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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 95 No. 6 (March 15), 2000:
pp. 2104-2110
NEOPLASIA
c-Myc hot spot mutations in lymphomas result in inefficient
ubiquitination and decreased proteasome-mediated turnover
Fuad Bahram,
Natalie von der Lehr,
Cihan Cetinkaya, and
Lars-Gunnar Larsson
From the Department of Plant Biology, Uppsala Genetic Center,
Swedish University of Agricultural Sciences, and Department of Genetics
and Pathology, University of Uppsala, University Hospital, Uppsala,
Sweden.
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Abstract |
The c-myc proto-oncogene encodes a short-lived transcription
factor that plays an important role in cell cycle regulation, differentiation and apoptosis. c-myc is often rearranged in
tumors resulting in deregulated expression. In addition, mutations in the coding region of c-myc are frequently found in human
lymphomas, a hot spot being the Thr58 phosphorylation site, a mutation
shown to enhance the transforming capacity of c-Myc. It is, however, still unclear in what way this mutation affects c-Myc activity. Our
results show that proteasome-mediated turnover of c-Myc is substantially impaired in Burkitt's lymphoma cells with mutated Thr58
or other mutations that abolish Thr58 phosphorylation, whereas endogenous or ectopically expressed wild type c-Myc proteins turn over
at normal rates in these cells. Myc Thr58 mutants expressed ectopically
in other cell types also exhibit reduced proteasome-mediated degradation, which correlates with a substantial decrease in their ubiquitination. These results suggest that
ubiquitin/proteasome-mediated degradation of c-Myc is triggered by
Thr58 phosphorylation revealing a new important level of control of
c-Myc activity. Mutation of Thr58 in lymphoma thus escapes this
regulation resulting in accumulation of c-Myc protein, likely as part
of the tumor progression.
(Blood. 2000;95:2104-2110)
© 2000 by The American Society of Hematology.
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Introduction |
The myc family of proto-oncogenes encodes
transcription factors that play a central role in the regulation of
various aspects of cell growth (for review see Henriksson and
Lüscher1). Chromosomal aberrations involving
myc family loci have been implicated in the generation of a
variety of human tumors and are often strongly correlated to a poor
prognosis. Among these are the translocations involving c-myc
and the immunoglobulin loci that occur in a high percentage of human
Burkitt's lymphoma, AIDS-associated lymphomas, and certain acute
lymphoblastic leukemias, resulting in deregulated expression of
c-myc (for review see Klein and Klein2 and Marcu et
al3). In addition, as shown in Figure 1,
point mutations are frequently found in the coding region of
c-myc both in primary tumors and in cell lines derived from
these lymphomas.4-12 Hot spots for these mutations are
found within or near the evolutionary conserved Myc box 1, the residue
most frequently mutated being Thr58, a mutation also common to 3 avian
viral myc genes and which has been shown to enhance the
transforming activity of both v- and c-Myc proteins.13-17
Thr58 is an in vivo phosphorylation site suggested to be targeted by
glycogen synthase kinase 3 (GSK3).16-18 The phosphorylation
is abolished by mutation of Pro57 and is dependent on prior
phosphorylation of Ser62 by MAP and/or cyclin-dependent kinases,16-20 2 other frequently mutated residues in the
lymphomas (Figure 1). Although it seems clear that phosphorylation of
Thr58 mediates a negative regulatory signal to c-Myc and that mutation of this residue may be involved in lymphoma tumor progression it is
still unclear in what way the mutations affect the activity of c-Myc.
Recent reports have suggested that Myc-family proteins are
degraded by the ubiquitin/proteasome pathway in vivo.21-23 Because proteasome-mediated turnover of several transcription factors
and cell cycle regulatory proteins are modulated by
phosphorylation/dephosphorylation,24,25 we investigated the
possibility that Thr58 phosphorylation may be involved in
ubiquitin/proteasome-mediated degradation of c-Myc.
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| Fig 1.
c-myc missense mutations in lymphomas.
