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1
Departments of
* Cellular and Structural Biology and
Molecular Medicine/Institute of Biotechnology, University of Texas Health Science Center, San Antonio, Texas 78245-3207, USA
1Correspondence: Department of Molecular Medicine/Institute of Biotechnology, University of Texas Health Science Center, 15355 Lambda Dr., San Antonio, TX 78245-3207, USA. E-mail: christy{at}uthscsa.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: helix-loop-helix • dominant-negative regulators • 26S proteasome
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 2)
. The ubiquitin-conjugated proteins are recognized
by the large multisubunit proteasome complex, which degrades them into
small peptides. The 26S proteasome complex has been implicated in the
proteolysis of transcription factors, cell cycle regulatory proteins,
oncogenes, tumor suppressors, and proteins involved in cellular
differentiation (1
, 3
4
5)
. The rapid degradation of
certain regulatory proteins is important in the stringent control of
their signaling activity (1
, 3)
.
Id proteins are small helix-loop-helix proteins that do not possess
basic DNA binding domains, and are thought to act as negative
regulators by heterodimerizing with DNA binding bHLH proteins and
preventing their DNA binding and transcriptional activities
(6)
. The four mammalian members of the Id protein family
are 69–78% identical at the amino acid level within their HLH
dimerization domains, but other parts of the proteins are essentially
unrelated (6
, 7)
. Recent studies have implicated the Id
family proteins in the regulation of important cellular processes,
including cell growth and cell cycle progression (8
9
10
11
12)
,
cellular differentiation (6)
, embryonic development
(13
14
15)
, and cell death (12
, 16
17
18)
. We
have recently identified an Id3-interacting protein by using the yeast
two-hybrid screening system (M. A. Bounpheng, I. N.
Melnikova, S. G. Dodds, and B. A. Christy, unpublished
results). This protein, the mouse homologue of the human JAB1 putative
coactivator protein (19)
, appears to be present in a large
complex that may be related to proteasome function (20)
.
In addition, JAB1 is homologous to the Poh1 protein, which was shown to
be a component of the 19S regulatory complex of the 26S proteasome
(20)
. Another group has recently isolated a subunit of the
26S proteasome complex, the S5a protein, as an Id1-interacting protein
(21)
. Because of these associations with proteins involved
in protein degradation, we were interested in determining whether Id3
and other Id family proteins are degraded by the ubiquitin–proteasome
pathway. In this study, we show that the Id family proteins are
short-lived proteins whose degradation is dependent on the
ubiquitin–proteasome pathway. Id4 protein degradation appears to be
regulated differently from that of the other three family members.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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:NeoE47
(22)
and inserting it into the XbaI site of
pcDNA3.1. pGL2, a plasmid containing the luciferase cDNA under control
of the SV40 promoter, was obtained from Promega (Madison, Wis.). HA-Ub
and His6-Ub are described elsewhere
(23)
.
Cell culture
(HEK) 293 human embryonal kidney cells were obtained from the
American Type Culture Collection and maintained in low glucose
Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal
bovine serum (FBS), and 100 µg/ml of penicillin and streptomycin. The
temperature-sensitive ts20tg Balb/C 3T3 cells and the E1-transfected
derivative (H38–5) were maintained at 35°C in DMEM supplemented with
10% newborn calf serum and 100 µg/ml of penicillin and streptomycin,
as described previously (24)
.
Transient transfection assays and radiolabeling of cells
All transfections were done using the FUGENE transfection
reagent according to the manufacturer’s recommended procedure
(Boehringer Mannheim Corp., Indianapolis, Ind.). 293 cells were plated
at 2 x 105 cells/35 mm plate and ts20tg and
H38–5 cells were plated at 1.25 x 105
cells/35 mm plate ~24 h prior to transfection and transfected with 2
µg of expression plasmid. Experiments that included coexpression of
CMV-E47 also contained pGL2 (a plasmid containing the SV40 promoter
driven luciferase gene) for determining transfection efficiency. For
these experiments, an aliquot of the 293 cells were used to prepared
cell extracts and assayed for luciferase activity according to
manufacturer’s protocol (Promega). Metabolic labeling of transfected
293 cells and subsequent lysis of cells for direct immunoprecipitation
were performed as described previously (25)
. For metabolic
labeling in the presence of proteasome inhibitors, 293 cells were
transfected with CMV-Id3 for ~36 h, after which the cells were washed
with serum-free DMEM (low glucose) to remove the DNA and incubated with
medium without cysteine and methionine supplemented with 5% dialyzed
FBS for 1 h. The cell were then incubated in cysteine- and
methionine-free media with 10% dialyzed FBS and 100 µCi/ml of
[35S]methionine for 2 h. Proteasome
inhibitors, the lysosomal protease inhibitor chloroquine (100 µM,
Sigma, St. Louis, Mo.), or DMSO vehicle were added 30 min after
addition of labeled media and were present during the chase as well. To
inhibit 26S proteasome activity, two different inhibitors—10 µM
lactacystin (Calbiochem-Novabiochem Corp., San Diego, Calif.)
