(Received for publication, March 4, 1997)
From the Unit of Biochemistry, the B. Rappaport Faculty of Medicine and the Rappaport Institute for Research in the Medical Sciences, Technion-Israel Institute of Technology, Haifa 31096, Israel and the § Institute for Cancer Research, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111
Previous studies have indicated that a ~1,500-kDa complex, designated the cyclosome or anaphase-promoting complex, has a regulated cyclin-ubiquitin ligase activity that targets cyclin B for degradation at the end of mitosis. The cyclosome is inactive in the interphase of the embryonic cell cycle and is converted to the active form in late mitosis in a phosphorylation-dependent process initiated by protein kinase Cdc2-cyclin B. We show here that the active, phosphorylated form of the cyclosome from clam oocytes binds to p13suc1, a protein known to associate with Cdc2. The following evidence indicates that the binding of the cyclosome to p13suc1 is not mediated via the Cdc2-cyclin B complex: (a) activated cyclosome binds to p13suc1-Sepharose following its separation from Cdc2-cyclin B by gel filtration chromatography; (b) cyclosome from interphase extracts, activated by a kinase in which cyclin B has been replaced by an N-terminally truncated derivative fused to glutathione S-transferase, binds well to p13suc1-Sepharose but not to glutathione-agarose. An alternative possibility, that the phosphorylated cyclosome binds directly to a phosphate-binding site of p13suc1, is supported by the observation that the cyclosome is efficiently eluted from p13suc1-Sepharose by phosphate-containing compounds. This information was utilized to develop a procedure for the affinity purification of the cyclosome. A factor abundant in the fraction not adsorbed to p13suc1-Sepharose stimulates the activity of purified cyclosome. It is suggested that binding of Suc1 may have a role in the regulation of cyclosome activity.
Recent studies have indicated that a large ~1,500-kDa complex, referred to as the "cyclosome" (1) or anaphase-promoting complex (APC)1 (2), plays an important role in the degradation of cyclin B at the end of mitosis. Cyclin B is the activating subunit of protein kinase Cdc2. The activation of Cdc2 is required for cells to undergo mitosis, while the inactivation of Cdc2, caused by the specific and regulated proteolysis of its cyclin B subunit, is essential for exit from mitosis (reviewed in Ref. 3). Studies in cell-free extracts that reproduce embryonic cell cycles showed that cyclin B is degraded by the ubiquitin pathway (4, 5), a system in which proteins are targeted for degradation by ligation to ubiquitin (reviewed in Ref. 6). Cyclin ubiquitinylation and degradation depends on a partially conserved 9-amino acid motif, the "destruction box," which is usually located ~40-50 amino acid residues from the N terminus of mitotic cyclins (4).
We have been studying the mechanisms of cyclin degradation by fractionation of a clam oocyte cell-free system (7). Three components were identified to be involved in the ligation of ubiquitin to cyclin B: the ubiquitin-activating enzyme E1, a specific ubiquitin-carrier protein E2-C (7, 8), and a cyclin ubiquitin ligase activity associated with particulate material. E1 and E2-C are constitutively active, but the particle-associated ligase activity is cell cycle-regulated; it is inactive in the interphase, but becomes activated at the end of mitosis (7). The particle-associated component was extracted with salt, partially purified, and found to be a ~1,500-kDa complex, the cyclosome (1). The cyclosome acts on both cyclin A and cyclin B and requires intact destruction box sequences of both cyclins. The activity of the cyclosome is regulated by reversible phosphorylation, as indicated by the findings that the active, mitotic form of the cyclosome can be converted to the inactive form by treatment with an okadaic acid-sensitive phosphatase (9) and that the inactive, interphase form of the cyclosome can be converted to the active form by incubation with protein kinase Cdc2-cyclin B (1, 9). Activation of the cyclosome by protein kinase Cdc2-cyclin B includes a time lag (1, 9), which may serve to prevent the premature inactivation of the kinase in the cell cycle. Based on these findings, we suggested the cyclosome has a regulated cyclin-ubiquitin ligase activity, which targets cyclin B for destruction at the end of mitosis (1).
A similar particle, termed the APC, was identified in Xenopus egg extracts by King et al. (2). By the use of antibodies that cross-react with Xenopus proteins, these investigators furthermore identified two subunits of the complex as homologues of the products of budding yeast CDC16 and CDC27 genes, which are required for exit from mitosis and the degradation of mitotic cyclins (10). Other subunits of the cyclosome/APC particle have been identified in a variety of organisms (11-16) and appear to be strongly conserved in evolution. The cyclosome is also involved in the degradation of anaphase inhibitor proteins, which contain destruction box sequences (17-19). The subunits of the complex responsible for its specific actions, as well as those responsible for the regulation of its activity, have not yet been identified.
