* Department of Biology and Geosciences, Faculty of Science, Shizuoka University, Shizuoka 422, Japan; Division of Biological
Sciences, Graduate School of Science, Hokkaido University, Sapporo 060, Japan; and § Laboratory of Reproductive Biology,
National Institute for Basic Biology, Okazaki 444, Japan
Immediately before the transition from metaphase to anaphase, the protein kinase activity of maturation or M-phase promoting factor (MPF) is inactivated by a mechanism that involves the degradation of its regulatory subunit, cyclin B. The availability of biologically active goldfish cyclin B produced in Escherichia coli and purified goldfish proteasomes (a nonlysosomal large protease) has allowed the role of proteasomes in the regulation of cyclin degradation to be examined for the first time. The 26S, but not the 20S proteasome, digested recombinant 49-kD cyclin B at lysine 57 (K57), producing a 42-kD truncated form. The 42-kD cyclin was also produced by the digestion of native cyclin B forming a complex with cdc2, a catalytic subunit of MPF, and a fragment transiently appeared during cyclin degradation when eggs were released from metaphase II arrest by egg activation. Mutant cyclin at K57 was resistant to both digestion by the 26S proteasome and degradation at metaphase/anaphase transition in Xenopus egg extracts. The results of this study indicate that the destruction of cyclin B is initiated by the ATP-dependent and ubiquitin-independent proteolytic activity of 26S proteasome through the first cutting in the NH2 terminus of cyclin (at K57 in the case of goldfish cyclin B). We also surmise that this cut allows the cyclin to be ubiquitinated for further destruction by ubiquitin-dependent activity of the 26S proteasome that leads to MPF inactivation.
PROTEOLYSIS plays an important role in the regulation of the eukaryotic cell cycle. The termination of
mitosis and meiosis, the transition from metaphase
to anaphase, is induced by the degradation of cyclin B, a
regulatory subunit of maturation or M phase promoting factor (MPF;1 Murray et al., 1989 Fish oocytes provide an appropriate experimental system with which to investigate the molecular mechanisms
controlling meiosis and the embryonic cell cycle. Several
factors responsible for the regulation of meiotic maturation of fish oocytes have been identified. These include the
isolation and characterization of a fish maturation-inducing hormone (17 Materials
Goldfish were purchased from a local supplier and maintained at 15°C until use. The 20S and 26S proteasomes were purified from immature goldfish oocytes by conventional column chromatography as described (Tokumoto et al., 1995a Electrophoresis and Immunoblot Analysis
Electrophoresis proceeded as described by Laemmli (1970) Determination of the Digestion Site of Cyclin B
Electroblotted 42-kD fragments of cyclin B were prepared as described
(LeGendre and Matsudaira, 1989 Production of Recombinant Cyclin Bs
Full length ( Recombinant proteins were produced in E. coli BL21 (DE3) and purified by SDS-PAGE followed by electroelution from the gel, as described
previously (Hirai et al., 1992 Restricted Digestion of Cyclin B by 26S Proteasome
Although the 26S proteasome is a ubiquitin-dependent
protease in general (Armon et al., 1990
We determined whether the 26S proteasome digests the
NH2- or COOH-terminal region of the cyclin B. We used
monoclonal anti-cyclin B63, which recognizes the COOH
terminus of cyclin B, and the anti-cyclin B112, which recognizes the NH2 terminus (Hirai et al., 1992
The NH2-terminal sequences of cyclins, including a consensus sequence called the destruction box, play a critical
role in targeting cyclins for degradation (Glotzer et al.,
1991
The 26S proteasome also digested native cyclin B that
had been isolated as a complex with cdc2, yielding a truncated cyclin of ~42-kD (Fig. 5 A). This was the same size
as the fragment produced by digestion of recombinant, full
length cyclin B with the 26S proteasome. The digestion of
the cdc2-cyclin B complex with 26S proteasome, however,
did not cause kinase inactivation of cdc2 (Fig. 5 B).