(26
, 27)
or 5 µM Z-L3VS
(28)
, kindly provided by Dr. Hidde Ploegh, Harvard Medical
School—were used. After labeling for 2 h, cells were chased with
growth media without labeled amino acids for 2 h before harvest.
Western blot analysis
Twenty-four hours after transfection, 293 cells were washed once
with serum-free media and refed with growth media containing either
DMSO vehicle, the proteasome inhibitors lactacystin (10 µM) and
Z-L3VS (5 µM or 10 µM), or the lysosomal
protease inhibitor chloroquine (100 µM) for varying amounts of time.
The cells were harvested at the indicated times, lysed in 100 µl of
50 mM Tris pH 7.5/0.5% sodium dodecyl sulfate (SDS), boiled for 10
min, and clarified by centrifugation. Nuclear extracts were prepared as
described previously (29)
. ts20tg and H38–5 cells were
transfected and incubated with DNA precipitate at 35°C for ~16 h,
after which they were either reincubated at 35°C or shifted to 39°C
before harvest at the indicated time points. The cells were harvested
in the same manner as 293 cells. For all Western blots, ~50 µg of
protein was loaded onto a 12% SDS-polyacrylamide gel electrophoresis
(PAGE) gel, transferred to nitrocellulose, and analyzed with
appropriate primary antibodies. Detection of the antigen–antibody
complexes was performed using either enhanced chemiluminescence
(Pierce, Rockford, Ill.) or alkaline phosphatase (Kirkegaard & Perry
Labs, Gaithersburg, Md.) according to manufacturer’s protocol.
HIS6-tagged protein purification
293 cells were plated at a density of 8 x
105 cells/10 cm plate and transfected with 8 µg
of total DNA; 4 µg of each expression construct was used for each DNA
precipitate. Twenty-four hours after DNA precipitate incubation, the
cells were rinsed with serum-free media and refed with growth media
containing 5 µM Z-L3VS for ~18 h. Cells were
harvested and His6-tagged proteins were
precipitated using methods described previously (30)
. The
precipitated proteins were electrophoresed on 12% SDS-PAGE gels,
blotted to nitrocellulose, and analyzed with appropriate antibodies.
Antibodies
Rabbit polyclonal anti-Id1(JC-FL) and anti-Id2(C-20)
antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz,
Calif.). Rabbit polyclonal anti-Id3 antibody (BC302) was generated
against the carboxyl-terminal region of the Id3 protein produced in
bacteria (31)
, and rabbit polyclonalanti-Id4
antibody (JH563) was generated against the
carboxyl-terminal portion of the Id4 protein produced in
bacteria (H. P. Chen, R. Parvari, R. J. Christy, and B. A. Christy, unpublished results). The mouse monoclonal anti-Id3
antibody (2F2) was generated by the UTHSCSA Institutional Hybridoma
Facility. The immunogen was full-length Id3 protein produced in
bacteria. Rabbit polyclonal anti-ubiquitin antibody was purchased from
StressGen Biotechnologies Corp. (Victoria, B.C., Canada).