To define the mode of action and mechanisms of regulation of the cyclosome, its extensive purification is essential. The Xenopus cyclosome/APC particle was purified by immunoprecipitation (2, 15), but such preparations are not suitable for biochemical studies. In the present report we describe the binding of the cyclosome to p13suc1 and its use for affinity purification. p13suc1 was originally identified in the fission yeast by its ability to suppress certain temperature-sensitive mutations of Cdc2 (20). A homologous protein in budding yeast, designated Cks, was found to bind strongly to the cyclin-dependent kinase (21). The Suc1/Cks family of proteins is essential for viability and is highly conserved in evolution (reviewed in Refs. 22 and 23), but its exact functions remained unknown. Genetic evidence in yeasts indicates multiple roles in the cell cycle, including entry into mitosis, exit from mitosis, and transition between G1 and S phases of the cell cycle (24, 25). Recent biochemical studies with immunodepleted extracts of Xenopus eggs further indicated that Suc1/Cks is required in at least two stages of the embryonic cell cycle: in the activation of the Cdc2-cyclin B complex by tyrosine dephosphorylation of Cdc2, and in exit from mitosis due to cyclin B degradation (26). The crystal structures of Suc1/Cks proteins (27, 28) and of their complex with Cdc2 (29) have been solved. In addition to the Cdk binding site they contain a highly conserved phosphate-binding site. It has been suggested (but not yet demonstrated) that the latter site may bind to some phosphorylated proteins, and thus Suc1/Cks may have a role in targeting Cdk-cyclin kinases to certain phosphorylated proteins (26, 29).
We show here that p13suc1 selectively binds the active, phosphorylated form of the cyclosome. Our evidence suggests that this binding is due to interaction of the cyclosome with the phosphate-binding site of p13suc1. This information was utilized to develop an affinity procedure for the purification of the cyclosome.
Ubiquitin from bovine erythrocytes, rcm-BSA, STI, and
p-nitrophenylphosphate (pNPP) were obtained from Sigma, and
okadaic acid was obtained from Boehringer Mannheim. Ubiquitin aldehyde was prepared as described (30). E1 was purified from human erythrocytes (31). Recombinant clam E2-C was expressed in bacteria as described (8)
and purified by gel filtration on Superdex 50. p13suc1 was
expressed in E. coli and purified by gel filtration as
described (32). p13suc1 was coupled to cyanogen
bromide-activated Sepharose-4B at a concentration of 11-13 mg of
protein/ml of swollen gel. For control beads, BSA was coupled to
Sepharose at a similar concentration. Glutathione-agarose was purchased
from Sigma (G-4510). Recombinant human GST-88-cyclin B was expressed
as described (33) and purified by affinity chromatography on
glutathione-agarose. The expression and purification of clam cyclin A
and its N-terminally truncated derivative (1, 34) and of N-terminal
fragments of sea urchin cyclin B fused to protein A (4, 17)
have been described previously. Proteins were
radioiodinated as described (4).
Extracts of M phase and interphase clam oocytes were prepared as described previously (7). Both types of extracts were subjected to fractionation on DEAE-cellulose (7), and the fraction not adsorbed to the column, fraction 1, was extracted with 0.25 M KCl, as described (1). This salt extract of fraction 1, which contains the cyclosome (1), served as the source for affinity purification.
Affinity Purification of CyclosomePrior to affinity
purification, the cyclosome was converted to the active form by
preincubation with ATP (1, 9) as follows. The incubation mixture
contained the following in a volume of 1.2 ml: 50 mM
Hepes-KOH (pH 7.2), 1 mM DTT, 1 mM
MgCl2, 0.5 mM ATP, 10 mM
phosphocreatine, 100 µg/ml creatine phosphokinase, 12 mg of protein
of salt extract of fraction 1 from M phase clam oocytes, and 1 µM okadaic acid. Following incubation at 18 °C for 60 min, the sample was mixed with an equal volume of a solution consisting
of 500 mM KCl, 40 mM Tris-HCl (pH 7.2) and 1 mM DTT and then added to 1.2 ml of
p13suc1-Sepharose beads. The beads had been washed previously
three times with 10-ml portions of buffer A (50 mM
Tris-HCl, pH 7.2, 0.25 M KCl, and 1 mM DTT).