Upper panel, summary of reported missense mutations in the
c-myc coding region in Burkitt's lymphomas, AIDS-related
lymphomas, B-cell acute lymphoblastic leukemias and the 3 avian
v-myc-containing acute transforming retroviruses MC29, OK10,
and MH2, representing 32 primary tumors and 28 cell
lines.4-13 Amino acid positions of the most frequently
mutated residues are indicated. Below is shown the c-Myc structure with
indicated conserved regions. MB1; Myc box 1, MB2; Myc box 2, TAD;
transactivation domain, Acidic; central acidic region, NLS; nuclear
localization signal, BR; basic region, HLH; helix-loop-helix motif,
Zip, leucine zipper motif, P; clusters of in vivo phosphorylation
sites.1 The box at upper right shows the amino acid
sequence of the conserved Myc box 1, including the Thr58 and Ser62
phosphorylation sites. GSK3; glycogen synthase kinase 3, MAPK; mitogen
activated protein kinase, CDK; cyclin-dependent kinase. Asterisk (*)
denotes the positions of frequent missense mutations. Lower panel,
amino acid positions of c- or v-myc missense mutations in cell
lines or constructs used in the study.
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Materials and methods |
Cell culture and transfection
The Burkitt's and BJAB lymphoma cell lines and the U-937-myc-6
monoblasts were cultured in RPMI-1640 medium supplemented with 10%
fetal calf serum and antibiotics. The U2OS osteosarcoma cells were
grown in DMEM containing 10% calf serum. Daudi, Jijoye, Raji, Mutu
1148 and Rael are EBV-positive African Burkitt's lymphoma, CA46 and
ST486 are EBV negative American Burkitt's lymphoma cell lines.7,11,12 BJAB is a non-Burkitt's human B-cell
lymphoma cell line12 and U-937-myc-6 is an OK10
v-myc-containing subclone of the human monoblastic cell line
U-937.26 For transfection of Raji, 2 × 107
cells were mixed with 20 µg of DNA in a 0.4 cm electroporation
cuvette (Biorad), and electroporated at 960 µF, 300 V in a Biorad
gene pulser as described.26 For transfection of U2OS,
subconfluent cells grown on 10-cm cell culture dishes were incubated
with 2 µg of DNA in 6 µL of FuGENE6 (Boehringer Mannheim) per dish.
Cells were harvested 16 hours after transfection. The following DNA
constructs were used: CMV-Myc and CMV-MycT58A were generated by
subcloning EcoRI-fragments containing full-length human c-myc
cDNAs from the plasmids pspMyc0/1 and pspMycC,16 respectively, kindly provided by Drs M. Henriksson and B. Lüscher, into the CMV-driven expression vector pCIneo. The
CMV-MycT58A c-myc gene contains a mutation leading to
substitution of Thr58 to Ala. pcDNA3-FLAG-Myc-wt, kindly provided by Dr
B. Lüscher, contains an N-terminal FLAG-epitope fused in frame
with a full-length human c-myc cDNA in the CMV-driven
expression vector pcDNA3. The CMV-His6-Ub and CMV-HA-Ub
constructs contain His6-and HA-tagged ubiquitin gene
octamers,27 respectively, kindly provided by Dr D. Bohmann.
Pulse chase, immunoprecipitation, and immunoblot analysis
Five × 106 cells were pulsed for 40 minutes in 1 mL of methionine-free RPMI-1640 medium containing 5.55 MBq
of 35S-methionine followed by chase in medium containing 1 mmol/L cold methionine and 10 µg/mL cycloheximide. Cells were lysed
in AB buffer and immunoprecipitated with specific antibodies,
whereafter the samples were analyzed on 10% SDS-PAGE gels as
described.28 The signals were quantitated using a Fuji bas
image analyzer. For studies of turnover of unlabeled proteins,
5 × 106 cells were treated with 100 µg/mL
cycloheximide before harvest, whereafter cell lysates were
immunoprecipitated as above and subjected to immunoblot analysis as
described.28 IG-13 rabbit pan-Myc antiserum26 and biotinylated C-33 monoclonal pan-Myc antibodies (Santa Cruz Biotechnology [SCB]) were used for immunoprecipitation and immunoblot analysis, respectively, of Myc. M2 FLAG antibodies (Sigma) was used to
detect FLAG-epitope tagged c-Myc. For analysis of  -catenin, p27Kip1
and -actin, H102, C19, and H196 antisera (SCB), respectively, were used.
