addition of NIK caused a
decrease in cytosolic levels of GFP-p100N, implying that
GFP-p100N is being degraded in a manner that is dependent on NIK activity.
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DISCUSSION |
While RelB and the other NF-B family member RelA share many
overlapping functions, RelB can also activate, inhibit, or regulate gene transcription differently from RelA (56). It is therefore important to dissect potential differences between how RelB and other
NF-B family members, RelA or c-Rel, are regulated. In this study, we
have demonstrated that RelB cellular localization is regulated by p100
and not by other IB proteins. We have further demonstrated that RelB
is not regulated by IKK but by NIK or NIK activation of IKK
through its targeting of p100. Such findings strongly argue for a
difference in regulation of RelA and RelB. Furthermore, we have
identified the regions of p100 required for effective association with
and retention of RelB in the cytosol, and determined that p100 contains
one or more nuclear export signals which are crucial for the cytosolic
localization of p100, and thus, functional inhibition of RelB.
Previous studies had demonstrated that IB binds weakly to RelB as
compared with RelA (47), that the COOH terminus of p100 can inhibit
transcriptional activity mediated by RelB (57), and that p100 retains
RelB in the cytosol of specific breast cancer cell lines (58). Results
from the present study extend those observations by formally
demonstrating the differences in affinity of the different IB
molecules toward RelA and RelB, and further identifying molecular
mechanisms whereby p100 inhibits RelB nuclear localization.
Until now, p100 and p105 have always been considered to similarly
function as alternative inhibitors of NF-B, but this
study highlights significant functional differences between them.
First, while significant homology exists between these two IB-like
molecules, RelB has a significant affinity toward p100 and much less
toward p105, yet both inhibitors can bind RelA. This suggests that RelB may seek a unique protein conformation in p100, most likely conferred by ankyrin region 4, that favors its interaction with p100. Second, recent data indicate that TNF stimulation of the IKK complex can
lead to the inducible phosphorylation of not only IB and IB
but also p105, but not p100 (19). It appears p100 phosphorylation and
degradation is dependent upon specific IKK activity, possibly induced via NIK (19, 21, 22). This data is further highlighted by the
inability of TNF-, which induces IKK activity and leads to the
translocation of RelA to the nucleus, to result in the nuclear
translocation of RelB. This corresponds to the finding that TNF
stimulation leads to p105 degradation, but does not affect p100
(59).
The demonstration that p100 prevents the nuclear
localization and transcriptional activity of RelB, dependent upon the
nuclear export of p100 is, to our knowledge, the first report of an
atypical inhibitor of NF-B containing a nuclear export signal. The
precise identification, and mutational analysis, of this NES will
undoubtedly be the next step in describing this mechanism of p100
nuclear and cytosolic shuttling. Efforts are underway to identify this sequence, and have been thus far delayed only by the identification of
more than one potential NES in the carboxyl-terminal portion of p100
between amino acids 744 and 900. It is possible that a series of NES
located in this portion of p100 act in concert to facilitate nuclear
shuttling. To our knowledge, the identification of a sequence acting as
a nuclear localization signal has yet to be identified on the p100
protein, and may prove an interesting area of investigation.
There is an ongoing debate regarding how p52 and p100 are generated
from the same RNA transcript. The established mechanism is that p100
protein is translated, and p52 is simply a product of p100 protein
processing (20). This mechanism offers the possibility that the
ankyrin-containing carboxyl-terminal region of p100, i.e.
p100N or IB
, may be generated from this protein processing. A
second mechanism has recently been offered which instead describes p100
and p52 as being alternatively generated from the same transcript in a
co-translational manner, dependent upon proteolytic processing by the
proteasome and a glycine-rich region in the carboxyl-terminal portion
of p52 (60). Regardless of the functional mechanism, in this study we
have demonstrated that both full-length p100 and its COOH terminus are
effective in inhibiting RelB nuclear localization. We believe that the
experimental results from the coimmunoprecipitation of native p100 with
RelB (Fig. 1A), the confocal microscopy results in Fig. 4,
and the ability of full-length p100 to inhibit the transcriptional
activity of RelB or RelB/p52 on an NF-B-driven luciferase reporter
(Fig. 7C) is compelling evidence that the full-length p100
protein is indeed the physiological inhibitor of RelB. Thus, the
p100N constructs we have created may be useful reagents to inhibit
RelB in cell and disease models in which RelB protein is predominantly
present in the nucleus, such as in B cell malignancies including
Epstein-Barr virus-infected B cells or in other antigen presenting
cells, such as macrophages and dendritic cells.
Finally, the finding that RelB cellular localization is controlled by
p100 lends potential insight into a set of lymphomas that correlate
with a translocation or deletion of the NFKB2 gene, which
codes for p100. The mutations that occur within this class of lymphomas
lead to a truncation of the p100 protein that can no longer repress
certain NF-B activity (61-64). We hypothesize that this unrepressed
NF-B activity may correspond to unregulated RelB activity, and that
p100s lack of repression on RelB activity may be what drives the
lymphoproliferation seen in these diseases. This is initially
supported by the finding that the shorter 3' p100 deletions tested in
the present study are less able to inhibit RelB nuclear translocation
or its transcriptional activity by virtue of their inability to exit
the nucleus of the cell. If this hypothesis holds true and these
lymphomas are found to have elevated RelB activity, then the finding
that p100 is RelB's physiological inhibitor will have clinical
relevance in certain lymphomas, and future treatments for these
lymphomas could be appropriately directed to controlling RelB nuclear
presence and transactivation.
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ACKNOWLEDGEMENTS |
We thank Nancy Rice (National Cancer
Institute, Frederick, MD) for her generous gift of antibodies, Minoru
Yoshida (The University of Tokyo, Tokyo, Japan) for his generous gift
of leptomycin B, members of the Paya laboratory for helpful discussion,
and Teresa Hoff for preparation of the manuscript.
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FOOTNOTES |
*
This work was supported by National Institutes of Health
Grant R01 AI36076 and the Mayo Foundation.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: Mayo Clinic, 200 First St. SW, Guggenheim 501, Rochester, MN 55905. Tel.: 507-284-3747; Fax: 507-284-3757; E-mail: paya@mayo.edu.
Published, JBC Papers in Press, October 31, 2001, DOI 10.1074/jbc.M109619200
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ABSTRACT
INTRODUCTION