The sample was mixed with beads at 0 °C for 60 min and then
transferred to a column (0.7 cm diameter) at 4 °C. The flow-through
fraction was collected, together with a 5-ml wash with buffer A. The
column was washed with 30 ml of buffer B (50 mM Tris-HCl
(pH 7.2), 250 mM KCl, 20% (v/v) glycerol, 1 mM
DTT, and 0.5% (v/v) Nonidet P-40) and then with 10 ml of buffer C (50 mM Tris-HCl, pH 7.2, 1 mM DTT, and 20% (v/v)
glycerol). The cyclosome was eluted either with 30 ml of 50 mM Tris-HCl, pH 9.0, containing 1 mM DTT and
0.2 mg/ml STI, or with 20 ml of 50 mM pNPP in 50 mM Tris-HCl, pH 7.2, 1 mM DTT, and 0.2 mg/ml STI. The addition of STI was necessary to prevent the adsorption of the
dilute enzyme to surfaces. STI was chosen as the carrier protein, since
it has a relatively low molecular mass (20 kDa), and thus it does not
interfere with the detection of cyclosome subunits, which are of larger
size, in SDS-polyacrylamide gel electrophoresis. Elution was at a flow
rate of 1-1.5 ml/min. When the pH 9 buffer was used for elution, the
eluate was collected to a tube containing Tris-HCl, pH 7.2 (0.1 M, final concentration), to decrease the pH. Both eluate
and flow-through fractions were concentrated with Centriprep-10
concentrators (Amicon), diluted at least 10-fold with 50 mM
Tris-HCl (pH 7.2) containing 1 mM DTT and concentrated
again to ~0.5 ml. Glycerol was added to 20% (v/v), and samples were
stored at 70 °C. p13suc1-Sepharose beads could be
regenerated by washing with 30 ml of 50 mM Tris-HCl (pH
9.0), followed by a wash with 30 ml of 1 M KCl. The beads
were stored in 50 mM Tris-HCl (pH 7.2) containing 0.02%
sodium azide.
GST-88-cyclin
B-Cdc2 was prepared by the incubation of interphase extracts of clam
oocytes (in which cyclin B is absent) with recombinant GST-
88-cyclin
B and ATP, followed by the purification of the active kinase. The
reaction mixture contained the following in a volume of 3.6 ml: 15 mg/ml protein of extract of interphase clam oocytes, 0.2 mg/ml
GST-
88-cyclin B, and other ingredients similar to those described
above for preincubation with ATP and okadaic acid, except that the
concentration of MgCl2 was 5 mM. Following
incubation at 18 °C for 60 min, the formation of active kinase was
verified by an assay of histone H1 kinase activity. It was found that
H1 kinase activity rose from very low initial levels (~0.5 units/µg
of protein) to levels comparable with those in mitotic extracts (~60
units/µg of protein). The kinase was then purified by adsorption to
p13suc1-Sepharose, elution with free p13suc1, and
removal of free p13suc1 by gel filtration, as described
(7).
Reaction mixtures contained the following in a volume of 10 µl: 40 mM Tris-HCl (pH 7.6), 1 mg/ml rcm-BSA, 1 mM DTT, 5 mM MgCl2, 10 mM phosphocreatine, 50 mg/ml creatine phosphokinase, 50 µM ubiquitin, 1 µM ubiquitin aldehyde, 1 pmol of E1, 5 pmol of E2-C, 1 µM okadaic acid, enzyme source as specified, and 1-2 pmol (~105 cpm) of 125I-labeled cyclin B-(13-91)/protein A. This N-terminal fragment of cyclin B has been shown to be a suitable substrate for destruction box-specific, cell cycle-regulated ubiquitinylation and degradation of mitotic cyclins (1, 4, 7). When enzyme activity associated with Sepharose beads was determined, samples were agitated during incubation. Following incubation at 18 °C for 1 h, the samples were subjected to electrophoresis on a 12.5% polyacrylamide-SDS gel. Results were quantified with a PhosphorImager (Molecular Dynamics). The amount of radioactivity in all cyclin-ubiquitin conjugates was expressed as the percentage of total radioactivity in each lane (1). Reactions were conducted in the range linear with enzyme concentration, which was 5-40% 125I-cyclin ligated to ubiquitin. One unit of ligase activity was defined as that converting 1% 125I-cyclin to ubiquitin conjugates under the conditions described above.
Miscellaneous AssaysActivity of protein kinase cyclin B-Cdc2 was measured by the phosphorylation of histone H1 following adsorption to p13suc1-Sepharose. Virtually all such activity in extracts of meiotic M phase clam oocytes is due to cyclin B-Cdc2 complexes (data not shown). Histone H1 kinase assays were conducted as described (35), except that the concentration of ATP was 300 µM, and reactions were conducted at 18 °C. One unit of enzyme activity is defined as that causing the incorporation of 1 pmol of phosphate into histone H1 under these conditions. Protein concentration was determined with the Bio-Rad assay, using bovine serum albumin as standard. To estimate the amount of protein bound to beads, proteins were first eluted from beads by mixing with 4 M guanidine hydrochloride for 1 h at room temperature.
p13suc1 from fission yeast and homologous Cks proteins from other organisms bind strongly to Cdc2 and to some other members of the Cdk family of proteins (22, 23). p13suc1-Sepharose beads are therefore commonly used to isolate Cdk-cyclin complexes (36). In preliminary experiments, we found that the active form of the cyclosome2 bound tightly to p13suc1-Sepharose. We then tried to examine the nature of the interaction between p13suc1 and the cyclosome and to exploit this binding for the affinity purification of the cyclosome.
In the experiment shown in Fig. 1, a crude fraction from
M phase clam oocytes (1) was preincubated with ATP, or not
preincubated, and then was applied to p13suc1-Sepharose beads.
