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J. Biol. Chem., Vol. 278, Issue 3, 1817-1823, January 17, 2003
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From the
Received for publication, August 28, 2002, and in revised form, October 1, 2002
The ubiquitin-proteasome system is responsible
for the regulation and turnover of many short-lived proteins both in
the cytoplasm and in the nucleus. Degradation can occur via two
distinct pathways, an N terminus-dependent pathway and a
lysine-dependent pathway. The pathways are characterized by
the site of initial ubiquitination of the protein, the N terminus or an
internal lysine, respectively. MyoD, a basic helix-loop-helix
transcription factor, is a substrate for the ubiquitin-proteasome
pathway and is degraded in the nucleus. It is preferentially tagged for
degradation on the N terminus and thus is degraded by the N
terminus-dependent pathway. Addition of a 6× Myc
tag to the N terminus of MyoD can force degradation through the
lysine-dependent pathway by preventing ubiquitination at
the N-terminal site. Modifications of the nuclear localization signal
and nuclear export signal of MyoD restrict ubiquitination and
degradation to the cytoplasm or the nucleus. Using these mutants, we
determined which degradation pathway is dominant in the cytoplasm and
the nucleus. Our results suggest that the lysine-dependent pathway is the more active pathway within the cytoplasm, whereas in the
nucleus the two pathways are both active in protein degradation.
Degradation of many short-lived cellular proteins, such as
transcription factors, tumor suppressors, and cell cycle regulators, occurs via the ubiquitin-proteasome pathway (1-4). Through this pathway, proteins are targeted for degradation by the 26 S proteasome via the formation of a polyubiquitin chain. The process begins with
activation of ubiquitin by the ubiquitin-activating enzyme (E1),1 followed by transfer
of ubiquitin to E2, a ubiquitin-conjugating enzyme. E2 shuttles
the ubiquitin molecule to the substrate-specific ubiquitin ligase (E3),
which then delivers the ubiquitin to the substrate to be degraded.
Initially, it was thought that ubiquitination occurred only through a
lysine-dependent ubiquitination pathway in which ubiquitin
is covalently attached to the substrate protein via an amide linkage to
the Among the short-lived proteins degraded by the ubiquitin-proteasome
system are several transcription factors, including MyoD. MyoD, a
nuclear basic helix-loop-helix transcription factor necessary for
skeletal muscle differentiation (9), is rapidly degraded by the
ubiquitin-proteasome system both in vitro and in
vivo (10). Ubiquitination on the N terminus of MyoD appears to
occur in preference to ubiquitination on internal lysines (8), and MyoD
appears to be ubiquitinated and degraded in the nucleus by the
ubiquitin-proteasome system (11).
A nuclear ubiquitin-proteasome system may also be responsible for the
degradation of several other transcription factors, including Smad2 and
the basic helix-loop-helix/Per-ARNT-Sim homology domain dioxin receptor
(12, 13), as the activated forms of these proteins require
translocation into the nucleus for degradation. In addition, Far1, a
protein required for establishing cell polarity of mating yeast and for
bringing about cell cycle arrest, is ubiquitinated and degraded
in the nucleus (14). In contrast, cyclin D1 (15), p53 (16),
p27kip1 (17), I Transport of cellular proteins, including transcription factors such as
MyoD, into and out of the nucleus is facilitated by nuclear
localization and nuclear export sequences (NLS and NES), which
facilitate transport of such proteins across the nuclear envelope
(21-24). NLS exist as single sequences (5-12 amino acids) of basic
amino acids (lysine or arginine; Ref. 25) or as bipartite regions of basic residues separated by 10-12 nonbasic residues (26).
Less is known about NES, but they are generally comprised of regions of
hydrophobic amino acids.
Thus, ubiquitin-mediated protein degradation occurs in both the
cytoplasm and the nucleus, and both lysine-dependent and N terminus-dependent ubiquitination pathways exist for
several proteins, including MyoD. The aim of the present study was to
determine the relationship of these ubiquitin-mediated degradation
pathways using NLS and NES mutants and inhibitors of nuclear uptake and export. Herein, we report that for MyoD, both the N
terminus-dependent and the lysine-dependent
pathways function within the nucleus, although the
lysine-dependent pathway appears to be more active in the
cytoplasm. The importance of this observation lies in the elucidation
of alterations in cellular protein degradation in physiological and
pathophysiological states.
