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Molecular Cancer Research 1:280-289 (2003)
© 2003 American Association for Cancer Research

Cell Cycle, Cell Death, and Senescence

Phosphorylation of the Cyclin B1 Cytoplasmic Retention Sequence by Mitogen-Activated Protein Kinase and Plx1

Susan Walsh1, Seth S. Margolis1 and Sally Kornbluth1

1 Duke University Medical Center, Durham, NC

Requests for reprints: Sally Kornbluth, C370 LSRC, Research Drive, Box 3813, Duke University Medical Center, Durham, NC 27710. Phone: (919) 613-8624; Fax: (919) 681-1005. E-mail: kornb001{at}mc.duke.edu


   Abstract
Top
 Notes
 Abstract
Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 
The cyclin B1/Cdc2 complex regulates many of the dramatic cellular rearrangements observed at mitosis. Although predominantly cytoplasmic during interphase, this kinase complex translocates precipitously to the nucleus at the G2-M transition. The interphase cytoplasmic location of cyclin B1/Cdc2 reflects continuous, albeit slow, nuclear import and much more rapid nuclear export. In contrast, the sudden nuclear accumulation of the complex before entry into mitosis reflects a marked increase in the import rate, with a concomitant inhibition of cyclin B1 nuclear export. These dynamic changes in cyclin B1/Cdc2 localization are regulated by phosphorylation of four serines within a region of cyclin B1 known as the cytoplasmic retention sequence (CRS). Phosphorylation of all four serines is required for rapid nuclear entry, whereas phosphorylation of only the last in the series (Ser 113) is required to prevent nuclear export by CRM1. As these residues represent key loci of regulation, it is important to identify the kinases acting on these sites. Here we report that Xenopus cyclin B1 is regulated by both Erk and Plx kinases, and that Cdc2, counter to previous speculation, is not required for CRS phosphorylation. Phosphorylation of the first two of the CRS serines (Ser 94 and Ser 96) is catalyzed by Erk in the Xenopus system. Although it was previously reported that Ser 113 is a Plx substrate, we were unable to observe phosphorylation of this residue in isolation by purified Plx. Rather, in contrast to previously published data, we have found that the penultimate CRS serine (Ser 101) is a Plx substrate. Collectively, these data demonstrate a new role for Erk in mitotic regulation, identify the Ser 101-directed kinase, and provide a picture of cyclin B1/Cdc2 regulation by the combinatorial action of distinct kinases.


   Introduction
Top
 Notes
 Abstract
 Introduction
Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 
Entry into mitosis is regulated by the activity of cyclin B/Cdc2 complexes. Through activation of other mitotic kinases and direct phosphorylation of additional cellular components, these complexes promote the dramatic rearrangement of cellular structures observed in mitosis. Because inappropriate activation of cyclin B/Cdc2 complexes can lead to catastrophic premature mitosis, these complexes are tightly regulated. Cyclin B/Cdc2 enzymatic activity is controlled by phosphorylation on Cdc2 at two sites, Thr 14 and Tyr 15 (1–3). Phosphorylation of these sites is catalyzed by the nuclear tyrosine kinase Wee1 and the membrane-associated dual-specificity kinase Myt1 (1, 4–13). Dephosphorylation of these sites, promoted by the Cdc25 phosphatase, then allows Cdc2 activation and consequent mitotic entry (12–15).

While phosphorylation of cyclin B does not appear to regulate the enzymatic activity of the complex, the subcellular localization of the cyclin B1/Cdc2 complex is controlled through phosphorylation of four serines (94, 96, 101, and 113 in Xenopus) within a region of cyclin B1 known as the cytoplasmic retention sequence (CRS) (16–24). Although cyclin B1 is primarily cytoplasmic during interphase of the cell cycle, deletion of the CRS region (aa 76–126 in Xenopus) results in its inappropriate nuclear accumulation (21). Furthermore, mutation of the four CRS serines to alanines suppresses M phase induction by cyclin B1 injected into Xenopus oocytes, and activity can be restored through appendage of a strong nuclear localization sequence (NLS) (18, 19, 24).

Although the nuclear accumulation of cyclin B1 lacking a CRS was originally thought to indicate a role for the CRS in promoting cyclin B1 cytoplasmic anchoring, it is now believed that this behavior reflects, at least in part, the presence of a nuclear export sequence (NES) within the CRS (16, 22, 23). During interphase, cyclin B1 shuttles in and out of nuclei continuously, with the rate of nuclear export exceeding the rate of nuclear import. However, at the time of entry into mitosis, phosphorylation of the four CRS serines results in an inhibition of nuclear export and a marked increase in cyclin B1 nuclear import rate. This acceleration of nuclear import raises the possibility that the CRS may, as initially hypothesized, regulate anchoring to a cytoplasmic constituent as well as cyclin B1 nuclear export. Although cyclin B1 enters nuclei slowly during interphase through a direct interaction with the nuclear transporter importin-ß, it has also been suggested that CRS phosphorylation may create a novel nuclear localization sequence for a more efficient interaction with an alternative nuclear import receptor (17, 25, 26).

Analysis of the individual sites of cyclin B1 phosphorylation revealed, surprisingly, that phosphorylation of all four Ser sites within the CRS is required for the rapid nuclear import rate seen at mitosis, but phosphorylation of only Ser 113 is required to inhibit nuclear export of cyclin B1 (24). On the basis of these data, we hypothesized that rapid mitotic nuclear entry of cyclin B1 at the time of mitosis required the concerted action of kinases acting on all four of these sites, suggesting that kinases acting on these sites would be critical for regulating the timing of mitotic entry. Therefore, we wished to ask which kinases were involved in promoting these phosphorylations. Recently, it was reported that the Plx kinase, known to regulate various aspects of mitosis, including activation of Cdc25, was responsible for phosphorylation of Ser 147 in human cyclin B1, the equivalent of Xenopus Ser 113 (27). It has also been widely speculated that Cdc2 can phosphorylate cyclin B1, thereby helping to accelerate nuclear entry of the complex (28).

Using purified kinases, complete Xenopus egg extracts, and Xenopus oocytes, we have now reexamined the identity of kinases acting on the first three sites within the CRS. We show here that cyclin B1 is phosphorylated in Xenopus egg extracts (either arrested in meiotic metaphase or driven into mitosis by the addition of recombinant cyclin B1) at Ser 94 and Ser 96 by the MEK-activated mitogen-activated protein kinase (MAPK) Erk. Experiments in intact Xenopus oocytes suggest that these are also Erk sites in progesterone-treated oocytes. Furthermore, we present data to suggest that Cdc2 is not responsible for phosphorylation of any of the Ser residues of Xenopus cyclin B1 within the CRS, raising the possibility that the rapid entry of cyclin B1/Cdc2 at mitosis in Xenopus does not require autophosphorylation of the complex by Cdc2. Finally, in contrast to previously reported findings, we did not observe phosphorylation of Ser 113 in isolation by purified Plx (27); rather, we have found that Ser 101 is a Plx substrate.


