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Oncogene (1999) 18, 7552 ± 7558
1999 Stockton Press All rights reserved 0950 ± 9232/99 $15.00
http://www.stockton-press.co.uk/onc
Protein stabilization: a common consequence of mutations in independently
derived v-Myc alleles
Paul R Gavine1, James C Neil2 and Dorothy H Crouch*,1
1
Biomedical Research Centre, University of Dundee, Ninewells Hospital and Medical School, Dundee DD1 9SY, UK; 2Department
of Veterinary Pathology, University of Glasgow Veterinary School, Bearsden, Glasgow G61 1QH, UK
Myc is overexpressed in many cancers as a result of gene
rearrangement or ampli®cation, but coding sequence
changes which cluster in the N-terminal transactivation
domain also appear to play a role in tumour progression.
The prototypic v-Myc gene of MC29 virus di€ers from
avian c-Myc by a series of mutations, including a change
at a regulatory phosphorylation site within the mutational hotspot (thr-61) which is known to potentiate
transformation in vitro. We now show that the mutation
at thr-61 stabilizes the v-Myc protein (turnover
di€erence) and that this single mutation is both necessary
and sucient for the phenotype. A major involvement of
the proteasome in Myc degradation was con®rmed, but
surprisingly, a dilysine motif adjacent to thr-61 proved
not to be the ubiquitin target. Two other v-Myc genes
which carry a mutation at thr-61 (avian MH2) or a large
deletion encompassing this domain (feline T17) were
found to be stabilized to a similar extent as MC29,
showing that stabilization is a common feature of
independently derived Myc oncogenes. These results
suggest a common selective process in the genesis of
these three viral oncoproteins and a mechanistic link with
Jun, Fos and Myb oncoproteins which are also stabilized
relative to their cellular counterparts.
Keywords: Myc; viral mutations; stabilization
Introduction
Myc, originally identi®ed as the transforming component of avian leukosis virus, MC29, functions as a
sequence speci®c nuclear transcription factor, working
in association with its obligate partner, Max. Both
dimerization with Max through the C-terminal helix ±
loop ± helix/leucine zipper (HLH-LZ) domain and
sequence-speci®c DNA binding mediated by the
adjacent basic region (B) are prerequisites for Myc to
function, and the resulting Myc : Max complex
transactivates target genes through an N-terminal
transactivation domain in Myc (Henriksson and
Luscher, 1996).
myc expression is strictly controlled in the cell
ensuring regulated growth through the correct expression of its downstream target genes. Escape from this
control through retroviral transduction, chromosome
ampli®cation or retroviral insertion, however, results in
elevated levels of Myc, aberrant gene expression and
*Correspondence: DH Crouch
Received 26 April 1999; revised 21 July 1999; accepted 21 July 1999
deregulated growth. Mutational changes in Myc, either
somatic or virally-derived, can also contribute signi®cantly to the development of the neoplastic
phenotype, and intriguingly, these are clustered in a
hotspot region in the amino terminal transactivation
domain (Henriksson and Luscher, 1996; Hoang et al.,
1995; Smith-Sorensen et al., 1996).
As a consequence of oncogenic activation, the
normal signalling mechanisms which regulate cellular
protooncogenes are subverted. Unravelling the molecular consequences of these activating events will
therefore contribute both to our understanding of
how cellular proteins are controlled by normal cell
signalling and how viral oncoproteins escape this.
Examples of activating mutations are deletion of key
regulatory regions (d domain in Jun (Gillespie, 1991))
or mutation of speci®c phosphorylation sites (Myc, Fos
(Curran et al., 1994; Henriksson et al., 1993; Okazaki
and Sagata, 1995; Pulverer et al., 1994)).
A common feature of c-Myc and other nuclear
transcription factors (Jun, Fos) is their short half life,
which ensures the maintenance of low steady state
levels required for controlled growth. Degradation of
these, and other cell cycle regulators and tumour
suppressors, is mediated by the 26S proteasome
(Ciechanover, 1998; Gross-Mesilaty et al., 1998; Musti
et al., 1997), following conjugation of ubiquitin to
lysine residues of the target protein (Ciechanover,
1998).