Detection of Intermediate Cyclin B during
Egg Activation
Mature goldfish oocytes are arrested at metaphase of meiosis II (metaphase II) and can be activated by the contact
with water (Yamashita et al., 1992a
Destruction Analysis in Xenopus Egg Extracts
Although the results so far suggested that the 26S proteasome interacts with the NH2-terminal region of cyclin B
and then cuts it at K57, digestion of cyclin B by the 26S
proteasome in vitro is limited to cleavage of a single peptide bond and does not induce kinase inactivation of MPF
(Fig. 5 B). We believe that the incomplete digestion of cyclin B by the 26S proteasome in vitro is due to the absence
of factors responsible for further degradation of cyclin B. The most likely candidate is ubiquitin and its ligase. In
fact, goldfish oocytes contain high levels of free ubiquitins
(Tokumoto et al., 1993b We purified and characterized the 26S proteasome from
immature Xenopus oocyte extracts (Tokumoto and Ishikawa, 1995 Cyclin
Digestion by the 26S Proteasome and Degradation of In
Vitro Translated Cyclin Bs in Xenopus Egg Extract
Our experiment using E. coli-produced cyclins demonstrated that the 26S proteasome restrictively digests the
NH2 terminus of cyclin B and that only cyclins digestible
or digested by 26S proteasome are degradable in Xenopus
egg extracts activated with Ca2+. To confirm that these results are not artifacts derived from the incorrect form of
cyclins produced in E. coli, we performed a similar experiment using cyclin B proteins translated in vitro in rabbit
reticulocyte lysate. Cyclin B proteins produced in E. coli
and translated in vitro gave the same results. Cyclin
Immediately before the transition from metaphase to
anaphase, the kinase activity of MPF is inactivated through
the degradation of its cyclin B subunit. The mechanism of
cyclin degradation, which must be a highly selective process since few other proteins are degraded only at this
time, is poorly understood. Using recombinant goldfish cyclin B and purified 26S proteasomes, we investigated the
role of proteasomes in the regulation of cyclin degradation during egg activation. We found that purified 26S proteasome digests not only recombinant cyclin B, but also native cyclin B at its NH2-terminal portion, producing a 42-kD intermediate form. Since the 42-kD cyclin B appears
transiently during the initial phase of normal egg activation, this digestion should not be an artifact but rather an
initial step in cyclin B degradation upon egg activation. Using Xenopus egg extracts, we also showed that the initial digestion and further degradation of cyclin B are
tightly linked. Cyclins The NH2-terminal sequences of cyclin B, including a
consensus sequence that is called the destruction box, play
a critical role in targeting cyclins for degradation, since
truncated sea urchin (Murray et al., 1989 In this study, we proposed that the initial reaction of cyclin B destruction is the restricted cleavage of its NH2-terminal portion (K57 in the case of goldfish cyclin B) by the
26S proteasome. As well as cyclin The mechanism of cyclin B degradation after initial
cleavage by the 26S proteasome remains to be determined.
The most likely candidate will be ubiquitin-dependent
proteolysis. Proteins to be degraded by the ubiquitin pathway are ligated to ubiquitin through their lysine amino
acid groups and then degraded by the 26S proteolytic complex (Hershko and Ciechanover, 1982 The cyclin B subunit of MPF must be ubiquitinated immediately before the onset of its destruction at the metaphase/anaphase transition. The findings of the present
study indicate that the restricted cleavage of cyclin B triggers its ubiquitination. It is likely that the digestion of the
NH2-terminal restricted portion by the 26S proteasome
(K57 in goldfish cyclin B) changes the cyclin structure
available for further chemical modifications including ubiquitination, which leads to the complete destruction of
the cyclin at metaphase/anaphase transition. Since cyclin
Cyclin B is absent in immature (prophase I-arrested)
goldfish oocytes. Cyclin B is de novo synthesized during
oocyte maturation and forms an MPF complex with extant
cdc2, which drives the prophase I-arrested oocytes to
metaphase II (mature oocytes; Hirai et al., 1992 Stewart et al. (1994) Our present data suggest that the NH2-terminal restricted cleavage of cyclin B by 26S proteasome allows cyclin to be ubiquitinated. Compared with the idea that the
initiation of cyclin destruction is dependent on the activity
of ubiquitin ligase, irrespective of 26S proteasome activity
(Sudakin et al., 1995 Ubiquitination of cyclins has been well studied genetically and biochemically (for review see Murray, 1995). Further, it is suggested
that cyclin is degraded by a ubiquitin-dependent proteolytic pathway (Glotzer et al., 1991
; Hershko et al., 1991
;
Sudakin et al., 1995
). Proteins subject to ubiquitin-dependent proteolysis are ligated to ubiquitin through their
lysine residues and then degraded by a nonlysosomal large
protease called the proteasome (or the multicatalytic protease), which is found in all eukaryotes from yeast to humans (Armon et al., 1990
; Driscol and Goldberg, 1990
;
Kanayama et al., 1992
; for reviews see Hershko and Ciechanover, 1982
; Orlowski, 1990
). Thus proteasomes must
play an important role in cyclin degradation. Indeed, a genetic approach has revealed that mutation of the gene encoding one proteasome subunit causes G2/M arrest (Ghislain et al., 1993
; Gordon et al., 1993
). To date however,
direct biochemical evidence for the involvement of proteasomes in cyclin degradation is poor.
,20
-dihydroxy-4-pregnen-3-one; Nagahama and Adachi, 1985
) and the components of MPF
(cdc2, the catalytic subunit and cyclin B, the regulatory
subunit; Yamashita et al., 1992a
,b; Kajiura et al., 1993
).
