(Received for publication, August 22, 1995)
From the
Recombinant c-Jun and c-Fos were ubiquitinylated by the
ubiquitin carrier enzymes E2, E2
, or
E2
in the presence of the ubiquitin-activating enzyme,
E1. Addition of ubiquitin protein ligase E3 substantially enhanced the
E2
-mediated ubiquitinylation of c-Jun and c-Fos.
Truncated c-Jun and c-Fos mutant proteins including wbJun and wbFos
were also ubiquitinylated under the same conditions, suggesting the
sites of ubiquitinylation are located within the dimerization and DNA
binding domains of c-Jun and c-Fos. The E3-dependent ubiquitinylation
of c-Jun was inhibited upon the heterodimerization of c-Jun with c-Fos.
Further addition of E2
significantly enhanced
ubiquitinylation of c-Jun in the heterodimer suggesting a regulatory
role of E2
. Polyubiquitinylated c-Jun, wbFos, and wbJun,
but not E2
-ubiquitinylated c-Jun, were readily degraded
by the ATP-dependent 26 S multicatalytic proteases. These results
suggest that the temporal control of c-Jun and c-Fos may be regulated
through the ubiquitinylation pathways, and the ubiquitinylation of
c-Jun and c-Fos may in turn be regulated in response to the
heterodimerization between them and the cooperation between E2
and E3 mediated polyubiquitinylation.
Ubiquitin is a 76-residue protein that can be covalently
attached to a number of cytoplasmic, nuclear, and integral membrane
proteins (for reviews, see (1, 2, 3) ).
Ubiquitinylation of proteins has attracted increasing attention, since
genetic analyses have implicated important roles of ubiquitinylation in
a number of cellular regulatory processes including DNA repair, induced
mutagenesis, sporulation, cell cycle transitions from G to
S and from G
to M, stress resistance and peroxisome
biogenesis(4) , and programmed cell death(5) . The
molecular events that lead to the phenotypes exhibited by mutants of
ubiquitinylation enzymes are not well understood, largely because most
of the protein substrates involved have not been identified and the
substrate specificity of the ubiquitinylation isozymes are not known.
Thus far, a few, but important, in vivo substrates have been
identified including phytochrome photoreceptor from plants(6) ,
cyclins from Xenopus(7) , and cell type-specific
transcript repressor MAT
2 of yeast(8) . In
these cases, ubiquitinylation was demonstrated to play pivotal roles in
the turnover of these proteins.
Several enzymes involved in
ubiquitinylation have been isolated and characterized(9) . The
ubiquitin-activating enzyme E1 ()activates ubiquitin and
transfers the activated ubiquitin to one of the ubiquitin-conjugating
isozymes, E2, which can then covalently attach the ubiquitin to various
protein substrates. One of the E2 isozymes, E2
,
apparently modifies the ubiquitin protein ligase E3, which then
catalyzes the transfer of ubiquitin to substrates in a processive
manner and forms polyubiquitinylated chains(10) . The
polyubiquitinylated substrates are selectively degraded by an ATP and
ubiquitin-dependent 26 S proteasome(11) .
Jun and Fos are nuclear proteins encoded by immediate-early genes with transcription activation activities. They are involved in the signaling pathways in regulating cellular growth, differentiation, and neuronal responses(12, 13) . These proto-oncoproteins display very short half-lives(14) , a feature shared by a number of other proto-oncoproteins, such as Myc, Myb, Erb, and E1a(12) , whose degradation might exert a regulatory control of their activities. Inasmuch as cellular transformation results from continuous or deregulated expression of oncoproteins(15, 16) , the exact mechanism that controls the turnover of these proteins is important for our understanding of an array of activities regulated by oncoproteins. The mechanism of the temporal control of these proteins is not well understood. The involvement of ubiquitin conjugation in the turnover of nuclear regulatory proteins was suggested by the E1-dependent degradation of in vitro translated proteins in the reticulocyte lysate(17) . The ubiquitinylation and degradation of c-Jun (18) and p53 (19) have recently been demonstrated in vivo. In the case of c-Jun, hemagglutinin epitope or oligohistidine labeled proteins were produced in vivo and c-Jun but not v-Jun was found to be selectively ubiquitinylated and degraded in vivo. The enzymes involved in the ubiquitinylation and degradation of c-Jun have not been identified. More recently, ATP-dependent but ubiquitin-independent degradation of c-Jun by the 26 S proteasome was demonstrated in vitro, and opened the possibility of multiple pathways of the degradation of c-Jun(20) . In the case of p53, a papilloma viral E6-activated E3 was found to selectively ubiquitinylate p53(21) .
