From the Department of Molecular Biology, Graduate School of Medical Science, Kyushu University, Fukuoka 812-8582, Japan
Received for publication, December 11, 2000, and in revised form, January 30, 2001
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ABSTRACT |
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Mitotic cyclins A and B contain a
conserved N-terminal helix upstream of the cyclin box fold that
contributes to a significant interface between cyclin and
cyclin-dependent kinase (CDK). To address its contribution
on cyclin-CDK interaction, we have constructed mutants in conserved
residues of the N-terminal helix of Xenopus cyclins B2 and
A1. The mutants showed altered binding affinities to Cdc2 and/or Cdk2.
We also screened for mutations in the C-terminal lobe of CDK that
exhibited different binding affinities for the cyclin-CDK complex.
These mutations were at residues that interact with the cyclin
N-terminal helix motif. The cyclin N-terminal helix mutations have a
significant effect on the interaction between the cyclin-CDK complex
and specific substrates, Xenopus Cdc6 and Cdc25C.
These results suggest that the N-terminal helix of mitotic cyclins is
required for specific interactions with CDKs and that to interact with
CDK, specific substrates Cdc6 and Cdc25C require the CDK to be
associated with a cyclin. The interaction between the cyclin N-terminal
helix and the CDK C-terminal lobe may contribute to binding specificity
of the cyclin-CDK complex.
Eukaryotic cell cycle progression is regulated by the activity of
cyclin-dependent kinases
(CDKs)1 bound to an
activating cyclin subunit. CDK activation is temporally controlled by
association with specific cyclins during the cell cycle (1). Cyclin A
is found to form a complex with Cdk2 in S phase and with Cdc2 in
G2-M phase. Cdc2 binds to cyclin A or cyclin B, and Cdk2
binds to cyclin A or cyclin E. Recent work has shown that the cyclin
subunit plays a major role in substrate recognition by the cyclin-CDK
complex (2, 3). A single CDK associated with different cyclins can
recognize different substrates (4-6). In the complex, a conserved
hydrophobic patch, which includes residues from the degenerate
MRAIL sequence in the cyclin box fold (CBF), serves as a docking
site on the cyclin A molecule for the RXL motif present in certain
substrates (7). Similarly, the VxCxE sequence present in D
cyclins is a putative Rb-interacting motif (8, 9).
The structure of human Cdk2 complexed with residues 173-432 of cyclin
A is known (10, 11). The cyclin-CDK complex is primarily stabilized by
interactions between the cyclin N-terminal CBF and the PSTAIRE
motif in the N-terminal lobe of the CDK (10). In addition to the two
CBFs, cyclin A has another N-terminal Both cyclin A and cyclin B have the conserved N-terminal helix in their
N-terminal domains. We have shown previously that deletion of the
N-terminal helix of cyclin A1 abolishes both cyclin A1 binding to and
activation of a CDK (17, 18). Several reports have shown that cyclin A
and cyclin B behave differently during mitotic events and have distinct
functions in the cell cycle (4, 19-24). These different functions may
depend upon the sequences upstream of the CBF (25-27).
To further understand structure-function relationships of the cyclin A-
and cyclin B-CDK complexes, we have extended our mutational analysis of
the N-terminal helix of mitotic cyclin B to the N-terminal helix of
cyclin A and to the interacting site of this N-terminal helix on the
CDK subunit. In this study we have generated a series of mutations in
the N-terminal helix of cyclin B and of cyclin A that alter the ability
of the cyclin to bind to Cdc2 and Cdk2. We have also identified
specific mutations in the C-terminal lobe of Cdc2 and Cdk2 both by
genetic screens suppressing the toxicity of vertebrate cyclin
expression in yeast and by site-directed mutagenesis. We show here that
the N-terminal helix of cyclin B, as well as that of cyclin A, is
required for specific interactions between cyclin and CDK, because
these mutations prevented the formation of a complex between the cyclin
and a CDK subunit. Furthermore, we found that the association of cyclin
A/B with a preferred substrate Cdc6/Cdc25C, respectively, was impaired
by mutations in the N-terminal helix. The N-terminal helix of mitotic
cyclins may contribute to the binding specificity of the
cyclin-CDK complex through interaction with the CDK C-terminal lobe.