Plasmids and Construction of MyoDNLS and
MyoDNES--
Wild-type MyoD and lysine-less MyoD, each of
which is in the pCIneo vector, and N-terminal-blocked (6× Myc-tagged)
MyoD, which is in the pCS2+MT vector, have been described previously
(8, 11). Preparation of NLS and NES mutants of MyoD was
accomplished using the QuikChange site-directed mutagenesis kit
(Stratagene) according to the manufacturer's instructions. DNA
sequencing using Big Dye Version 2.0 (Applied Biosystems) was used to
confirm all sequences.
Cell Culture--
HeLa cells, which were selected because they
do not express MyoD, were grown in Dulbecco's modified Eagle's
medium, which was supplemented with 10% fetal calf serum, 2 mM L-glutamine, and antibiotics (100 units/ml
penicillin G and 100 µg/ml streptomycin) (Invitrogen) and was
maintained in a humidified chamber at 37 °C and 5% CO2.
Transient transfections (efficiency ~40-60%) were performed using
the FuGENE 6 reagent (Roche Molecular Biochemicals), and cells were
analyzed 16-24 h later.
Immunofluorescent Localization of MyoD--
Subcellular
localization of MyoD and its mutants in HeLa cells was determined by
direct immunofluorescence using the mouse monoclonal anti-MyoD antibody
(1:100 dilution, NCLMyoD1; NovoCastra) followed by incubation with a
TRITC-conjugated donkey anti-mouse IgG (heavy and light chain; Jackson
ImmunoResearch) as described previously (11). MG132 (10 µM; Peptides International) or LMB (10 nM;
Sigma) were added to cells where indicated 2 h prior to fixation.
MG132 was prepared as a 10 mM stock solution in
Me2SO, and LMB was prepared as a 10 µM
solution in ethanol. Cells were observed using a Zeiss Axioskop
microscope, and 10-20 random fields of each culture condition were
photographed (magnification ×40) using a Zeiss AxioCam digital camera.
Cells were scored, and the percentage of cells expressing
nuclear MyoD was determined by methods similar to those of Sachdev
et al. (27) and Yagita et al. (28). Specifically,
cells were scored according to the cellular localization of MyoD as
predominately nuclear, predominately cytoplasmic, or distributed
equally between the nucleus and the cytoplasm. For each determination
over 100, cells were scored from 3 to 6 independent transfections.
Determination of Degradation of MyoD and MyoD Mutants in
Vivo--
As described previously (11), 16-24 h after transfection,
the HeLa cells were incubated with CHX (100 µg/ml; Sigma) to inhibit further protein synthesis. MG132 (10 µM) or LMB (10 nM) was added along with CHX as necessary. Following
incubation for 0, 0.5, 1, 2, and 3 h, the cells were lysed for at least
30 min in phosphate-buffered saline containing 5% Igepal, 1 mM EDTA, 1 mM dithiothreitol, and 2 mM phenylmethylsulfonyl fluoride. Afterward the cells were
sonicated and then centrifuged at 14,000 rpm for 10 min at 4 °C in
an Eppendorf microcentrifuge to remove cellular debris. The
lysates were mixed with an equal amount of 2× Laemmli sample buffer
(Bio-Rad), and equal amounts of each sample were run on a 12% Tris-HCl
gel (Bio-Rad) and were electroblotted onto nitrocellulose (Osmonics).
The blots were probed with monoclonal anti-MyoD antibody (1:100
dilution; NovoCastra) followed by incubation with a secondary
horseradish peroxidase-conjugated antibody and detection by
chemiluminescence (Amersham Biosciences). The resulting bands were
quantitated using the EADS system (Eastman Kodak Co.), and the data
were graphed using the Excel graphing program (Microsoft). The
degradation rate is expressed as half-life (t1/2), the time for degradation of 50% of the MyoD. Each of the constructs was evaluated by 3-8 independent determinations of
t1/2. The data are expressed as ± S.D.
MyoD is localized to the cell nucleus in growing myoblasts (29)
and in a variety of cells following transfection (11). To determine the
subcellular localization of lysine-less MyoD (Lys We next compared the rates of degradation of wild-type, lysine-less,
and N-terminal-blocked MyoD in vivo. 16-24 h following transfection, HeLa cells were treated with CHX to inhibit further protein synthesis, and the amount of MyoD at 0, 0.5, 1, 2, and 3 h
was determined via Western blot analysis. MG132 or LMB was added
together with CHX in replicate samples to determine the degradation
rate of these proteins under conditions in which the proteasome or
nuclear export was inhibited. As seen in Fig.
