The RRASK Motif in Xenopus Cyclin B2 Is Required for the Substrate Recognition of Cdc25C by the Cyclin B-Cdc2 Complex*
Tadahiro Goda
,
Takashi Ishii
,
Nobushige Nakajo ¶,
Noriyuki Sagata ¶ and
Hideki Kobayashi
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From the
Department of Molecular Biology, Graduate School of Medical Sciences, Kyushu University, Maidashi, Fukuoka 812-8582, Japan,
¶ Department of Biology, Graduate School of Sciences, Kyushu University, Hakezaki, Fukuoka 812-8582, Japan
Received for publication, January 8, 2003
, and in revised form, March 20, 2003.
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ABSTRACT
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The FLRRXSK sequence is conserved in the second cyclin box fold of B-type cyclins. We show that this conserved sequence in Xenopus cyclin B2, termed the RRASK motif, is required for the substrate recognition by the cyclin B-Cdc2 complex of Cdc25C. Mutations to charged residues of the RRASK motif of cyclin B2 abolished its ability to activate Cdc2 kinase without affecting its capacity to bind to Cdc2. Cdc2 bound to the cyclin B2 RRASK mutant was not dephosphorylated by Cdc25C, and as a result, the complex was inactive. The cyclin B2 RRASK mutants can form a complex with the constitutively active Cdc2, but a resulting active complex did not phosphorylate a preferred substrate Cdc25C in vitro, although it can phosphorylate the non-specific substrate histone H1. The RRASK mutations prevented the interaction of Cdc25C with the cyclin B2-Cdc2 complex. Consistently, the RRASK mutants neither induced germinal vesicle breakdown in Xenopus oocyte maturation nor activated in vivo Cdc2 kinase during the cell cycle in mitotic extracts. These results suggest that the RRASK motif in Xenopus cyclin B2 plays an important role in defining the substrate specificity of the cyclin B-Cdc2 complex.
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INTRODUCTION
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The activities of cyclin-dependent kinase (CDK)1 are essential for eukaryotic cell cycle progression. Monomeric CDKs are inactive and associate with the cyclin subunits to form active complexes. The activities of the cyclin-CDK complexes are controlled by their interactions with several proteins, such as activating and inhibitory kinases, CDK inhibitors, and adaptors. Cyclin-CDK complexes interact with specific substrates and phosphorylate them temporally. Different types of cyclins associate with distinct CDKs to form active complexes, and these cyclin-CDK complexes phosphorylate different substrates in cell cycle progression.
Recognition of specific substrates by the cyclin-CDK complex is determined in part by interactions of the cyclin subunit with the substrate (1, 2). How the cyclin confers CDK substrate selectivity is one of the fundamental problems in cell cycle regulation. Some CDK substrates, such as pRb, Cdc25, Cdc6, and the CDK inhibitor p21, contain an RXL motif (3, 4, 5, 6). The RXL motif of these substrates binds directly to the MRAIL motif of the cyclin. The MRAIL motif is present in a hydrophobic patch region in the cyclin box fold 1 (CBF1). This motif in cyclin A has been shown to be a docking site for cyclin A-Cdk2 substrates (7). In cyclin D, the LXCXE motif present in its N-terminal sequence binds directly to pRb, and mutations in this sequence inhibit efficient pRb phosphorylation (8, 9). Likewise, mutations within a similar LXCXE motif in cyclin E prevent phosphorylation of pRb by the cyclin E-Cdk2 complexes (10). These motifs enhance the activity of the G1 cyclin-CDK complex toward specific substrates. These findings suggest that the substrate recognition is defined in part by specific motifs in the cyclin subunit. In comparison with G1 cyclin, it is rather difficult to detect the interaction of mitotic substrates with cyclin B, probably due to high substrate turnover. So far, the interactions of cyclin B-Cdc2 with the RXL motif of Myt1 (11) and of Cdc25C (12, 13) have been investigated.
Several distinct domains and motifs that are involved in CDK regulation characterize cyclin proteins. The N-terminal CBF1 in the middle region of cyclins is required for CDK association (14, 15), and the N-terminal domain including a destruction box is necessary for the ubiquitin-dependent cyclin degradation (16, 17). A cytoplasmic retention signal for subcellular localization of the cyclin is located between the destruction box and the cyclin box (18). The N-terminal helix motif upstream of the cyclin box contributes to specific binding of cyclins A and B to CDKs (19). The MRAIL motif that contributes to a hydrophobic patch on the surface of cyclin A serves as a substrate-docking site (7). In this study, we describe that the degenerate RRASK motif of cyclin B is required for the substrate recognition of Cdc25C by the cyclin B-Cdc2 complex. The RRASK motif is found in the N-terminal region of the second cyclin box fold (CBF2) in cyclin B but is not present in cyclin A. We have generated mutations in the RRASK motif to analyze its role in mediating substrate-cyclin B interaction. Mutations within the RRASK motif in Xenopus cyclin B2 did not affect its binding to Cdc2, but abolished its ability to activate Cdc2 kinase in vivo and in vitro, as a result of a failure of Cdc25C to dephosphorylate the cyclin B2-Cdc2 complex. Furthermore, a constitutively active Cdc2 mutant (Cdc2-AF) bound to the cyclin B2 RRASK mutant failed to phosphorylate the preferred Cdc2 kinase mitotic substrates, Cdc25C and Myt1. The affinity of the cyclin B-Cdc2 for these substrates was decreased by RRASK mutations. The RRASK motif of cyclin B thus affects Cdc2 activity by mediating critical interactions between the cyclin B-Cdc2 complex and the mitotic substrates. We discuss the role of the RRASK motif of cyclin B in substrate recognition by CDK.