Both truncated and full length recombinant goldfish cyclins are biologically active (Hirai et al., 1992
; Katsu et al.,
1993
). To understand the role of the proteasome in meiotic maturation, particularly in cyclin degradation, we
have purified and characterized SDS-dependent (20S) and
-independent (26S) proteasomes from goldfish oocyte cytosol (Tokumoto et al., 1995a
,b). The availability of biologically active goldfish cyclin B produced in Escherichia
coli and of purified goldfish proteasomes allows the role of
proteasome in the regulation of cyclin degradation to be
examined for the first time. Here we propose that the 26S proteasome initiates cyclin degradation through the first
cut in its NH2 terminus.
Materials and Methods
,b). Xenopus laevis were obtained from a dealer and
maintained at 20°C. Xenopus CSF-arrested egg extracts were prepared by
the method of Murray et al. (1989)
.
, using 12.5%
gels under denaturing conditions. Cyclin B degradation was assessed by
immunoblotting against anti-goldfish cyclin B (B63 and B112) monoclonal
antibodies (Yamashita et al., 1992b
). Immunocomplexes were visualized
using the ECL detection kit (Amersham Intl., Arlington Heights, IL).
). The NH2-terminal amino acids were
determined using a protein sequencer (470A; Applied Biosystems, Chiba,
Japan).
0) and NH2-terminal truncated (
41 and
68) goldfish cyclin Bs were produced as described (Hirai et al., 1992
; Katsu et al., 1993
;
Yamashita et al., 1995
). Mutant cyclin B in which lysine 57 was replaced
by arginine (
0K57R) was produced as follows. A cDNA clone encoding
full length goldfish cyclin B (Hirai et al., 1992
) was mutated using a site-
directed mutagenesis system (Mutan K; Takara, Tokyo, Japan), following
a strategy based on the method of Kunkel (1985)
, according to manufacturer's instructions. Double-strand, mutated cDNA was prepared by T3
polymerase using single-strand cDNA and the following oligonucleotide:
AAGAAGGAAGTGAGGGTGGCGCCCAAGGTGGAG. This oligonucleotide was designed to produce a mutation site (bold type) and a restriction enzyme site (underlined) as described (Yamashita et al., 1995
).
Mutant clones were screened by digestion with the restriction enzyme and
confirmed by sequencing.
). 35S-labeled cyclins were produced using a
TNT T7-coupled Reticulocyte Lysate System (Promega Biotech, Madison, WI) according to the manufacturer's instructions.
Results
; Driscol and Goldberg, 1990
; Kanayama et al., 1992
), it also catalyzes an
ATP-dependent and ubiquitin-independent proteolysis (Tanaka et al., 1983
; Matthews et al., 1989
; Murakami et al.,
1992
). Therefore, we initially investigated whether or not
the 26S proteasome can degrade in vitro nonubiquitinated,
full length goldfish cyclin B produced in E. coli (cyclin
0).
Goldfish proteasomes (20S and 26S) were purified from
immature goldfish oocytes by sequential chromatography (Tokumoto et al., 1995a
,b). SDS-PAGE has demonstrated
that the 26S proteasome consists of multiple subunits with
a molecular mass ranging from 23.5 to 140 kD, whereas
the 20S proteasome includes subunits ranging from 23.5 to
31.5 kD (Fig. 1 A). The 26S, but not the 20S proteasome,
digested 49-kD cyclin
0 and produced a 42-kD cyclin
(Fig. 1 B). Protease inhibitors of microbial origin (antipain, chymostatin, and leupeptin) blocked the cyclin digestion at high (200 µM) but not at low concentrations (50 µM). Proteasome inhibitors (MG115, MG132, and PSI)
that inhibit the chymotrypsin-like activity and ubiquitin-dependent protein degradation (Figueiredo-Pereira et al.,
1994
; Rock et al., 1994
; Jensen et al., 1995
), blocked the digestion at 50 µM (Fig. 1 C). No digestion proceeded when the 26S proteasome was depleted with an anti-proteasome
antibody (Fig. 2, A and B). The reaction was also prevented when ATP was depleted from the reaction mixture
(Fig. 2 C). These results indicate that the digestion of cyclin B is not due to a contaminating protease in the 26S
proteasome fraction, but is catalyzed by the 26S proteasome itself.
Fig. 1.
Digestion of E. coli-produced goldfish cyclin
B by 26S proteasome purified from immature goldfish
oocytes. (A) Subunit composition of 20S and 26S proteasomes. Purified 20S (10 µg)
and 26S (27 µg) proteasomes
were resolved by electrophoresis and stained with Coomassie brilliant blue
R-250. (B) Digestion of full
length cyclin B by purified
proteasomes. Cyclin 0 (5 µg/ml) was incubated at room temperature with purified 20S or 26S proteasomes
(60 µg/ml) in reaction buffer
(100 mM Tris-HCl, 5 mM
MgCl2, 0.04 mM ATP, pH
7.6). Samples were exposed to Laemmli's SDS sample buffer at the indicated times during incubation. Cyclin B was detected by immunoblotting against an anti-goldfish cyclin B (B63) monoclonal antibody. The position to which the digested cyclin B migrated is indicated by an asterisk. (C) Effect of protease and proteasome inhibitors on cyclin B digestion by the 26S proteasome. Cyclin
0 was incubated for 60 min without (None) or with the 26S proteasome in the absence (Control) or presence of various inhibitors at 50 µM. Cyclin
B was detected by the B63 antibody. The position of the digested cyclin B is indicated by an asterisk.