Construction of Cyclin and CDK Mutants by in Vitro
Mutagenesis--
Internal deletions of Xenopus cyclin B2
were constructed as described previously (17). Xenopus
cyclin B2 S90A in pGEM1 was cleaved at the unique NcoI site
between Ala90 and Pro91 and then the linearized
cyclin B2 was digested by Bal31 endonuclease to make a series of
internal deletions. The DNA was repaired with T4 DNA polymerase,
ligated with T4 DNA ligase, and introduced into
Escherichia coli strain TG1. Mutant DNA was then
prepared from individual colonies and sequenced. Selected mutants were transcribed using an in vitro system (Stratagene) and
translated in a rabbit reticulocyte lysate system (RPN3151;
Amersham Pharmacia Biotech).
[35S]Methionine-labeled products were analyzed by
SDS polyacrylamide gel electrophoresis (SDS-PAGE).
A series of point mutants of Xenopus cyclin A1 and cyclin B2
were constructed according to the Kunkel method (28). The
myc-tagged cyclin A1 mutants were constructed by
subcloning from cyclin A1 mutants in pGEM1 to myc-tagged
cyclin A1 in pGEM1. Xenopus Cdc2 and Cdk2 mutants were
constructed by polymerase chain reaction-based mutagenesis
(29).
Isolation of Suppressor Mutations in CDC28 in Budding Yeast That
Suppress the Growth Toxicity Caused by Xenopus Cyclin
Expression--
Saccharomyces cerevisiae 15Dau carrying
Xenopus wild-type cyclin B2 or cyclin A1 in pNV7 were
incubated on minimal plates containing galactose at 26 °C for about
10 days. Colonies growing at 26° C but not at 37 °C were isolated
as temperature-sensitive suppressor mutants (30). Colonies on galactose
plates harboring the vector pYS91 that contains a galactose-inducible
actin gene ACT1, whose expression is lethal to the wild-type
strain, were excluded as mutants in galactose-promoter function. Tetrad
analysis was employed to show that both suppression of growth toxicity and temperature sensitivity for the growth are caused by a mutation of
a single locus. To identify the suppressor gene, each
temperature-sensitive strain was transformed with a yeast genomic
library contained in the YCp50 vector. Colonies that were no longer
temperature-sensitive for growth and that were sensitive to
Xenopus cyclin expression were isolated. After the plasmid
DNA was recovered from these colonies, the minimal length of DNA
required for complementation was determined and sequenced to identify
the mutated gene. Among them, only cdc28 mutations were
isolated as suppressors of Xenopus cyclin expression. The
mutation sites in the CDC28 gene of the suppressor strains
were determined by the gap-repair method (31).
In vitro Translation, Immunoprecipitation, and
p13suc1 Bead Assay--
Xenopus CSF extracts
and interphase extracts were prepared by the standard method (26, 32).
An mCAPTM RNA capping kit (Stratagene) was used for
in vitro transcription of cyclins and CDKs and their
mutants. 0.5 µCi of [35S]methionine and mRNAs to a
final concentration of 0.1 µg/µl were added to 10 µl of
RNase-treated or non-treated Xenopus egg extracts containing
50% rabbit reticulocyte lysate. After incubating at 23 °C for 90 min, 1 µl of samples was analyzed by SDS-PAGE and autoradiography.
For immunoprecipitation, reactions (10 µl) were diluted with 300 µl
of bead buffer (50 mM Tris-HCl, pH 7.4, 5 mM
NaF, 250 mM NaCl, 5 mM EDTA, 5 mM
EGTA, 0.1% Nonidet P-40, 1 µg/ml leupeptin, 2 µg/ml aprotinin, 10 µg/ml soybean trypsin inhibitor, 15 µg/ml benzamidine) and
incubated with anti-Cdc2, anti-Cdk2, anti-cyclin B2, or
anti-myc antibody at 4 °C for 1 h with rotation.
Protein A-Sepharose beads were added and then incubated at 4 °C for
1 h with rotation. The beads were recovered after washing with
bead buffer three times. [35S]-labeled cyclins or CDKs
were detected by SDS-PAGE and autoradiography. For precipitation with
p13suc1 beads, reactions (10 µl) were diluted with 300 µl of bead buffer, mixed with p13suc1 beads, and
incubated at 4 °C for 1 h with rotation. The beads were washed
with bead buffer three times and then [35S]-labeled
cyclins were detected by SDS-PAGE and autoradiography.
Pull-down Assay with GST-XCdc6 Protein--
Xenopus Cdc6 (a gift
from J. Blow) in pGEX-KG was transformed into E. coli BL21
cells. Protein expression was induced by addition of 1 mM
isopropyl-1-thio-
For GST pull-down assays, Xenopus cyclin and mutant cyclin
mRNAs were in vitro-translated with
[35S]methionine in the RNase-treated CSF/reticulocyte
lysate mixture (1:1). 10 µl of this reaction mixture diluted with 200 µl of ELB+ was incubated with 2 µg of either GST-XCdc6
or GST for 1 h on ice. GSH-agarose beads were then added,
incubated for 2 h at 4 °C with rotation and washed three times
with ELB+, and bound proteins were separated by SDS-PAGE.