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EXPERIMENTAL PROCEDURES
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Construction of Cyclin B2 Mutants, Cdc25C, Myt1, and Cdc2The RRASK mutants of Xenopus laevis cyclin B2, K272Q (Lys-272 mutated to Gln), R268A/R269A (Arg-268 to Ala, Arg-269 to Ala), and R268L/R269Q/K272Q (Arg-268 to Leu, Arg-269 to Gln, Lys-272 to Gln), were made by PCR-based in vitro mutagenesis (20). R163A was constructed as described previously (19). Myc-tagged and GST-tagged cyclin B2 constructs were also made by PCR-based in vitro mutagenesis (20). In brief, the Myc tag sequence was subcloned at the N terminus of Xenopus cyclin B2 in pT7GUK+ by NcoI digestion of Xenopus cyclin B2 in pT7GUK+ and of the Myc tag sequence with an NcoI site at the end. To prepare GST-tagged cyclin B2, a GST tag sequence that has a BglII site at the N terminus and an NcoI site at the C terminus was subcloned into Xenopus cyclin B2 in pT7GUK+. Both constructs were confirmed by sequencing. Xenopus Cdc2-AF-4Myc was constructed as follows: Cdc2-AF (21) in pT7GUK+ and Cdc2-4Myc in pT7GUK+ were digested with NdeI and HindIII, and then Cdc2-AF fragment was subcloned into Cdc2-4Myc in pT7GUK+. Xenopus Cdc25C and Xenopus Myt1 were described elsewhere (21, 22).
Xenopus Egg Extracts, in Vitro Translation, Immunoprecipitation Xenopus CSF extracts and mitotic extracts were prepared by the standard method (23). The mCAP RNA capping kit (Stratagene) was used for in vitro transcription of cyclin B2 mutants and Cdc2. 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. After incubating at 23 °C for 90 min, 1-µl samples were 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-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 and/or Cdc2 were detected by SDS-PAGE and autoradiography.
GST Pull-down Assay and ImmunoblottingGST-tagged cyclin B2 mRNAs to a final concentration of 0.1 µg/µl were added to 10 µl of Xenopus CSF extracts. After incubating at 23 °C for 90 min, 1-µl samples were analyzed by SDS-PAGE followed by immunoblotting with anti-GST antibody. For GST pull-down assays, reactions (10 µl) were diluted with 300 µl of bead buffer, mixed with GSH-agarose beads, and incubated at 4 °C for 1 h with rotation. The beads were washed with bead buffer three times, and then co-precipitated Cdc2 was detected by SDS-PAGE and immunoblotting with anti-Cdc2 or anti-phospho-Cdc2 antibody.
Preparation of GST-Cdc25C and GST-Myt1 ProteinXenopus Cdc25C in pGEX-KG and a C-terminal fragment of Xenopus Myt1 (residues 392548) in pGEX-KG were transformed into Escherichia coli BL21 cells. Protein expression was induced by addition of 1 mM isopropyl-1-thio-
-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 GSH-agarose beads, and bound proteins were eluted with 10 mM glutathione (pH 7.0).
Phosphorylation of Histone H1, GST-Cdc25C, and GST-Myt1For in vitro phosphorylation assays, mRNAs coding for Myc-tagged Xenopus cyclin B2 and its mutants were translated in the mitotic extracts supplemented with [35S]methionine at 23 °C for 2 h and then immunoprecipitated with anti-Myc antibody and protein A-Sepharose beads. In the case of cyclin-Cdc2-AF complex, mRNAs of Xenopus cyclin B2 mutants were co-translated with Cdc2-AF-4Myc mRNA in RNase-treated mitotic extracts supplemented with [35S]methionine at 23 °C for 2 h. Cyclin-Cdc2-AF complex was immunoprecipitated with anti-Myc antibody and protein A-Sepharose beads. Beads were then washed with bead buffer three times and incubated with the reaction mixture (20 mM HEPES, pH 7.8, 15 mM MgCl2, 5 mM EGTA, 0.2 mg/ml bovine serum albumin, 1 mM dithiothreitol, 0.2 mM ATP, 10 µCi of [
-32P]ATP, 0.1 µg/µl histone H1) at 23 °C for 30 min. Samples were analyzed by SDS-PAGE and autoradiography. For phosphorylation of Xenopus Cdc25C or Myt1, GST-Cdc25C or GST-Myt1 (residues 392548) replaced histone H1 and were assayed as described above. To assay in vivo Cdc2 kinase activity in mitotic extracts, mRNAs of Xenopus wild-type or mutant cyclin B2 were translated in RNase-treated mitotic extract with [35S]methionine at 23 °C. 1 µl of the extracts was incubated with 9 µl of reaction mixture (20 mM HEPES, pH 7.8, 15 mM MgCl2, 5 mM EGTA, 0.2 mg/ml bovine serum albumin, 1 mM dithiothreitol, 0.2 mM ATP, 10 µCi of [
-32P]ATP, 0.1 µg/µl histone H1) at 23 °C for 30 min. The samples were analyzed by SDS-PAGE and autoradiography.