[View Larger Version of this Image (30K GIF file)]
Fig. 2.
Inhibition of cyclin
B digestion by proteasome or
ATP depletion. Cyclin B
was detected by the B63 antibody. The position to which
the digested cyclin B migrated is indicated by an asterisk. (A) Immunodepletion of 26S proteasome from
purified 26S proteasome fraction. 26S Proteasome was
immunoprecipitated by affinity-purified anti-proteasome
IgG (Anti) or control IgG
(Cont), as described (Tokumoto and Ishikawa, 1993).
Supernatants (S) and precipitates (P) were immunoblotted
with a mixture of three monoclonal antibodies against
goldfish 20S proteasome
(GC4/5, 3
and 3
; Tokumoto et al., 1995a
). (B) Digestion of cyclin B in the mock- (Cont) or proteasome-depleted (Anti) 26S proteasome fraction. Cyclin
0 (5 µg/ml) was incubated at room temperature with the supernatant after immunoprecipitation with control or
anti-proteasome IgG. Samples were exposed to Laemmli's SDS sample buffer at the indicated times during incubation. (C) Effect of
ATP depletion on cyclin B digestion by the 26S proteasome. Cyclin
0 (5 µg/ml) was incubated at room temperature for 60 min without
(Control) or with the 26S proteasome in the presence of an ATP-depleting system (10 mM glucose and 1 µg/ml hexokinase,
ATP) or
2 mM ATP (+ATP).
[View Larger Version of this Image (23K GIF file)]
; Katsu et al.,
1993
). The B63 antibody recognized the 42-kD intermediate and the NH2-terminal deletion mutants of cyclin B,
while the B112 did not react with the intermediate but recognized the full length cyclin B (Fig. 3 B). Therefore, we
concluded that cyclin B was digested in the NH2 terminus.
To define the cleavage site, the NH2-terminal amino acid
sequence of the electroblotted 42-kD cyclin was directly
determined. The site of cyclin B cleavage by the 26S proteasome was the COOH-terminal peptide bond of lysine
57 (K57; Fig. 3 A).
Fig. 3.
NH2-terminal sequence of goldfish cyclin B
and digestion of recombinant
cyclin B by the 26 S proteasome. (A) Amino acid sequence of the NH2-terminal
region of goldfish cyclin B. The site digested by the 26S
proteasome (COOH terminus of K57) and truncated
sites of deletion mutants
(41,
68) are indicated. The
destruction box and lysine-rich stretch are also indicated. (B) Digestion of full
length and truncated cyclin
Bs by the 26S proteasome. Cyclins
0,
41, and
68
were incubated in the absence (
) or presence (+) of
the 26S proteasome for 120 min at room temperature. Cyclin degradation was assessed by immunoblotting
against two kinds of anti-
cyclin B (B63 and B112)
monoclonal antibodies. B112
recognizes the NH2-terminal
portion of goldfish cyclin B. The position of the digested
cyclin B is indicated by an asterisk.
[View Larger Versions of these Images (19 + 14K GIF file)]
; Lorca et al., 1991
; Luca et al., 1991
; Kobayashi et al.,
1992
). We therefore examined the role of NH2-terminal
sequences in cyclin digestion by the 26S proteasome in
vitro. We produced two NH2-terminal truncated cyclins:
cyclin
41 lacking the destruction box and cyclin
68 lacking the destruction box and half of the lysine-rich stretch (Fig. 3 A). Neither cyclin
68 nor cyclin
41 were digested
by 26S proteasome (Fig. 3 B). This finding suggests that
the NH2-terminal region of cyclin B, including the destruction box, supplies an interaction site between cyclin B and
26S proteasome that is necessary for the subsequent cutting of cyclin B at K57. Interaction between the NH2 terminus of cyclin B and the 26S proteasome was also suggested by an experiment with a truncated protein containing
the first 89 amino acids of Xenopus cyclin B2 (B2Nt, a gift
from Dr. M.J. Lohka, University of Calgary, Calgary, Canada; Velden and Lohka, 1993
). B2Nt inhibited the digestion of cyclin B by the 26S proteasome in a dose-dependent manner (Fig. 4). Control lysozyme, a basic and low
molecular weight protein like B2Nt, did not inhibit the cyclin digestion (Fig. 4).