   Results
Top
 Notes
 Abstract
 Introduction
 Results
Discussion
 Materials and Methods
 Acknowledgements
 References
 
Ser 94 and Ser 96 Are Not Cdc2 Phosphorylation Sites in Xenopus Egg Extracts
As phosphorylation of all four sites within the CRS region proved to be critical for regulating cyclin B1 nuclear localization (24), we wished to further explore the identification of kinases acting on these residues. To examine the phosphorylation of each of these sites in isolation, we generated a panel of GST-CRS fusion proteins, each containing a single serine (at 94, 96, 101, or 113) and alanine substitutions at the other three phosphorylation sites. Because Ser 94 and Ser 96 are canonical Cdc2 Ser/Pro phosphorylation sites (Fig. 1A), it has been widely assumed that Cdc2 is the relevant enzyme phosphorylating at least one of these sites. Indeed, in an in vitro kinase assay using purified Cdc2/cyclin B1 and our panel of GST-CRS substrates, the wild-type protein (CRSWT) and the proteins retaining Ser at residue 94 (CRS94S) or Ser at residue 96 (CRS96S) were all efficiently phosphorylated (Fig. 1B).



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  		FIGURE 1. In vitro, Cdc2/cyclinB1 phosphorylates Ser 94 and Ser 96. A. Sequence of Xenopus cyclin B1 CRS (aa 76–126) with the phosphorylation sites highlighted. This sequence was fused to GST for CRS expression constructs. For numbering of residues within the CRS, the human equivalents of Xenopus Ser 94, Ser 96, Ser 101, and Ser 113 are Ser 126, Ser 128, Ser 133, and Ser 147, respectively. B. Purified cyclin B1/Cdc2 kinase was incubated with 1 µg of GST-CRS proteins in kinase buffer with radiolabeled ATP for 20 min at room temperature. Samples were then washed once with ELB with 0.1% Triton X-100, and SDS-PAGE sample buffer was added. Samples were boiled and resolved by SDS-PAGE. Phosphorylated proteins were detected by autoradiography.

 
To determine whether Cdc2 was truly responsible for phosphorylation of these sites in a complex cellular mixture, we converted an interphase Xenopus egg extract to mitosis by the addition of recombinant nondegradable His-cyclinB1{Delta}13 protein. After the extract had clearly entered mitosis (as judged by microscopic visualization of mitotic chromatin condensation), we added the phosphatase inhibitor microcystin to preserve the mitotic phosphorylation and depleted Cdc2 from extracts. As shown in Fig. 2A, three rounds of depletion rendered Cdc2 undetectable by Western blotting. These depleted extracts, along with control mitotic and interphase extracts, were then assayed for kinase activity using either the well-characterized Cdc2 substrate, histone H1 (29), the GST-CRS94S protein, or the GST-CRS96S protein as substrates (Fig. 2, B–D). As shown in Fig. 2, histone H1-directed kinase activity dropped dramatically in the Cdc2-depleted extract, but Ser 94 and Ser 96 phosphorylations were virtually unaffected.



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  		FIGURE 2. Depletion of Cdc2 from mitotic extracts does not significantly affect Ser 94- or Ser 96-directed kinase activity. Crude interphase extract with energy regenerating mix was converted to mitosis by the addition of His-cyclinB1{Delta}13 for 1 h at room temperature. Anti-Cdc2 antibody was coupled to protein A-Sepharose for 1 h at 4°C and then used in three rounds of depletion of the mitotic extract in combination with Ni-NTA agarose. A. Extract samples were diluted into SDS-PAGE sample buffer and resolved by SDS-PAGE. The proteins were transferred to polyvinylidene difluoride (PVDF) membrane and blotted for Cdc2 with an anti-PSTAIRE antibody. B–D. Samples from the above extracts were incubated with 1 µg of GST-CRS94S protein (B), GST-CRS96S protein (C), or histone H1 (D) protein in kinase buffer with radiolabeled ATP for 10 min at room temperature. The reaction was then stopped with SDS-PAGE sample buffer. Samples were boiled and resolved by SDS-PAGE. Phosphorylated proteins were detected and quantitated by phosphorimager analysis.

 
To confirm the data, we treated mitotic Xenopus egg extracts with the Cdc2 kinase inhibitor roscovitine (30) (Fig. 3). Mitotic extracts treated with vehicle alone (DMSO) exhibited high levels of histone H1-, GST-CRS94S-, and GST-CRS96S-directed kinase activity (Fig. 3). However, with increasing concentrations of roscovitine, histone H1-directed kinase activity was progressively repressed, but Ser 94- and Ser 96-directed kinase activities were largely unaffected (Fig. 3). Therefore, contrary to our expectations and published hypotheses (28), neither of the Ser/Pro sites in the Xenopus CRS seems to be dependent on Cdc2 for its phosphorylation. This raises the possibility that Cdc2/cyclin B1 autophosphorylation activity may not be required for nuclear entry of cyclin B1, and that another mitotically activated kinase is responsible for phosphorylation of these sites.



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  		FIGURE 3. The Cdc2 inhibitor, roscovitine, does not affect Ser 94- or Ser 96-directed kinase activity. Mitotic extracts were treated with vehicle (DMSO) or with roscovitine at varying concentrations. Samples from the treated extracts were then incubated with 1 µg of GST-CRS94S protein (A), GST-CRS96S protein (B), or histone H1 (C) protein in kinase buffer with radiolabeled ATP for 10 min at room temperature. The reaction was then stopped with SDS-PAGE sample buffer. Samples were boiled and resolved by SDS-PAGE. Phosphorylated proteins were detected and quantitated by phosphorimager analysis.

 
Phosphorylation of Ser 94 and Ser 96 Is Regulated by the Mos/MEK/Erk MAPK Pathway
The Erk MAP kinases are also Ser/Pro-directed kinases implicated in cell cycle progression. Indeed, it has been speculated that MAPK might regulate cyclin B1/Cdc2 localization through phosphorylation of either the Ser 94 or Ser 96 site (28). In vitro, purified MAPK can phosphorylate cyclin B1 within the CRS region, although the question of whether Ser 94 or 96 is the target has not been investigated. To address this issue directly, we first examined the ability of the purified MAPK Erk1 to phosphorylate individual residues within the CRS. As shown in Fig. 4A, when we incubated purified Erk1 and radiolabeled ATP with each protein in our panel of GST-CRS proteins, the constructs containing Ser at residues 101 or 113 were not detectably phosphorylated by Erk. However, the CRSWT, CRS94S, and CRS96S were all phosphorylated, consistent with the notion that Ser 94 and Ser 96 are Erk substrates.



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  		FIGURE 4. In vitro, MAPK phosphorylates Ser 94 and Ser 96. A. Purified Erk1 kinase was incubated with 1 µg of GST-CRS proteins in kinase buffer with radiolabeled ATP for 20 min at room temperature. Samples were then washed, boiled in SDS-PAGE sample buffer, and resolved by SDS-PAGE. Phosphorylated proteins were detected by phosphorimager analysis. To better view the CRS96S phosphorylation, the lower panel shows six times the amount of substrate as the upper panel. B. The Ser 96 site is phosphorylated in the CRSWT protein by Erk1, as detected by the phosphoserine 96S antibody. Purified Erk1 kinase was incubated with 5 µg CRSWT substrate in kinase buffer with ATP. At the noted time points, a fraction of the reaction was removed and diluted into SDS-PAGE sample buffer. These reactions were resolved by SDS-PAGE and immunoblotted with the phosphoserine 96 antibody.

 
Although Ser 96 was phosphorylated by Erk, the GST-CRS construct used to monitor this activity (containing Ser at position 96 and Ala at positions 94, 101, and 113) was phosphorylated considerably less well than the equivalent construct used to monitor Ser 94 phosphorylation. This raises the possibility that Ser 96 may not be as efficient a substrate for Erk as Ser 94. However, it is also possible that the particular construct used was not in an optimal configuration for Erk-mediated phosphorylation of Ser 96. Therefore, to monitor Ser 96 phosphorylation more easily, we produced a phosphoserine 96-specific antibody able to recognize phosphorylated Ser 96 in the context of the CRSWT. As shown in Fig. 4B, the CRSWT reacted with this antibody only after Erk phosphorylation. Hence, for further analysis of Ser 96 phosphorylation, as described below, we used the antibody for detection of Ser 96 phosphorylation.