Interaction of GST-Cdc25C and GST-Myt1 with the Cyclin B-Cdc2 ComplexXenopus cyclin B2 and mutant mRNAs were translated in vitro with [35S]methionine added to the RNase-treated CSF/reticulocyte lysate mixture (1:1). 10 µl of this reaction mixture was incubated with 4 µg of GST-Cdc25C, GST-Myt1 (residues 392548), or GST for 10 min at 23 °C and then diluted with 300 µl of bead buffer containing with 0.1 or 2% Tween 20 instead of 0.1% Nonidet P-40. GSH-agarose beads were then added, incubated for 1 h at 4 °C with rotation, and finally washed three times with bead buffer containing with 0.1 or 2% Tween 20 instead of 0.1% Nonidet P-40. Bound proteins were separated by SDS-PAGE. The gel was dried and exposed to x-ray film.
MicroinjectionXenopus stage VI oocytes were injected with 2.7 ng of cyclin B2 or the mRNAs of its mutant and cultured at 20 °C in modified Barth's solution (24). Maturation was assessed by appearance of a white spot in the animal pole of injected oocytes.
AntibodiesAnti-Cdc25C antibody was prepared as described (22). Anti-Cdc2 monoclonal (A17) and anti-cyclin B2 monoclonal (X121) antibodies were provided by Tim Hunt (Cancer Research UK, London, UK). Anti-c-Myc antibody (A14) was purchased from Santa Cruz Biotechnology, and anti-phospho-Cdc2 antibody (Tyr-15) was purchased from Cell Signaling Technology, Inc.
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RESULTS
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Mutations in the RRASK Motif of Cyclin B2 Abolish Its Ability to Activate Cdc2 Kinase without Affecting Binding to Cdc2Cyclin B contains the amino acid sequence FLRRXSK in the first
-helix of cyclin box fold 2, which we refer to as the RRASK motif. This degenerate RRASK motif is well conserved in B-type cyclins but not in cyclin A (Fig. 1A). To explore its role in the cyclin-CDK complex, we made a number of mutants at the charged amino acids of the RRASK motif in Xenopus cyclin B2, in which basic residues RRXXK were substituted to either non-charged or structurally similar residues or to alanine. A single mutant K272Q, a double mutant R268A/R269A (designated as RR268AA), and a triple mutant R268L/R269Q/K272Q (designated as RRK268LQQ) were prepared.

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FIG. 1. Sequence alignment of the RRASK motif in cyclin B. A, schematic drawing of the conserved domains in cyclin B. Bold letters indicate identical residues. Asterisks on the top of the amino acid sequences denote the residues mutated in this work. XL represents X. laevis. As shown in B, mutations within the cyclin B2 RRASK motif abolish its ability to activate Cdc2 kinase without affecting Cdc2 binding. Cyclin B2 mRNA was translated in the nuclease-treated CSF extracts with [35S]methionine (upper panel), and [35S]-labeled cyclins bound to endogenous Cdc2 were immunoprecipitated by anti-Cdc2 antibody followed by SDS-PAGE and autoradiography (middle panel). For phosphorylation, Myc-cyclin B2 and its RRASK mutants were translated and pulled down by immunoprecipitation with anti-Myc antibody. The Myc-precipitated cyclin B2-Cdc2 complex was assayed for phosphorylation of histone H1 (lower panel). Lane 1, wild-type (WT)-cyclin B2; lane 2, RR268AA; lane 3, RRK268LQQ; lane 4, K272Q; lane 5, R163A; lane 6, no mRNA.
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First, we examined the effect of mutations in the RRASK motif on the ability of cyclin B2 to bind to Cdc2 and to activate the Cdc2 kinase in Xenopus egg extracts. Cyclin mRNAs were translated in nuclease-treated CSF extracts supplemented with [35S]methionine, and the cyclin B2-Cdc2 complexes were immunoprecipitated with anti-Cdc2 antibody. Fig. 1B shows that RRASK mutants bound to Cdc2 (RR268AA, RRK268LQQ, K272Q; lanes 24, middle panel). Next, Myc-tagged versions of cyclin B2 and its mutants were translated and pulled down from the extracts by immunoprecipitation with anti-Myc antibody. The Myc-cyclin B-Cdc2 complex was then assayed for histone H1 kinase activity (lower panel). The RRASK mutant-Cdc2 complexes failed to phosphorylate histone H1 (RR268AA (lane 2) and RRK268LQQ (lane 3)). As a control, the MRAIL motif mutant R163A neither bound to Cdc2 nor activated Cdc2 kinase (lane 5). Histone H1 was phosphorylated by the cyclin B2 single mutant K272Q-Cdc2 (lane 4), as well as by the wild-type cyclin B2-Cdc2 (lane 1) complexes. The result shows that the RRASK mutants of cyclin B2 abolish its ability to activate Cdc2 kinase, without affecting binding to Cdc2 in egg extracts.