The gel was dried and exposed to x-ray film.
Phosphorylation of Histone H1, XCdc6, and
XCdc25C--
Xenopus cyclin A1 mRNA was translated in
the RNase-treated CSF/reticulocyte mixed extracts (1:1) with
[35S]methionine at 23 °C for 2 h. In the case of
Xenopus cyclin B2, the CSF extract was replaced with
interphase extracts (9:1). 1 µl of the reaction were incubated with 9 µl of reaction mixture (20 mM Hepes, pH 7.8, 15 mM MgCl2, 5 mM EGTA, 1 mM dithiothreitol, 0.2 mg/ml bovine serum albumin, 0.2 mM ATP, 0.5 mCi/ml [
For histone H1 kinase assay with the precipitated cyclin-CDK complex,
mRNAs of Xenopus cyclin mutants were cotranslated with Cdc2 or Cdk2 mRNA in RNase-treated and Ca2+-treated
CSF/reticulocyte mixed extracts (1:1) with
[35S]methionine at 23 °C for 2 h. Cyclin mutants
were immunoprecipitated with anti-Cdc2 or anti-Cdk2 antibody and
protein A-Sepharose beads. Beads were washed with bead buffer three
times and then incubated with the reaction mixture (20 mM
Tris-HCl, pH 7.5, 7.5 mM MgCl2, 0.2 mM ATP, 10 µCi [ Antibodies--
Anti-Cdc2 monoclonal (A17) and anti-cyclin B2
monoclonal (X121) antibodies were provided by T. Hunt, and anti-Cdk2
antibody was provided by N. Sagata. Anti-c-myc antibody
(A14) was purchased from Santa Cruz Biotechnology.
The N-terminal helix in cyclin B2 is required for binding to
Cdc2
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-helix upstream of the
CBF. In the cyclin A-CDK complex, the cyclin N-terminal helix faces to
the CDK C-terminal lobe and is topologically separated from the other
ten
-helices that compose the CBF (11) (see Fig. 1A).
Based on structural analysis (11-15), the cyclin N-terminal helix is
thought to be an independent structural unit that may be
important for cyclin-CDK interaction (16). However, to date there is no
experimental evidence supporting the role of the N-terminal helix of cyclin.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-D-galactopyranoside for 8 h at
26 °C. The bacteria were lysed in ELB+ (50 mM Hepes, pH 7.0, 250 mM NaCl, 0.1% Nonidet
P-40, 5 mM EDTA, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml aprotinin, and
1 µg/ml leupeptin) and sonicated. After centrifugation, the
supernatant was incubated with glutathione-agarose beads, and bound
proteins were eluted with 10 mM glutathione (pH 7.0). Xenopus Cdc25C (a gift from N. Sagata) in pGEX-KG was
prepared similarly.
-32P]ATP, 0.1 µg/µl histone H1) at 23 °C for 30 min. The samples were analyzed
by SDS-PAGE and autoradiography. In the case of assay of
Xenopus Cdc25C phosphorylation, GST-XCdc25C (0.1 µg/µl) was replaced by histone H1 and assayed for phosphorylation as described above.
-32P]ATP, 0.1 mg/ml
histone H1) at 23 °C for 30 min. Samples were analyzed by SDS-PAGE
and autoradiography. XCdc6 phosphorylation was assayed similarly,
except that 4myc-Cdc2 and mutant cyclin A were cotranslated
and that GST-XCdc6 (0.1 µg/µl) was replaced by histone H1 as substrate.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
The cyclin N-terminal helix is well conserved in all mitotic A- and B-type cyclins (Fig.
1B). To explore its role in
the cyclin-CDK interaction, we first made internal deletions within
Xenopus cyclin B2 and tested the ability to bind to Cdc2 in
Xenopus egg extracts by coimmunoprecipitation with anti-Cdc2
antibody. Fig. 2A summarizes our results, which show that B2 cyclins that lack the N-terminal helix
(
86-131 and
73-156) are unable to bind to Cdc2, whereas B2
cyclins with smaller deletions (
87-96,
86-110, and
87-122) bind to Cdc2. Therefore, there is a possibility that the sequence upstream of the N-terminal CBF that includes the sequence encoding the
N-terminal helix is required for Cdc2 binding.