Proteasome inhibitors and purification of ubiquitin
conjugates
The proteasome inhibitors Z-leu-leu-NVA-H aldehyde (MG132),
Z-leu-leu-leu-H aldehyde (MG115) (Peptides International) and N-acetyl-leucinyl-leucinyl-norleucinal-H (LLnL) (Sigma), the lysosome inhibitor chloroquine, the cysteine protease inhibitor
L-trans-epoxysuccinic acid (E64), the acid protease inhibitor pepstatin
A (Boehringer) or dimethyl sulfoxide (DMSO) (Sigma) vehicle were added
to the cells 2 hours before harvest at a concentration of 50 µmol/L
or for pepstatin, 10 µg/mL. For detection of ubiquitin conjugates, U2OS cells from three 10-cm dishes transfected with
CMV-His6-Ub were lysed in 2 mL of 6 mol/L guanidinium-HCl,
0.1 mol/L
Na2HPO4/NaH2PO4 pH 8.0, 5 mmol/L imidazole and sonicated whereafter
His6-Ub-conjugated proteins were purified on a
Ni2+-NTA-agarose (Qiagen) column as
described.27 After washing, eluted proteins were analyzed
by immunoblotting using C33 Myc antiserum (SCB) and monoclonal
ubiquitin antibodies (Zymed).
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Results |
Decreased turnover of c-Myc mutated at Thr58 or Pro57 in Burkitt's
lymphoma cells
The top panel of Figure 1 shows point mutations in the coding region
of c-myc found to date in primary Burkitt's and
AIDS-associated lymphomas and certain acute lymphoblastic leukemias and
in cell lines derived from these tumors.4-12 The most
frequently mutated residue is the in vivo phosphorylation site Thr58
followed by Pro57, which is required for Thr58 phosphorylation, both
located in the evolutionary conserved Myc box 1. c-Myc has a short
half-life of about 30 minutes.29 To investigate whether the
hot spot mutations in c-myc might affect protein turnover, the
Burkitt's lymphoma (BL) cell lines Daudi (wild type [wt]
c-myc), Raji, and CA46 (c-myc mutations, including
Thr58 and Pro57, see Figure 1), were treated with cycloheximide to
block protein synthesis during which the decay of the c-Myc protein was
measured by immunoblot analysis. Figure 2 shows that the
steady state level of c-Myc in Daudi declined rapidly as expected. In
contrast, the mutated c-Myc proteins in Raji and CA46 decayed at a much
slower rate. To ensure that the prolongation of c-Myc half-life in
these cells was not a result of a general impairment of proteasomal
degradation, we measured the stability of -catenin and p27Kip1, 2 other proteasomal substrates.30-32 The
ubiquitin/proteasome-mediated degradation of -catenin is regulated
through phosphorylation by GSK3,31,32 the same kinase suggested to phosphorylate c-Myc at Thr58, suggesting that the degradation of these 2 proteins potentially may occur through the same
pathway. The half-life of -catenin seemed to be less than 1 hour in
agreement with previous reports31,32 (Figure
2, middle panel). No major differences in its rate of degradation were
observed between the 3 cell lines, thus excluding a general defect in
ubiquitin/proteasome-mediated turnover in the BL cells. The
cyclin-dependent kinase inhibitor p27Kip1 turns over at a slower
rate33 and was essentially stable in all 3 cell lines
during this time frame (Figure 2, lower panel).
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| Fig 2.
Stabilization of endogenous mutated c-Myc proteins in
Burkitt's lymphoma cells.
Immunoblot analysis of c-Myc (upper panel), -catenin (middle panel)
and p27Kip1 (lower panel) after cycloheximide (CHX) treatment of Daudi,
Raji, and CA46 Burkitt's lymphoma cells for the indicated time points
was performed using specific antibodies as described in "Materials
and Methods."