Such M phase extracts are made about 10-15 min before the anaphase of
meiosis I (7), when protein kinase Cdc2-cyclin B is already active but
the cyclosome is still inactive. When such extracts are incubated in
the presence of ATP and okadaic acid, the cyclosome becomes activated
by endogenous protein kinase Cdc2-cyclin B (1, 9). Following mixing of
preincubated extracts with p13suc1-Sepharose or control beads,
the beads were thoroughly washed, and cyclin-ubiquitin ligation
activity associated with the beads or remaining in the supernatants was
assayed as described under "Experimental Procedures."
Cyclin-ubiquitin conjugates are the ladder of bands of molecular size
higher than free 125I-cyclin. As shown in Fig. 1, without
preincubation, very little cyclin-ubiquitin ligation activity was
associated with p13suc1-Sepharose beads (lane 1),
and most of it remained in the supernatant (lane 4).
Following preincubation of extracts with ATP, considerable activity of
cyclin-ubiquitin ligation was associated with p13suc1-Sepharose
beads (lane 2), and it disappeared from the supernatant almost completely (lane 5). These findings suggest that the
conversion of the cyclosome to the active form increases its affinity
to p13suc1. Although almost all ligase activity disappeared
from the supernatants of preincubated extracts, only 15-25% of the
activity was recovered associated with p13suc1-Sepharose beads.
Binding of cyclosome was apparently specific for
p13suc1-Sepharose, because under similar conditions, there was
no significant binding to control (BSA-Sepharose) beads (Fig. 1,
lane 3), and all activity remained in the supernatant
(lane 6).
Mode of Binding of Cyclosome to p13suc1
We have considered several alternative possibilities to account for the binding of the cyclosome to p13suc1. It is possible that binding is not direct but that the cyclosome binds Cdc2-cyclin B, which in turn is bound to p13suc1. The cyclin B subunit of the Cdc2-cyclin B protein kinase complex is a substrate of the cyclosome for ubiquitin ligation, and thus cyclin B-Cdc2 may be tightly bound to the active site of the ligase. The Cdc2-cyclin B protein kinase is also an activator (although not necessarily a direct one) of the cyclosome, and it is possible that the kinase is tightly bound to site(s) of the cyclosome that it may phosphorylate. In both of these cases, p13suc1 would bind to the Cdc2 subunit of the Cdc2-cyclin B complex, which is tightly associated with the cyclosome. A third possibility is that p13suc1 may bind directly to the active, phosphorylated form of the cyclosome, possibly by its phosphate-binding site (27-29).
We have first examined the possibility that the cyclosome is bound to
p13suc1 via Cdc2-cyclin B by asking the question whether,
following activation of the cyclosome, the presence of Cdc2-cyclin B is
still required for binding to p13suc1-Sepharose. In the
experiment shown in Fig. 2, the cyclosome was first
converted to the active form by preincubation of M phase extract with
ATP and then was separated from Cdc2-cyclin B by gel filtration on
Superose-6 in the presence of salt. As shown previously (9), this
procedure separates the active cyclosome (~1,500 kDa) from most of
Cdc2-cyclin B (~100 kDa). Following gel filtration, the binding of
each fraction to p13suc1-Sepharose beads was examined. It may
be seen in Fig. 2 that the profile of cyclin-ubiquitin ligation
activity adsorbed to p13suc1-Sepharose closely followed that
before adsorption. The recovery of activity was about 20% in each
fraction, regardless of the amount of residual Cdc2-cyclin B. Thus, for
example, cyclin-ubiquitin ligase activity bound to p13suc1
beads in fractions 20-22, which had no detectable Cdc2-cyclin B kinase
activity, to an extent similar to that in fractions 24-26, which
contained a low amount of residual kinase activity. In all fractions,
cyclin-ubiquitin ligase activity was completely removed from the
supernatants following adsorption to p13suc1 beads (data not
shown). These findings suggest that following the activation of the
cyclosome, the continued presence of Cdc2-cyclin B is not required for
the binding of the cyclosome to p13suc1.
It is possible that the active form of the cyclosome is associated with
a tightly bound molecule of Cdc2-cyclin B, which is below the detection
limit of our kinase assay and is not dissociated during gel filtration.
We therefore continued to examine this problem by another approach,
using interphase extracts. In interphase extracts (prepared from
emetine-arrested two-cell clam embryos; Ref. 7) cyclin B is mostly
degraded, levels of protein kinase Cdc2-cyclin B are about 100-fold
lower than in M phase extracts, and the cyclosome is inactive. The
cyclosome can be converted to the active form by incubation of
interphase extracts with protein kinase Cdc2-cyclin B and ATP (1, 7).