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RESULTS |
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S-methionine for 2 h, chased for 0–7 h, lysed, and
immunoprecipitated with a mouse monoclonal antibody against the
amino-terminal portion of Id3. As shown in Fig. 1A
, the half-life of Id3 is estimated to be ~20 min in 293
cells. A previous study had also shown the half-life of Id3 to be
relatively short: ~1 h in COS-7 cells (25)
. Since the
ubiquitin-mediated proteolytic system is involved in the targeted
degradation of many short-lived regulatory proteins (1)
,
we hypothesized that Id3 and possibly other Id family proteins would
also be degraded by the 26S proteasome. Consistent with this
hypothesis, the S5a protein (thought to be a regulatory subunit of the
26S proteasome) was recently identified as an Id1-interacting protein
(21)
. Thus, we investigated the role of the
ubiquitin–proteasome pathway in Id3 protein degradation. Pulse-chase
experiments were performed in Id3-transfected 293 cells, which were
incubated a specific irreversible inhibitor of the 26S proteasome
(lactacystin or Z-L3VS) or with an inhibitor of
lysosomal proteolysis (chloroquine) (33)
. As shown in Fig. 1B
, we found that treatment of 293 cells with inhibitors of
the 26S proteasome blocked Id3 degradation, whereas treatment with the
lysosomal proteolysis inhibitor chloroquine had no effect. This
result suggests that Id3 protein turnover in the cell is mediated
through the ubiquitin–proteasome pathway. Accumulation of Id3 protein
was analyzed at various times after cells were treated with the
proteasome inhibitor Z-L3VS (Fig. 2A
). 293 cells were transiently transfected with Id3 and
incubated in 5 µM Z-L3VS for 0–24 h prior to
harvest. Detection of the Id3 protein by Western blotting with anti-Id3
antibody showed a dramatic increase in accumulation of Id3 protein as
early as 5 h after treatment with proteasome inhibitor (Fig. 2A
). No Id3 protein was detected in similarly treated
mock-transfected cells (data not shown).
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Since a previous reported indicated that coexpression of the
Id3-interacting bHLH protein E47 greatly increased the half-life of Id3
(25)
, we tested whether the cotransfection of E47 could
protect the Id3 protein from degradation by the proteasome. 293 cells
were transfected with Id3 alone or Id3 plus E47; some of the plates
were treated with 5 µM Z-L3VS for 8 h
prior to harvest and processing as described above. Both transfection
sets contained pGL2, an SV40 promoter driven luciferase expression
constructs, which was included to assess transfection efficiency. As
shown in Fig. 2B
, coexpression of E47 with Id3 caused a
large increase in the amount of Id3 protein detected in whole cell
extracts in the absence of proteasome inhibitor. In the presence of a
proteasome inhibitor, the proportion of accumulated Id3 was
significantly less with the coexpression of E47. These results indicate
that coexpression of E47 protected Id3 protein from degradation by the
proteasome. Examination of the luciferase activity from the cell
extracts prepared from the same transfection indicated that all the
cells from each set transfected equally well. Surprisingly, when
nuclear protein from the same experiment was examined, the Id3 protein
present in the nucleus was not protected from degradation by the
proteasome by coexpression of E47 in the absence of proteasome
inhibitors. However, when proteasome inhibitor was added, it appears
that significantly less Id3 protein accumulated when it was coexpressed
with E47. A similar effect of E47 coexpression was observed in CV-1
cells treated with lactacystin for 24 h (data not shown).
Since these results suggested that accumulation of Id3 protein was
regulated by the ubiquitin–proteasome pathway, we wanted to determine
whether the remaining Id family proteins (Id1, Id2, and Id4) were
degraded by a similar mechanism. We therefore tested whether
accumulation of these proteins was affected by inhibiting the 26S
proteasome with lactacystin or Z-L3VS. 293 cells
were transiently transfected with expression plasmids containing
full-length Id cDNAs and then treated with DMSO vehicle only or with
the inhibitors lactacystin, Z-L3VS, or
chloroquine. Cells were harvested at 8 and 24 h after inhibitor
treatment. As shown in Fig. 3
, accumulation of Id1, Id2, and Id3 protein was dramatically increased
in cells treated with inhibitors of the 26S proteasome, but not with
the lysosomal proteolysis inhibitor chloroquine. In contrast,
accumulation of Id4 protein was not increased by inhibition of the 26S
proteasome (Fig. 3
, last row). These data suggest that the Id1, Id2,
and Id3 proteins are all degraded by the ubiquitin–proteasome pathway,
but this pathway does not appear to be a major one for the degradation
of the related Id4 protein.