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To measure c-Myc turnover more precisely, we performed
35S-methionine pulse chase experiments using the lymphoma
cell lines listed in Figure 1. Immunoprecipitation of c-Myc, followed
by SDS-PAGE analysis showed an expected short half-life of c-Myc in
Daudi and BJAB cells (both wt c-myc, Figure
3), estimated to be 35 and 25 minutes,
respectively, by image analysis (see also Figure 5A). In
contrast, the mutated c-Myc in Raji cells exhibited a significantly
slower turnover rate (half-life of approximately 105 minutes). Further,
c-Myc was at least as stabilized in the endemic Epstein-Barr virus
(EBV)-positive BL cell lines Mutu and Rael, both with a single mutation
of Thr58, and in the sporadic EBV-negative BL cell lines CA46 and ST486
with common mutations in Pro57 (Figures 1, 3, and 5A). In Jijoye BL
cells with wt c-myc, the turnover rate of c-Myc was normal. We
concluded from these experiments that c-Myc was stabilized in the 5 BL
cell lines that have in common the 2 most frequent c-myc
missense mutations in lymphomas, affecting amino acid positions 58 or
57, both resulting in abolishment of Thr58 phosphorylation.
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| Fig 3.
Pulse chase analysis of c-Myc turnover in Burkitt's and
BJAB lymphoma cells.
Cells were pulse labeled with 35S-methionine followed by a
chase in medium containing cold methionine for indicated time points.
Cell lysates were immunoprecipitated with pan-Myc antiserum and
analyzed by SDS-PAGE as described in "Materials and Methods."
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Ectopically expressed wt c-Myc is degraded at a normal rate in
Burkitt's lymphoma cells with mutated endogenous c-Myc
To provide further evidence that the stabilization of c-Myc in BL
cells is due to the mutations in c-myc rather than a result of
other oncogenic events or cell-type specific factors in these cells, we
transfected Raji cells with an expression vector containing a
FLAG-epitope tagged wt c-myc gene. The turnover of the
transfected wt c-Myc protein as well as of the endogenous mutated c-Myc
protein was determined by CHX treatment, followed by sequential
immunoprecipitation of cell lysates by  -FLAG and -Myc sera and
immunoblot analysis of the immunoprecipitates using Myc antiserum.
Figure 4A shows that the FLAG-wt c-Myc
protein turned over at the expected normal rate, whereas the half-life
of the endogenous mutated protein was prolonged as shown previously. We
therefore conclude that the stabilization of c-Myc observed in the BL
cells is indeed a result of the mutations in c-myc. Whether in
addition also other genetic alterations are required for the
stabilization of mutated c-Myc in these cells remains an open question
(see also "Discussion").
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| Fig 4.
Turnover of ectopically expressed Myc proteins in
Burkitt's and non-Burkitt's cells.
(A), Stability of FLAG-epitope tagged wild type (wt) c-Myc in Raji
Burkitt's lymphoma cells. 5 µg of pcDNA3-FLAG-Myc-wt (FLAG-wt c-Myc)
or pcDNA3 (Mock) were electroporated into 2 × 107
Raji cells together with 15 µg of carrier DNA. 16 hours after
electroporation aliquots of cells were treated with CHX for the
indicated times or left untreated. FLAG-tagged wt c-Myc was
immunoprecipitated from the cell extracts with M2 FLAG antibodies,
followed by immunoprecipitation of the endogenous mutated c-Myc protein
from the same extracts with IG-13 -Myc serum. The immunoprecipitated
material was then subjected to immunoblot analysis using C33 Myc
antibodies. Note that the exposure time of the film presented at the
left -FLAG panel is longer for than for the right -Myc panel. (B,
C), pulse chase analysis of v- and c-Myc turnover in U-937 monoblasts
stably expressing the OK10 v-Myc protein (B) and of wt c-Myc and the
T58A mutant after transient transfections of U2OS osteosarcoma cells
(C). The pulse chase was performed at the indicated time points as
described in the legend to Figure 3. In (C), semiconfluent U2OS
osteosarcoma cells were transfected with 2 µg of CMV-driven
c-myc per 10-cm dish. The pulse chase was performed 16 hours
after transfection.