Protein stabilization as a result of disruption in
proteasome-directed degradation of key regulatory
proteins may represent a potential mechanism of
oncogenic activation since inappropriate levels of
these proteins in the cell would result. Indeed, some
key nuclear transcription factors are stabilized as a
consequence of oncogenic activation. Both viral Fos
and the leukaemia-speci®c form of c-Myb are more
stable than their cellular counterparts due to carboxy
terminal deletions (Bies and Wol€, 1997; Curran et al.,
1994). v-Jun, however, is stabilized through a deletion
termed the d domain in the N-terminus, which
abrogates ubiquitin dependent degradation (Treier et
al., 1994).
We set out to establish whether the prototypic vMyc from the avian MC29 virus is stable. We now
report that MC29 v-Myc is considerably more stable
than c-Myc as a consequence of a single point
mutation in a major phosphorylation site, and
propose that loss of phosphorylation at this site
stabilizes the Myc protein. Furthermore, we show
that two independently derived v-Myc isolates with
analogous mutations at thr-61 (avian virus MH2) or a
deletion spanning this domain (feline virus T17) are
Stabilization of viral Myc proteins
PR Gavine et al
also stable. Finally, we propose that protein stabilization is a common mechanism of oncogenic activation
of nuclear transcription factors such as Myc, Fos, Jun
and Myb (Bies and Wol€, 1997; Curran et al., 1994;
Isaksson et al., 1996; Okazaki and Sagata, 1995).
Results
MC29 v-Myc oncoprotein is stable
To address whether the MC29 v-Myc oncoprotein is
more stable than c-Myc, we generated avian retroviral
vectors which encode either v-myc sequences derived
from the avian leukosis virus, MC29 (Walter et al.,
1986) or avian c-Myc (La Rocca et al., 1994). The
location of the MC29 v-Myc point mutations with
respect to the structure of c-Myc is outlined in Figure
1a. MC29 v-Myc di€ers from c-Myc by ®ve point
mutations, which are scattered throughout the protein
(Walter et al., 1986). Signi®cantly, thr-61, found within
the highly conserved N-terminal Myc Box 1 (MBI:
amino acids 45 ± 63), is the most commonly mutated
amino acid in tumour-derived Myc genes (Henriksson
and Luscher, 1996; Hoang et al., 1995; Smith-Sorenson
et al., 1996).
The constructs were transfected into chick embryo
®broblasts (CEF) and subjected to G418 selection. To
measure protein turnover, the resulting cultures were
treated with the protein synthesis inhibitor, emetine,
for the appropriate lengths of time, and Western blots
used to analyse Myc degradation in the total cellular
pool of Myc. This was felt to preclude di€erential
labelling of c-Myc and v-Myc in pulse-chase experiments due to di€erences in half life and methionine
content of the mutants. Equal amounts of protein
lysates were analysed by Western blot, and Myc
proteins visualized using an anti-myc antibody (Hann
Figure 1 MC29 v-Myc is more stable than c-Myc. (a) Schematic
representation of MC29 v-Myc-derived point mutations relative
to c-Myc. The domain structure of Myc shows an N-terminal
transactivation domain and C-terminal basic (B), helix ± loop ±
helix (HLH) and leucine zipper (LZ) domains. Thr-61, the most
mutated residue in tumour-derived Myc genes (Henricksson and
Luscher, 1996; Hoang et al., 1995; Smith-Sorensen et al., 1996) is
highlighted. Each construct was cloned into the avian retroviral
expression vector, SFCV-sa7 (La Rocca et al., 1994) and
transfected into chick embryo ®broblasts (CEF). (b) The
turnover of avian MC29 v-Myc and c-Myc proteins in CEF
was detected by Western blot of total cell lysates using an antimyc antibody following treatment with the protein synthesis
inhibitor, emetine, for the indicated periods of time
et al., 1983). From Figure 1b, it can clearly be seen
that MC29 v-Myc is considerably more stable than cMyc, suggesting that the virally-derived point mutations stabilize the v-Myc protein. Densitometric
scanning reveals that the half life of MC29 v-Myc
protein is extended to 61 min, compared to 20 min for
c-Myc.
The Ras/Raf/MAPK pathway has been shown to
result in stabilization of Myc (Sears et al., 1999).