To assay Ser 96-directed kinase activity across the cell cycle, we used a cycling egg extract (one which oscillates from S to M phase based on the synthesis and degradation of cyclins) as a source of Ser 96-directed kinases. To distinguish oscillations in the kinase activities from oscillations in cyclin B1 abundance, we used exogenous GST-CRSWT as a substrate in these reactions. At 10-min intervals after the start of room temperature incubation, aliquots of cycling extract were allowed to react with recombinant GST-CRSWT and immunoblotted with the anti-phosphoserine 96 antibody. Parallel samples supplemented with nuclei were withdrawn at each time point and stained with Hoechst 33258 for microscopic observation of mitotic entry (nuclear envelope breakdown, chromatin condensation) or immunoblotted for active MAPK, as evidenced by the presence of activating phosphorylations. As shown in Fig. 5A, phosphorylation at Ser 96 fluctuated with the cell cycle, increasing at 60 min, coincident with chromatin condensation (data not shown) and activation of MAPK.



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  		FIGURE 5. Phosphorylation of Ser 94 and Ser 96 is regulated by MAPK. A. A cycling extract was made and supplemented with energy regenerating mix and sperm chromatin. At 10-min intervals, 50 µl of cycling extract was removed and incubated with GST-CRSWT for 10 min at room temperature. After the 10-min incubation, SDS-PAGE sample buffer was added to stop the reaction. The samples were resolved by SDS-PAGE, and the proteins were transferred to PVDF membrane and blotted with the phospho-specific Ser 96 antibody (lower panel). In parallel, 1-µl samples were taken at each time point and stained with Hoechst's dye. These samples were analyzed by fluorescence microscopy for changes in cell cycle. Mitotic chromosome condensation occurred from 60 to 80 min (data not shown). In addition, parallel extract samples were removed at the appropriate time points and immunoblotted for endogenous phospho-MAPK (upper panel). B, C, D. Crude interphase extracts with energy regenerating mix were treated with DMSO (vehicle) or 5 µM U0126 for 15 min at room temperature. Treated extracts were then converted to mitosis by the addition of His-cyclinB1{Delta}13 for 1 h at room temperature, treated with Mos for 30 min to activate the MAPK pathway, or treated with Mkp-1 (40 µg/ml) to inactivate MAPK. B. Samples were diluted into SDS-PAGE sample buffer and resolved by SDS-PAGE. The proteins were transferred to PVDF membrane and blotted for phospho-MAPK. C. Samples from the above extracts were incubated with 1 µg of GST-CRS94S protein in kinase buffer with radiolabeled ATP for 10 min at room temperature. The reaction was stopped with SDS-PAGE sample buffer. Samples were boiled and resolved by SDS-PAGE. Phosphorylated proteins were detected and quantitated by phosphorimager analysis. D. One microgram of GST-CRSWT was incubated with the above extracts for 10 min at room temperature. SDS-PAGE sample buffer was added, and samples were boiled and resolved by SDS-PAGE. The proteins were transferred to a PVDF membrane and blotted with the phosphoserine 96 antibody. S, interphase extract; M, interphase extract converted to mitosis by the addition of His-cyclinB1{Delta}13; M/U0126, interphase extract first treated with 5 µM U0126 and then converted to mitosis by the addition of His-cyclinB1{Delta}13; S/Mos, interphase extract treated with Mos; Buffer, ELB buffer; M/Buffer, interphase extract converted to mitosis by the addition of His-cyclinB1{Delta}13 and then ELB added; M/Mkp-1, interphase extract converted to mitosis by the addition of His-cyclinB1{Delta}13 and then Mkp-1 added.

 
Whereas the ability of purified Erk to phosphorylate Ser 94 and Ser 96 was consistent with a role for this kinase in controlling cyclin B1 localization, we wished to determine whether Erk was indeed the kinase responsible for phosphorylating these sites in the complex extract system. Therefore, we performed kinase assays using GST-CRS94S or GST-CRSWT (for subsequent recognition by the phosphoserine 96 antibody) as substrates and Xenopus egg extracts as a source of kinases. Not surprisingly, egg extracts arrested in interphase of the cell cycle (S extracts) phosphorylated both sites inefficiently, consistent with the cytoplasmic localization of hypophosphorylated cyclin B1 at this phase of the cell cycle (Fig. 5, C and D). However, when interphase extracts were driven into mitosis by the addition of recombinant His-cyclinB1{Delta}13 protein, Ser 94 and Ser 96 were both phosphorylated by kinases present in the extract (Fig. 5, C and D).

Although MAPK activity is low during interphase, it has been reported that the addition of cyclin B1 to interphase egg extracts promotes MAPK activation (for reviews: Refs. 31, 32). Moreover, this activation requires the upstream MAPK activator MEK (Ref. 33 and Fig. 5B, left-hand panel). Accordingly, to examine the role of MAPK in Ser 94 and Ser 96 phosphorylation, we treated an interphase egg extract with the MEK inhibitor U0126 before the addition of His-cyclinB1{Delta}13 (34). Extracts treated in this manner failed to activate MAPK (Fig. 5B, left-hand panel) but still activate Cdc2 (data not shown). Notably, Ser 94- and Ser 96-directed kinase activities dropped to almost interphase levels when MAPK activation was prevented (Fig. 5, C and D, left-hand panels). Therefore, we conclude that MAPK is essential for Ser 94 and Ser 96 phosphorylation of Xenopus cyclin B1 in the egg extract system. To further demonstrate that activation of the MAPK pathway was essential for this activity, in the absence of other mitotic kinase activation, we supplemented interphase extracts with the MEK activator Mos (33, 35) (Fig 5B). This treatment alone was sufficient to increase Ser 94 and Ser 96 phosphorylation to mitotic levels, presumably through the activation of the downstream MAPK (Fig. 5, C and D, left-hand panels). Furthermore, U0126 treatment abolished this increased phosphorylation due to the Mos addition (data not shown).

To assess more directly the effect of MAPK on Ser 94 and Ser 96 phosphorylation, we inactivated the MAPK through the use of the MAPK-directed phosphatase, Mkp-1, which removes the activating phosphorylations from MAPK (36). To first activate MAPK, we created mitotic extracts by the addition of recombinant His-cyclinB1{Delta}13 protein to interphase extracts. Mitotic entry was confirmed microscopically (data not shown), and then control buffer or Mkp-1 protein was added to the extracts. After 45 min of room temperature incubation, kinase assays were performed on the CRS94S (radiolabeled) and CRS96S substrates (immunoblot). In addition, to confirm the maintenance of mitosis in the extract, samples were stained with Hoechst 33258 and examined for mitotic chromosome condensation after Mkp-1 treatment (data not shown). As shown in Fig. 5B (right-hand panel), Mkp-1 removed the activating phosphorylations from MAPK, leading to a marked diminution in both Ser 94- and Ser 96-directed kinase activities (Fig. 5, C and D, right-hand panels). Collectively, these data argue strongly that MAPK is the Ser 94- and Ser 96-directed kinase.