Cdc2 Bound to the Cyclin B2 RRASK Mutants Is Not Dephosphorylated by Cdc25CWe next tested the phosphorylation state of Cdc2 bound to the RRASK mutants (Fig. 2). Nucleasetreated mitotic extracts containing [35S]-labeled Cdc2 (Fig. 2A, lane 1) were incubated with cyclin B2 or RR268AA mRNAs to drive the cell cycle, and the cyclin-Cdc2 complex was analyzed by immunoprecipitation with anti-cyclin B2 antibody followed by SDS-PAGE and autoradiography. In control experiments, Cdc2 bound to wild-type cyclin B2 was phosphorylated and then dephosphorylated during the cell cycle (lanes 25), whereas Cdc2 bound to RR268AA was phosphorylated but not subsequently dephosphorylated (lanes 69). This result indicates that Cdc2 bound to RR268AA failed to be dephosphorylated by Cdc25C. To determine whether Cdc2 bound to the RRASK mutant is phosphorylated or not, Cdc2 was immunoblotted with anti-phospho-Cdc2 antibody (Fig. 2B). GST-tagged cyclins were used in this experiment. Following incubation in the extracts for 90 min (Fig. 2A), GST-bound Cdc2 was immunoblotted with anti-phospho-Cdc2 antibody. Cdc2 bound to wild-type cyclin B2 (lane 2) and to K272Q (lane 4) were dephosphorylated, but Cdc2 bound to RRK268LQQ was not dephosphorylated at Tyr-15 (lane 3). Thus, Cdc2 bound to the cyclin B2 RRASK mutant, which is once phosphorylated by Myt1/Wee1 inhibitory kinase, fails to be dephosphorylated at Tyr-15 by Cdc25C. This result was confirmed by chase experiments in which the phosphorylated form of Cdc2 (samples in Fig. 2A, lanes 2 or 6) was incubated with an active Cdc25C, and dephosphorylation of Cdc2 was followed at entry into mitosis by adding active Cdc25C to the extracts (Fig. 2C). Cdc2 bound to wild-type cyclin B2 was dephosphorylated by active Cdc25C (lanes 14), whereas Cdc2 bound to RR268AA was not (lanes 58).

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FIG. 2. Cdc2 bound to cyclin B2 RRASK mutant is not dephosphorylated by Cdc25C. A, failure in dephosphorylation of Cdc2 at M-phase. Cdc2 mRNA was translated in nuclease-treated mitotic extracts with [35S]methionine. After a 45-min incubation (lane 1), cyclin B2 or RR268AA mRNAs was added to the extracts. The extracts were incubated to drive the cell cycle, and the cyclin B2-Cdc2 complex was immunoprecipitated with anti-cyclin B2 antibody at intervals of 45 min. A band shift of [35S]-labeled Cdc2 was followed by SDS-PAGE and autoradiography. Lanes 25, wild-type (WT) cyclin B2; lanes 69, RR268AA. As shown in B, the RRASK mutant fails to dephosphorylate Tyr-15 of Cdc2. GST-tagged cyclin B2 mRNA was translated in mitotic extracts and incubated for 90 min (lane 1). GST-cyclin B2 was precipitated with GSH agarose beads and immunoblotted with anti-GST antibody (upper panel). Bound Cdc2 was immunoblotted with anti-Cdc2 (middle panel) and anti-phospho-Cdc2 (lower panel) antibodies. Lane 2, wild-type cyclin B2; lane 3, RRK268LQQ; lane 4, K272Q; lane 5, R163A; lane 6, no mRNA. As shown in C, Cdc2 bound to the RRASK mutant is not dephosphorylated by addition of an active Cdc25C. Phosphorylated Cdc2 was prepared from the extracts after 45 min incubation (panel A, lanes 2 and 6). These extracts were incubated with the CSF-arrested extracts containing the active Cdc25C (data not shown). At time intervals, Cdc2 was immunoblotted with anti-Cdc2 antibody (upper panel), and the phosphorylated form of Cdc2 was immunoblotted with anti-phospho-Cdc2 antibody (bottom panel). Lanes 14, Cdc2 bound to wild-type cyclin B2; lanes 58, Cdc2 to RR268AA.
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The Constitutively Active Cdc2-AF Complexed with the Cyclin B2 RRASK Mutant Phosphorylates a General Substrate Histone H1 but Not Mitotic Substrates Cdc25C and Myt1We next investigated the ability of phosphorylation by the constitutively active Cdc2 bound to the cyclin B2 RRASK mutant in vitro (Fig. 3). To do this, two Cdc25C-dependent dephosphorylation sites of Cdc2 (Thr-14 and Tyr-15) were substituted to Ala and Phe (Cdc2-AF), respectively. Cdc2-AF is constitutively active and therefore does not require dephosphorylation by Cdc25C to phosphorylate its substrates (21). Both Myc-tagged Cdc2-AF and the cyclin B2 RRASK mutant were translated in nuclease-treated mitotic extracts, and the cyclin B2-Cdc2-AF complex was immunoprecipitated with anti-Myc antibody (Fig. 3A). Cdc2-AF bound to RR268AA (lane 2) and to RRK268LQQ (lane 3). The active complex was then assayed for in vitro phosphorylation against a general substrate histone H1 and a preferred substrate Cdc25C (Fig. 3, B and C). Active Cdc2-AF bound to RR268AA or to RRK268LQQ phosphorylated a general substrate histone H1 (Fig. 3B, upper panel, lanes 2 and 3). However, even the active complex failed to phosphorylate Cdc25C, the preferred cyclin B2-Cdc2 substrate (middle panel, lanes 2 and 3). In comparison, Cdc2-AF complexed with the MRAIL mutant (R163A) phosphorylated neither histone H1 nor Cdc25C (lane 4). As a control, the K272Q-Cdc2-AF complex, as well as the wild-type cyclin B2-Cdc2-AF complex, can phosphorylate both Cdc25C and histone H1 (Fig. 3C). These results show that the RRASK motif of cyclin B2 is required for phosphorylation of the preferred mitotic substrate Cdc25C, indicating the failure of Cdc25C binding to the cyclin B-Cdc2 complex.