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Stabilization of ectopically expressed v- and c-Myc with mutated
Thr58
To investigate whether Myc proteins mutated at amino acid position
58 were stabilized also in other cells than BL, we used human
U-937-myc-6 monocytic cells containing the avian OK10 virus v-myc gene,28 which is mutated at Thr58 and at 1 C-terminal residue not found in other v-myc genes or in tumors
(Figure 1, lower panel). Pulse chase experiments showed that the OK10
v-Myc protein, which migrates slightly faster than the human protein, exhibited a prolonged half-life similar to that of the mutated c-Myc
proteins in BL (Figures 4B and 5B), whereas the endogenous c-Myc
protein was degraded at a normal rate (half-lives 120 and 40 minutes,
respectively). Unlike v-Myc, normal chicken c-Myc was not stabilized
when transiently transfected into human HeLa cervical carcinoma or U2OS
osteosarcoma cells (data not shown).
To provide further evidence for a general role of Thr58 in c-Myc
turnover, we transiently transfected U2OS cells with full-length c-myc cDNA-constructs with a single mutation substituting Thr58 for Ala (T58A) or with the wt sequence. Pulse chase experiments showed
that the T58A mutant was stabilized (estimated half-life 80 minutes),
whereas the wt c-Myc protein turned over at a normal rate (half-life 30 minutes) (Figures 4C and 5C). Quantitations of some of the Myc pulse chase experiments in Figures 3 and 4 are
summarized in Figure 5. We conclude from these results that Myc
proteins with common mutations at Thr58 or Pro57 in BL cells have
significantly slower turnover rates than the wt proteins and that Thr58
mutants have increased half-lives also in 2 other cell types.
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| Fig 5.
Quantitation of c- and v-Myc turnover in Burkitt's and
non-Burkitt's cells.
(A), Burkitt's lymphoma cells lines, (B) U-937-myc-6 cells, (C)
transiently transfected U2OS cells. The results shown in Figures 3 and
4B, C were quantitated by image analysis. The cell lines and the status
of their respective endogenous c-Myc proteins or constructs used for
transfection are indicated in the figure. The quantitation is presented
in a logarithmic scale as percentage of remaining labeled protein
during the times of chase indicated.
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Decreased proteasome-mediated turnover and inefficient
ubiquitination of mutated Myc proteins
c- and N-Myc, like many other transcription factors and cell cycle
regulatory molecules, are degraded in vitro by the ubiquitin-proteasome pathway,34 recently shown also in vivo.21-23 We
therefore investigated whether inhibitors of the proteasome affected
the turnover of Thr58 and Pro57 Myc mutants. Immunoblot analysis of v-
and c-Myc in U-937-myc-6 cells showed that c-Myc decayed rapidly after
blocking protein synthesis with cycloheximide, whereas a substantial
amount of v-Myc was still present (Figure
6A, upper panel) in agreement with the
pulse chase experiments in Figure 4B. Addition of the proteasome
inhibitors MG115, MG132, or LLnL together with cycloheximide stabilized
c-Myc, whereas the lysosome inhibitor chloroquine (CQ), the cysteine
protease inhibitor E64, the acid protease inhibitor pepstatin, or the
vehicle (DMSO) had little effect, thus confirming the involvement of
the proteasome in Myc turnover. A faint smear of high molecular weight
bands was observed in cells treated with proteasome inhibitors, which
might represent polyubiquitinated forms of c-Myc. Although v-Myc was
more stable than c-Myc, the addition of proteasome inhibitors lead to
some further accumulation of v-Myc. Again chloroquine, E64 and
pepstatin had little effect. Immunoblot analysis of -actin (Figure
6A, lower panel) confirmed that equal amounts of protein were applied
to the gel.
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| Fig 6.
Retarded proteasome-mediated turnover and inefficient
ubiquitination of Myc mutants.