Fig. 4.
Inhibition of 26S proteasome-catalyzed cyclin B digestion in vitro by the NH2-terminal fragment of Xenopus cyclin B2
(B2Nt). Cyclin 0 was incubated with purified 26S proteasome
(60 µg/ml) for 120 min in the absence (Control) or presence of
various concentrations of B2Nt or lysozyme. Cyclin B was detected with the B63 antibody. The migrating position of the digested cyclin B is indicated by an asterisk.
[View Larger Version of this Image (28K GIF file)]
Fig. 5.
Digestion of native cyclin B by 26S proteasome. The
truncated cyclin B produced by the 26S proteasome digestion is
indicated by an asterisk. (A) Digestion of cyclin B in MPF complex by the 26S proteasome. The MPF complex in mature carp
oocytes was prepared using suc1 beads (Yamashita et al., 1992b).
The beads were washed with buffer (50 mM Tris-HCl, 20% glycerol, 10 mM 2-mercaptoethanol, 0.1 mM ATP, pH 7.5) and
shaken in the absence (
) or presence (+) of 60 µg/ml of the 26S
proteasome at room temperature with agitation. Samples were
treated with SDS sample buffer at the indicated times and immunoblotted against the B63 antibody. Two cyclin bands were detected, and only the upper band was digested by the 26S proteasome. It is unlikely that these two bands correspond to different
phosphorylation states of cyclin B (Yamashita et al., 1992b
). In C,
only a single band of cyclin B was detected when oocytes were directly exposed to SDS sample buffer. Therefore, the lower band
is probably produced by undesirable proteolysis during treatment
with the suc1 beads. (B) Protein kinase activity of suc1 precipitates before and after the digestion with 26S proteasome. The kinase activity of suc1 precipitates incubated for 60 min in the absence (
) or presence (+) of 26S proteasome was measured with
a synthetic peptide substrate for cdc2, as described (Yamashita et
al., 1992a
). Activities are indicated as a percentage of the activity
at 0 min for each condition. (C) Detection of a truncated cyclin B
during goldfish egg activation. Ovulated eggs (2 ml) were placed
in 3 ml goldfish Ringer's solution (Yamashita et al., 1992b
) and
immediately homogenized in 5 ml SDS sample buffer at the indicated times. Before detecting truncated cyclin B by immunoblotting with B63 or B112, proteins with a molecular mass of 40-50
kD were separated by SDS-PAGE (Prep Cell Model 491; Bio
Rad, Richmond, CA) and concentrated.
[View Larger Versions of these Images (38 + 16 + 16K GIF file)]
,b). Cyclin B was degraded within a few minutes after egg activation (Nagahama et al., 1995
). A 42-kD fragment of cyclin B transiently appeared during the initial phase of goldfish egg
activation. In a partially purified and highly concentrated
fraction from egg extracts, intermediate cyclin B was detected 3 min after egg activation (Fig. 5 C). The monoclonal antibody B112, which recognizes the NH2-terminal
region of cyclin B, did not react with the intermediate (Fig.
6 C). These results suggest that NH2-terminal digestion of
cyclin B by the 26S proteasome is not an artifact of in vitro
proteolysis but an initial reaction of cyclin B degradation
that proceeds upon egg activation.
Fig. 6.
Digestion of goldfish cyclin B by the Xenopus
26S proteasome. Cyclin was
visualized by immunoblotting with B63 (A-C) and
B112 (C). The position of the
digested cyclin B is indicated
by asterisks. (A) Digestion of
full length cyclin B. Cyclin 0
(5 µg/ml) was incubated at
room temperature with purified 20S or 26S proteasomes (60 µg/ml) in the reaction
buffer (100 mM Tris-HCl, 5 mM MgCl2, 0.04 mM ATP,
pH 7.6). Samples were exposed to Laemmli's SDS
sample buffer at the indicated times during incubation. (B) Digestion of truncated cyclin B. Cyclins
0,
41, and
68 were incubated in the absence (
) or presence (+) of 60 µg/ml of 26S proteasome for 120 min at room temperature. (C) Cyclin was digested by Xenopus 26S proteasomes at the
NH2 terminus. Goldfish cyclin
0 was digested by 26S proteasome for the indicated times and then stained with B63 or B112 antibody.
[View Larger Version of this Image (32K GIF file)]
).
). We then examined whether or not this proteasome digests goldfish cyclin B, like the goldfish proteasome can. Xenopus 26S, but not 20S proteasomes, digested
the NH2 terminus of goldfish cyclin B and produced the
42-kD intermediate (Fig. 6, A and C). NH2-terminal truncated cyclins were not digested by the Xenopus 26S proteasome (Fig. 6 B). These results indicate that the Xenopus
26S proteasome can digest goldfish cyclin B, suggesting a
similar role of goldfish and Xenopus proteasomes in the
regulation of cyclin degradation. We then examined the
involvement of 26S proteasome in cyclin B degradation
using a Xenopus cell-free system widely used for cell cycle
studies, which contains the complete system necessary for cyclin degradation (Murray et al., 1989
).