Therefore, to exclude any emetine-mediated e€ects on
MAP kinase signalling, we showed that no alterations
in MAPK activity occurred during emetine treatment
using an anti-phospho-p44/p42 MAPK antibody (New
England Biolabs) which detects the active p44/p42
MAPK. In addition, in combination with the use of the
speci®c MAPK inhibitor, U0126 (Promega), we
conclude that the stabilization observed in v-Myc was
not due to emetine-induced activation of MAPK which
speci®cally targeted v-Myc (data not shown).
Threonine 61 regulates Myc degradation
To focus on the roles of individual point mutations in
MC29 v-Myc, we generated each point mutation in
isolation (Figure 2a). The resulting constructs were
transfected into CEF and analysed for the stability of
the Myc proteins following emetine treatment. The
thr-61 to met-61 mutation (c-MycT61M) is sucient to
stabilize the protein to levels seen in v-Myc (Figure
2b). To discover whether the other mutations
contribute to altered Myc turnover, we generated a
construct, v-MycM61T, which contained, in addition to
the four other virally-derived point mutations, a
backmutation of met-61 to thr-61 (Figure 3a). These
results clearly show that the thr-61 to met-61 mutation
is sucient to stabilize the c-Myc protein, whilst the
met-61 to thr-61 mutation destabilizes the v-Myc
protein, even in the presence of the other viral
mutations (Figure 3b).
As has previously been reported (Ciechanover, 1998;
Gross-Mesilaty et al., 1998; Salghetti et al., 1999), we
con®rm a major involvement of the proteasome in Myc
degradation in the avian system using the speci®c
proteasome inhibitor, lactacystin (Fenteany et al., 1995;
Gross-Mesilaty et al., 1998). Treatment with lactacystin
resulted in a clear stabilization of both v-Myc and cMyc proteins (Figure 4). Use of the less speci®c
proteasome inhibitor, ALLnL (Krappmann et al.,
1996) also resulted in stabilization of the Myc
oncoproteins (data not shown).
Mutation of thr-61 does not completely prevent
proteasomal degradation of v-Myc, which could be
interpreted in a number of ways. Firstly, it is likely that
the thr-61 to met-61 mutation a€ects the rate of
proteasomal degradation of v-Myc resulting in reduced
degradation of v-Myc, but not its complete stabilization. Alternatively, we cannot rule out a nonproteasomal mechanism of degradation, although this
has not previously been reported for Myc.
Proteasome function is una€ected by Myc
transformation
Oncogenes can interfere with the activity of the
ubiquitin-proteasome pathway (Nakajima et al.,
1998), and it has been suggested that Myc may
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Stabilization of viral Myc proteins
PR Gavine et al
7554
a€ect proteasome function (Henriksson and Luscher,
1996). Therefore, transformation by v-Myc, but not cMyc, may have a general e€ect on proteasome
function. To address this possibility, we investigated
the turnover of a well characterized proteasomedegraded protein, c-Jun (Musti et al., 1997; Treier et
al., 1994). Our results show that c-Jun is unstable in
both c-Myc- and v-Myc-transformed CEF (Figure 5),
suggesting that transformation by v-Myc does not
a€ect proteasome function.
A dilysine motif within Myc Box I is not the ubiquitin
target
Proteasomal degradation of IkB results when TNF-adirected phosphorylation signals ubiquitination of an
Figure 2 The single MC29 mutation, Thr-61 to Met-61, stabilizes c-Myc. (a) Point mutations of MC29 v-Myc and comparison
with avian c-Myc. Each MC29 v-Myc point mutation was generated in isolation, cloned into the avian retroviral vector, SFCV-sa7
for turnover studies in CEF. (b) The e€ect of each MC29 point mutation on the turnover of the Myc protein was performed by
Western blot analysis as outlined in Figure 1b
Stabilization of viral Myc proteins
PR Gavine et al
7555
Figure 5 Proteasome function is una€ected by Myc transformation. The turnover of c-Jun, another proteasome-degraded nuclear
oncoprotein, in v-Myc and c-Myc-transformed CEF was shown
by Western blot using an anti-jun antibody
B.