In the experiment shown in Table I, interphase extract
was incubated with a derivative of Cdc2 kinase containing recombinant
cyclin B that lacked the N-terminal 88 amino acid residues and was
fused to glutathione S-transferase (GST). This truncated
derivative of cyclin B can form active protein kinase with Cdc2, which
can activate the cyclosome, but it cannot be ubiquitinylated by the
cyclosome because it lacks the "destruction box" region at the
N-terminal part (4, 34). N-terminally truncated cyclin B does not
interfere with cyclin-ubiquitin ligation, even at high concentrations
(Ref. 4 and see below), so it presumably cannot bind to the active site
of the cyclosome involved in cyclin-ubiquitin ligation. Following
incubation of interphase extract with ATP and without added protein
kinase Cdc2, only a small amount of cyclin-ubiquitin ligase activity
was found to be bound to p13suc1-Sepharose beads, which was
presumably due to the action of the low amounts of Cdc2-cyclin B
(assayed as H1 kinase) that remain in such extracts. After incubation
of interphase extracts with protein kinase GST-cyclin B-Cdc2, which
activates the cyclosome, there was a marked increase in the amount of
cyclin-ubiquitin ligation activity associated with
p13suc1-Sepharose beads (Table I). This was accompanied by a
complete disappearance of cyclin-ubiquitin ligase activity from the
supernatant (data not shown). This finding confirms the conclusion that
the active form of the cyclosome binds preferentially to
p13suc1. This observation also appears to be incompatible with
the notion that the cyclosome is bound to p13suc1 via cyclin B
bound to the active ubiquitination site, since 88-cyclin B is
neither a substrate nor a competitive inhibitor, and thus it cannot be
bound to the ubiquitination active site of the cyclosome.
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In this experiment, we have also taken advantage of the fact that
GST-88-cyclin B-Cdc2 can be bound to glutathione-agarose beads via
its GST moiety. If binding of the cyclosome to p13suc1 is
mediated by Cdc2-cyclin B bound to any site or subunit of the cyclosome, it would be expected that, following incubation of
interphase extracts with GST-
88-cyclin B-Cdc2, the cyclosome would
be bound to GSH-agarose beads along with the kinase. However, no
significant binding of cyclin-ubiquitin ligase activity to GSH beads
could be detected under conditions identical to those promoting
cyclosome binding to p13suc1-Sepharose (Table I). By contrast,
the binding of GST-
88-cyclin B-Cdc2 to GSH beads (assayed by H1
kinase activity) was as efficient as its binding to
p13suc1beads. The cumulative evidence from the above
experiments does not support the notion that Cdc2-cyclin B mediates an
indirect binding of the cyclosome to p13suc1. A direct binding
of active, phosphorylated cyclosome to the phosphate-binding site of
p13suc1 is suggested by the nature of compounds that promote
the elution of the cyclosome from p13suc1 beads, as described
below.
We have next examined different experimental conditions for the elution of the cyclosome from p13suc1 beads. The aim of these experiments was 2-fold: to gain an insight into the mode of the interaction of p13suc1 with the cyclosome and to establish experimental conditions for the affinity purification of the cyclosome. In the experiments shown in Table II, activated cyclosome was first bound to p13suc1-Sepharose, and then samples of the beads were suspended in various solutions. The elution of the enzyme was monitored both by the decrease of activity associated with beads and by its appearance in the eluate. At pH 7.2 and at low ionic strength, the enzyme was strongly bound to p13suc1-Sepharose beads, and no significant activity was eluted in this solution. Treatment with 300 mM KCl caused a partial loss of enzyme activity from beads, while 600 mM KCl caused a drastic loss of cyclin-ubiquitin ligase activity associated with p13suc1-Sepharose. However, this was not accompanied by a corresponding increase in enzyme activity in the eluate (Table II, experiment 1). It rather seems that high salt concentration causes the inactivation of cyclin-ubiquitin ligase. In contrast to the effects of KCl, 150 mM potassium phosphate caused significant elution of enzymatically active cyclosome from p13suc1-Sepharose (Table II, experiment 1). A similar elution was obtained with 150 mM potassium sulfate (data not shown). Phosphate and sulfate are known to bind to the anion-binding site of Cks/Suc1 proteins (27-29), and thus the results suggest that elution by these compounds may be due to competition with the cyclosome on binding to the anionic site of p13suc1.
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Under the conditions described above, elution of the cyclosome by phosphate and sulfate was only partial, and thus was not suitable for affinity purification. Higher concentrations of phosphate or sulfate caused the loss of enzyme activity, presumably due to inactivation by high ionic strength. We therefore searched for other phosphate compounds that may elute the cyclosome and found that pNPP at significantly lower concentration (50 mM), caused an almost complete loss from p13suc1 beads and a relatively high yield in the eluate (Table II, experiment 1). The role of the phosphate moiety of pNPP in elution was indicated by the lack of influence of p-nitrophenyl-glycerol (50 mM) on this process (data not shown). Efficient elution of the enzyme from p13suc1 beads was also obtained by raising the pH to 9.0 (Table II, experiment 2). In subsequent experiments we therefore used elution by either pH 9 or pNPP for affinity purification of the cyclosome.