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To directly determine whether Id family proteins are ubiquitinated, we
coexpressed Id cDNAs along with ubiquitin that was epitope-tagged with
six histidine residues (His6-Ub). Cells were
treated with 5 µM Z-L3VS for 18 h prior to
harvest in order to promote accumulation of ubiquitinated
intermediates. Transfected cell lysates were incubated with
nickel-agarose resin to bind the His6-tagged
protein. Bound proteins were washed, fractionated on SDS-PAGE, and
transferred to nitrocellulose. Duplicate blots were probed with
antibodies to the appropriate Id protein and anti-ubiquitin antibody.
As shown in Fig. 4A
(left), Id1 immunoreactive protein was precipitated with
the nickel-agarose beads after transfection with both
His6-Ub and Id1, but not when either construct
was transfected alone. Thus, the precipitation of Id1 protein depends
on the coexpression of His6-tagged ubiquitin
protein. As a control, a different epitope-tagged ubiquitin construct
(HA-Ub, which will not bind to nickel-agarose beads) was cotransfected
with the Id1 expression construct, but no Id1 immunoreactive protein is
detected after precipitation with nickel-agarose. In mock-transfected
cells or cells transfected with His6-Ub alone
(without Id1), no Id1 immunoreactive protein is detected after
precipitation with nickel-agarose beads. As expected for a protein
modified by the covalent addition of
His6-ubiquitin, a ladder of Id1 immunoreactive
bands with a higher molecular weight than unmodified Id1 protein are
detected. When an identical blot is probed with anti-ubiquitin antibody
(Fig. 4A
, right side), multiple precipitated ubiquitinated
proteins can be detected in the cells that are conjugated to His-tagged
ubiquitin. As shown in Fig. 4B
, similar results were
obtained with Id3 and His6-Ub transfected cells.
Similar results were obtained for Id2 and Id4 proteins (data not
shown).
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To confirm that the increased accumulation of Id1, Id2, and Id3
proteins in inhibitor-treated cells is specifically due to inhibition
of the 26S proteasome complex, we used a mutant cell line containing a
temperature-sensitive deficiency in protein degradation by the
ubiquitin–proteasome pathway (34)
. The ts20tg cell line
is a temperature-sensitive mutant line derived from Balb/c 3T3 mouse
fibroblast cells after mutagenesis with N-methyl-N-nitrosoguanidine
(34)
. At the permissive temperature of 35°C, ts20 mutant
cells grow and behave normally, but display a growth defect when
shifted to the nonpermissive temperature (39°C). The defect in ts20tg
cells has been found to be a temperature-sensitive mutation in the
ubiquitin-activating enzyme E1 (24)
that can be corrected
by introduction of the human E1 gene into the cells. The E1 enzyme
activates ubiquitin in an ATP-dependent process necessary for the
ubiquitination of proteins that targets them for degradation by the 26S
proteasome. Since only one E1 enzyme has been found in mammals, its
inactivation inhibits ubiquitination completely and leads to an
increased accumulation of proteins normally degraded rapidly by the 26S
proteasome (24)
. When Id family cDNAs were transfected
into ts20tg cells and maintained at the permissive temperature
(35°C), a low level of each transfected protein could be seen
(Fig. 5
). When transfected ts20tg cells were shifted to the nonpermissive
temperature (39°C), an increased accumulation of all Id proteins was
observed (Fig. 5)
. In mock-transfected cells, no Id proteins could be
detected under these experimental conditions at either the permissive
or nonpermissive temperature (data not shown). When Id family cDNAs
were transfected into the H38–5 cells in which the
temperature-sensitive defect has been corrected by stable ectopic
expression of a human E1 gene (24)
, there was no increased
accumulation of Id proteins at the nonpermissive temperature. These
results are consistent with the hypothesis that degradation of all four
of the Id proteins is dependent on the E1 ubiquitin-activating enzyme.