(A) Immunoblot analysis of v- and c-Myc (upper panel) and -actin
(lower panel) in U-937-myc-6 cells after 2 hours treatment with the
proteasome inhibitors MG115, MG132, and LLnL, the protease inhibitors
chloroquine (CQ), E64, and pepstatin or DMSO vehicle in the presence of
cycloheximide (CHX) as indicated. (B) Pulse chase analysis of c-Myc
turnover in the presence of the proteasome inhibitor MG115. Daudi (wt
c-Myc) and CA46 (mutated c-Myc) Burkitt's lymphoma cells were treated
with the proteasome inhibitor for 1.5 hours, pulsed with
35S methionine followed by chase in the presence of MG115
for the indicated time points or were pulse labeled in the absence of
the inhibitor. Cell lysates were then immunoprecipitated with pan-Myc
antibodies and analyzed as in Figure 3. (C) Reduced ubiquitination of
the T58A c-Myc mutant. U2OS cells were transfected with wt c-Myc, the
T58A c-Myc mutant, His6-Ub or HA-Ub vectors alone or in
combination as indicated in the figure. The transfected cells were
treated with MG115 during the last 2 hours before harvest.
His6-Ub-conjugated proteins were purified as described in
"Materials and Methods" and subjected to Western blot analysis
using Myc antibodies (upper panel) or dot blot analysis using ubiquitin
antibodies (lower panel). The smears of
c-Myc-His6-Ub-conjugates are indicated. The middle panel
shows c-Myc immunoblot analysis of 1/10 of the input material (note
that the exposure time for this blot is shorter than for the upper
panel).
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We next investigated the effects of proteasome inhibitors on c-Myc
stability in BL cells. Pulse chase experiments revealed that wt c-Myc
in Daudi was unstable with a half-life of 30 minutes, whereas mutated
c-Myc in CA46 turned over slowly with a half-life of 95 minutes (Figure
6B). The addition of MG132 stabilized wt c-Myc in Daudi significantly,
although only a small effect was seen in CA46 cells. In both cases the
increased half-life was in the order of 125 minutes. Together these
results suggested that Myc proteins with mutations in amino acid
positions 58 or 57 have an impaired proteasome-mediated turnover but
are still degraded by this pathway albeit at a slower rate.
Because degradation by the 26S proteasome is dependent on covalent
ubiquitination of target proteins by ubiquitin (Ub)-conjugating enzymes
(for review see references 24 and 25), we investigated whether the
retarded proteasome-mediated turnover of mutated Myc proteins was
correlated with decreased ubiquitination. Ubiquitinated c-Myc proteins
are very difficult to detect due to their rapid turnover. To visualize
these intermediates, U2OS cells were transiently transfected with wt or
T58A mutant c-Myc together with an expression vector containing
histidine-tagged ubiquitin (His6-Ub) octamers27 and treated with the proteasome inhibitor MG115 before harvest. Proteins conjugated with His6-Ub were purified over a
Ni-agarose column and the presence of His6-Ub-conjugated
c-Myc was analyzed by immunoblotting. Figure 6C shows that a high
molecular weight smear indicative of polyubiquitinated proteins was
detected by c-Myc antibodies in cells transfected with wt c-Myc
together with His6-Ub, but not in cells transfected with
c-Myc plus hemagglutinin-tagged ubiquitin (HA-Ub), with c-Myc alone or
with His6-Ub or HA-Ub alone. We therefore conclude that
c-Myc became ubiquitinated in vivo in agreement with recent
reports.21-23 The c-Myc band migrating at its normal
molecular weight represents unmodified c-Myc protein binding
unspecifically to the column, since it was present also in lanes
lacking His6-Ub. In contrast to the prominent smear of polyubiquitinated wt c-Myc protein, significantly less
His6-Ub-c-Myc was detected in cells transfected with the
T58A c-Myc mutant together with His6-Ub (Figure 6C).
Immunoblot analysis of ubiquitin showed that approximately equal
amounts of total ubiquitinated proteins from His6-Ub
transfected cells were eluted from the column (Figure 6C, lower panel).
The middle panel of Figure 6C shows that approximately equal amounts of
transfected c-Myc were applied to the column. We conclude from these
results that mutation of amino acid position 58 results in inefficient
ubiquitination of c-Myc.
| |
Discussion |
In summary, our results show that endogenous c-Myc proteins with
mutated Thr58, with or without additional mutations, or with Pro57
mutation as the common denominator display substantially decreased
proteasome-mediated turnover in Burkitt's lymphoma cells. Because
phosphorylation of Thr58 is dependent on an intact Pro57,17 this mutation probably has the same consequence as mutation of Thr58.