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FIG. 3. Constitutively active Cdc2-AF bound to RRASK mutant fails to phosphorylate Cdc25C and Myt1, although it can phosphorylate histone H1 in vitro. A, translation of Myc-Cdc2-AF and cyclin B2 mutants. Both Myc-tagged Cdc2-AF and cyclin mRNAs were translated in nuclease-treated mitotic extracts with [35S]methionine followed by SDS-PAGE and autoradiography. WT, wild type. B, in vitro phosphorylation assay by the RRASK mutant-Cdc2-AF complex. The Myc-Cdc2-AF that bound to [35S]-labeled cyclins was immunoprecipitated with anti-Myc antibody from the extracts in panel A. The complex was assayed for in vitro phosphorylation against histone H1 (upper panel), Cdc25C (middle panel), and the Myt1 C-terminal segment (lower panel). Lane 1, wild-type cyclin B2; lane 2, RR268AA; lane 3, RRK268LQQ; lane 4, R163A; lane 5, no mRNA. C, relative levels of phosphorylation of histone H1 and Cdc25C. The cyclin B2 RRASK mutants including K272Q were tested for phosphorylation assays of histone H1 and Cdc25C as in panel B. The mean of the activity in two separate experiments is shown in the histogram, normalizing the value in wild-type cyclin B2 to 100%.
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We also examined phosphorylation of another mitotic substrate, Myt1. Because Myt1 kinase inhibits the cyclin B-Cdc2 complex (25), we removed the kinase domain from the central portion of Myt1 and used its C-terminal segment as substrate (residues 392548). Similarly, the Cdc2-AF complexed with RRASK mutants altered the phosphorylation state of the C-terminal Myt1 (Fig. 3B, lower panel, lanes 2 and 3), suggesting that some specific site(s) of the multiphosphorylation sites within the C-terminal Myt1 failed to be phosphorylated (see "Discussion").
Physical Interaction of Mitotic Substrates Cdc25C and Myt1 with the Cyclin B2-Cdc2 Complex Is Prevented by the Mutations within the RRASK MotifThen, we investigated the effects of the RRASK mutation on physical interaction of Cdc25C with the RR268AA-Cdc2 complex (Fig. 4). GST-Cdc25C was incubated with the CSF/reticulocyte mixed extracts containing [35S]-labeled cyclin B2 or RR268AA. After incubating the extracts for 10 min, the GSH-agarose beads were added to precipitate GST-bound materials followed by washing in the presence of Tween 20 (see "Experimental Procedures"). By using these conditions, we can detect a transient interaction between Cdc25C and the cyclin B2-Cdc2 complex in Xenopus (Fig. 4, lanes 310). GST-Cdc25C associated with wild-type cyclin B2-Cdc2 complex (Fig. 4A, lanes 3 and 7) but not with GST alone (lanes 5 and 9). In contrast to wild-type cyclin B2, the association between the RR268AA-Cdc2 and GST-Cdc25C was decreased to about 25% that bound to the wild-type cyclin B2-Cdc2 complex (lanes 4 and 8). Similarly, the association of the Myt1 C-terminal segment with RR268AA was decreased 50% (Fig. 4B, lanes 4 and 8). Thus, the mutant RR268AA impaired the interaction of Cdc25C and Myt1 with the cyclin B2-Cdc2 complex, indicating that the RRASK motif of cyclin B2 affects specific interactions of mitotic substrates with the cyclin BCdc2 complex. Interestingly, both substrates bound less well to the phosphorylated form of RR268AA than to the non-phosphorylated form (cf. Fig. 4, upper and lower bands, lanes 4 and 8; see also "Discussion").

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FIG. 4. Physical interaction of Cdc25C and Myt1 with the cyclin B2-Cdc2 complex is prevented by mutations within the RRASK motif. Association of Cdc25C or Myt1 with the RRASK mutant-Cdc2 complex was assayed by GST pull-down followed by SDS-PAGE and autoradiography. GST-Cdc25C (A) or GST-Myt1 (residues 392548) (B) was incubated for 10 min with the CSF/reticulocyte mixed extracts (1:1) containing [35S]-labeled cyclin B2 or RR268AA. WT, wild type. The GSH-Sepharose beads was then added to the extracts to precipitate GST-bound materials followed by washing the beads in the presence of Tween 20 (0.1 and 2%, respectively). Note that phosphorylated form of cyclin B2 was precipitated with the substrate in both assays, indicating that the substrate interacts with the cyclin B2-Cdc2 complex in this assay.