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Fig. 1.
Sequence alignment of the N-terminal helix
motif in mitotic cyclins. A, schematic drawing of the
location of the cyclin N-terminal helix in the cyclin-CDK complex. The
cyclin N-terminal helix is shown by a helical bar. Note that
the crystal structure of the human cyclin A-CDK2 complex was determined
using an N-terminally truncated (residues 173-432) human cyclin A
(10-11). B, N-terminal helix motif sequences.
Xenopus cyclins B2 and A1 tested in this work are shown at
the top of the sequence alignment. Bold letters
highlight conserved residues in either cyclin A or cyclin B. Identical
residues in the N-terminal helix of cyclins A and B are indicated by
dots. An asterisk denotes the conserved glutamic
acid residue (see also Fig. 3B). Residue numbers of the
Xenopus B2 and A1 cyclins and the human cyclins are shown at
the top and the bottom of each sequence
alignment, respectively. D-box denotes the destruction
box.
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Fig. 2.
Analysis of a series of mutants of the
N-terminal helix in Xenopus cyclin B2.
A, internal deletions of the N-terminal domain. Cdc2 binding
was tested by coimmunoprecipitation with anti-Cdc2 antibody. The
results are summarized at the right of figure. Cyclin box
mutant R163A was used as a negative control. B,
alanine-scanning mutants of the N-terminal helix. Binding to Cdc2 is
shown at the right of figure. C, Cdc2 binding
assay of the N-terminal helix mutants. Examples of the series of
alanine-scanning mutants are shown. [35S]-labeled protein
was translated from each mRNA in the extracts as described under
"Experimental Procedures" (Translation), and the ability
to bind to Cdc2 was tested by p13suc1 bead assay
(Binding). WT, wild-type. D,
activation of histone H1 kinase activity by the N-terminal helix
mutants. Each mutant was produced in the nuclease-treated extracts and
assayed for histone H1 kinase. Relative levels of the activity are
shown in the histogram, in which the activity of each mutant was
normalized by subtraction with a negative control.
Next we examine whether the conserved residues in the cyclin B2 N-terminal helix are actually required for Cdc2 binding. To do this, we generated a series of alanine mutants of these residues and tested their ability to bind to Cdc2. Mutations in the conserved residues in the cyclin B2 N-terminal helix (Y131A, I135A, Y136A, Y138A, L139A, and E143A), with the exception of V132A and D134A, yielded proteins that were unable to bind to Cdc2 (Fig. 2, B and C). In contrast, mutants both upstream (D121A, L127A, and S129A) and downstream (Y152A and L153A) of the N-terminal helix bound Cdc2. The cyclin box mutant R163A, which was used as a control, was unable to bind Cdc2. From this analysis we concluded that the N-terminal helix in cyclin B2 is required for binding to Cdc2.
To determine whether the cyclin N-terminal helix mutants were able to activate CDK activity, mRNAs encoding mutant cyclins were translated in nuclease-treated interphase extracts, and phosphorylation of histone H1 was assayed. Fig. 2D shows that those N-terminal helix mutants that failed to bind Cdc2 also little activate histone H1 kinase. Four of the mutants tested are at residues in the N-terminal helix of cyclin B (Tyr131, Ile135, Tyr138, and Glu143) that are highly conserved in all known mitotic cyclins (see Fig. 1B).
Comparison of the Abilities of the Cyclin A and B N-terminal
Helices to Bind to CDK--
It has been demonstrated previously that
the N-terminal deletions of cyclin A abolish CDK binding (17-18). To
assess the requirement for conserved residues in the cyclin A
N-terminal helix for CDK binding, we constructed a series of alanine
mutants of Xenopus cyclin A1 and assayed the ability to bind
Cdc2. Fig. 3A shows a list of
the constructs and a summary of binding, and Fig. 3B shows a
representative binding assay. Mutants in the N-terminal helix of cyclin
A1 (Y164A, I168A, and E176A) with one exception (E167A) failed to bind
to Cdc2 (see also Fig. 4). In control
experiments, mutants outside the N-terminal helix (V159A, S162A, Y185A,
and M186A) bound Cdc2. The results using the cyclin A1 mutants
paralleled our earlier results with mutants of cyclin B2.