Figure 3 Back mutation of Met-61 to Thr-61 in v-Myc is
sucient to destabilize MC29 v-Myc. (a) Myc constructs were
generated which contained the mutations, thr-61 to met-61
(T61M) and met-61 to thr-61 (M61T) in a c-Myc and v-Myc
background respectively. (b) The e€ect of thr-61 to met-61 (T61M)
and met-61 to thr-61 (M61T) mutation on the turnover of c-Myc
and v-Myc respectively was shown by Western blot of total cell
lysates as outlined in Figure 1b. Densitometric scanning shows
that T61M mutation results in a 1.7 fold stabilization (half lives of
c-Myc and c-Myc (T61M) are 14 and 24 min respectively). The
M61T mutation results in a 2.2 fold destabilization (half lives of vMyc and v-Myc (M61T) are 47 and 20.7 min respectively)
Figure 6 A dilysine motif within MBI is not the ubiquitin target
in Myc. (a) Myc Box I sequence surrounding the phosphorylation
sites, ser-65 and thr-61, which are subject to hierarchical
phosphorylation by MAPK and GSK-3a respectively (Lutterbach and Hann, 1994). A dilysine motif, KK54,55, adjacent to
these phosphorylation sites was mutated to diarginine residues,
RR54,55 (Rodriguez et al., 1996) to determine the e€ect on Myc
turnover. (b) The e€ect of the KK54,55RR54,55 mutation on the
turnover of c-Myc was shown by Western blot of total cell lysates
as outlined in Figure 1b
adjacent dilysine motif (Rodriguez et al., 1996). The
arrangement of a dilysine motif with respect to known
phosphorylation sites in IkB is similar to that in the Nterminus of Myc (Rodriguez et al., 1996) (Figure 6a).
In view of this similarity, we asked whether the two
lysine residues adjacent to the known MBI phosphorylation sites, thr-61 and ser-65 (Figure 6a) (Lutterbach
and Hann, 1994) are ubiquitin targets in Myc. We
generated a mutant, Myc (KK54,55RR54,55,) in which the
dilysine motif, KK54,55, was mutated to a diarginine
motif, RR54,55 (Rodriguez et al., 1996). The half life of
Myc (KK54,55RR54,55,) in CEF was determined and
Figure 6b shows that these lysines are not ubiquitin
targets, since this mutation does not stabilize the
protein. Indeed, the protein appears more unstable
than c-Myc, which was con®rmed by analysis of earlier
time points (data not shown).
Two independently isolated v-Myc alleles, avian MH2
and feline T17, are also stable
Figure 4 A major component of Myc in chick embryo
®broblasts is degraded by the proteasome. The speci®c
proteasome inhibitor, lactacystin, was added to v-Myc and cMyc-transfected CEF prior to emetine treatment to con®rm
proteasome-directed degradation of Myc. Stabilized Myc proteins
were detected by Western blot as described in Figure 1b
To establish whether stabilization is a common feature
of independently derived v-Myc alleles, we generated
avian retroviral vectors encoding the v-myc sequences
derived from the avian leukosis virus, MH2 (Walter et
al., 1986) and feline leukaemia virus T17 (Fulton et al.,
Stabilization of viral Myc proteins
PR Gavine et al
7556
Figure 7 Two independently isolated v-Myc isolates, avian MH2
and feline T17, are more stable than c-Myc. (a) Schematic
representation of avian MH2 and feline T17 v-Myc relative to cMyc. The T17 v-Myc mutations are localized as indicated (Fulton
et al., 1996), however, due to the extensive nature of the
mutations in MH2 v-Myc (Walter et al., 1986), these are not
accurately represented. Thr-61, the only mutation in common
between MC29 v-Myc (Figure 1a) and MH2 v-Myc, is also
deleted in T17 v-Myc, is highlighted. Each viral construct was
cloned into the avian retroviral expression vector, SFCV-sa7 (La
Rocca et al., 1994) and transfected into chick embryo ®broblasts
(CEF). (b) The turnover of MH2 and T17 v-Myc proteins in CEF
detected by Western blot of total cell lysates as described in
Figure 1b. Chimaeras between T17 v-Myc and feline c-Myc
con®rm that the T17 deletion is solely responsible for the
stabilization (data not shown)
1996) (Figure 7a). In addition to extensive point
mutations, MH2 has a small deletion. T17 v-Myc,
however, contains an extensive 74 amino acid deletion
in the transactivation domain, together with two point
mutations in the C-terminus (Fulton et al., 1996).