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The RRASK Mutants Neither Induce Germinal Vesicle Breakdown in Oocyte Maturation nor Activate in Vivo Cdc2 Kinase in Xenopus Egg ExtractsTo confirm the effects of the RRASK mutations on cells in vivo, we first injected mRNAs encoding mutant cyclins into Xenopus stage VI oocytes and examined the occurrence of germinal vesicle breakdown in meiotic maturation. As evident in Fig. 5A, oocytes injected with RR268AA mRNA did not undergo germinal vesicle breakdown, whereas those injected with the K272Q did. Next, we investigated the ability of RRASK mutants to activate Cdc2 kinase in mitotic extracts in vivo (Fig. 5B). mRNAs of cyclin B2 or RR268AA were translated in the nuclease-treated mitotic extracts to drive the cell cycle, and in vivo Cdc2 kinase was followed by histone H1 phosphorylation during cell cycle progression. Consistent with the result of microinjection, addition of RR268AA mRNA did not activate Cdc2 kinase during mitosis (lanes 610). As a control, cyclin B2 mRNA can activate Cdc2 kinase (lanes 15), in accordance with its pattern of phosphorylation. Both sets of in vivo data in Fig. 5 clearly show that mutations in the RRASK motif abolished the ability to entry into M-phase in Xenopus egg system.

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FIG. 5. Mutations within the RRASK motif neither induce meiotic maturation nor activate Cdc2 kinase in vivo in Xenopus egg extracts. A, microinjection. Xenopus stage VI oocytes were injected with cyclin mRNAs (wild-type cyclin B2, RR268AA, K272Q, and R163A) and scored for maturation by the occurrence of germinal vesicle breakdown. B, activation of Cdc2 kinase in mitotic extracts. Wild-type cyclin B2 or RR268AA mRNA was translated in the nuclease-treated mitotic extracts with [35S]methionine, and the extracts were incubated to drive the cell cycle. At the time intervals indicated, the Cdc2 kinase activity of the extracts was assayed for histone H1 phosphorylation. These samples were analyzed by SDS-PAGE and autoradiography. Lanes 15, wild-type cyclin B2; lanes 610, RR268AA; lanes 1115, no mRNA.
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DISCUSSION
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Roles of the RRASK Motif and the MRAIL Motif in the Substrate Recognition of the Cyclin B-Cdc2 ComplexCyclin plays a primary role in the substrate recognition by the cyclin-CDK complex (2). For example, the substrate Cdc25C binds in vitro to cyclin monomer with high affinity and with less affinity to the CDK-cyclin complexes (12). Cdc25C possesses a cyclininteracting motif (residues 195244 in human Cdc25C) (13). In this study, we show that the RRASK motif in CBF2 is required for the recognition of Cdc25C by cyclin B2-Cdc2. Two basic residues Arg-268 and Arg-269 of the RRASK motif are essential for interaction with the substrate Cdc25C but not for the interaction of cyclin B2 with Cdc2. A prediction from the crystal structure of the cyclin A/CDK2 (26, 27) is that the RRASK motif would be exposed on the cyclin B surface near the catalytic cleft between the Cdc2 N-terminal and C-terminal lobes. The specificity of substrate recognition is increased by preassociation of the substrate with a kinase at a docking site adjacent to its catalytic domain (28, 29). It is thus conceivable that the RRASK motif in the first
-helix of CBF2 may aid in a docking interaction with Cdc25C. The RRASK mutant-Cdc2 complex is also impaired in its ability to bind and phosphorylate Myt1 (Figs. 3 and 4), a Cdc2 regulator that also contains a cyclininteracting RXL motif in its C-terminal sequence (11). Phosphorylation of a kinase-dead Wee1 by the RRASK mutant-Cdc2-AF complex was also decreased (data not shown). Thus, the RRASK motif may participate in the recognition of a subset of mitotic substrates that includes at least Cdc25C, Myt1, and Wee1. The RRASK motif is well conserved not only in cyclin B2 but also in cyclins B1, B4, and B5 (30), but less in cyclin B3.
Besides the RRASK motif in CBF2 of cyclin B, the MRAIL motif is present at the N-terminal region in CBF1 of cyclins A, B, D, and E (Fig. 1). The MRAIL motif is located at the opposite surface of the cyclin from the catalytic site of the cyclin-CDK complex (1, 27). In the cyclin A-Cdk2 complex, a hydrophobic patch containing the MRAIL motif (residues Met-210, Leu-214, and Trp-217 in human cyclin A) is important for binding of the substrate p107 to cyclin A (7). Moreover, other CDK-interacting proteins such as kinase inhibitors and the adapter Cdc6 utilize this motif to bind directly on cyclins A and E (5, 6, 31, 32). The MRAIL motif in the cyclin B-Cdc2 complex has a similar role in substrate binding. In the cyclin B structure, the hydrophobic patch that includes the MRAIL sequence was first identified as the P box, which is required for Cdc25 activation. P box mutants (residues Arg-202, Asp-231 in human cyclin B) could bind to Cdc2 but could not activate Cdc2 kinase, as a result of the inactivation of Cdc25 (33). In comparison, the RRASK mutant can bind to Cdc2 but cannot activate Cdc2 kinase, as a result of the failure of binding to Cdc25C. Thus, in this context, the RRASK motif must play a more direct role in the interaction of Cdc25C with the cyclin B2-Cdc2 that is distinct from its previously characterized role as a part of the P box. Recently, the P box region of human cyclin B1 has been also shown to interact with Cdc25C (13). Taken together with these data, it appears that the substrate recognition of Cdc25C by the cyclin B-Cdc2 complex requires not only the RRASK motif but also the MRAIL motif.