2, wild-type MyoD was degraded with a
t1/2 of 0.8 ± 0.1 h, which was markedly
increased to ~11 ± 4 h in the presence of MG132. The
half-life was slightly increased in the presence of LMB to 1.3 ± 0.2 h (legend to Fig. 2). The half-lives of lysine-less MyoD and
N-terminal-blocked MyoD were significantly increased over the
half-lives of wild type, with t1/2 of ~2.7 ± 0.6 h and 2.6 ± 0.6 h, respectively, as seen in Table I. As with wild-type MyoD, the half-lives
of lysine-less MyoD and N-terminal-blocked MyoD were markedly increased
by incubation with MG132, although they were minimally affected by
incubation with LMB (data not shown). Together these data suggest that
wild-type MyoD, lysine-less MyoD, and N-terminal-blocked MyoD are each
degraded via the ubiquitin-proteasome system within the nucleus.
Determinants of Nuclear and Cytoplasmic Ubiquitin-mediated
Degradation of MyoD*
,,¶
Edward Mallinckrodt Department of Pediatrics
and Molecular Biology and Pharmacology, Washington University School of
Medicine and St. Louis Children's Hospital, St. Louis, Missouri 63110 and the § Department of Biochemistry and Rappaport Institute
for Research in Medical Sciences, Faculty of Medicine, Technion-Israel
Institute for Technology, Haifa 31096, Israel
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-amino group of an internal lysine (5). However, recent studies
have shown that the N terminus of a protein substrate may also serve as
the site of ubiquitination (6-8), a pathway termed N
terminus-dependent ubiquitination. Via either
ubiquitination pathway, polyubiquitin chain formation continues by the
conjugation of subsequent ubiquitin moieties to the attached ubiquitin,
and the substrate-ubiquitin conjugate is then degraded by the 26 S
proteasome in an ATP-dependent manner. Isopeptidases cleave
the ubiquitin chain, and the single ubiquitin molecules are recycled
(5). Currently, the relative contribution of each of these two pathways
is unknown.
B
(18), and the aryl
hydrocarbon receptor (19) all require nuclear export prior to
degradation by the ubiquitin-proteasome system. Ligand-activated Smad3,
although ubiquitinated in the nucleus, is also exported to the
cytoplasm for degradation by the 26 S proteasome (20).
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Arg
mutations) and N-terminal-blocked (6× Myc-tagged) MyoD, MyoD
constructs were transfected into HeLa cells and were visualized 24 h later by immunofluorescence (Fig. 1).
Transfected cells were also treated either with MG132 or with LMB
2 h prior to fixation. MG132 inhibits the proteasome, whereas LMB
inhibits CRM-1-dependent nuclear export to retain MyoD
within the nucleus (30). As seen in Fig. 1, wild-type MyoD,
lysine-less MyoD, and N-terminal-blocked MyoD were all localized to the
nucleus under basal conditions as well as in the presence of MG132 or
LMB. Thus, at steady state, MyoD and its lysine-less and
N-terminal-blocked mutants were found exclusively in the cell
nucleus.
View larger version (48K):
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Fig. 1.
Localization of wild-type MyoD, lysine-less
MyoD, and N-terminal-blocked MyoD. 24 h following
transfection of HeLa cells, cells were treated with MG132 or LMB, and
the localization of each construct was visualized by
immunofluorescence. Wild-type MyoD, lysine-less MyoD, and
N-terminal-blocked MyoD were localized exclusively to the nucleus under
all conditions.
View larger version (27K):
[in a new window]
Fig. 2.
Half-life of wild-type MyoD with MG132.
A, 24 h after transfection with wild-type MyoD, HeLa
cells were treated with CHX or CHX plus MG132. Cells were lysed at 0, 0.5, 1, 2, or 3 h and were evaluated via SDS-PAGE gel and Western
blot for MyoD. B, the pixels for each band were measured and
normalized so that the number of pixels at t = 0 was
100%. The log10 of the % pixels was plotted
versus time for each time point, and the
t1/2 was calculated as the log of 50% (1.7 h). The t1/2 for MyoD was 0.8 h and
11.2 h when MG132 was added. Treatment of the cells with CHX plus
LMB increased the t1/2 to 1.3 h (data not
shown).