To examine the role of MAPK in triggering cyclin B1 CRS phosphorylation in an intact cell physiologically stimulated to enter M phase, we treated primed Xenopus oocytes with progesterone in the presence and absence of the MEK inhibitor U0126 to prevent MAPK activation. At the concentrations used, this inhibitor did not prevent germinal vesicle breakdown (GVBD), as was recently reported by others (37). Lysates prepared from the treated and untreated oocytes were then assayed for Ser 94-directed kinase activity using radiolabeled ATP. In the absence of U0126, Ser 94 phosphorylation became detectable 2 h after progesterone stimulation and continued to increase with time (Fig. 6A). However, U0126 treatment rendered Ser 94 phosphorylation undetectable until 12 h after progesterone stimulation. Inhibition of MAPK produced a similar result for Ser 96-directed kinase activity, as observed by immunoblotting endogenous full-length cyclin B1 in the oocyte lysates with the anti-phosphoserine 96 antisera (Fig. 6B). The residual signals in the 12-h time point may reflect the presence of a low-level CRS-directed non-MAPK pathway kinase or, perhaps more likely, result from loss of U0126 potency over time. Nonetheless, taken together with the extract data, the oocyte findings strongly suggest that phosphorylation of Ser 94 and Ser 96 can be modulated by the Mos/MEK/MAPK pathway.



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  		FIGURE 6. U0126 inhibition of the MAPK pathway prevents phosphorylation at both the Ser 94 and Ser 96 sites in Xenopus oocytes. Oocytes were isolated from primed frogs and cultured in modified Barth's buffer with calcium. The medium was supplemented with either 50 µM U0126 or DMSO for 1 h at 18°C. Oocytes were then treated with 1 µM progesterone in the presence of the above medium. At the noted times (where t = 0 is equivalent to addition of progesterone), oocytes were frozen in liquid nitrogen. A. Oocyte lysate from three oocytes was obtained and used to phosphorylate 1 µg of CRS94S protein in the presence of radiolabeled ATP for 10 min at room temperature. The reaction was stopped with SDS-PAGE sample buffer and resolved by electrophoresis and autoradiography. B. Oocyte lysate was made from six oocytes, and the equivalent of two oocytes was resolved by SDS-PAGE and immunoblotted with the phosphoserine 96 antibody.

 
Phosphorylation of Ser 101 Is Regulated by Plx
Previously published work has implicated the Plx kinase as a regulator of cyclin B1 nuclear trafficking (27). Through analysis of cyclin B1 mutants and generation of a phospho-specific antibody directed against the equivalent of Ser 113 in human cyclin B1 (Ser 147 of human cyclin B1), Toyoshima-Morimoto et al. (27) concluded that Plk1 (the human homologue of Xenopus Plx1) phosphorylates Ser 147, and not the human cyclin B1 equivalents of Ser 94, 96, or 101. However, using our series of Xenopus CRS mutant derivatives, we found that purified baculovirus-produced Plx was capable of phosphorylating only the constructs containing Ser at position 101, and not those containing Ser at position 113 (Fig. 7, A and D).



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  		FIGURE 7. Plx phosphorylates the Ser 101 site but not Ser 113. A. Baculovirus-produced Plx kinase was incubated with 1 µg of GST-CRS proteins in kinase buffer with radiolabeled ATP for 20 min at room temperature. Samples were then washed once with buffer, and then SDS-PAGE sample buffer was added. Samples were boiled and resolved by SDS-PAGE. Phosphorylated proteins were detected by autoradiography. B. A cycling extract was made and supplemented with energy regenerating mix and sperm chromatin. At 10-min intervals, 50 µl of cycling extract was removed and incubated with human His-cyclinB1{Delta}13 for 10 min at room temperature. After the 10-min incubation, SDS-PAGE sample buffer was added to stop the reaction. The samples were resolved by SDS-PAGE, and the proteins were transferred to PVDF membrane and blotted with the phospho-specific Ser 133 antibody. In parallel, 1-µl samples were taken at each time point and stained with Hoechst's dye. These samples were analyzed by fluorescence microscopy for changes in cell cycle. Mitotic chromosome condensation occurred from 50 to 60 min (data not shown). C. Anti-Plx antibody or preimmune sera were coupled to protein A-Sepharose for 1 h at 4°C and then used in four rounds of depletion of mitotic extract. Samples were diluted into SDS-PAGE sample buffer and resolved by SDS-PAGE. The proteins were transferred to PVDF membrane and blotted for Plx. Lane 1, total mitotic extract; lane 2, Plx-depleted extract; lane 3, preimmune-depleted extract. Mitotic extract, Plx-depleted extract, and preimmune-depleted extract were incubated with human His-cyclinB1{Delta}13 for 10 min at room temperature. After the 10-min incubation, reactions were diluted into mitotic buffer with 2 µM microcystin and incubated for 1 h at 4°C with Ni-NTA agarose. The samples were then washed three times with mitotic buffer, and SDS-PAGE sample buffer was added. The samples were resolved by SDS-PAGE, and the proteins were transferred to PVDF membrane and blotted with the phospho-specific Ser 133 antibody. Lane 1, total mitotic extract; lane 2, Plx-depleted extract; lane 3, preimmune-depleted extract. D. Baculovirus-produced Plx kinase was incubated with 1 µg of GST-CRS proteins, which includes serine to glutamate mutations to mimic phosphorylation, in kinase buffer with radiolabeled ATP for 5 or 20 min at room temperature. SDS-PAGE sample buffer was added, and samples were boiled and resolved by SDS-PAGE. Phosphorylated proteins were quantitated by phosphorimager analysis. A coomassie stain of the kinase assays is shown below the graph to show equal protein loading.

 
To further analyze Ser 101 phosphorylation and the role of Plx in this process, we used an antibody prepared against a peptide derived from the human CRS phosphorylated at residue Ser 133 (equivalent to phosphoserine 101 of Xenopus) (38). As was done for Ser 96, above, we assayed Ser 101-directed kinase activity across the cell cycle, using Xenopus egg cycling extracts as a source of kinases. In this case, however, we used exogenous, recombinant human His-cyclinB1{Delta}13 as a substrate. At 10-min intervals after the start of room temperature incubation, aliquots of cycling extract were incubated with recombinant His-cyclinB1{Delta}13 and then immunoblotted with the anti-phosphoserine 133 antibody. Microscopic observation of parallel samples supplemented with nuclei was done to monitor nuclear envelope breakdown and chromatin condensation. As shown in Fig. 7B, phosphorylation at this site fluctuated with the cell cycle, reaching a peak at 40 min, 10 min before chromatin condensation was evident microscopically (data not shown).

To determine whether Plx was required for phosphorylation of Ser 101 in the complete egg extract, we immunodepleted Plx from mitotic extracts (Fig 7C) and performed kinase assays using the recombinant His-cyclinB1{Delta}13 protein as a substrate. Samples were then immunoblotted with the anti-phosphoserine 133 antibody. As shown in Fig. 7C, depletion of Plx prevented mitotic extracts from phosphorylating this residue, further supporting the hypothesis that Plx is responsible for phosphorylating Ser 101.

In addition, we tested a cycling extract for phosphorylation of a GST-CRS construct containing Ser 101 and Glu at the other CRS serines (GST-CRS-EESE). Phosphorylation of the Ser 101 site in this construct fluctuated with the cell cycle, reaching a peak at mitosis, when nuclear evidence of mitosis was detected (data not shown). Furthermore, when we immunodepleted Plx from the peak fraction (along with a separate, non-peak fraction) and reassayed these depleted extracts for GST-CRS-EESE-directed kinase activity, we found that Plx depletion reduced the mitotic-derived peak phosphorylation to near interphase levels. These data fully support the idea that Plx phosphorylates Ser 101, thereby contributing to cyclin B1 nuclear import.