Relationship between Mitotic Substrate and High Cdc2 Kinase Activity in M-phaseAssociations between the cyclin BCdc2 and its mitotic substrates are rather unstable as compared with those between the cyclin A/E-Cdk2 and their substrates. In this regard, it is intriguing that CDK activity in S-phase is at a low level and that a further increase to a high level would initiate mitosis (34). This result suggests that phosphorylation of mitotic substrates requires high kinase activity, perhaps achieved in part by cyclin B-Cdc2 having a high turnover as a result of shortening the time of a contact between the substrate and the cyclin-CDK complex. Two cooperative substrate-interacting motifs, the MRAIL in CBF1 and the RRASK in CBF2, might contribute to high levels of Cdc2 kinase activity through a transient but efficient interaction with the substrate. Based on previous observations and our findings in this report, we propose that the RRASK motif of cyclin B plays a critical role in increasing the affinity of mitotic substrates captured by the MRAIL motif and thereby enhances cyclin B-Cdc2 substrate specificity.
Rapid Cdc2 kinase activation at the entry into mitosis involves increased Cdc25 phosphatase activity and a coordinate reduction in the activity of Myt1 kinase (35). Cdc2 kinase phosphorylates Cdc25C, and the resulting phosphorylated Cdc25C activates Cdc2 kinase (25). In contrast to the positive feedback loop on Cdc2 activity by Cdc25C phosphorylation, Myt1 kinase is hyperphosphorylated by Cdc2 kinase concomitant with a decrease in its kinase activity and creates a negative feedback on Cdc2 activity by Myt1 phosphorylation (36). Although Myt1 could also interact with the cyclin B-Cdc2 complex through the RRASK motif, its interaction with the RRASK motif appears to be distinct from that of Cdc25C. Judging from Figs. 3 and 4, Cdc2 bound to the RRASK mutant can still phosphorylate Myt1 in vitro with an alteration of its pattern, although the association with Myt1 substrate is impaired (about 50%). This suggests that some of phosphorylation sites within the Myt1 C-terminal sequence could be selectively phosphorylated by an efficient interaction between Myt1 and the cyclin B-Cdc2 via the RRASK motif. In addition, we have noticed that a phosphorylated form of the cyclin B2 RRASK mutants associates less well with the substrates Cdc25C and Myt1 (Fig. 4). This result may reflect a dependence of cyclin B-substrate interaction on cyclin B phosphorylation. Presumably, the cyclin B2-Cdc2 complex "intra"-phosphorylates its cyclin B subunit, and then the autophosphorylated cyclin B could attach preferably to the mitotic substrate.
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FOOTNOTES
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* This work was supported by grants-in-aid 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. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 
Present Address: National Institute for Medical Research, Mill Hill, London NW7 1AA, UK. 
|| To whom correspondence should be addressed. Tel.: 81-92-642-6179; Fax: 81-92-642-6183; E-mail: hkobaya{at}molbiol.med.kyushu-u.ac.jp.
1 The abbreviations used are: CDK; cyclin-dependent kinase; CBF; cyclin box fold, CSF; cytostatic factor, GST; glutathione S-transferase. 
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ACKNOWLEDGMENTS
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We thank Dr. Jane Endicott for critical reading and valuable comments on the manuscript, and Dr. Minoru Funakoshi for technical help and advice. We also thank Drs. Julian Gannon and Tim Hunt for antibodies and Dr. Takeharu Nishimoto for support on this work.