Localization and t1/2 of wild-type MyoD and N terminus-blocked
and lysine-less MyoD mutants
Nuclear transcription factors, including MyoD, are synthesized in the cytoplasm and are targeted to the nucleus via NLS. To determine whether MyoD or its mutants are degraded within the cytoplasm either prior to nuclear uptake or during nucleocytoplasmic recycling, we sought to identify the NLS within MyoD to generate NLS-deficient mutants.
Based on results from Vandromme et al. (31), we mutated the
basic residues within two regions of MyoD (amino acid regions 110-112
and 130-135) to alanine, as Vandromme et al. report that deletion of both of these regions is necessary to prevent nuclear localization of MyoD. However, as seen in Fig.
3, substitution mutagenesis yielded
distinctly different results. Mutagenesis of K102A, R103A, K104A,
R110A, R111A, and K112A (MyoDNLS1) was sufficient to
markedly inhibit nuclear import, whereas mutagenesis of K133A and R134A
(MyoDNLS2) did not affect subcellular localization. Given
these results, the entire amino acid sequence of MyoD was evaluated for
additional basic residue-containing regions which may serve as NLS.
Mutation of R117A, R119A, R120A, R121A, and K124A
(MyoDNLS3) proved to be an additional NLS (Fig. 3). This
same region was examined by Vandromme et al. (31) using
deletion mutagenesis and was found not to serve as an NLS. Inhibition
of nuclear import of MyoDNLS3 (79% cytoplasmic) was
comparable with that seen with MyoDNLS1 (83% cytoplasmic).
Furthermore, mutation of both NLS1 and NLS3 (MyoDNLS1+3)
did not inhibit import substantially more (i.e. ~87%
cytoplasmic) than that observed with the single mutation. Therefore it
is likely that NLS1 and NLS3 represent subdomains of a single large
targeting sequence (amino acid region 102-124) within MyoD. Evaluation
of three additional regions in MyoD (R143A, K146A, and R151A; R220A and
R221A; R235A, R238A, and K241A) failed to reveal any additional NLS.
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The rates of degradation of several of these NLS mutants were examined. The t1/2 of wild-type MyoD and MyoDNLS2, both of which localized to the nucleus (96 and 92% nuclear, respectively), were each found to be 0.8 ± 0.1 h. However, the t1/2 of MyoDNLS1, MyoDNLS3, and MyoDNLS1+2, each of which localized predominately to the cytoplasm (83, 79, and 79% cytoplasmic, respectively), were found to be 1.3 ± 0.3, 1.2 ± 0.1, and 1.2 ± 0.2 h, respectively. Thus, it appears that the rate of MyoD degradation correlates with its subcellular localization, in that nuclear localization is associated with more rapid degradation.
The NLS1 mutation identified above did not result in complete absence
of MyoD from the nucleus. It may be that MyoD undergoes nucleocytoplasmic shuttling and that the mutagenesis of NLS1 causes MyoD to enter the nucleus more slowly than wild-type MyoD, or it may be
that the rate at which MyoD exits the nucleus is accelerated when NLS1
is mutated. To test if MyoDNLS1 undergoes nucleocytoplasmic
shuttling, 16-24 h after transfection of HeLa cells with
MyoDNLS1, LMB was added, and MyoD localization was
visualized via immunofluorescence. As seen in Fig.
4, incubation with LMB led to a dramatic
accumulation of MyoDNLS1 within the nucleus (17-93%
nuclear). This suggests that the MyoDNLS1 mutant cycles
between the cytoplasm and the nucleus, although it does not indicate
whether MyoDNLS1 is entering the nucleus more slowly or
exiting the nucleus more quickly than wild-type MyoD. Localization of
this mutant to the nucleus in the presence of LMB was associated with a
decrease in the half-life of MyoDNLS1
(t1/2 = 1.0 ± 0.1 h) compared with that
seen in the absence of LMB (1.3 ± 0.3 h; Fig. 4).