While these data showed that Ser 101 is phosphorylated by Plx, regardless of the phosphorylation state of other CRS Ser residues, we considered it possible that prior phosphorylation at residues 94 and 96 might enable us to visualize the previously reported Plx-mediated Ser 113 phosphorylation (27). Moreover, we wished to know whether phosphorylation of other CRS residues might enhance Ser 101 phosphorylation. To test this hypothesis, we created a panel of GST-CRS mutants in which serine residues were changed to glutamates to simulate phosphoserine. However, we were unable to detect phosphorylation of Ser 113 in isolation in any of our assays. Even when Ser 94, 96, and 101 were all changed to Glu, Ser 113 could not be phosphorylated by Plx in our in vitro kinase assays (Fig. 7D). Indeed, the CRS-EESE and CRS-EESS proteins were phosphorylated to similar levels by Plx, suggesting that the only phosphorylation in these proteins was due to Ser 101-directed kinase activity. These results suggest that Plx may not be the responsible kinase for phosphorylating Ser 113, consistent with the fact that the Ser 101 site fits the previously published Plx consensus motif, while the Ser 113 site does not (39). An inability of Plx to phosphorylate residue 113 has also been recently observed for human cyclin B1 (38). The other potentially interesting information to emerge from the data in Fig. 7D concerns the enhancement of Ser 101 phosphorylation when any of the other Ser residues are changed to Glu. Although Glu residues may not always precisely mimic phosphorylation, these data raise the possibility that prior phosphorylation by MAPK or the Ser 113-directed kinase may facilitate Plx-induced Ser 101 phosphorylation.


   Discussion
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
Materials and Methods
 Acknowledgements
 References
 
The timing of mitotic events is controlled, at least in part, by the nuclear accumulation of cyclin B1 (16–24). Cyclin B1 trafficking has also been implicated as a locus of control for DNA-responsive checkpoints, preventing mitotic entry in the presence of incompletely replicated or damaged DNA (40). Having previously demonstrated a role for CRS phosphorylation in regulating both nuclear import and export of cyclin B1 protein (24), we now assign specific kinases to phosphorylation of Ser 94, Ser 96, and Ser 101. Moreover, we have shown that Cdc2 autophosphorylation is not required for either Ser 94 or 96 phosphorylation, the Ser/Pro sites postulated to be substrates of this enzyme.

Ser 94 and Ser 96 Are Substrates of the MAPK Pathway
Although it was previously reported that purified Erk could phosphorylate cyclin B1 in vitro (28), the site of this phosphorylation was not established. Moreover, it was not clear that MAPK pathways were required for phosphorylation within the CRS. Our data indicate that in mitotic extracts of Xenopus eggs and in Xenopus oocytes, the MAPK pathway is primarily responsible for phosphorylation at Ser 94 and Ser 96. Activation of the MAPK pathway in the absence of other active mitotic kinases sufficed to promote Ser 94 and 96 phosphorylation. Additionally, inhibition of this pathway prevented Ser 94 and 96 phosphorylation even in the presence of other mitotically activated kinases, supporting a role for MAPK in cyclin B1 nuclear localization.

MAPK has been extensively characterized as a component of M phase entry in Xenopus oocytes (for reviews: Refs. 31, 32). While regulating cyclin B1 localization is clearly not the only role of MAPK in promoting mitotic entry in this system, our data suggest that it is likely to be one cell cycle function of this enzyme. As observed by Ferrell (41), MAPK activation can promote Cdc2 activation in a cell-free extract lacking nuclei, clearly suggesting other roles for MAPK in regulating the Cdc2/cyclin B1 complex. Indeed, it was recently reported that there is an important role for MAPK, primarily derived from its ability to activate p90Rsk, in promoting the G2-M transition in Xenopus oocytes (42–44). Previously published work established that all of the Ser residues in the CRS are important in inducing meiotic maturation of Xenopus oocytes, where MAPK activity rises at the G2-M transition (24). Nuclear translocation of Cdc2/cyclin B1 may be particularly important for the timing of M phase in this system, which lacks nuclear Wee1 to prevent Cdc2 activation. We note, however, that Roberts et al. (45) have recently reported that U0126 treatment delayed cyclin B1 nuclear localization and subsequent mitotic entry in HeLa cells, suggesting a possible conserved role for MAPK pathways in regulating cyclin B1 localization.

Cdc2 Is Not Required for CRS Phosphorylation in Xenopus Egg Extracts
Although both Ser 94 and 96 could be phosphorylated by purified cyclin B1/Cdc2, the fact that MAPK inhibition could prevent Ser 94 and Ser 96 phosphorylation ruled them out as candidate Cdc2 sites. Furthermore, immunodepletion of Cdc2 from egg extracts or inhibition of Cdc2 using roscovitine had virtually no effect on either Ser 94 or Ser 96 phosphorylation.

Previously, we reported that a cyclin B1 mutant unable to bind Cdc2 could enter the nucleus slowly during interphase (25), but we did not examine the kinetics of import of this mutant at the time of entry into mitosis, when the cyclin B1 import rate is considerably more rapid. Moreover, we used a truncated cyclin B1 in these studies, which may not behave identically to the wild-type protein. Recently, Takizawa et al. (26) reported that Cdc2 activity is required for mitotic import of full-length human cyclin B1. Taking into consideration the fact that CRS phosphorylation was required for nuclear import, they proposed that autophosphorylation by the complex might be a prerequisite for cyclin B1 nuclear entry. Coupling these observations with our findings, we would suggest the possibility that a kinase activated downstream of Cdc2/cyclin B1, the activity of which is maintained following Cdc2 immunodepletion (e.g., a Ser 113-directed kinase distinct from Plx), is responsible for the CRS-directed activity in our extracts. This may not be the case in all species/cell types. Indeed, phosphorylation of cyclin B in meiotic starfish oocytes seems to be catalyzed largely by Cdc2 (46).

Regulation of Cyclin B1 by Plx
As reported by Toyoshima-Morimoto et al. (27), Plx can phosphorylate the cyclin B1 CRS, thereby regulating cyclin B1 nuclear accumulation. However, we propose that the site of Plx regulation is not Ser 113, but rather is Ser 101. Indeed, we were unable to demonstrate phosphorylation of Ser 113 in isolation, as has been reported, raising the possibility that Ser 113 is phosphorylated by a novel kinase. The original purification of Plx as a cyclin B1-directed kinase by Toyoshima-Morimoto et al. was performed using a construct in which both Ser 101 and 113 (Ser 133 and 147 of human cyclin B1) were wild type, raising the possibility that some of the purified activity was directed against 101, and not 113. Indeed, this work did not assign any specific kinase to phosphorylation of Ser 101. Our work indicates strongly that the relevant kinase acting on this residue is Plx. Although the discrepancy between our work and that of the Toyoshima-Morimoto group concerning phosphorylation of Ser 113 (Ser 147 in human cyclin B1) could possibly be attributed to species differences, we note that the Pines laboratory, working with human cyclin B1, has also found Ser 101 (human Ser 133) to be the site of Plx-mediated phosphorylation using full-length human cyclin B1 (38). In addition, as reported here, cell-cycle-dependent Ser 101 phosphorylation of cyclin B1 is absolutely dependent on the presence of Plx in egg extracts. Because it is difficult to separate phosphoserine 101 and 113 by phosphopeptide mapping, further examination of this issue awaits the production of a phosphoserine 113 antibody reactive against Xenopus cyclin B1. In any event, our data obtained using either isolated CRS fusion proteins or human His-cyclinB1{Delta}13 and a phospho-specific antibody in a Plx-depleted extracts clearly demonstrate that Ser 101 is a bona fide Plx substrate. Therefore, regardless of how it affects Ser 113, the Plx kinase is essential for the rapid mitotic import of cyclin B1, as Ser 101 phosphorylation, like that of the other CRS serines, is critical for this process.