The observation that Id4 protein accumulated at the nonpermissive
temperature was somewhat surprising, since we did not observe a
dramatic accumulation of Id4 in the presence of proteasome inhibitors
(Fig. 3)
. The fact that the Id4 protein turnover is dependent on the E1
enzyme suggest that Id4’s degradation is dependent on ubiquitination,
but it appears to be much less sensitive to degradation by the 26S
proteasome than are the other Id family proteins. Perhaps Id4 protein
is ‘rescued’ from proteolytic degradation by the action of a
deubiquitinating enzyme. Alternatively, the bulk of Id4 protein in the
cell may be degraded by an alternative mechanism. In any case, it is
clear from the data presented here that the degradation of Id4 protein
is regulated differently from that of the other three Id family
proteins.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. In that study, the half-life of the transfected
Id3 protein in COS-7 cells was ~60 min. When E47 was cotransfected
with Id3, the half-life of the Id3 protein increased to ~4 h,
suggesting that interaction with its binding partner was protecting the
Id3 protein from degradation. Second, while searching for
Id3-interacting proteins using the yeast two-hybrid screen, we
identified the mouse JAB1 homologue, which is related to factors
thought be present in the 19S regulatory complex of the 26S proteasome.
Third, the proteasome subunit S5a was previously isolated as an
Id1-interacting protein (21)
. Fourth, data from our
laboratory indicate that the Id4 protein also has a relatively short
half-life (70–80 min) in 3T3-L1 cells (H. P. Chen, R. Parvari,
R. J. Christy, and B. A. Christy, unpublished results).
Finally, we have noticed that it is difficult to express large amounts
of Id proteins in many cell types even after transient transfection
under the control of a strong viral promoter. For all these reasons, we
suspected that the Id proteins are quite unstable inside cells and that
their degradation may be tightly regulated in order to avoid
deleterious consequences.
Since the accumulation of many regulatory factors involved in cell
growth, differentiation, and transcriptional regulation is controlled
at the level of degradation by the 26S proteasome [including some
Id-interacting proteins such as E2A (35)
and MyoD
(32
, 36)
], we initiated the current study in order to
determine whether Id3 and other Id family proteins were degraded by the
ubiquitin–proteasome pathway. Initially, we examined the stability of
Id3 in 293 cells using pulse-chase labeling and estimated that in these
cells Id3 had a very short half-life of ~20 min. Although this value
is less than the half-life previously determined in a different cell
line (20 min vs. 60 min; 25
), both studies determined a
relatively short half-life for the Id3 protein. We also show that Id3
protein degradation can be blocked by addition of specific inhibitors
of the 26S proteasome, leading to a dramatic increase in accumulation
of Id3 protein. Consistent with the previous report (25)
,
we show that the Id3 protein was less susceptible to degradation by the
ubiquitin–proteasome pathway when coexpressed with an interacting
protein, E47 (Fig. 2)
. Since Id3 is a member of a family of four
related proteins, we were interested to determine whether the other Id
family proteins were also degraded by a similar mechanism. Id1, Id2,
and Id4 were transfected into 293 cells and treated with lactacystin or
Z-L3VS to inhibit the 26S proteasome, chloroquine, to inhibit lysosomal
proteolysis or DMSO vehicle. As shown in Fig. 3
, inhibition of the 26S
proteasome by either lactacystin or Z-L3VS resulted increased
accumulation of Id1, Id2, and Id3 protein. In contrast, Id4 protein
accumulation was not dramatically increased in the presence of
inhibitors of 26S proteasome activity. This suggests that the Id1, Id2,
and Id3 proteins are normally degraded rapidly by the proteasome, but
that Id4 protein degradation is regulated very differently. Inhibition
of lysosomal proteolysis by chloroquine treatment did not affect
accumulation of any of the Id family proteins, indicating that none of
these proteins are degraded significantly through this pathway. We next
tested whether Id proteins can form ubiquitin conjugates in
vivo by cotransfection of His6-tagged
ubiquitin along with Id cDNAs. As shown in Fig. 4A, B
, when
Id1 or Id3 was cotransfected into cells along with
His6-tagged ubiquitin (but not HA-tagged
ubiquitin, which was used as a control), both Id1 and Id3 proteins were
found bound to nickel-agarose beads. Much of the Id1 and
Id3-immunoreactive protein bound to nickel-agarose in the presence of
His6-ubiquitin is larger than the unmodified Id1
or Id3 proteins, suggesting that ubiquitination has occurred. Similar
results were obtained with Id2 and Id4 (data not shown). These results
suggest that all four Id family proteins are modified by ubiquitination
in 293 cells, suggesting that their degradation depends on ubiquitin
modification. Although the Id4 protein degradation is not sensitive to
inhibitors of the 26S proteasome, it does appear to be modified by
ubiquitination in vivo.