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The ability of MyoD to cycle in and out of the nucleus suggests either
the presence of an intrinsic NES or of association with a chaperone
(32) that supports MyoD nuclear export. Typically, NES are regions high
in leucine content. Comparison of the MyoD sequence to other known NES
within various proteins, such as protein kinase A inhibitor (33),
cyclin B1 (34), human immunodeficiency virus-1 Rev protein (35), or
human Cdc25C (36), suggested a candidate NES within the N terminus
region of MyoD. Thus, using the MyoDNLS1 mutant as a
template, Leu-3, Leu-4, Leu-8, Ile-11, and Leu-13 were mutated to
alanines (MyoDNLS1NES), and the construct was transfected
into HeLa cells. A marked increase in nuclear localization of MyoD
(17-42% nuclear) was observed (Fig. 5)
consistent with the notion that amino acid sequence 3-13 contains a
nuclear export signal. Two other regions high in leucine content were
mutated on the MyoDNLS1 background (L268A, L269A, and
L270A; L248A, L251A, and I254A) to identify other potential NES within
MyoD; however, no others were identified.
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The NES of wild-type MyoD was then mutated to determine the half-life
of MyoDNES. As expected, MyoDNES localized
exclusively to the nucleus (100% nuclear) (Fig.
6). The half-life of MyoDNES
(t1/2 = 1.05 ± 0.04 h) decreased compared
with that seen for MyoDNLS1 (t1/2 = 1.3 ± 0.3 h) and slightly increased compared with that seen
for wild-type MyoD (t1/2 = 0.8 ± 0.1 h).
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Having identified the major NLS and NES signals in MyoD, we next sought to determine which degradation pathway (N terminus-dependent or lysine-dependent) predominates in the nuclear and cytoplasmic compartments. To localize the various mutants to the cytoplasm or the nucleus, we prepared NLS1 or NES mutations on N-terminal-blocked MyoD and lysine-less MyoD.
The N-terminal-blocked species of MyoD are degraded via the
lysine-dependent pathway. The N-terminal-blocked NLS1
mutant (N-terminal-blocked MyoDNLS1) was degraded with a
shorter t1/2 (1.6 ± 0.3 h) compared with
the N-terminal-blocked wild-type MyoD (2.7 ± 0.6 h), whereas
the N-terminal-blocked NES mutant (N-terminal-blocked MyoDNES) was degraded with a t1/2
(2.8 ± 0.5 h) similar to the N-terminal-blocked wild-type
MyoD (Fig. 7A). As expected,
N-terminal-blocked wild-type MyoD and N-terminal-blocked MyoDNES were nuclear, whereas N-terminal-blocked
MyoDNLS1 was predominately cytoplasmic. The more rapid
degradation of the N-terminal-blocked species localized to the
cytoplasm (N-terminal-blocked MyoDNLS1) suggests that the
lysine-dependent pathway may be more active for MyoD in the
cytoplasm than in the nucleus.
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The lysine-less species of MyoD are degraded via the N
terminus-dependent pathway. Whereas lysine-less wild-type
MyoD and lysine-less MyoD with the NES mutation (lysine-less
MyoDNES) were both nuclear as expected, lysine-less MyoD
with the NLS1 mutation (lysine-less MyoDNLS1) was only 40%
cytoplasmic. This result is somewhat unexpected because the NLS1
mutation on either the wild-type MyoD or N-terminal-blocked MyoD
background yielded species with 83 or 75% cytoplasmic localization, respectively (Figs. 3, 4, and 7A). Lysine-less
MyoDNLS1 had a shorter t1/2 (1.5 ± 0.4 h) than lysine-less wild-type MyoD (2.6 ± 0.6 h).
Lysine-less MyoDNES was degraded at a substantially slower
rate (t1/2 = 5.8 ± 0.9 h). The slower
degradation of the lysine-less species localized to the nucleus
(lysine-less MyoD and lysine-less MyoDNES) suggests that
the N terminus-dependent pathway is less active in the nucleus.
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DISCUSSION |
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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The ubiquitin-proteasome system exists in both the cytoplasm and
the nucleus and is responsible for the degradation of many short-lived
cellular proteins. Ubiquitination of the target protein can occur on an
-amino group of an internal lysine or on the N terminus of the
protein tagged for destruction. MyoD, a basic helix-loop-helix
transcription factor, is ubiquitinated and degraded by the
ubiquitin-proteasome system in the nucleus (11). Ubiquitination of the
protein occurs preferentially on the N terminus, leading to N
terminus-dependent degradation. Blocking of the N terminus with a 6× Myc tag, via methylation, or through carbamylation of the
-amino group leads to preferential degradation by the
lysine-dependent pathway (8). Using NLS and NES mutants of
N-terminal-blocked and lysine-less MyoD, we determined that the
lysine-dependent pathway is more active in the cytoplasm,
whereas both degradation pathways have equal activity in the nucleus.