Either forcible localization of cyclin B1 to the nucleus or inhibition of cyclin B1 nuclear export has been reported to affect operation of DNA damage-responsive checkpoints (40). Furthermore, the coordinate nuclear accumulation of Cdc25 and cyclin B1 appears to accelerate mitotic onset (47). Hence, the identification of kinases controlling Cdc2/cyclin B1 localization and an elucidation of their regulation is of continuing importance for understanding the molecular mechanisms governing entry into mitosis.


   Materials and Methods
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
Acknowledgements
 References
 
Protein Expression and Reagents
The construction of the GST-CRS constructs has been described previously (24). Briefly, primers with BamHI or HindIII restriction sites were designed to the 5' and 3' ends, respectively, of the CRS region of cyclin B1 (nucleotides 223–381) with the following sequences: 5'-CGGGATCCATGCCTCTTAAAGTGATAGAAG-3' and 5'-GGGAAGCTTATCATCATCAGCATCAAC-3'. The PCR product was digested and ligated into the bacterial expression vector pGEX-KG. All GST-CRS constructs and GST-Mkp-1 were expressed in BL21 Escherichia coli. Bacteria were grown to an OD600 of 0.5 and then induced with 1 mM isopropyl-1-thio-ß-D-galactopyranoside for 3 h at 37°C. Bacteria were pelleted and washed with 0.9% NaCl solution. The pellets were resuspended and lysed in buffer [0.5 M sucrose, 50 mM Tris (pH 7.5), 1 mM EDTA, 8 mM KCl, and 1 mM DTT] with lysozyme. The lysate was spun at 17,000 x g for 30 min, and the protein supernatant was incubated with glutathione-Sepharose 4B (Amersham Pharmacia, Piscataway, NJ) for 1 h at 4°C. The Sepharose was then washed three times in wash buffer [10 mM HEPES (pH 8.0), 300 mM NaCl, and 1 mM DTT] and stored in this buffer at 4°C.

The baculovirus expression vector for nondegradable human His-cyclinB1{Delta}13 was generously provided by David O. Morgan (Department of Physiology, University of California, San Francisco). The His-Plx baculovirus clone was described previously (48). MBP-Mos was a gift from Katherine Swenson-Fields (Department of Pharmacology, Duke University). Ni-NTA agarose was purchased from Qiagen, Valencia, CA. Histone H1 substrate was purchased from Roche Molecular Biochemicals, Indianapolis, IN.

Preparation and Treatment of Xenopus Oocytes
Stage VI oocytes were prepared from frogs primed with 25 IU of pregnant mare serum gonadotropin 3–5 days before manual excision of ovaries. Ovaries were digested using 2.8 units of liberase (Roche Molecular Biochemicals) in OR-2 buffer [82.5 mM NaCl, 2 mM KCl, 1 mM MgCl2, 5 mM HEPES (pH 7.5)] for 1.5 h at room temperature. Oocytes were then washed extensively with OR-2 buffer and stored in OR-2 buffer + 1% fetal bovine serum + 0.2% gentamicin overnight at 18°C. Healthy stage VI oocytes were then selected for drug treatment. Eighty oocytes were placed in modified Barth's (MB) buffer with calcium in the presence of DMSO or 50 µM U0126 for 1 h at 18°C. Progesterone was then added to a final concentration of 1 µM. At each time point, nine oocytes were collected and frozen in liquid nitrogen.

Preparation of Xenopus Egg Extracts
Interphase and mitotic egg extracts were prepared according to the protocols of Smythe and Newport (49). To visualize mitotic chromosome condensation, extracts were supplemented with demembranated sperm chromatin (~2000/µl) and energy regenerating mix (2 mM ATP, 5 µg/ml creatine kinase, and 20 mM phosphocreatine). Extract samples were taken at various time points and subjected to formaldehyde fixation and staining with Hoechst 33258. Changes in chromatin morphology associated with cell cycle progression were monitored by fluorescence microscopy.

The kinase inhibitors, roscovitine and U0126, were both purchased from Calbiochem, San Diego, CA, and dissolved in DMSO. Mitotic extracts were treated with roscovitine at concentrations ranging from 1 to 25 µM for 25 min before kinase assays were performed. Interphase extracts were treated with U0126 at 5 µM for 15 min before adding recombinant His-cyclinB1{Delta}13 to convert the extract to mitosis.

In Vitro Kinase Assays
One microgram of substrate and 2 µl of extract or kinase were used in each 50 µl kinase reaction. The protein was quantitated by a Bradford assay, and the kinase assays were coomassie stained after SDS-PAGE and before drying and exposure to film. The kinases used were Erk1 (Calbiochem), Cdc2/cyclin B1 (Calbiochem), or baculovirus-produced Plx1 (48). The kinase reaction buffer consisted of 10 mM HEPES (pH 7.2), 5 mM MgCl2, 50 mM NaCl, 12.5 mM DTT, and 100 µM ATP. Radioactive labeling was performed by adding ATP-{gamma}-32P to the kinase buffer. Reactions were incubated at room temperature and then stopped with 2x SDS-PAGE sample buffer. Substrate phosphorylation was analyzed after SDS-PAGE and autoradiography. For quantitation, samples were analyzed by phosphorimager analysis.

For oocyte kinase activity, oocytes were resuspended in 20 µl oocyte lysis buffer [20 mM HEPES (pH 7.5), 80 mM ß-glycerophosphate, 15 mM MgCl2, 20 mM EGTA, 50 mM NaF, 1 mM Na3VO4, 10 µg/ml aprotin/leupeptin, 1 mM phenylmethylsulfonyl fluoride] per oocyte and lysed by pipetting. Samples were then microfuged at 4°C at 14,000 rpm, and the supernatant was collected. For radiolabeled kinase assays, 10 µl of supernatant was added to 9 µl oocyte kinase buffer [20 mM HEPES (pH 7.3), 10 mM EGTA, 20 mM MgCl2, 200 µM ATP, ATP-{gamma}-32P] and 1 µg substrate protein (histone H1 or CRS94S). The reaction was incubated at room temperature for 10 min and stopped with SDS-PAGE sample buffer. Radioactive ATP incorporation was measured by SDS-PAGE and autoradiography. For kinase activity against the Ser 96 site, oocytes were lysed in 10 µl oocyte lysis buffer per oocyte, and SDS-PAGE sample buffer was added. Endogenous cyclin B1 phosphorylation was measured by immunoblotting with the phosphoserine 96 antibody.

Immunodepletions
Xenopus anti-Cdc2 antibody was produced in rabbits using a KLH-conjugated peptide (KSSLPDNQIRN) as the antigen. Protein A-Sepharose resin (Sigma-Aldrich, St. Louis, MO) was washed three times with PBS and subsequently incubated with anti-Cdc2 IgG or anti-Plx IgG for 1 h at 4°C. The Sepharose was then washed three times with mitotic buffer [240 mM ß-glycerophosphate (pH 7.3), 60 mM EGTA, 45 mM MgCl2, and 1 mM DTT]. Microcystin (2 µM; Calbiochem) was added to the egg extracts to maintain mitotic phosphorylations, and these extract samples (150 µl) were depleted with the protein A-antibody resins three times at 4°C for 45 min each for Cdc2 or Plx antisera [generously provided by James Maller (Howard Hughes Medical Institute and Department of Pharmacology, University of Colorado School of Medicine)]. Depleted extracts were then used in kinase assays.