Signi®cantly, the T17 deletion spans the mutational
hotspot in Myc and the highly conserved Myc Box I
(MBI: amino acids 45 ± 63).
Analysis of MH2 and T17 v-Myc degradation show
both viral products are more stable than their cellular
counterparts (Figure 7b), leading us to conclude that
stabilization is a common occurrence in viral Myc
isolates. Using chimaeras between T17 v-Myc and cMyc, we con®rmed that the T17 deletion was solely
responsible for stabilizing the protein (data not shown).
In addition, the common mutation at thr-61 (MC29
and MH2 v-Myc) or deletion spanning this domain
(T17 v-Myc) suggests a common mechanistic basis for
the stabilization.
Discussion
Three independent viral isolates of Myc (two avian and
one feline) encode proteins which are considerably
Figure 8 Mutations in MBI regulate Myc degradation. (a)
Hierarchical phosphorylation of the Myc Box I (MBI ± amino
acids 45 ± 63) amino acids, ser-65 and thr-61 by MAPK and GSK3a (Lutterbach and Hann, 1994) respectively regulate Myc
degradation. MBI is depicted by the hatched area. The
proteasome inhibitor, lactacystin, signi®cantly stabilizes Myc in
the avian system, which is consistent with current evidence
suggesting Myc is degraded through the proteasome (Ciechanover, 1998; Gross-Mesilaty et al., 1998). (b) MC29 and MH2 vMyc mutations within MBI, and speci®cally the GSK-3a
phosphorylation site, thr-61, reduce Myc degradation. Thr-61 is
clearly pivotal in controlling Myc degradation, but on the basis of
current available data, a role for phosphorylation on ser-65
cannot be discounted. Unlike IkB degradation, ubiquitination of
Myc does not occur through a dilysine motif upstream of key
regulatory phosphorylation sites. (c) Deletion of MBI residues
49 ± 124 in T17 v-Myc also results in reduced degradation. This
may be analogous to the d domain of c-Jun, which serves as a
recognition site for the ubiquitination machinery. The d domain
deletion in ASV17 v-Jun therefore abrogates ubiquitin-dependent
degradation of c-Jun (Isaksson et al., 1996)
more stable than c-Myc, suggesting that reduced
degradation may be a common mechanism for
enhancing the oncogenicity of the Myc protein.
Indeed, stabilization may be a common mechanism
for all nuclear transcription factor oncoproteins, since
activated forms of Jun, Fos and Myb are also more
stable than their cellular counterparts (Bies and Wol€,
1997; Curran et al., 1994; Okazaki and Sagata, 1995;
Treier et al., 1994). Posttranscriptional mechanisms
which result in reduced degradation may therefore
contribute to the development of the neoplastic
phenotype.
Loss of thr-61, a major phosphorylation site, is a
key feature of three independently derived viral Myc
isolates suggesting a common selective process, and
implicating MBI as a major determinant in degradation of Myc (Salghetti et al., 1999). Mutation at thr-61
also potentiates Myc transformation in vitro, suggesting a correlation between stabilization and transforming function (Henriksson et al., 1993; Pulverer et al.,
1994). In chick cells, the appearance of colonies in soft
agar produced by Myc(T61M) more closely resembles vMyc than c-Myc (data not shown), further supporting
the hypothesis that stabilization potentiates Myc
transformation. However, the role of the other MC29
point mutations in regulating Myc transforming
function cannot be discounted, particularly since a
CKII phosphorylation site is also mutated in MC29 vMyc (ser-325 to leu-325) (Luscher et al., 1989; Walter
Stabilization of viral Myc proteins
PR Gavine et al
et al., 1986). In the case of T17 v-Myc, the MBI
deletion is responsible for stabilizing T17 v-Myc in
both ®broblasts and T cells (Fulton et al., 1996), but
the protein is inactive for in vitro transformation and
apoptosis in ®broblasts, while retaining the potential to
induce T cell lymphomas in vivo. These results suggest
that Myc has pleiotropic e€ects, which contribute
di€erentially to transformation of di€erent cell types.