This determination was based on t1/2 calculations of the different N-terminal-blocked and lysine-less mutants, and although the differences in the t1/2 may be small, they are likely to be biologically significant because a 2-4-fold change in the half-life of a protein may underlie a significant change in a protein steady-state level and hence in a biological function. It should also be noted that only the subcellular localization and t1/2 were assayed and that kinetic determinations of MyoD nucleocytoplasmic shuttling were not evaluated. Furthermore, it is certainly possible, if not likely, that these changes have multiple effects on MyoD, affecting such aspects as DNA binding, acetylation, and/or dimerization. Although the results presented herein are specific to MyoD, future studies will focus on these pathways of cellular localization and degradation and on how the pathways relate to other proteins degraded by the ubiquitin-proteasome system.
To determine the ability of the ubiquitin-proteasome system to degrade MyoD in the cytoplasm, the NLS of MyoD was mutated in an attempt to inhibit nuclear localization. NLS are characterized by single or bipartite regions of basic amino acids. In the present study NLS mutants of MyoD were prepared by mutagenesis of clusters of basic amino acids (Lys and Arg) to alanines. Among the six regions evaluated, MyoDNLS1 and MyoDNLS3 were found to significantly reduce nuclear localization of MyoD (17 and 21% nuclear, respectively). Mutation of NLS1 and NLS3 together did not significantly inhibit nuclear import to a greater degree than that seen with either single mutation alone. Therefore, it is likely that MyoD possesses one NLS signal (amino acid region 102-124) and that NLS1 and NLS3 function as subdomains of this sequence. These results differ from those previously reported by Vandromme et al. (31) in which they identify two NLS regions (amino acid regions 100-112 and 130-135) that appear to function independently in that deletion of both regions is required to inhibit nuclear localization of MyoD. We find that substitution mutagenesis of NLS1 (amino acid region 102-112) alone is sufficient to inhibit import and that NLS2 (amino acid region 133-134) is not a nuclear targeting sequence. Furthermore, MyoDNLS1+2 was no more cytoplasmic than MyoDNLS1 alone (Fig. 3).
Using MyoDNLS1 as a background, we identified a single NES signal within the N terminus of MyoD (Fig. 5). Although MyoDNLS1NES was 42% nuclear compared with 17% nuclear for MyoDNLS1, there was still a considerable amount of MyoD in the cytoplasm. Treatment of MyoDNLS1NES with LMB caused the protein to be greater than 88% nuclear (data not shown), suggesting that mutagenesis of the NES does not fully inhibit nuclear export. One reason for this may be that MyoD associates with cofactors that facilitate nuclear export regardless of the mutated NES, or it may be that a separate NES, consisting of residues other than leucine, exists.
Examination of the rates of degradation of the various MyoD mutants yields a strong correlation between degradation rate and subcellular localization. Constructs that were localized to the nucleus, (96-100%) wild-type MyoD, MyoDNLS2, and MyoDNES, were degraded with a t1/2 of 0.8-1.05 h, whereas MyoDNLS1, MyoDNLS1+2, or MyoDNLS3, each of which was predominately cytoplasmic (17-21% nuclear), displayed t1/2 of 1.2-1.3 h (Fig. 3).
MyoDNES had a slightly longer t1/2 than wild-type MyoD (1.05 versus 0.8 h; Fig. 6). One potential reason for this finding may be that we had introduced modifications to the protein at the N terminus, which appears to be its main ubiquitination site (8). Another possibility is that the nuclear degradation machinery is saturated when MyoD is exclusively nuclear. Consistent with this latter possibility, we found that the t1/2 for the degradation of wild-type MyoD in the presence of LMB is somewhat slower than that seen without the inhibitor (legend to Fig. 2).