In an attempt to further understand the substrate specificity of the E2 and E3 isozymes and to characterize the ubiquitinylation substrates, we have examined the ubiquitinylation of c-Jun and c-Fos using purified enzymes. In this study, we report the in vitro ubiquitinylation of c-Jun and c-Fos catalyzed by reconstituted enzymes purified from reticulocytes. Two enzyme systems that efficiently ubiquitinylated c-Jun and c-Fos are found. Interestingly, both c-Jun and c-Fos can be ubiquitinylated efficiently by the same E2 isozymes directly as well as via the protein ubiquitin ligase E3. Furthermore, the E3-ubiquitinylated Jun and Fos are selectively degraded by the 26 S proteasome. Preliminary results of these studies have been reported earlier(22) .
Rabbit reticulocytes were purchased from Green Hectares (Oregon, WI). Leupeptin, bestatin, and pepstatin A were from Boehringer Mannheim, and TLCK, TPCK, antipain, and chymostatin were from Fluka. Ubiquitin, aprotinin, AEBSF, Arg-Ala, Phe-Ala, and succinyl-Leu-Leu-Val-Tyr-Methyl coumarin were from Sigma. Human c-Jun was from Promega. Wild type and mutated c-Jun and c-Fos proteins were expressed in Escherichia coli, purified under denaturing conditions by affinity chromatography on nickel-nitriloacetic acid, and slowly renatured by dialysis as described previously(23) . Rabbit antibodies specifically against rat c-Jun and rat c-Fos were as described previously(23) . Ubiquitin was covalently attached to CH-activated Sepharose 4B (Pharmacia Biotech Inc.) according to the manufacturer's instruction. SDS-polyacrylamide gel electrophoresis was carried out using the precast Novex 8% or step-gradient 8-16% polyacrylamide gels. Proteins were assayed using the Bio-Rad protein assay.
Figure 1:
FPLC Mono Q chromatography of the
AMP/PP and DTT/pH 9 elutes. The AMP/PP
(a) and DTT/pH 9 (b) eluates from covalent
affinity chromatography on ubiquitin-Sepharose were loaded separately
onto a Mono Q column at 4 °C. The column was washed with 5 ml of
buffer A (50 mM Hepes, pH 7.5, 1 mM DTT, 1 mM EDTA, 1 mM EGTA), eluted with a 40 ml linear gradient of
0-0.5 M KCl in buffer A, a 4-ml linear gradient of
0.5-1 M KCl and 5 ml of 1 M KCl in buffer A.
Fractions of 2 ml were collected at a flow rate of 1 ml/min. Absorbance
at 280 nm was monitored.
Figure 2:
Ubiquitinylation of c-Jun and c-Fos.
Ubiquitinylation of c-Jun (A) and c-Fos (B) was
carried out under the standard ubiquitin conjugation assay conditions
using I-ubiquitin, followed by SDS-polyacrylamide gel
electrophoresis and PhosphorImager analysis. A, lane
1, 1 µM c-Jun and 50 nM each of E1,
E2
, and E3; lane 2, 1 µM c-Jun with
50 nM E1 and 50 nM E2
. The
radioactively labeled 55-kDa protein band between Jun(ub
)
and Jun(ub
) was immunoprecipitated by monospecific
anti-c-Jun antibodies and was evidently due to impurity in the c-Jun
preparation. B, lane 1, 1 µM c-Fos and
50 nM each of E1, E2
, and E3; lane 2, 1
µM c-Fos with 50 nM E1 and 50 nM E2
.