Immunoblotting
Immunoblotting was performed after SDS-PAGE and transfer to polyvinylidene difluoride membranes (Millipore, Bedford, MA). Blots were incubated with appropriate primary antisera, either anti-PSTAIRE monoclonal, anti-Plx polyclonal [generously provided by Erich Nigg (Department of Cell Biology, Max-Planck-Institute for Biochemistry)], anti-phospho-MAPK 44/42 (Cell Signaling Technologies, Beverly, MA), anti-phosphoserine 133 human cyclin B1 [generously provided by Jackman et al. (38), or anti-phosphoserine 96 Xenopus cyclin B1. The anti-phosphoserine 96 Xenopus cyclin B1 antibody was produced in rabbits using a KLH-conjugated phosphoserine peptide (QVEPSSPSPMETSGC) as the antigen. Blots were then incubated with the appropriate secondary antibody, either horseradish peroxidase-linked goat anti-rabbit or goat anti-mouse antibody (Jackson ImmunoResearch Laboratories, West Grove, PA). Blots were developed using an enhanced chemiluminescence kit (Renaissance, Dupont, PA).


   Acknowledgements
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
References
 
We are grateful to Drs. Mark Jackman and Jonathon Pines for their generous provision of anti-phosphoserine 133 antibody and to Dr. Jim Maller for his kind gift of anti-Plx sera. We are also grateful to Dr. Erich Nigg for his kind gift of Plx antibody, Dr. Katherine Swenson-Fields for her MBP-Mos construct, and Dr. David Morgan for the nondegradable human His-cyclinB1{Delta}13. We are grateful to Melinda Miller and Dr. Tom Guadagno for their kind gift of the GST-Mkp-1 plasmid. We thank Douglas C. Weiser for his critical review of the manuscript and technical discussions.


   Notes
Top
 Notes
Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 
1 NIH RO1 GM60500 to S.K. S.K. is a Scholar of the Leukemia and Lymphoma Society. Back

Received August 22, 2002; revised January 3, 2003; accepted January 6, 2003.