An analysis of the amino acid sequences of the four Id family proteins
does not reveal exact PEST consensus sequences or destruction box
sequences, which are the signals for ubiquitination and degradation of
cell cycle regulators (1)
. Compilation of destruction box
sequences from many B- and A-type cyclins from various organisms showed
that they have the loose consensus sequences R (A/T) (A) L (G) x (I/V)
(G/T) (N), where the only invariable residues are R and L in positions
1 and 4, respectively, but the rest of the sequences are quite variable
(1)
. Close examination of the amino acid sequences of the
Id proteins (6)
reveals a small homology domain with the
consensus sequence R (A/T) (P/R) L (S/T) (A/T) L N in the carboxyl
terminus of Id1, Id2, and Id4 that shows partial homology to the
destruction box sequence. However, this region of homology is not found
in the Id3 protein sequence. To determine which domain of the Id
proteins targeted them for degradation by the 26S proteasome, we
examined the accumulation of Id1 deletion mutant proteins in
Z-L3VS-treated cells. We found that deletion of
the carboxyl terminus, the NH2 terminus, or the
HLH domain of Id1 resulted in greater accumulation of Id1 protein in
the presence of proteasome inhibitors, suggesting that all of the
mutants are still unstable and are targeted for destruction by the 26S
proteasome (data not shown). The rapid degradation of Id proteins after
ectopic expression most likely accounts for the difficulty in
expressing large amounts of these proteins in the cell that we and
others have encountered in the past.
In addition to the studies performed in 293 cells (described above), we
used a well-characterized mutant fibroblast cell line (ts20tg) that
contains a temperature-sensitive defect in the E1 ubiquitin-activating
enzyme (24)
. ts20tg cells grow and behave normally at the
permissive temperature (35°C), but show reduced growth at the
nonpermissive temperature (39°C). The block in E1 activity leads to
an increase in accumulation of the proteins that are normally degraded
by the ubiquitin pathway (24)
. When ts20tg cells are
transiently transfected with Id cDNAs and shifted to the nonpermissive
temperature, increased accumulation of all four Id proteins was
observed (Fig. 5)
. We were somewhat surprised to see increased Id4
accumulation, since treatment with proteasome inhibitors did not
dramatically alter accumulation of Id4. It is possible that the
degradation of Id4 is dependent on ubiquitination, but may be largely
‘rescued’ from degradation by a deubiquitination mechanism.
Alternatively, other proteolytic systems could be involved in Id4
protein degradation. Recently, mouse EL4 lymphoma cells adapted to grow
in the presence of toxic levels of proteasome inhibitors were isolated
(37)
. Although the activity of the 26S proteasome is very
low in these cells, they can traverse through the cell cycle normally,
suggesting that the cells are still able to carry out this vital
function normally influenced by the 26S proteasome (37)
. A
protease identified as tripeptidyl peptidase II whose hydrolytic
activities were resistant to specific proteasome inhibitors was
recently identified (38)
, which may provide an alternative
pathway. These studies suggest that alternative protein complexes are
important in protein degradation, although their normal role in
cellular processes is yet to be determined. It is possible that
degradation of Id4 protein occurs through one of these alternative
pathways or through an unidentified proteolytic pathway. In previous
experiments in another cell type, we determined that the half-life of
Id4 protein is ~75 min (H. P. Chen, R. Parvari, R. J.
Christy, and B. A. Christy, unpublished results). Therefore, even
though the Id4 protein is also relatively short-lived, its degradation
appears to be regulated differently than that of the other Id family
proteins. We have noticed in several cell types that FLAG
epitope-tagged Id4 protein accumulates to a higher level than FLAG
epitope-tagged Id1, Id2 or Id3 proteins (when detected using anti-FLAG
antibody), possibly because Id4 protein is degraded more slowly within
the cells. Further experiments to clarify this issue are in progress.
Recent studies have reported that some Id-interacting proteins are also
degraded by the ubiquitin–proteasome pathway. Kho et al.