In the
presence of E3, the E2-mediated ubiquitinylation of c-Jun
or c-Fos was appreciably enhanced, while E2
-mediated
ubiquitinylation of c-Jun as expected was not affected. Fig. 2, A and B, show the ubiquitinylation of c-Jun (lane
1) and c-Fos (lane 1), respectively, by E2
and E3. Quantitation of the ubiquitinylated species showed that
8% of c-Jun and 4% of c-Fos were ubiquitinylated in 30 min by 50 nM E1/E2
and E3, as compared to 2.5% and 1.9%,
respectively, in the absence of E3 (Table 1). Furthermore, four
distinct ubiquitinylated c-Jun and c-Fos species together with some
high molecular weight polyubiquitinylated species were observed (Fig. 2). The total amount of ubiquitin covalently attached to
c-Jun in the presence of E3 was at least 10-fold higher than that in
the absence of E3, when high molecular weight species were included in
the quantitation.
A number of truncated proteins of c-Jun were
previously constructed to examine their DNA binding and the
dimerization characteristics(23) . These truncated c-Jun
proteins were found to be good substrates of ubiquitinylating enzymes.
As shown in Fig. 3A, truncated c-Jun proteins were
ubiquitinylated by E2 to similar extents as the
full-length c-Jun. However, primarily monoubiquitinylated species were
obtained from the truncated proteins. When the amounts of truncated
c-Jun proteins that were ubiquitinylated by E2
were
quantitated, amounts very similar to those for c-Jun were found (Table 2, right column). Since the extent of ubiquitinylation of
c-Jun by E2
changed little after truncation, the primary
sites of ubiquitinylation in intact c-Jun and those in mutant c-Jun
proteins are likely to be the same.
Figure 3:
Ubiquitinylation of truncated c-Jun and
c-Fos mutants. A, C-Jun mutant proteins were ubiquitinylated
with 50 nM E1, 50 nM E2, and 50 nM E3 (lanes 1-3) or at 50 nM E1 and 50
nM E2
(lanes 4-6) under the
standard ubiquitin-conjugating conditions and analyzed as described
under ``Materials and Methods.'' The lanes labeled as 187, 199, and 241 correspond to reaction
products from Jun(187-334), Jun(199-334), and
Jun(241-334), respectively. B, c-Fos mutant proteins
were ubiquitinylated by E1, E2
, and E3 (lanes
1-3) or by E1 and E2
(lane 4). Lane 1, c-Fos; lane 2, wbFos(116-211); lane
3, wbFos(
basic); and lane 4,
wbFos(C204S).
Ubiquitinylation of c-Jun
truncation mutants by E3 was appreciably more efficient than those of
the full-length protein. As shown in Table 2, 50% of wbJun was
ubiquitinylated by E2 and E3. The majority of the
ubiquitinylated proteins appeared as high molecular weight conjugates,
suggesting the polyubiquitinylation of the truncated c-Jun proteins.
The high efficiency of ubiquitinylation exhibited by wbJun suggested
that the sites of ubiquitinylation in c-Jun are likely located within
the 110 residues in wbJun that contain the DNA binding domain and the
leucine repeats, although sites within the deleted regions cannot be
excluded. The large increases of ubiquitinylation mediated by
E2
and E3 in the truncated c-Jun indicated that either
additional sites in c-Jun became available in the truncated proteins or
the presence of additional E3 isozymes in the E3 preparation which
catalyzed the ubiquitinylation of the truncated c-Jun and c-Fos
proteins. As shown in Table 2, deletion of N-terminal 90 residues
in c-Jun was sufficient to bring about the enhanced ubiquitinylation of
c-Jun. The partial deletion of the basic region in
wbJun(
260-266) (Table 2) did not appreciably affect
the extents of ubiquitinylation or numbers of ubiquitin moieties
conjugated to wbJun catalyzed by E3. Thus the deleted residues in the
basic region were not required for the ubiquitinylation of wbJun.