0 was completely degraded within 30 min after
adding Ca2+ to Xenopus egg extracts, although it was stable in the absence of Ca2+ (Fig. 7 A). NH2-terminal truncated cyclins
41 and
68 were not degraded in Xenopus
egg extracts even after activation with Ca2+ (Fig. 7 B). In
contrast to cyclins
41 and
68, the 42-kD cyclin fragment
(cyclin
57), which had been produced by the prior digestion of cyclin
0 with the purified 26S proteasome, was degraded in Xenopus extracts after adding Ca2+ (Fig. 8). To
exclude the possibility that the 42-kD cyclin fragment remained in complex with the NH2-terminal portion after digestion with the 26S proteasome, we performed gel chromatography under the buffer conditions that were used for
cyclin digestion. When cyclin treated with 26S proteasome
was chromatographed, 42-kD cyclin and NH2-terminal
fragment (~9 kD) were clearly separated (Fig. 9). This result indicates that there is no significant interaction between 42-kD cyclin and the NH2-terminal fragment after
digestion.
Fig. 7.
Degradation of goldfish cyclin B in Xenopus egg extracts. Cyclin B was detected with B63 antibody. (A) E. coli-produced goldfish cyclin 0 was added to Xenopus egg extract at a final concentration of 5 µg/ml. Incubations proceeded in the
absence (
Ca2+) or presence (+Ca2+) of 0.4 mM CaCl2 for the
indicated times. (B) E. coli-produced cyclin
0,
41, and
68
were added to Xenopus extracts at the final concentration of 5 µg/ml. Cyclin degradation was induced by 0.4 mM Ca2+ and terminated by adding SDS sample buffer at the indicated times.
[View Larger Version of this Image (45K GIF file)]
Fig. 8.
Degradation of intermediate cyclin B (cyclin 57) in
Xenopus egg extracts. Cyclin
57 was obtained by digesting cyclin
0 with 26S proteasome. 1/20 vol of the digestion mixture
containing cyclins
0 and
57 was added to Xenopus extracts,
and cyclin degradation was examined in the absence (
Ca2+) or
presence (+Ca2+) of 0.4 mM Ca2+. Samples were exposed to SDS
sample buffer at the indicated times. Cyclin degradation was assessed by immunoblotting with B63 antibody. The position of cyclin
57 is indicated by an asterisk.
[View Larger Version of this Image (41K GIF file)]
Fig. 9.
Separation of intermediate cyclin B and NH2-terminal
fragment by gel chromatography. (A) Sephadex G-50 column
chromatography. The 35S-labeled cyclin 0 was produced in vitro
in rabbit reticulocyte lysate. Digestion of 35S-labeled cyclin
0
was performed for 60 min at room temperature with (
) or without (
) 26S proteasome. Samples were then separated on Sephadex G-50 column (1.0 × 19.0 cm) in 100 mM Tris-HCl, 5 mM
MgCl2, pH 7.6. Fractions of 0.5 ml were collected. Arrows indicate the eluted positions of molecular weight standards as follows: 1, bovine serum albumin; 2, myoglobin; 3, ubiquitin; 4, total column volume (Vt). (B) SDS-PAGE analysis of gel chromatography fractions. Sephadex G-50 column chromatography fractions from 26S proteasome-treated cyclin
0 and untreated (Control) were separated by SDS-PAGE (15% gel) followed by
autoradiography on Imaging plates (Fuji Film). The positions of
the digested cyclin B is indicated by an asterisk, and the positions
of NH2-terminal portion of cyclin B is indicated by an arrowhead.
[View Larger Version of this Image (21K GIF file)]
0 translated in vitro was digested by the 26S , but not the 20S proteasome (Fig. 10 A), and degraded in Xenopus egg extracts (Fig. 10, C and D). NH2-terminal truncated cyclins
41 and
68 were resistant to 26S proteasome digestion
(Fig. 10 B) and to degradation in Xenopus egg extracts
(Fig. 10, C and D). Furthermore, the point mutant, cyclin
0K57R (in which the position of cleavage by the 26S proteasome, lysine 57, was converted to arginine), was neither
digested by the 26S proteasome nor degraded in Xenopus
extracts (Fig. 10, B-D).
Fig. 10.
Digestion and
degradation of in vitro translated cyclin B. The 35S-
labeled cyclins 0,
0K57R,
41, and
68 were produced
in vitro in rabbit reticulocyte
lysate. After the translation
of each cyclin, the lysate was
incubated in the presence of
100 µg/ml of cycloheximide at room temperature under
the indicated conditions.