(35)
showed that the bHLH protein E12 interacts with the
ubiquitin-conjugating enzyme UbcE2A, is ubiquitinated, and normally is
turned over rapidly (half-life ~60 min). Treatment with the
proteasome inhibitor MG132 increases accumulation of E12 in transfected
cells. The myogenic transcription factor MyoD is also a short-lived
protein (half-life 20–30 min); a recent report implicates the
ubiquitin–proteasome pathway in its degradation (32
, 36)
.
Regulation of the stability of the Id proteins within the cell provides
another mechanism to control the balance between E-box binding bHLH
protein dimers and inactive dimers, allowing the cell to fine-tune the
regulatory activities of these transcription factors.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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domain. Cell 78,787-798[Medline]
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H. Kurooka and Y. Yokota Nucleo-cytoplasmic Shuttling of Id2, a Negative Regulator of Basic Helix-Loop-Helix Transcription Factors J. Biol. Chem., February 11, 2005; 280(6): 4313 - 4320. [Abstract] [Full Text] [PDF]
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F. Vinals and F. Ventura Myogenin Protein Stability Is Decreased by BMP-2 through a Mechanism Implicating Id1 J. Biol. Chem., October 29, 2004; 279(44): 45766 - 45772. [Abstract] [Full Text] [PDF]
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 C. N. Papandreou and C. J. Logothetis Bortezomib as a Potential Treatment for Prostate Cancer Cancer Res., August 1, 2004; 64(15): 5036 - 5043. [Abstract] [Full Text] [PDF]
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J. S. Trausch-Azar, J. Lingbeck, A. Ciechanover, and A. L. Schwartz Ubiquitin-Proteasome-mediated Degradation of Id1 Is Modulated by MyoD J. Biol. Chem., July 30, 2004; 279(31): 32614 - 32619. [Abstract] [Full Text] [PDF]
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 J. Tang, G. M. Gordon, M. G. Muller, M. Dahiya, and K. E. Foreman Kaposi's Sarcoma-Associated Herpesvirus Latency-Associated Nuclear Antigen Induces Expression of the Helix-Loop-Helix Protein Id-1 in Human Endothelial Cells J. Virol., May 15, 2003; 77(10): 5975 - 5984. [Abstract] [Full Text] [PDF]
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C. A. Whittaker and R. O. Hynes Distribution and Evolution of von Willebrand/Integrin A Domains: Widely Dispersed Domains with Roles in Cell Adhesion and Elsewhere Mol. Biol. Cell, October 1, 2002; 13(10): 3369 - 3387. [Abstract] [Full Text] [PDF]
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 K. C. Park, J. H. Kim, E.-J. Choi, S. W. Min, S. Rhee, S. H. Baek, S. S. Chung, O. Bang, D. Park, T. Chiba, et al. Antagonistic regulation of myogenesis by two deubiquitinating enzymes, UBP45 and UBP69 PNAS, July 23, 2002; 99(15): 9733 - 9738. [Abstract] [Full Text] [PDF]
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 C.-j. Liu, B. Ding, H. Wang, and P. Lengyel The MyoD-Inducible p204 Protein Overcomes the Inhibition of Myoblast Differentiation by Id Proteins Mol. Cell. Biol., May 1, 2002; 22(9): 2893 - 2905. [Abstract] [Full Text] [PDF]
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 M. H. Glickman and A. Ciechanover The Ubiquitin-Proteasome Proteolytic Pathway: Destruction for the Sake of Construction Physiol Rev, April 1, 2002; 82(2): 373 - 428. [Abstract] [Full Text] [PDF]
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T. Lopez-Rovira, E. Chalaux, J. Massague, J. L. Rosa, and F. Ventura Direct Binding of Smad1 and Smad4 to Two Distinct Motifs Mediates Bone Morphogenetic Protein-specific Transcriptional Activation of Id1 Gene J. Biol. Chem., January 25, 2002; 277(5): 3176 - 3185. [Abstract] [Full Text] [PDF]
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 K J Vincent, E Jones, M J P Arthur, D E Smart, J Trim, M C Wright, and D A Mann Regulation of E-box DNA binding during in vivo and in vitro activation of rat and human hepatic stellate cells Gut, November 1, 2001; 49(5): 713 - 719. [Abstract] [Full Text] [PDF]
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