When the ubiquitinylation of truncated c-Fos proteins by E2 and by E2
and E3 were examined (Fig. 3B), results similar to those obtained with the
truncated c-Jun proteins were observed (Table 3). The truncated
c-Fos proteins, similar to full-length c-Fos, were good substrates of
E2
. Similar to that observed in c-Jun, the truncated
c-Fos proteins including wbFos(116-224) and wbFos(
basic)
were more efficiently ubiquitinylated by E3 than the full-length c-Fos.
These results suggest that residues deleted from c-Fos were not
required for efficient ubiquitinylation by E2
or by
E2
and E3.
Figure 4:
Effects of Arg-Ala and Phe-Ala on the
ubiquitinylation of wb-Jun and wb-Fos. a, ubiquitinylation in
the absence (lanes 1-3) and the presence of oxidized
ribonuclease A (lanes 4-6), and b,
ubiquitinylation of wb-Fos(116-211) (lanes 7-9)
and wb-Jun(224-334) (lanes 10-12) were carried out
separately in the presence of 50 nM each of E1,
E2, and E3 and
I-ubiquitin under the
standard ubiquitin conjugating assay conditions in the absence (lanes 1, 4, 7, and 10) and
presence of 1 mM Arg-Ala (lanes 2, 5, 8, and 11) or 1 mM Phe-Ala (lanes
3, 6, 9, and 12). Reaction products
were analyzed by SDS-polyacrylamide gel electrophoresis followed by
PhosphorImager analysis.
Figure 5:
Degradation of ubiquitinylated wbJun and
wbFos by the 26 S proteasome. Protein was ubiquitinylated using I-ubiquitin, and 50 nM each of E1,
E2
, and E3 under the standard ubiquitin conjugating assay
conditions, and the ubiquitinylated protein was incubated with 2 mM ATP in the presence of 26 S proteasome or 20 S proteasome.
Aliquots were withdrawn at the specified time points shown, and the
reaction products were analyzed by SDS-polyacrylamide gel
electrophoresis and PhosphorImager analysis. a, oxidized
ribonuclease with 26 S proteasome (left panel) or 20 S
proteasome (right panel); b, wbJun and wbFos with 26
S proteasome; c, c-Jun with 26 S proteasome with
ubiquitinylated c-Jun that obtained from E1, E2
, and E3 (left panel) and that obtained with E1 and E2
(right panel).
The present investigation provides direct evidence for the
ubiquitinylation of c-Jun and c-Fos. The E2 and E3 isozymes that
ubiquitinylated this family of transcription factors are identified.
The recombinant c-Jun and c-Fos are evidently good substrates for
E2 and E3 ubiquitinylation enzyme systems. The high
levels of ubiquitinylation of c-Jun or c-Fos suggest that inherent
structural features in c-Jun and c-Fos were recognized by both
E2
and E3. The same two enzyme systems, E1/E2
and E1/E2
/E3 were found to be the most efficient
among various reconstituted enzyme systems for both c-Jun and c-Fos.
Ubiquitinylation of deletion mutant proteins of c-Jun and c-Fos
suggested that most of the sites of multi- and polyubiquitinylation are
most likely located in the heterodimerization and the DNA binding
domains. Heterodimerization of c-Jun and c-Fos affected the
ubiquitinylation of c-Jun by E3. The polyubiquitinylated c-Jun and
c-Fos are susceptible to the 26 S but not to the 20 S proteasome for
degradation.
With the inclusion of a battery of protease inhibitors
in the reticulocyte lysate, E1 and E2 were copurified
throughout the purification suggesting that at least part of the E1 and
E2
may be physically associated as a multienzyme complex.
Chemical cross-linking using a cleavable heterobifunctional
cross-linking reagent, 3,3`-dithiobis(sulfosuccinimidylpropionate),
suggested that indeed E1 and E2
were physically
associated. However, the association appears to be weak since gel
filtration of the complex partially separated E1 and E2
.
Gel filtration of the complex in the presence of 0.5 M NaSCN
completely separated E1 and E2
(data not shown) (22) . The association of E1 and E2
is
biochemically important since it may provide a mechanism for the direct
transfer of ubiquitin from E1 to E2
without first
dissociating from E1 followed by reassociation with E2
in
solution. We consistently obtained somewhat higher levels of
ubiquitinylation using the copurified E1/E2
preparation
than those using recombined purified E1 and E2
.