It is notable that free p13suc1, at concentrations of 1-5 mg/ml, did not cause significant elution of cyclosome from p13suc1-Sepharose beads, although it eluted protein kinase Cdc2-cyclin B at high efficiency (data not shown). On the other hand, free p13suc1 did prevent the binding of the cyclosome to p13suc1-Sepharose beads. It might be that the binding of the cyclosome particle to the beads sterically hinders the access of free p13suc1 to the binding site.
The Fraction Not Adsorbed to p13suc1-Sepharose Contains Factor(s) That Stimulate the Activity of Affinity-purified CyclosomeAs noted above, although cyclin-ubiquitin ligase
activity disappeared from the supernatants following adsorption of the
cyclosome to p13suc1-Sepharose beads (Fig. 1), only 15-25% of
the activity was recovered associated with beads, and around 10% of
initial activity was recovered following elution with pNPP or pH 9 (Table I). We wondered, therefore, whether the enzyme was separated
from a stimulatory factor during affinity purification. Indeed, we
found that the addition of small amounts of the flow-through fraction
to the affinity-purified enzyme strongly stimulated cyclin-ubiquitin ligation activity (Fig. 3A). The extent of
the stimulation varied between 3- and 6-fold, depending on the
preparation of affinity-purified cyclosome. Similar stimulation of
activity by the flow-through fraction was observed with enzyme eluted
from beads by pH 9 buffer (Fig. 3A) or with pNPP or
phosphate (data not shown). When the flow-through fraction was
subjected to gel filtration chromatography on Superose-6, stimulatory
activity eluted in two peaks: a sharp peak at about 100 kDa and a
higher molecular mass broad peak at about 400-800 kDa (Fig.
3B). At least part of the higher molecular mass peak may be
an aggregate of the lower molecular mass factor, since when the higher
molecular mass peak was subjected to repeated separation on Superose-6
in the presence of 0.25 M KCl, part of activity was
converted to the lower molecular mass form (data not shown).
The mode of action of the stimulatory factor from the flow-through fraction is not known. It does not seem to be involved in the phosphorylation process responsible in the conversion of the interphase form of the cyclosome to the mitotic form (see "Discussion"). The purification and characterization of this factor is the subject of continued work. In the present study, the stimulatory activity of this factor was used for the detection of purified cyclosome at high sensitivity.
Purification of the Cyclosome by Affinity Chromatography and Gel FiltrationThe purification obtained by the affinity procedure is summarized in Table III. Affinity chromatography was carried out as described under "Experimental Procedures," using elution at pH 9. Cyclin-ubiquitin ligase activity of the cyclosome was followed in the various fractions in the presence or absence of the flow-through stimulatory factor. In addition, the amounts of active protein kinase Cdc2-cyclin B (assayed as H1 kinase) and of total protein were also estimated in the various fractions. As expected, the flow-through fraction contained most of protein and lacked appreciable cyclin-ubiquitin ligase and H1 kinase activities. About 5% of the initial protein was bound to p13suc1-Sepharose beads (before elution at pH 9), along with 26% of the cyclin-ubiquitin ligase, and essentially all H1 kinase activities were recovered. Most of the protein bound to p13suc1-Sepharose is probably phosphorylated, since without preincubation with ATP and okadaic acid, only about 2% of the initial protein was bound (data not shown). Elution with pH 9 buffer released most of the cyclin-ubiquitin ligase activity from p13suc1-Sepharose. However, only about one-third of the total protein that was adsorbed to the beads and a small fraction of the H1 kinase were eluted at pH 9, and the rest remained adsorbed to p13suc1-Sepharose beads. This relative selectivity in the elution of the cyclosome at pH 9 caused its enrichment in the eluate and its separation from most of protein kinase Cdc2-cyclin B. The recovery of cyclin-ubiquitin ligation activity in the pH 9 eluate was about 13% when assayed without the flow-through and 66% when assayed in the presence of the flow-through. Thus, the overall purification achieved by this procedure was around 30-fold when assayed in the presence of the flow-through stimulatory factor (Table III).
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It is notable that while the flow-through stimulated the activity of the cyclosome in the pH 9 eluate about 5-fold, it stimulated only slightly the activity of the enzyme bound to p13suc1 beads (Table III and see "Discussion"). It is also noteworthy that when assayed in the presence of the flow-through, the amount of cyclin-ubiquitin ligation activity in the pH 9 eluate was 2-fold higher than that associated with the p13suc1-Sepharose beads prior to elution (Table III). It might be that the catalytic efficiency of immobilized enzyme associated with beads is lower than that of the free cyclosome in solution or that immobilized enzyme is not accessible to added flow-through factor.
To further purify the cyclosome, the affinity-purified preparation was
subjected to gel filtration chromatography on Superose-6 (Fig.
4). The activity of cyclin-ubiquitin ligation was
assayed in the presence or absence of the flow-through stimulatory
factor. Following gel filtration, activity without the flow-through was very low and was stimulated more than 10-fold by the flow-through (Fig.