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The cyclin A1 mutants Y171A and L172A behaved differently in Cdc2 binding assay to the equivalent cyclin B2 mutants Y138A and L139A. Both Y171A and L172A mutants bound normally to Cdc2, whereas the equivalent cyclin B2 mutants did not. To confirm this difference, we used nuclease-treated egg extracts, from which endogenous cyclin mRNAs had been eliminated to translate the cyclin, and tested the binding to endogenous Cdc2 (Fig. 3C). Cdc2 bound to the cyclin A1 Y171A and L172A mutants (lanes 2 and 3) but did not bind to the equivalent cyclin B2 mutants Y138A and L139A (lanes 6 and 7). Therefore, Tyr138 and Leu139 in the N-terminal helix of cyclin B2 are essential for the interaction with Cdc2, but the equivalent residues in cyclin A1 (Tyr171and Leu172) are not required for the cyclin A1-Cdc2 interaction.
The cyclin A1 N-terminal helix mutants possess residual histone H1 kinase activity in accordance with the binding ability, when assayed in the nuclease-treated extracts (Fig. 3D, Y164A and I168A). Therefore, in contrast to our results with mutants in the N-terminal helix of cyclin B, the N-terminal helix mutants of cyclin A1 can, but not fully, activate histone H1 kinase. This result suggests that these mutations cause a decrease in the stability of the cyclin-CDK complex.
A Mutant in the N-terminal Helix of Cyclin A1, E167A, Can Bind to Cdc2 But Not to Cdk2-- Cyclin A binds to both Cdc2 and Cdk2. To test whether a specific residue in the N-terminal helix of cyclin A can affect the ability of cyclin A to bind to Cdc2 and Cdk2, we compared the abilities of cyclin A1 mutants to bind to Cdk2 and Cdc2. Because the level of endogenous Cdk2 is relatively low in the extracts used in the assay, Cdk2 protein was expressed from its mRNA to increase its concentration to a similar level to that of the endogenous Cdc2. We then tested a series of alanine mutants of cyclin A1 for the ability to bind to Cdk2 by immunoprecipitation with anti-Cdk2 antibody (Fig. 3, A and B). The mutants in the cyclin A1 N-terminal helix that failed to bind to Cdc2 also abolished cyclin A1 binding to Cdk2 (Fig. 3B, see I168A). However, one mutation, E167A, showed greatly reduced affinity for Cdk2 but still bound Cdc2 (denoted by an asterisk in Fig. 3B). The cyclin A1 mutant Y164A also showed reduced Cdk2 binding.
To confirm this result, the cyclin A1 mutant, E167A, was analyzed
further. Cdk2 or Cdc2 and myc-tagged E167A were
[35S]-labeled by cotranslation in nuclease-treated
extracts, and the ability of myc-tagged E167A to bind to
Cdk2 or Cdc2 was tested by coimmunoprecipitation with
anti-myc-antibody, followed by autoradiography. As shown in
Fig. 4A, the cyclin A1 E167A mutant was able to bind Cdc2
(lane 2) but not to Cdk2 (lane 6). In the control
experiments, wild-type cyclin A1 bound to both Cdc2 and Cdk2
(lanes 1 and 5), and the cyclin box mutant
231-232 bound neither CDK (lanes 3 and 7).
The same results were obtained by coimmunoprecipitation of cyclin A1
E167A with anti-Cdc2 and -Cdk2 antibodies (see Fig. 4B,
lanes 2 and 6). To show that the E167A
specifically lacks Cdk2-dependent kinase activity, the
kinase activity of E167A coimmunoprecipitated with Cdc2 and Cdk2 was
compared (Fig. 4B, lower panel). Consistent with
the observed difference in binding affinity, E167A associated with Cdc2
displayed histone H1 kinase activity (lane 2), but the E167A
with Cdk2 showed reduced activity (lane 6). Therefore,
residue Glu167 in the cyclin A1 N-terminal helix is
important for interaction of cyclin A with Cdk2.
Mutations in the C-terminal Lobe of CDK Affect the Preferential Binding Affinities with Cyclins A and B-- Next we investigated CDK mutations at residues that interact with the cyclin N-terminal helix. To do this, we applied a genetic approach using budding yeast system. Ectopic expression of vertebrate A- or B-type cyclins inhibits growth, and the inhibition is dependent upon association with endogenous yeast Cdc28 (30, 33). By taking advantage of this effect, we have isolated temperature-sensitive cdc28 mutants that suppress the growth inhibition induced by ectopic cyclin expression. The procedure for isolation of the cdc28 mutations is described under "Experimental Procedures." With reference to the results of this yeast cdc28 mutant screen and the structure of the cyclin A-CDK2 complex (11), we constructed equivalent Xenopus CDK (Cdc2 and Cdk2) mutants by site-directed mutagenesis (Table I). Some of these CDK mutations would be expected to have significant effects on the cyclin-CDK interaction, because the equivalent mutations in yeast Cdc28 have been shown to possess preferential affinities for different endogenous cyclins (30).