To localize N-terminal-blocked MyoD and lysine-less MyoD to the cytoplasm or to the nucleus, we mutated their NLS and NES. The t1/2 of N-terminal-blocked MyoD was significantly longer than that of wild-type MyoD (2.7 ± 0.6 h and 0.8 ± 0.1 h, respectively), which is in agreement with previously published data (Fig. 7A; Ref. 8). Cytoplasmically localized N-terminal-blocked MyoD (N-terminal-blocked MyoDNLS1) was degraded with a shorter half-life than either wild-type N-terminal-blocked MyoD or N-terminal-blocked MyoDNES, both of which localized to the nucleus (Fig. 7A). This supports the notion that the lysine-dependent pathway is more active in the cytoplasm than in the nucleus.
The locus of activity of the N terminus-dependent pathway is less clear. Mutation of the NLS within lysine-less MyoD did not yield a construct with predominant cytoplasmic localization. Although some cytoplasmic localization was seen, lysine-less MyoDNLS1 was ~60% nuclear (Fig. 7B). The reason or reasons for this finding are unclear. One reason may be that mutagenesis of lysines to arginines may affect DNA binding as lysine 146 has been shown to make contacts with the bound DNA duplex (37). The reduced ability of MyoD to induce myogenesis as a result of mutagenesis of lysine 124 (38) may also contribute to the nuclear localization and shorter half-life of lysine-less MyoDNLS1. Activation and the DNA-binding ability of MyoD are also regulated by acetylation of lysines 99, 102, and 104 (39, 40), which are mutated in the MyoD lysine-less mutant. In addition, lysine-less MyoDNLS1 was degraded more rapidly than wild-type lysine-less MyoD, perhaps consistent with this notion. Lysine-less MyoDNES was nuclear with a half-life considerably longer than that of wild-type lysine-less MyoD (5.8 ± 0.9 h versus 2.6 ± 0.6 h). This finding likely results from the lack of recognition sites for the ubiquitin-proteasome system, as all of the lysines had been mutated to arginines and had been mutated within MyoDNES. The N terminus (amino acid region 3-13) had also been mutagenized, leaving no known site for recognition and ubiquitination, as alterations of amino acids with the protein alter its three-dimensional structure and hence its potential for recognition by the ubiquitin system.
A scheme for the pathways of MyoD degradation is illustrated in Fig.
8. MyoD is synthesized in the cytoplasm
and rapidly targeted to the nucleus, where it exists both uncomplexed
and bound to DNA. Wild-type MyoD can be tagged for ubiquitination on
internal lysines or at its N terminus. Modification of either of these recognition sites decreases the degradation rate of MyoD. Wild-type MyoD was degraded more rapidly in the nucleus than in the cytoplasm. N-terminal-blocked MyoD, however, was degraded more rapidly in the
cytoplasm. This suggests that the lysine-dependent pathway of degradation is more active in the cytoplasm than in the nucleus. Because the half-lives of wild-type MyoDNLS1 and
N-terminal-blocked MyoDNLS1 were similar, it may be that
cytoplasmic MyoD is degraded only through a
lysine-dependent pathway, whereas both pathways function equally well in the nucleus. Equivalent activities of the two pathways
within the nucleus are also supported by the similar half-lives of
wild-type N-terminal-blocked MyoD and wild-type lysine-less MyoD.
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At present it appears that many more proteins are tagged for
ubiquitin-dependent degradation on internal lysines.
In addition, it currently seems that the majority of "nuclear"
proteins require nuclear export prior to degradation. Consistent with
these observations, the lysine-dependent degradation
pathway appears to be more active within the cytoplasm than in the
nucleus. This raises the possibility that nuclear proteins that are
degraded in the nucleus undergo degradation via an N
terminus-dependent pathway, as is the case with MyoD.
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FOOTNOTES |
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* This work was supported by grants from the National Institutes of Health.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: Dept. of Pediatrics, Washington University School of Medicine, 660 S. Euclid Ave., St. Louis, MO 63110. Tel.: 314-286-2868; Fax: 314-286-2894; E-mail: Schwartz@kids.wustl.edu.
Published, JBC Papers in Press, October 22, 2002, DOI 10.1074/jbc.M208815200
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ABBREVIATIONS |
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The abbreviations used are: E1, ubiquitin-activating enzyme; E2, ubiquitin-conjugating enzyme; E3, ubiquitin ligase; NLS, nuclear localization sequence(s); NES, nuclear export sequence(s); TRITC, tetramethylrhodamine isothiocyanate; LMB, leptomycin B; CHX, cycloheximide.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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