Ser-65 and thr-61 are subject to hierarchical
phosphorylation by MAPK and GSK-3a respectively
(Lutterbach and Hann, 1994). However, since an
increase in phosphorylation at ser-65 is detected when
thr-61 is mutated (Lutterbach and Hann, 1994), the
regulatory mechanism for Myc stabilization could
either be through loss of thr-61 phosphorylation or
an increase in ser-65 phosphorylation. Myc degradation would, therefore, be regulated by the balance
between these two regulatory kinases in the cell. In the
absence of GSK-3a, ser-65 phosphorylation by MAPK
may result in Myc stabilization (Sears et al., 1999), and
subsequent thr-61 phosphorylation by GSK-3a will
destabilize Myc (Figure 8a). However, as a consequence of altered thr-61 phosphorylation, viral Myc
mutants such as MC29 and MH2 v-Myc would be
stabilized due to reduced degradation (Figure 8b). The
T17 v-Myc deletion, however, removes MBI and the
N-terminal regulatory phosphorylation sites (Figure
8c). Intriguingly, this deletion is reminiscent of the d
domain deletion spanning the transactivation domain
in ASV17 v-Jun (Isaksson et al., 1996; Treier et al.,
1994), which serves as a recognition site for the
ubiquitin machinery and abrogates ubiquitin-dependent degradation of v-Jun. Therefore, by disrupting the
Myc degradation signal sequence, these viral Myc
mutations may interfere with the ability of the
ubiquitin machinery to recognize and degrade Myc
(Figure 8).
Phosphorylation di€erentially regulates proteasomal
degradation of a number of important transcription
factors. For example, phosphorylation destabilizes
MyoD (Song et al., 1998), whilst Fos and Jun are
stabilized (Chen et al., 1996; Musti et al., 1997;
Okazaki and Sagata, 1995). Degradation of Myc,
however, may be regulated by two phosphorylation
events, one which stabilizes the protein (possibly ser-65
(Sears et al., 1999) or an alternative (Colman and
Ostrowski, 1996) and one which destabilizes Myc (thr61) (Figure 8). The balance between signalling pathways, phosphorylation and degradation of transcription factors is clearly important for maintaining
appropriate levels in the cell, and viral oncoproteins
subvert this control through the acquisition of point
mutations (May et al., 1998; Musti et al., 1997).
Despite having a similar organization of dilysine
residues and phosphorylation sites which are required
for the signal-induced degradation of IkB, Myc
degradation does not appear to be through a similar
mechanism. In fact, when mutated, the lysine residues
adjacent to MBI can appear to destabilize c-Myc even
further, suggesting that the primary MBI sequence can
regulate the rate of Myc degradation (Salghetti et al.,
1999). MBII, a domain which is also critical for Myc
transformation and plays a role in Myc degradation
(Flinn et al., 1998; Stone et al., 1987) contains two
lysine residues which may be the potential ubiquitin
targets. Alternatively, ubiquitination may involve a
novel target residue (Breitschopf et al., 1998), and we
are currently determining the key ubiquitin targets in
Myc.
In summary, we have described three independently
derived v-Myc alleles which are considerably more
stable than their cellular counterparts, suggesting
stabilization may be a common consequence of
mutations in Myc. We have discussed possible
mechanisms of how Myc degradation may be
regulated through mutation of either key phosphorylation sites or ubiquitin regulating sequences in view of
current evidence suggesting that Myc is degraded
through the proteasome (Ciechanover, 1998; GrossMesilaty et al., 1998). Experiments are therefore
currently underway to determine the precise biological
and biochemical consequence of these virally derived
mutations of Myc.
Materials and methods
Cell culture and transfections
Cell culture and transfection of appropriate SFCV ± Myc
constructs (10 mg) together with RCAN (A) helper (4 mg) into
secondary chick embryo ®broblasts (CEF) was performed
essentially as described (La Rocca et al., 1994). Following
G418 selection, cultures were expanded and used to analyse
Myc turnover.