4A). The elution position of affinity-purified cyclosome was
similar to that observed previously with cruder preparations (1, 9),
with an apparent molecular size of about 1,500 kDa. This similarity in
size indicates that most of the cyclosome is bound to p13suc1
and is eluted as an entire complex, although the possible loss of some
loosely bound subunits cannot be ruled out (see "Discussion"). The
purity of the preparation was examined by silver staining. As expected,
the pH 9 eluate (Fig. 4B, OR lane) contained
numerous protein bands. However, many of these were removed from the
region at the cyclosome by the gel filtration procedure. While the
preparation following gel filtration is not homogenous, at least nine
protein bands in the subunit region of 60-200 kDa (marked by
dots in Fig. 4B) coincided with cyclosome
activity centered in fractions 22-24. Some of these protein bands may
be subunits of the clam cyclosome. We could not determine the extent of
further purification in the gel filtration procedure due to the very
low amounts of material obtained in this step.
Selectivity of Action of Affinity-purified Cyclosome
It was
important to examine whether the affinity-purified cyclosome
preparation retains selectivity for the destruction box of cyclin,
because a subunit of the complex responsible for the selectivity of
cyclin recognition may be lost or inactivated during purification. For
example, immunopurified APC/cyclosome from Xenopus eggs has
lost part of its destruction box selectivity (2). In the experiment
shown in Fig. 5, the action of affinity-purified cyclosome in the ligation to ubiquitin of different derivatives of
cyclin was compared with that of a crude preparation. As may be seen,
an N-terminal fragment of cyclin B consisting of amino acid residues
13-66 (that contains the destruction box; Ref. 4) was as effectively
ligated to ubiquitin by the pure enzyme as by the crude preparation
(Fig. 5, lanes 2 and 3). A similar construct, in
which the RAAL sequence in the destruction box has been scrambled to
AARL (17) is very poorly ligated to ubiquitin by both crude and pure
cyclosome preparations (Fig. 5, lanes 5 and 6).
In the latter case, only a small amount of the monoubiquitinylated
derivative was found, indicating a low affinity of the destruction box
mutant for the enzyme, resulting in poor processivity in the addition of multiple ubiquitin molecules. The selectivity of affinity-purified cyclosome for destruction box-containing cyclins was further examined by the competition of different unlabeled cyclin derivatives on the
ligation to ubiquitin of 125I-labeled cyclin B-(13-91)
fragment (Fig. 5, lanes 7-13). If the purified enzyme
retains destruction box selectivity, it is expected that only cyclins
that contain intact destruction box sequences would compete. It was
indeed found that while unlabeled cyclin B-(13-66) fragment strongly
inhibited cyclin-ubiquitin ligation by the purified cyclosome, the
corresponding AARL destruction box mutant did not (Fig. 5, lanes
9 and 10). Similarly, N-terminally truncated
derivatives of cyclin B and cyclin A, which lack the destruction box
regions, did not compete on cyclin-ubiquitin ligation by the purified
enzyme (Fig. 5, lanes 11 and 13), while
full-length cyclin A competed strongly (lane 12). These data
indicate that the affinity-purified cyclosome retains selectivity for
destruction box-containing cyclins, as observed previously with a
partially purified preparation (1).
This study was initiated by the observation that the active form
of the cyclosome binds strongly to p13suc1-Sepharose beads
(Fig. 1). Since the best known property of p13suc1 and of
homologous Cks proteins is their ability to bind Cdks (22, 23, 29), we
have first examined the possibility that cyclosome binding is mediated
via Cdc2-cyclin B, which is both a substrate of the enzyme and its
activator. We did not find evidence for such an indirect binding, since
activated cyclosome bound to p13suc1-Sepharose following its
separation from Cdc2-cyclin B (Fig. 2), and cyclosome from interphase
extracts activated by GST-88-cyclin B bound well to
p13suc1-Sepharose, but not to GSH-Sepharose (Table I). An
alternative possibility, that the cyclosome is bound directly to the
phosphate-binding site of p13suc1 (27-29) is suggested by its
elution by anions such as phosphate or sulfate and more effectively by
the phosphate ester p-nitrophenyl phosphate (Table II).
Since activation of the cyclosome is due to its phosphorylation (9,
15), the latter suggestion provides a straightforward explanation for
the observation that only the active form of the cyclosome binds to
p13suc1-Sepharose. A possible role of the anionic-binding site
of Cks proteins in their binding to phosphoproteins has been suggested (26, 29), but we are not aware of any previous instance in which this
has been shown to take place. It appears that many other phosphorylated
proteins may also bind to p13suc1-Sepharose, since incubation
of M phase extracts with ATP and okadaic acid, which promotes the
accumulation of phosphorylated proteins, caused a more than 2-fold
increase in the binding of total proteins to p13suc1-Sepharose
beads (data not shown).