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REFERENCES
|
---|
- Endicott, J. A., Noble, M. E., and Tucker, J. A. (1999) Curr. Opin. Struct. Biol. 9, 738744[CrossRef][Medline]
[Order article via Infotrieve]
- Millar, M. E., and Cross, F. R. (2001) J. Cell Sci. 114, 18111820[Abstract/Free Full Text]
- Adams, P. D., Sellers, W. R., Sharma, S. K., Wu, A. D., Nalin, C. M., and Kaelin, W. G., Jr. (1996) Mol. Cell. Biol. 16, 66236633[Abstract]
- Saha, P., Eichbaum, Q., Silberman, E. D., Mayer, B. J., and Dutta, A. (1997) Mol. Cell. Biol. 17, 43384345[Abstract]
- Adams, P. D., Li, X., Sellers, W. R., Baker, K. B., Leng, X., Harper, J. W., Taya, Y., and Kaelin, W. G., Jr. (1999) Mol. Cell. Biol. 19, 10681080[Abstract/Free Full Text]
- Wohlschlegel, J. A., Dwyer, B. T., Takeda, D. Y., and Dutta, A. (2001) Mol. Cell. Biol. 21, 48684874[Abstract/Free Full Text]
- Schulman, B. A., Lindstrom, D. L., and Harlow, E. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 1045310458[Abstract/Free Full Text]
- Dowdy, S. F., Hinds, P. W., Louie, K., Reed, S. I., Arnold, A., and Weinberg, R. A. (1993) Cell 73, 499511[Medline]
[Order article via Infotrieve]
- Lee, J.-O., Russo, A. A., Pavletich, N. P. (1998) Nature 391, 859865[CrossRef][Medline]
[Order article via Infotrieve]
- Kelly, B. L., Wolfe, K. G., and Roberts, J. M. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 25352540[Abstract/Free Full Text]
- Liu, F., Rothblum-Oviatt, C., Ryan, C. E., and Piwnica-Worms, H. (1999) Mol. Cell. Biol. 19, 51135123[Abstract/Free Full Text]
- Morris, M. C., and Divita, G. (1999) J. Mol. Biol. 286, 475487[CrossRef][Medline]
[Order article via Infotrieve]
- Morris, M. C., Heitz, A., Mery, J., Heitz, F., and Divita, G. (2000) J. Biol. Chem. 275, 2884928857[Abstract/Free Full Text]
- Kobayashi, H., Stewart, E., Poon, R., Adamczewski, J. P., Gannon, J., and Hunt, T. (1992) Mol. Biol. Cell 3, 12791294[Abstract]
- Lees, E. M., and Harlow, E. (1993) Mol. Cell. Biol. 13, 11941201[Abstract]
- Glozter, M., Murray, A. W., and Kirschner, M. W. (1991) Nature 349, 132138[CrossRef][Medline]
[Order article via Infotrieve]
- Stewart, E., Kobayashi, H., Harrison, D., and Hunt, T. (1994) EMBO J. 13, 584594[Abstract]
- Pines, J., and Hunter T. (1994) EMBO J. 13, 37723781[Abstract]
- Goda, T., Funakoshi, M., Suhara, H., Nishimoto, T., and Kobayashi, H. (2001) J. Biol. Chem. 276, 1541515422[Abstract/Free Full Text]
- Horton, R. M., and Pease, L. R. (1991) in Directed Mutagenesis (McPherson, M. J. ed), pp. 217247, IRL Press, Oxford
- Nakajo, N., Yoshitome, S., Iwashita, J., Iida, M., Uto, K., Ueno, S., Okamoto, K., and Sagata, N. (2000) Genes Dev. 14, 328338[Abstract/Free Full Text]
- Nakajo, N., Oe, T., Uto K, and Sagata, N. (1999) Dev. Biol. 207, 432444[CrossRef][Medline]
[Order article via Infotrieve]
- Murray, A. W. (1991) Methods Cell Biol. 36, 581605[Medline]
[Order article via Infotrieve]
- Sagata, N., Oskarsson, M., Copeland, T., Brumbaugh, J., and Vande Woude, G. F. (1988) Nature 335, 519525[CrossRef][Medline]
[Order article via Infotrieve]
- Coleman T. R., and Dunphy, W. G. (1994) Curr. Opin. Cell Biol. 6, 877882[Medline]
[Order article via Infotrieve]
- Brown, N. R., Noble, M. E., Endicott, J. A., Garman, E. F., Wakatsuki, S., Mitchell, E., Rasmussen, B., Hunt, T., and Johnson, L. N. (1995) Structure 3, 12351247[Medline]
[Order article via Infotrieve]
- Jeffrey, P. D., Russo, A. A., Polyak, K., Gibbs, E., Hurwitz, J., Massague, J., and Pavletich, N. P. (1995) Nature 376, 313320[CrossRef][Medline]
[Order article via Infotrieve]
- Holland, P. M., and Cooper, J. A. (1999) Curr. Biol. 9, R329-R331[CrossRef][Medline]
[Order article via Infotrieve]
- Tanoue, T., Adachi, M., Moriguchi, T., and Nishida, E. (2000) Nat. Cell Biol. 2, 110116[CrossRef][Medline]
[Order article via Infotrieve]
- Hochegger, H., Klotzbucher, A., Kirk, J., Howell, M., Guellec, K. I., Fletcher, K., Duncan, T., Sohail, M., Hunt, T. (2001) Development 128, 37953807[Abstract/Free Full Text]
- Calzada, A., Sacristan, M., Sanchez, E., and Bueno A. (2001) Nature 412, 355358[CrossRef][Medline]
[Order article via Infotrieve]
- Furstenthal, L., Kaiser, B. K., Swanson, C., and Jackson, P. K. (2001) J. Cell Biol. 152, 12671278[Abstract/Free Full Text]
- Zheng X.-F., and Ruderman, J. V. (1993) Cell 75, 155164[Medline]
[Order article via Infotrieve]
- Nurse, P. (1999) Biol. Chem. 380, 729733[Medline]
[Order article via Infotrieve]
- Kishimoto, T., and Okumura, E. (1997) Prog. Cell Cycle Res. 3, 241249[Medline]
[Order article via Infotrieve]
- Fattaey, A., and Booher, R. N. (1997) Prog. Cell Cycle Res. 3, 233240[Medline]
[Order article via Infotrieve]
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