The 35S-labeled proteins
were resolved by SDS-PAGE followed by autoradiography on Imaging plates
(Fuji Film). The position of
the digested cyclin B is indicated by an asterisk. (A) Digestion of full length cyclin B
by purified 20S and 26S proteasomes. The reticulocyte
lysate containing cyclin
0
was incubated with 60 µg/ml
of proteasomes. (B) Digestion of full length, point mutated, and NH2-terminal truncated cyclin Bs by purified
26S proteasome. The reticulocyte lysate containing cyclin
0,
0K57R,
41, or
68
was incubated in the absence
(
) or presence (+) of 60 µg/
ml of the 26S proteasome for
60 min. (C) Degradation of
cyclin B in Xenopus egg extracts. One ninetieth of the
lysate containing cyclin
0,
0K57R,
41, or
68 was
added to the Xenopus egg
extracts, and its degradation was induced by 0.4 mM Ca2+.
At the indicated times, the
reaction was terminated by
adding SDS sample buffer.
(D) The same sample as in C. Cyclin contents were quantified using an image analyzer
(BAS2000; Fuji Film).
[View Larger Version of this Image (36K GIF file)]
Discussion
41 and
68 that are indigestible by
the 26S proteasome are not degraded, whereas cyclins
0
and
57 digested by the 26S proteasome, are degraded.
These findings strongly suggest that the initial cutting of
the NH2-terminal region of cyclin B by 26S proteasome is
a prerequisite for the subsequent degradation that leads to the inactivation of MPF at the metaphase/anaphase transition.
), human (Lorca
et al., 1991
), and clam (Luca et al., 1991
) B-type cyclins
missing the first 90, 72, or 97 amino acids, respectively, and
clam (Luca et al., 1991
) and Xenopus (Kobayashi et al.,
1992
) A-type cyclins missing the NH2-terminal 60 or 62 amino acids are resistant to degradation. Each of these
truncated cyclins continuously activates cdc2, which prevents cells or cellular extracts from leaving mitosis. A truncated protein containing only the first 89 amino acids of
Xenopus cyclin B2 (B2Nt), including sequences essential
for cyclin degradation in other species, also inhibited cyclin degradation (Velden and Lohka, 1993
). These results
indicate interaction of the NH2-terminal portion of cyclin
with the destruction machinery.
68 that lacks the cutting site K57, cyclin
41 containing K57 was not cleaved,
indicating that the NH2-terminal region affords not only
the cutting site but also the interaction site necessary for
digestion by the 26S proteasome. This notion was confirmed by inhibiting the cyclin digestion with B2Nt that consists of the first 89 amino acid of Xenopus cyclin B2.
Cyclins
41 and
68 were neither digested by the 26S proteasome nor degraded in Xenopus egg extracts activated
by Ca2+, whereas cyclin
0 was digested at K57 by 26S
proteasome and degraded in the extracts. In addition, cyclin
57 produced by digesting cyclin
0 with the 26S proteasome, was degraded in the extracts. These results suggest that only cyclins that have undergone 26S proteasome
digestion at K57 can be degraded upon egg activation.
). The first evidence
that cyclin B degradation is mediated by ubiquitin-dependent proteolysis was provided by Glotzer et al. (1991)
.
Other support for the involvement of a ubiquitin-dependent pathway in the cyclin degradation arises from the observation that methylated ubiquitin, which prevents the
polyubiquitination of proteins destined for degradation,
delays cyclin degradation in an extract from clam embryos
(Hershko et al., 1991
). A complex containing cyclin-selective ubiquitin ligase activity has been identified in clam oocytes (Sudakin et al., 1995
). These findings suggest that the
cell cycle-specific cyclin degradation is mediated by a
ubiquitin-dependent proteolytic system.
57 was destroyed, whereas cyclin
68 was not, the lysine
residues between amino acids 58 to 68 constitute the most
likely ubiquitination site, and cutting by the 26S proteasome at K57 might be necessary to expose them to ubiquitinating enzymes. This notion should be verified by investigating the difference in the three-dimensional structure
of cyclins
0,
41,
57, and
68.
; Katsu et
al., 1993
; Yamashita et al., 1995
). As shown in this study
however, the 26S proteasome purified from immature goldfish oocytes can digest cyclin B. If the initial cleave of cyclin B by the 26S proteasome triggers cyclin destruction
as proposed in this study, the question remains why cyclin
B is stable during oocyte maturation and in mature oocytes but destroyed upon egg activation. Based on the results obtained from clam oocytes, Sudakin et al. (1995)
have suggested that the initiation of cyclin degradation is
triggered by ubiquitination caused by the activation of cyclin-selective ubiquitin ligase near the end of M-phase, which targets cyclin B for destruction by the 26S proteasome that is constitutively active during the cell cycle.