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To show that the mutated residues in CDK are actually required for
cyclin binding, Cdc2 mutants and wild-type cyclins A1 and B2 were
[35S]-labeled in nuclease-treated extracts and then the
abilities of Cdc2 mutants to bind to cyclins A and B were tested by
coimmunoprecipitation with cyclin A1 or cyclin B2 (Fig.
5A). The result is summarized in Table I. As expected, two Cdc2 mutants, C119Y and A281E, failed to
bind to both cyclin A1 and cyclin B2 (Fig. 5A, lanes
2 and 5). Equivalent mutations in Cdk2, C118Y and
A280E, also abolished the binding to cyclin A1 (Fig. 5B,
lanes 2 and 5). Thus, both Cys119/118
and Ala281/280 in Cdc2/Cdk2, respectively, are required for
the interactions of both Cdc2 and Cdk2 with cyclins A and B.
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Interestingly, the Cdc2 mutant S121A bound normally to cyclin A1 and B2, but the equivalent Cdk2 mutant S120A was impaired in its binding affinity to cyclin A1 (Fig. 5, A and B, lane 3). This result suggests that Ser120 may be involved in stabilizing the cyclin A-Cdk2 complex. In contrast, the Cdk2 mutant F152A bound strongly to cyclin A1, but the equivalent Cdc2 mutant F153A showed reduced binding to cyclin A1 and severely reduced to cyclin B (Fig. 5, A and B, lane 4). This result suggests that residue Phe153 in Cdc2 has a role in stabilizing in the cyclin B-Cdc2 complex.
The Effects of the Cyclin N-terminal Helix Mutants on the Binding
and Phosphorylation of Specific Substrates, XCdc6 and XCdc25C--
The
cyclin subunit plays a primary role in substrate binding and
recruitment to the catalytic site of the cyclin-CDK complex. To
investigate whether the cyclin N-terminal helix is important for the
interaction of the complex with substrate, we tested the effects of
the mutations in the N-terminal helix on substrate binding using
Xenopus Cdc6 and Cdc25C as a preferred substrate for cyclin
A and cyclin B, respectively. First, we confirmed that XCdc6 binds to
cyclin A (Fig. 6A, lanes
1 and 3) but not to cyclin B (lane 5). The
interaction of XCdc6 and cyclin A1 was impaired by the mutations in the
cyclin A1 N-terminal helix (Fig. 6B, lanes 2 and
4). The cyclin A1 N-terminal helix mutants that did not bind
CDK also failed to phosphorylate XCdc6 (Fig. 6C). XCdc6
bound to cyclin A1 and Cdc2 only when cyclin A1 was coexpressed in the extracts (Fig. 6D, lane 3) and not to free Cdc2
(lane 4). Free cyclin A1 also fails to bind to XCdc6,
because the C-terminal deletion C14 that cannot bind to Cdc2 (17)
did not bind to XCdc6 (Fig. 6E, lane 2).
Furthermore, cyclin A1 and Cdc2 that coprecipitated with XCdc6 was
proportional to the increasing amounts of Cdc2 present in the extracts
(data not shown), thereby indicating that the association of XCdc6 with
cyclin A depends upon cyclin A forming a complex with Cdc2. These
results suggest that the cyclin A1 N-terminal helix is required for
XCdc6 phosphorylation, because it is required for formation of the
cyclin-CDK complex.
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Next we investigated the effects of the cyclin B2 N-terminal helix
mutants on phosphorylation of Xenopus Cdc25C, a preferred substrate of the cyclin B2-Cdc2 complex (Fig.
7A). XCdc25C was phosphorylated preferentially by the cyclin B2-dependent
kinase (Fig. 7A, middle panel, lane
2), although the association of XCdc25C with cyclin B2 was not
detected in this condition (data not shown). By contrast, XCdc25C was
phosphorylated little by the cyclin A1-dependent kinase
(lane 1). As a control, histone H1 was phosphorylated both by cyclin A1- and B2-dependent kinases (lower
panel). These results suggest that, in contrast to XCdc6 for a
preferred cyclin A-Cdc2 substrate, XCdc25C is associated and rapidly
turned over on the cyclin B-Cdc2 complex. Then we tested the effects of
cyclin B2 N-terminal helix mutants on XCdc25C phosphorylation. The
cyclin B2 N-terminal helix mutants that did not bind Cdc2 also failed to phosphorylate XCdc25C (Fig. 7B). These results suggest
that the cyclin B2 N-terminal helix is required for XCdc25C
phosphorylation and thereby for the interaction of XCdc25C with the
cyclin B2-Cdc2 complex.