The
occurrence of E3 isozymes raised the possibility that the E3 isozyme
that acted on c-Jun and c-Fos may be different from that of wbJun and
wbFos. The inhibition patterns with respect to Arg-Ala and Phe-Ala
suggested that this may be the case. The observation that the
E3-mediated ubiquitinylation of wbFos and wbJun was apparently
stimulated by Arg-Ala indicated that there could be a different E3
isozyme that preferentially acts on the fragments of c-Fos and c-Jun.
The action of such additional E3 isozyme on wbFos and wbJun could play
a concerted role in the degradation of c-Fos and c-Jun along with
E3.
The susceptibility of the E3-ubiquitinylated c-Jun and
c-Fos to the 26 S multicatalytic proteasome is in accord with the
hypothetical in vivo function of ubiquitinylation in the
turnover of c-Jun and c-Fos. The regulation of the turnover of c-Jun
via ubiquitinylation in HeLa cells has recently been
demonstrated(18) . Although only 0.1-1% c-Jun was
ubiquitinylated in vivo, highly suggestive evidence was
obtained supporting the selective degradation of c-Jun via the
ubiquitinylation pathway. Furthermore, the retroviral counterpart v-Jun
cannot be polyubiquitinylated nor degraded efficiently in
vivo. The ubiquitinylation enzymes involved in the modification of
c-Jun in HeLa cells have not been identified. Since c-Jun but not v-Jun
was efficiently ubiquitinylated, the in vivo ubiquitinylation
is likely mediated through the -domain in c-Jun which is absent in
v-Jun. The present studies are consistent with the observed
ubiquitinylation of c-Jun and the degradation of ubiquitinylated c-Jun.
However, since wbJun, in which the
-domain was deleted, was also
efficiently ubiquitinylated by E3, our results suggest that c-Jun may
have undergone different ubiquitinylation pathways under different
conditions. The ubiquitinylating enzymes in reticulocytes that acted on
c-Jun may be different from those in HeLa cells. Alternatively,
different E3 isozymes or other cofactors may be involved in the in
vivo ubiquitinylation of c-Jun. Furthermore, the subcellular
location of the overexpressed recombinant c-Jun is not known at
present. The recent demonstration of ubiquitin-independent degradation
of c-Jun by the 26 S proteasome (20) provoked the question as
to the role of ubiquitination in the in vivo degradation of
c-Jun in HeLa cells. However, our results show that c-Jun and c-Fos can
be degraded by the 26 S proteases via the ubiquitin-dependent pathway.
The stability of c-Fos was shown to be regulated by phosphorylation (29) . Whether phosphorylation regulates the ubiquitinylation of c-Fos and c-Jun is not known. It is known in the case of the oncoprotein Mos that the stability is regulated by ubiquitinylation which is in turn regulated by phosphorylation(30, 31) . Preliminary studies of the effects of phosphorylation of c-Fos and c-Jun by a number of known protein kinases on the ubiquitinylation were inconclusive.
One of
the known mechanisms that regulate the DNA binding and transcription
activation activities of c-Jun and c-Fos is the heterodimerization via
the leucine repeats(28) . The present results show that
heterodimerization affected ubiquitination of c-Jun. The stimulatory
effect of E2 on the ubiquitinylation of the heterodimer
of c-Jun/c-Fos suggests that E2
could add an additional
level of regulation in the E3-dependent ubiquitinylation of the
heterodimeric c-Jun/c-Fos. It is intriguing that E2
was
found to be the most efficient isozyme among various E2 isozymes in the
ubiquitinylation of c-Jun and c-Fos. The present results open the
possibility of an interplay of the transcription activation by these
transcription factors and their ubiquitinylation, which may act in a
concerted fashion in the temporal regulation of transcription.
The reconstitution of the ubiquitinylation enzymes for c-Jun and c-Fos provided a route for a systematic studies of the mechanism and the regulation of ubiquitinylation. Further studies on the kinetics and mechanisms of E2- and E3-mediated ubiquitinylation of c-Jun will be reported.