Contrary to this, we found that the 26S proteasome purified from mature goldfish oocytes cannot digest cyclin B
and that at least two subunits in 26S proteasomes from immature and mature oocytes differ (Tokumoto, T., Horiguchi, R., Nagahama, Y., unpublished results). These findings suggest that some inhibitory mechanisms preventing
cyclin B degradation proceed on the proteasome itself at
least during metaphase II arrest. The amount of the proteasome in egg cytosol also changes dramatically during
oocyte maturation and egg activation; the lowest level is in
mature metaphase II-arrested oocytes, and there is a transient increase between the first and second meiotic cell cycles and upon egg activation in goldfish (Tokumoto et al.,
1993a
). Further studies should reveal how the subunit
composition of proteasomes and their contents during oocyte maturation and egg activation are involved in controlling the cell cycle by regulating cyclin stability.
have shown that binding with cdc2
is necessary for the degradation of Xenopus cyclins A and
B2 but not for that of cyclin B1. This implies that the
mechanisms of cyclin degradation vary according to the
types of cyclins. Since goldfish cyclin B exhibits higher homology to Xenopus cyclin B1 (66%) than to cyclin B2
(50%), the mechanism of cyclin degradation by the 26S
proteasome proposed in this study may be specific to cyclin B1.
), we propose that cyclin destruction is
primarily controlled by the activity of 26S proteasome.
). A
cyclin-specific ubiquitin ligase complex, the cyclosome, or
APC complex, has been characterized in clam and Xenopus, respectively (King et al., 1995
; Sudakin et al., 1995
).
These ubiquitin ligases (E3) catalyze ubiquitination using
a specialized ubiquitin carrier protein (E2). Among the
multiple species of E2s, UBC9 is required for cell cycle progression in late G2 or early M-phase (Seufert et al.,
1995
). UBC4 protein can ubiquitinate cyclins in Xenopus
egg extracts (King et al., 1995
). Recently, a novel cyclin-
selective UBC family member, E2-C, was reported which
can ubiquitinate cyclin B(13-91)/protein A fusion protein
in cyclosome-dependent manner (Aristarkhov et al., 1996
).
These reports have shown that destruction box mutants cannot be ubiquitinated or degraded after extract activation, suggesting that the destruction box is a recognition
sequence for the ubiquitinating system. However, we have
shown that mutants that lack the proteasome cleavage site
cannot be degraded after extract activation. The relative
importance of these two processes is unclear because of
the discrepancy between in vivo and in vitro results. In
vitro, proteasome cleavage and ubiquitination of cyclin seem independent of each other. Destruction box-dependent ubiquitination of cyclin by purified proteins does not
depend on the previous proteasome cleavage of cyclin B,
and cyclin cleavage by purified proteasome does not depend on previous ubiquitination of cyclin B. But, since
mutations that block in vitro ubiquitination and mutations
that block proteasome cleavage also block destruction in
extracts, the simple conclusion is that the ubiquitination and proteasome cleavage are both necessary for cyclin destruction in extracts. There are, however, no experimental
data available at present that provide information on the
relative order of these two steps in vivo. Therefore, there
are three possible in vivo scenarios. (a) Proteasome cleavage precedes ubiquitination to expose an NH2-terminal
lysine that is a good substrate for ubiquitination. An extreme view would be that destruction box-dependent
ubiquitination is an artifact that plays no role in vivo and
that the purpose of the destruction box is solely to induce
the initial ubiquitin-independent cleavage of cyclin. (b)
Destruction box-dependent ubiquitination precedes proteasome cleavage to recruit cyclin to the proteasome by
virtue of the proteasome's polyubiquitin binding subunit.
An extreme view would be that proteasome cleavage is
not necessary in vitro, and mutants like K57R are having a
direct effect on destruction box-dependent ubiquitination.
(c) There is no required order of proteasome cleavage and
destruction box-dependent ubiquitination, although both
events would be necessary for efficient cyclin destruction, they could occur in either order. Further studies are necessary to understand the molecular mechanism of cyclin degradation, especially identification of the lysine residue that
is destined to be ubiquitinated.
Received for publication 9 September 1996 and in revised form 22 June 1997.
Please address all correspondence to Dr. Y. Nagahama, Laboratory of Reproductive Biology, National Institute for Basic Biology, Okazaki 444, Japan. Tel.: (81) 564-55-7550; Fax: 81-564-55-7556; E-mail: nagahama{at}nibb.ac.jpWe are grateful to Dr. M.J. Lohka for the B2Nt and to Drs. H.A. Bern and A.W. Murray for critical reading of the manuscript.
This study was supported in part by Grants-in-Aid for Scientific Research on Priority Areas from the Ministry of Education, Science, Sports, and Culture of Japan (07283104 to Y. Nagahama) and by a Grant-in-Aid (Bio Media Program) from the Ministry of Agriculture, Forestry, and Fisheries (BMP 96-II-2-6). This study was performed under the National Institute for Basic Biology Cooperative Research Program (95-112 to T. Tokumoto).
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