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The binding of a cyclin to a CDK is essential for the activation of cyclin-dependent kinases. The crystal structure of the human cyclin A-CDK2 complex has shown that the interaction between the cyclin A CBF and the CDK2 PSTAIRE motif is required for complex formation. In this paper we show that the N-terminal helix of cyclin B, as well as that of cyclin A, is also essential for the cyclin-CDK interaction.
Interaction between the N-terminal Helix in Cyclin and the
C-terminal Lobe in CDK--
The crystal structure of the cyclin A-CDK2
complex revealed that the cyclin A N-terminal helix contacts the CDK
C-terminal lobe and that the CDK buries a number of hydrophobic
residues that are exposed on the cyclin A surface (11, 13). Our work shows that the cyclin B2 N-terminal helix is also important for formation of the cyclin-CDK complex. As illustrated in a helix wheel
projection of the cyclin N-terminal helix (Fig.
8), conserved residues, mutation of which
abolish CDK binding, lie along one side of -helical surface
(i.e. Tyr131, Ile135,
Tyr138, and Leu139 in cyclin B2). Analysis of
the cyclin A1 N-terminal helix mutants gave a similar result. Moreover,
Cdc2/Cdk2 mutations to residues Cys119/118,
Ser121/120, Phe153/152, and
Ala281/280, respectively, affected differentially their
ability to bind to cyclins (Table I). These residues are located in the
region of the CDK C-terminal lobe that contacts the cyclin
N-terminal helix (11).
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We have shown that these conserved residues of the N-terminal helix interact with this region located on the surface of the CDK C-terminal lobe and function in formation of specific cyclin-CDK complexes. These conserved residues are all hydrophobic (Tyr131, Ile135, Tyr138, and Leu139 in cyclin B2 and Tyr164 and Ile168 in cyclin A1), demonstrating the importance of hydrophobic interactions in the formation of these cyclin-CDK complexes. These N-terminal helix mutants can be degraded normally in Xenopus Ca2+-treated CSF extracts (data not shown), suggesting that they are correctly folded. By contrast, a mutation in conserved glutamic acid (Glu143 in cyclin B2 and Glu176 in cyclin A1) abolished not only the binding to CDK (see Figs. 2 and 3) but also its degradation in the extracts (data not shown). This result suggests that the mutations in these acidic residues have impaired the structural integrity of the protein. This glutamic acid does not appear to be in contact with CDK but to be deeply buried in the cyclin A structure.
Difference in the Interactions of Cyclins A and B with CDKs-- A CDK is composed of a C-terminal and an N-terminal lobe, and its active site is located at the catalytic cleft between the two (11-12). The interaction between the cyclin N-terminal CBF and the CDK PSTAIRE motif plays a major role in forming a tight complex. The interaction between the cyclin N-terminal helix and the C-terminal lobe of CDK may play a distinct role in determining the selectivity of cyclin-CDK complex formation. For example (a) the cyclin A1 E167A mutant bound to Cdc2 but not to Cdk2 (Fig. 4); (b) the Cdk2 S120A mutant showed reduced cyclin A binding, whereas an equivalent S121A mutant in Cdc2 bound both cyclin A and cyclin B (Fig. 5); (c) the cyclin B2 mutations Y138A and L139A specifically abolished Cdc2 binding, whereas an equivalent Y171A and L172A in cyclin A1 did not (Fig. 3C); (d) the F153A mutant of Cdc2 bound to cyclin A but not to cyclin B (Fig. 5). In agreement with the ternary structure of the complex (11-13), Glu167 of cyclin A and Ser120 of Cdk2 are interfaced with each other in the complex. Phe152 of human Cdk2 is buried in a hydrophobic pocket with the CDK structure.
Conserved residues on each of the four turns of the cyclin B N-terminal helix might contribute to the formation of the cyclin B-Cdc2 complex (see residues indicated by boxed bold letters in Fig. 8B). In contrast, conserved residues on only two turns of the helix seem to contribute to the cyclin A-Cdc2 complex formation (Fig. 8A). Therefore, the contacts within the cyclin B-Cdc2 complex appear to be more extensive in this region than those in the cyclin A-Cdc2 complex. These results support our previous study on differential stability of cyclin-CDK complexes, where we determined that cyclin A-Cdc2 dissociates more readily than cyclin B-Cdc2 (34). However, the cyclin A1-Cdc2 complex seems to be more stable than the cyclin A1-Cdk2 complex (data not shown). These differences might explain why the Cdc2 forms predominantly a complex with cyclin A1 in embryonic cells (35).