   References
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 

  1. Atherton-Fessler, S., Liu, F., Gabrielli, B., Lee, M. S., Peng, C. Y., and Piwnica-Worms, H. Cell cycle regulation of the p34cdc2 inhibitory kinases. Mol. Biol. Cell, 5: 989–1001, 1994.[Abstract]
  2. Borgne, A. and Meijer, L. Sequential dephosphorylation of p34(cdc2) on Thr-14 and Tyr-15 at the prophase/metaphase transition. J. Biol. Chem., 271(44): 27847–27854, 1996.[Abstract/Free Full Text]
  3. Gould, K. and Nurse, P. Tyrosine phosphorylation of the fission yeast cdc2+ protein kinase regulates entry into mitosis. Nature, 342: 39–45, 1989.[Medline]
  4. Kornbluth, S., Sebastian, B., Hunter, T., and Newport, J. Membrane localization of the kinase which phosphorylates p34cdc2 on Threonine 14. Mol. Biol. Cell, 5: 273–282, 1994.[Abstract]
  5. Krek, W., Marks, J., Schmitz, N., Nigg, E. A., and Simanis, V. Vertebrate p34cdc2 phosphorylation site mutants: effects upon cell cycle progression in the fission yeast Schizosaccharomyces pombe. J. Cell Sci., 102: 43–53, 1992.[Abstract/Free Full Text]
  6. McGowan, C. H. and Russell, P. Human Wee1 kinase inhibits cell division by phosphorylating p34cdc2 exclusively on Tyr15. EMBO J., 12: 75–85, 1993.[Medline]
  7. Mueller, P. R., Coleman, T. R., Kuamgai, A., and Dunphy, W. G. Myt1: a membrane-associated inhibitory kinase that phosphorylates cdc2 on both Threonine-14 and Tyrosine-15. Science, 270: 86–90, 1995.[Abstract/Free Full Text]
  8. Mueller, P. R., Coleman, T. R., and Dunphy, W. G. Cell cycle regulation of a Xenopus Wee1-like kinase. Mol. Biol. Cell, 6: 119–134, 1995.[Abstract]
  9. Norbury, C., Blow, J., and Nurse, P. Regulatory phosphorylation of the p34cdc2 protein kinase in vertebrates. EMBO J., 10: 3321–3329, 1991.[Medline]
  10. Solomon, M. J., Lee, T., and Kirschner, M. W. Role of phosphorylation in p34cdc2 activation: identification of an activating kinase. Mol. Biol. Cell, 3: 13–27, 1992.[Abstract]
  11. Watanabe, N., Broome, M., and Hunter, T. Regulation of the human Wee1Hu CDK tyrosine-15 kinase during the cell cycle. EMBO J., 14: 1878–1891, 1994.[Medline]
  12. Dunphy, W. G. and Kumagai, A. The cdc25 protein contains an intrinsic phosphatase activity. Cell, 67: 189–196, 1991.[Medline]
  13. Gautier, J., Solomon, M. J., Booher, R. N., Bazan, J. F., and Kirschner, M. W. cdc25 is a specific tyrosine phosphatase that directly activates p34cdc2. Cell, 647: 197–212, 1991.
  14. Millar, J. B. A. and Russell, P. The cdc25 M-phase inducer: an unconventional protein phosphatase. Cell, 68: 407–410, 1992.[Medline]
  15. Strausfeld, U., Labbe, J. C., Fesquet, D., Cavadore, J. C., Picard, A., Sadhu, K., Russell, P., and Doree, M. Dephosphorylation and activation of a p34cdc2/cyclin B complex in vitro by human CDC25 protein. Nature, 351: 242–245, 1991.[Medline]
  16. Hagting, A., Karlsson, C., Clute, P., Jackman, M., and Pines, J. MPF localization is controlled by nuclear export. EMBO J., 17(14): 4127–4138, 1998.[Medline]
  17. Hagting, A., Jackman, M., Simpson, K., and Pines, J. Translocation of cyclin B1 to the nucleus at prophase requires a phosphorylation-dependent nuclear import signal. Curr. Biol., 9(13): 680–689, 1999.[Medline]
  18. Li, J., Meyer, A. N., and Donoghue, D. J. Requirement for phosphorylation of cyclin B1 for Xenopus oocyte maturation. Mol. Biol. Cell, 6: 1111–1124, 1995.[Abstract]
  19. Li, J., Meyer, A. N., and Donoghue, D. J. Nuclear localization of cyclin B1 mediates its biological activity and is regulated by phosphorylation. Proc. Natl. Acad. Sci., 94: 502–507, 1997.[Abstract/Free Full Text]
  20. Pines, J. and Hunter, T. Human cyclins A and B1 are differentially located in the cell and undergo cell cycle-dependent nuclear transport. J. Cell Biol., 115: 1–17, 1991.[Abstract/Free Full Text]
  21. Pines, J. and Hunt, T. The differential localization of human cyclins A and B is due to a cytoplasmic retention signal in cyclin B. EMBO J., 13: 3772–3781, 1994.[Medline]
  22. Toyoshima, F., Moriguchi, T., Wada, A., Fukuda, M., and Nishida, E. Nuclear export of cyclin B1 and its possible role in the DNA damage-induced G2 checkpoint. EMBO J., 17(10): 2728–2735, 1998.[Medline]
  23. Yang, J., Bardes, E. S., Moore, J. D., Brennan, J., Powers, M. A., and Kornbluth, S. Control of cyclin B1 localization through regulated binding of the nuclear export factor CRM1. Genes Dev., 12(14): 2131–2143, 1998.[Abstract/Free Full Text]
  24. Yang, J., Song, H., Walsh, S., Bardes, E. S., and Kornbluth, S. Combinatorial control of cyclin B1 nuclear trafficking through phosphorylation at multiple sites. J. Biol. Chem., 276(5): 3604–3609, 2001.[Abstract/Free Full Text]
  25. Moore, J. D., Yang, J., Truant, R., and Kornbluth, S. Nuclear import of Cdk/cyclin complexes: identification of distinct mechanisms for import of Cdk2/cyclin E and Cdc2/cyclin B1. J. Cell Biol., 144: 213–224, 1999.[Abstract/Free Full Text]
  26. Takizawa, C. G., Weis, K., and Morgan, D. O. Ran-independent nuclear import of cyclin B1-Cdc2 by importin ß. Proc. Natl. Acad. Sci. USA, 96(14): 7938–7943, 1999.[Abstract/Free Full Text]
  27. Toyoshima-Morimoto, F., Taniguchi, E., Shinya, N., Iwamatsu, A., and Nishida, E. Polo-like kinase 1 phosphorylates cyclin B1 and targets it to the nucleus during prophase. Nature, 410(6825): 215–220, 2001.[Medline]
  28. Izumi, T. and Maller, J. L. Phosphorylation of Xenopus cyclins B1 and B2 is not required for cell cycle transitions. Mol. Cell. Biol., 11: 3860–3867, 1991.[Abstract/Free Full Text]
  29. Arion, D., Meijer, L., Brizuela, L., and Beach, D. cdc2 is a component of the M phase-specific histone H1 kinase: evidence for identity with MPF. Cell, 55: 371–378, 1988.[Medline]
  30. Meijer, L., Borgne, A., Mulner, O., Chong, J. P., Blow, J. J., Inagaki, N., Inagaki, M., Delcros, J. G., and Moulinoux, J. P. Biochemical and cellular effects of roscovitine, a potent and selective inhibitor of the cyclin-dependent kinases cdc2, cdk2 and cdk5. Eur. J. Biochem., 243(1–2): 527–536, 1997.[Medline]
  31. Abrieu, A., Doree, M., and Fisher, D. The interplay between cyclin-B-Cdc2 kinase (MPF) and MAP kinase during maturation of oocytes. J. Cell Sci., 114(Pt. 2): 257–267, 2001.[Abstract]
  32. Castro, A., Peter, M., Lorca, T., and Mandart, E. c-Mos and cyclin B/cdc2 connections during Xenopus oocyte maturation. Biol. Cell, 93(1–2): 15–25, 2001.[Medline]
  33. Posada, J., Yew, N., Ahn, N. G., Vande Woude, G. F., and Cooper, J. A. Mos stimulates MAP kinase in Xenopus oocytes and activates a MAP kinase kinase in vitro. Mol. Cell Biol., 13(4): 2546–2553, 1993.[Abstract/Free Full Text]
  34. Favata, M. F., Horiuchi, K. Y., Manos, E. J., Daulerio, A. J., Stradley, D. A., Feeser, W. S., Van Dyk, D. E., Pitts, W. J., Earl, R. A., Hobbs, F., Copeland, R. A., Magolda, R. L., Scherle, P. A., and Trzaskos, J. M. Identification of a novel inhibitor of mitogen-activated protein kinase kinase. J. Biol. Chem., 273(29): 18623–18632, 1998.[Abstract/Free Full Text]
  35. Shibuya, E. K. and Ruderman, J. V. Mos induces the in vitro activation of mitogen-activated protein kinases in lysates of frog oocytes and mammalian somatic cells. Mol. Biol. Cell, 4(8): 781–790, 1993.[Abstract]
  36. Minshull, J., Sun, H., Tonks, N. K., and Murray, A. W. A MAP kinase-dependent spindle assembly checkpoint in Xenopus egg extracts. Cell, 79(3): 475–486, 1994.[Medline]
  37. Gross, S. D., Schwab, M. S., Taieb, F. E., Lewellyn, A. L., Qian, Y. W., and Maller, J. L. The critical role of the MAP kinase pathway in meiosis II in Xenopus oocytes is mediated by p90(Rsk). Curr. Biol., 10(8): 430–438, 2000.[Medline]
  38. Jackman, M., Lindon, C., Nigg, E. A., and Pines, J. Active cyclin B1-CDK1 first appears on centrosomes at the end of prophase. Nat. Cell Biol., in press.
  39. Kelm, O., Wind, M., Lehmann, W. D., and Nigg, E. A. Cell cycle-regulated phosphorylation of the Xenopus polo-like kinase Plx1. J. Biol. Chem., 277(28): 25247–25256, 2002.[Abstract/Free Full Text]
  40. Jin, P., Hardy, S., and Morgan, D. O. Nuclear localization of cyclin B1 controls mitotic entry after DNA damage. J. Cell Biol., 141(4): 875–885, 1998.[Abstract/Free Full Text]
  41. Ferrell, J. E., Jr. Xenopus oocyte maturation: new lessons from a good egg. BioEssays, 21(10): 833–842, 1999.[Medline]
  42. Maller, J. L., Schwab, M. S., Roberts, B. T., Gross, S. D., Taieb, F. E., and Tunquist, B. J. The pathway of MAP kinase mediation of CSF arrest in Xenopus oocytes. Biol. Cell, 93(1–2): 27–33, 2001.[Medline]
  43. Gross, S. D., Lewellyn, A. L., and Maller, J. L. A constitutively active form of the protein kinase p90Rsk1 is sufficient to trigger the G2/M transition in Xenopus oocytes. J. Biol. Chem., 276(49): 46099–46103, 2001.[Abstract/Free Full Text]
  44. Gross, S. D., Schwab, M. S., Lewellyn, A. L., and Maller, J. L. Induction of metaphase arrest in cleaving Xenopus embryos by the protein kinase p90Rsk. Science, 286(5443): 1365–1367, 1999.[Abstract/Free Full Text]
  45. Roberts, E. C., Shapiro, P. S., Nahreini, T. S., Pages, G., Pouyssegur, J., and Ahn, N. G. Distinct cell cycle timing requirements for extracellular signal-regulated kinase and phosphoinositide 3-kinase signaling pathways in somatic cell mitosis. Mol. Cell Biol., 22(20): 7226–7241, 2002.[Abstract/Free Full Text]
  46. Borgne, A., Ostvold, A. C., Flament, S., and Meijer, L. Intra-M phase-promoting factor phosphorylation of cyclin B at the prophase/metaphase transition. J. Biol. Chem., 274(17): 11977–11986, 1999.[Abstract/Free Full Text]
  47. Takizawa, C. G. and Morgan, D. O. Control of mitosis by changes in the subcellular location of cyclin-B1-Cdk1 and Cdc25C. Curr. Opin. Cell Biol., 12(6): 658–665, 2000.[Medline]
  48. Winkler, K. E., Swenson, K. I., Kornbluth, S., and Means, A. R. Requirement of the prolyl isomerase Pin1 for the replication checkpoint. Science, 287(5458): 1644–1647, 2000.[Abstract/Free Full Text]
  49. Smythe, C. a. J. W. N. Systems for the study of nuclear assembly, DNA replication and nuclear breakdown in Xenopus laevis egg extracts. Methods in Cell Biology, pp. 449–468. San Diego, CA: Academic Press, Inc., 1991.



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