Although many different (presumably phosphorylated) proteins bind to p13suc1-Sepharose, considerable purification of the cyclosome is obtained by the present affinity procedure. This is aided by the fact that only a part of the proteins bound to p13suc1 are eluted at pH 9, along with the cyclosome (Table III). Furthermore, the large size of the cyclosome allows its efficient separation from many other proteins in a subsequent step of gel filtration chromatography (Fig. 4). The final preparation, although not homogenous, appears to be highly purified (Fig. 4B). It does not contain significant amounts of Cdc2-cyclin B protein kinase activity, and no Cdk was detectable by immunoblotting with anti-PSTAIRE antibody (data not shown). It thus appears that this purification procedure may be valuable for future studies on the mode of action of the cyclosome and its regulation by protein kinase Cdc2-cyclin B.
In the course of this study, we have observed that the activity of
affinity-purified preparations of the cyclosome is greatly stimulated
by a factor abundant in the fraction not adsorbed to p13suc1-Sepharose. In gel filtration chromatography of this
flow-through fraction, stimulatory activity eluted in two peaks, the
higher molecular size of which may be an aggregate of the smaller
factor (Fig. 3). At present, we do not know the mode of action of this stimulatory factor; its purification and characterization are being
pursued in our laboratory. The stimulatory factor is apparently not a
protein kinase based on the following observations: (a) the
activity of the factor is not inhibited by staurosporin, an agent that
inhibits completely the activation of the interphase form of the
cyclosome by protein kinase Cdc2-cyclin B; (b) the factor
stimulates cyclosome activity also when ATP is replaced by the
nonhydrolyzable -
analog AMPPNP (data not shown). This ATP analog
is not a substrate for protein kinases, but it can be used in the E1
reaction (which involves the scission of the
-
bond of ATP) that
is needed for the cyclin-ubiquitin ligation assay. We have furthermore
found that the activity of the stimulatory factor was not affected by
phosphatase treatment, while affinity-purified cyclosome was
inactivated by phosphatase treatment (data not shown), as observed
previously with partially purified cyclosome (9). It thus seems that
the stimulatory factor is not involved in the protein kinase pathway
that converts the interphase form of the cyclosome to the mitotic
phosphorylated form; rather, it stimulates the activity of
phosphorylated cyclosome. The factor is also not involved in the
selective recognition of destruction box-containing cyclins, since
similar selectivity was observed in the absence of the factor (Fig. 5)
as in its presence (data not shown). The stimulatory factor may be an
easily dissociable subunit of the cyclosome, which is dissociated or
inactivated during enzyme purification. Such a subunit may be present
in both cyclosome-associated and free forms, as reported previously for
some subunits of the cyclosome from fission yeast (16) and budding
yeast (14). If the factor is a dissociable subunit, it may be still
present in cyclosome associated with p13suc1-Sepharose, the
activity of which is stimulated only slightly by the flow-through
fraction (Table III). Alternatively, it is possible that cyclosome
immobilized on beads is not accessible to added factor. Further work is
required to characterize this stimulatory factor. In the present study,
the use of the flow-through factor was essential to detect highly
purified cyclosome following gel filtration, and the activity was
almost completely dependent on the addition of the stimulatory factor
(Fig. 4A).
Although the present data bear directly only on the characterization of the binding of the cyclosome to p13suc1 and its use for affinity purification, they may also give a clue as to the possible role of p13suc1 in cyclin degradation. As noted above, genetic evidence in yeasts indicated that p13suc1 and other homologous Cks proteins are required at multiple stages of the cell cycle, including exit from mitosis and the degradation of cyclin B (24, 25). Biochemical experiments in immunodepleted extracts of Xenopus eggs also indicated that Cks is required for cyclin degradation (26), but these experiments could not distinguish whether Cks is required for the activity of this system or for the activation of the cyclin degradation machinery. We found no evidence to indicate that p13suc1 is required for the activity of the mitotic, phosphorylated form of the cyclosome (data not shown), but it may be involved in the activation process. Since p13suc1 can bind both protein kinase Cdc2 and the phosphorylated cyclosome, it is possible that it may direct the kinase to the phosphorylated cyclosome. Assuming that multiple phosphorylations are required for full activation of the cyclosome, initial phosphorylations may cause tighter binding of protein kinase Cdc2 to the cyclosome via p13suc1 and thus may accelerate the rate of further phosphorylations. Such a model may also account, at least in part, for the lag kinetics of cyclosome activation by protein kinase Cdc2-cyclin B. Work in our laboratory is now aimed at examining the possible role of Suc1 protein in the regulation of cyclosome activity.
We thank Drs. Irwin A. Rose and Joan V. Ruderman for help and advice. The skillful technical assistance of Clara Segal and the expert secretarial help of Mary Williamson and Marie Estes are gratefully acknowledged. Thanks are due to Edward Enos and the collecting staff of the Marine Resources Facility at the Marine Biology Laboratory (Woods Hole, MA) for expert and enthusiastic help in the collection and maintenance of clams.