Our experiments further show that transfected wt c-Myc turns over at a
normal rate in BL cells containing mutated endogenous c-Myc and that
the half-lives of other proteasomal substrates are not altered. This
rules out a general impairment of the ubiquitin/proteasome-mediated degradation or other genetic alterations as the main explanation for
the c-Myc stabilization in these cells. Ectopically expressed v- and
c-Myc with Thr58 mutations are stabilized also in 2 other cell types,
in monoblastic tumor cells and osteosarcoma cells, respectively, which
is in agreement with recent observations in U2OS cells,23
published during the preparation of this manuscript. We further show
that the decreased turnover rate of the Thr58 mutant protein correlates
with inefficient ubiquitination, thus providing a rationale for the stabilization.
Our results suggest that phosphorylation of Thr58 triggers
ubiquitin/proteasome-mediated turnover. Escape from this regulation may
be sufficient to explain the increased transforming activity of Thr58
mutants 14-17 and their insensitivity to negative
regulation by the retinoblastoma-family protein p107.20,35
p107-associated cyclin/CDK complexes are suggested to phosphorylate
Ser62, another mutation in some lymphomas. This phosphorylation seems
to be a prerequisite for subsequent phosphorylation of
Thr58.16,18 Because incoming growth stimulatory or
inhibitory signals may affect phosphorylation of both these
phosphorylation sites, one can envisage a quite complex regulation of
Myc function in this region, which potentially could be influenced by
other oncogenic events. In agreement with this notion, a recent report
suggests that oncogenes like ras may affect c-Myc
stability.36 Mutation of Thr58 does, however, not
completely abolish ubiquitination and proteasome inhibitors somewhat
further stabilize the protein, indicating that there may be other
signals for ubiquitination of c-Myc. Recent studies of c-Myc
degradation in yeast37 and in HeLa cells23
indicate that there may be several independent signals for
proteasome-mediated turnover in the N-terminus. Such additional signals
in combination with differences in genetic and cellular backgrounds
might account for the apparent slight variations in the half-lives of
both wt and mutant c-Myc proteins in different cells (Figures 2 to 5).
Further studies are required to define these possible alternative
degradation motifs and how they are related to Thr58 and other
occurring mutations in c-Myc. Although other regions of c-Myc may also
participate in the ubiquitination and degradation of c-Myc our results
clearly suggest that Thr58 phosphorylation/dephosphorylation is a main
regulator of this process.
Recent studies suggest that phosphorylation is a frequently used
mechanism for controlling the degradation of cell cycle and growth
regulatory proteins through the ubiquitin/proteasome
pathway.24,25 It is intriguing that several viral forms of
oncoproteins, such as v-Jun and v-Fos like shown here for v-Myc, escape
degradation by the proteasome due to mutated destruction motifs
regulated by phosphorylation.27,38 Our results suggest that
development of Burkitt's and other lymphomas involves activation of
c-Myc by 2 sequential steps. First, the deregulation of expression of the c-myc gene through translocation to the immunoglobulin loci and, second, the mutation of a regulatory phosphorylation site leading
to escape from ubiquitin/proteasome-mediated turnover and accumulation
of c-Myc protein.
| |
Acknowledgments |
We thank Dr George Klein for providing Burkitt's lymphoma cell lines
and Drs Dirk Bohmann, Marie Henriksson, and Bernhard Lüscher for
plasmid constructs. We are also thankful to Drs Göran Akusjärvi, Kenneth Nilsson, Bernhard Lüscher, Fredrik
Öberg, Catharina Svensson, and Hans Ronne for valuable
discussions and criticism.
| |
Footnotes |
Submitted June 16, 1999; accepted November 15, 1999.
Supported by grants from the Swedish Cancer Society, the Children
Cancer Foundation of Sweden and the Lovisa & Thielman Foundation.
Reprints: Lars-Gunnar Larsson, Uppsala Genetic Center,
Department of Plant Biology, Swedish University of Agricultural Sciences, Box 7080, 750 07 Sweden; e-mail:
lars-gunnar.larsson{at}vbiol.slu.se.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
| |
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Abstract
Introduction