As well as the interactions made by the first two turns of the
N-terminal helix (Tyr164 in turn 1 and Ile168
in turn 2), the interaction of cyclin A1 with Cdk2 requires the acidic
residue Glu167, whereas the cyclin A1-Cdc2 complex does not
(Fig. 4). Ser120 in Cdk2 is 3.4 Å away from, and
forms a hydrogen bond with, Asp181 in human cyclin A, which
is equivalent to Glu167 of frog cyclin A (12-13). This
glutamic acid (Glu167) also forms a salt bridge with Cdk2
residue Lys278 that is predicted to be conserved in Cdc2
and Cdk2 complexes with cyclins A and B. Mutations that disrupt these
interactions have a differential affect on the overall stability of the
different cyclin-CDK complexes. In the case of cyclin A2-CDK binding, a similar affect was observed in the Tyr160 mutant of cyclin
A2 (data not shown). Tyr164 of cyclin A1, which is
equivalent to this Tyr160, is close to Glu167
in the -helix structure (Fig. 8A).
Comparison of the N-terminal Helix in Cyclin A with That in Cyclin H-- Cyclin H also contains the N-terminal helix. Cyclin H, from which this helix has been removed, can still bind to Cdk7, but the resulting complex does not exhibit kinase activity (36). The location of the N-terminal helix of cyclin H is different from that of cyclin A. In cyclin H, the N-terminal helix primarily contacts the N-terminal CBF (37). The hydrophobicity profile of the N-terminal helix of cyclin A is quite different to that of cyclin H. This observation suggests that the role of the N-terminal helix of cyclin H may be different from that of cyclin A and hence from that of cyclin B.
Possible Role of the N-terminal Helix Motif in Specificity of the
Cyclin-CDK Complex--
Cyclin plays a major role in substrate
recognition (3, 38). The best characterized docking site on cyclin A is
found on the opposite surface of the cyclin-CDK complex from that of the catalytic site. This hydrophobic patch can interact with an RXL
motif in substrates such as p107, pRb, and Myt1 (7, 39). However, this
hydrophobic patch is found in all A, B, D, and E cyclins, suggesting
that another motif must play a critical role in defining CDK substrate
selectivity. We have found that the binding of substrate XCdc6 to
cyclin A is dependent upon cyclin A forming a complex with a CDK; XCdc6
does not bind to either monomeric cyclin A or to monomeric CDK (Fig.
6). We also found that XCdc25C was a preferred substrate for the cyclin
B2-dependent kinase (Fig. 7). Furthermore, the binding
manner of XCdc25C for the cyclin B-Cdc2 substrate appears to be
different from that of XCdc6 for the cyclin A-Cdc2 substrate. In these
interactions, the cyclin N-terminal helix could contribute to the
binding site of substrates XCdc6/XCdc25C differently via a
conformational change in the complex, which enables it to recruit a
specific substrate into the catalytic cleft. The substrate specificity
might depend upon binding specificity of the cyclin-CDK complex, which
is due to the cyclin N-terminal helix.
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ACKNOWLEDGEMENTS |
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We are most grateful to Dr. Jane Endicott for valuable comments on the structure of the cyclin A-CDK2 complex and critical reading of the manuscript. We thank Drs. Tim Hunt and Julian Gannon for constructs and antibodies of cyclins, Cdc2, and Cdk2, Dr. Noriyuki Sagata for the use of frogs, XCdc25C, and anti-Cdk2 antibody, and Dr. Julian Blow for XCdc6. We also thank Drs. Mark Carrington and Tim Hunt for their support and discussions.
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FOOTNOTES |
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* This work was supported by grants from the Ministry of Education, Science, Sports and Culture of Japan.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.:
81-92-642-6179; Fax: 81-92-642-6183; E-mail:
hkobaya@molbiol.med.kyushu-u.ac.jp.
Published, JBC Papers in Press, January 30, 2001, DOI 10.1074/jbc.M011101200
2 Unpublished results.
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ABBREVIATIONS |
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The abbreviations used are: CDK(s), cyclin-dependent kinase(s), CBF(s); cyclin box fold(s), CSF; cytostatic factor, GST; glutathione S-transferase, PAGE; polyacrylamide gel electrophoresis.
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