Construction of retroviruses
The construction of SFCV ± c-Myc, SFCV ± v-Myc and
SFCV ± T17 v-Myc have previously been described (Fulton
et al., 1996; La Rocca et al., 1994). With the exception of
SFCV ± c-Myc(T61M), SFCV ± v-Myc(M61T) and SFCV ± cMyc(KK54,54RR54,55), all Myc mutants were generated by site
directed mutagenesis essentially as described (Crouch et al.,
1990; La Rocca et al., 1994). c-Myc(T61M) was generated by
replacing an 80 bp SacI ± PvuII fragment into pAT ± c-Myc
(Crouch et al., 1990) and subsequent cloning into SFCV-sa7
as previously described (Crouch et al., 1990; La Rocca et al.,
1994). To generate SFCV ± v-Myc(M61T), the 3' ClaI
fragment of c-Myc was replaced with a 607 bp ClaI
fragment from v-Myc. c-Myc(KK54,54RR54,55) was generated
by ligation of a PstI ± SacI linker containing the appropriate
mutation into spt ± c-Myc (Crouch et al., 1993), and the
subsequent HindIII fragment ligated into SFCV-sa7 (La
Rocca et al., 1994). For the construction of SFCV ± MH2 vMyc, a 285 bp amino terminal fragment and a carboxy
terminal 621 bp fragment were generated by PCR from a
pMH2 proviral cDNA template (Coll et al., 1983). The
primers pairs used were 5'-gacaagcttatcgataccatgccgctcagcgccagc-3' and 5'-cgtcaccatctccagctggtcggcgg-3' for the amino
terminal fragment, and 5'-gacaagcttctatgcacgagagttcttt-3' and
5'-gcggctctgctgggggtcgacacgccgccc-3' for the carboxy terminal
fragment. After HindIII/PstI digestion and HindIII/ClaI
digestion of the amino terminal and carboxy terminal
fragments respectively, the puri®ed fragments were ligated
into the HindIII site of SFCV-sa+, together with a central
584 bp PstI/ClaI spanning exon 2/exon 3 boundary, which
was isolated from pMH2 (Coll et al., 1983). All sequences
generated by PCR or mutagenesis were con®rmed by doublestranded sequencing using T7-Sequenase v2.0 essentially as
described by the manufacturers (Amersham).
Oligonucleotide site directed mutagenesis
Site directed mutagenesis was performed using the Sculptor
mutagenesis system (Amersham) as described previously
(Crouch et al., 1990). The mutagenic oligonucleotides used
7557
Stabilization of viral Myc proteins
PR Gavine et al
7558
were as follows: S325L: 5'-ccccgcacgttagact-3'; S350R: 5'ctgaagctgcgtttctttgcc-3', I383L: 5'-gttctgtctctccaatcggacgag-3';
K407N: 5'-ttgaaacacaaccttgaggagc-3'.
Western blotting
Cell lysates were prepared by lysing cultures in SDS-sample
bu€er containing 1% SDS, but no bromophenol blue or
mercaptoethanol. Protein concentrations were measured
using Micro BCA reagent (Pierce), and readjusted for
bromophenol blue and mercaptoethanol before loading onto
7.5% SDS ± PAGE gels. Transfer to nitrocellulose and
Western blotting was performed essentially as described (La
Rocca et al., 1994), except that incubation with the primary
antibody was performed in 2% BSA in TBST. Proteins were
detected using speci®c rabbit antibodies (anti-v-myc 12C
antibody (Hann et al., 1983) and anti-jun 730 (Kilbey et al.,
1996) and visualized using enhanced chemiluminescence.
Protein turnover analysis
Myc turnover was analysed in G418-selected cultures
following treatment with the protein synthesis inhibitor,
emetine (Sigma) at a ®nal concentration of 0.1 mM (Crouch
et al., 1990) for the indicated times. Proteasomal involvement
was determined using the inhibitor lactacystin (Aniti
Research Products) at a concentration of 10 mM (GrossMesilaty et al., 1998). The inhibitor was added 45 min prior
to the addition of emetine, and was present throughout the
experiment.
Acknowledgements
We wish to thank Jen Blake for technical assistance, RN
Eisenman and DAF Gillespie for providing the anti-v-myc
12C and anti-jun antibodies respectively, and CNA Palmer
and DW Meek for critical reading of the manuscript.
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