Article |
Address correspondence to Guowei Fang, Department of Biological Sciences, Stanford University, 385 Serra Mall, MC-5020, Stanford, CA 94305-5020. Tel.: (650) 725-2762. Fax: (650) 725-5807. E-mail: gwfang{at}stanford.edu
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Abstract |
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Key Words: Chfr; Cdc2; Plk1; mitosis; ubiquitin protein ligase
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Introduction |
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Progression from G2 to M is controlled by activation of the mitosis-promoting factor (MPF), a complex of the catalytic kinase subunit, Cdc2, and its positive regulatory subunit, cyclin B (King et al., 1994). As cells enter prophase, accumulation of mitotic cyclin B leads to formation of the Cdc2cyclin B complex, which is then transported into the nucleus (Murray and Kirschner, 1989; Solomon et al., 1990). However, MPF is held in an inactive state by the Wee1 and Myt1 kinases, which phosphorylate Cdc2 on tyrosine 15 and threonine 14 and inhibit MPF activity (Russell and Nurse, 1987; Draetta et al., 1988; Featherstone and Russell, 1991; Kornbluth et al., 1994; Mueller et al., 1995). Activation of the Cdc2 kinase and entry into prometaphase do not occur until the dual-specific phosphatase Cdc25C dephosphorylates Cdc2 (Russell and Nurse, 1986; Gautier et al., 1991; Kumagai and Dunphy, 1991, 1992; Osmani et al., 1991). Once activated, MPF triggers chromosome condensation, breakdown of the nuclear envelope, and formation of the mitotic spindle.
The activity of MPF is not only controlled by posttranslational modifications, such as phosphorylation and de-phosphorylation, but also by a ubiquitin-dependent proteolysis pathway (Fang et al., 1999). Cyclin B is degraded by such a pathway at late anaphase, which inactivates the Cdc2 kinase and allows cells to exit from mitosis.
Ubiquitin-dependent proteolysis involves cascade reactions of several enzymes, including a ubiquitin-activating enzyme (E1), a ubiquitin-conjugating enzyme (E2), and a ubiquitin ligase (E3). In this series of enzymatic reactions, the critical enzyme is the ubiquitin ligase that links E2 to substrates and provides specificity for the ubiquitination pathway (Hershko and Ciechanover, 1998). A group of ubiquitin ligases contains the ring finger domain (Lorick et al., 1999; Huang et al., 2000; Joazeiro and Weissman, 2000). The prototype of this group of E3s is the proto-oncogene c-Cbl that targets receptor tyrosine kinases for ubiquitination and degradation (Joazeiro et al., 1999). The RF domain in c-Cbl directly interacts with E2, and c-Cbl functions to bring the E2ubiquitin (Ub) conjugate adjacent to substrates (Yokouchi et al., 1999; Zheng et al., 2000). In the absence of substrates, the RF domain by itself has an auto-ubiquitination activity, a hallmark for this group of ubiquitin ligases (Joazeiro et al., 1999; Yokouchi et al., 1999).
There are at least two ubiquitin ligases involved in cell cycle control (King et al., 1996). The Skp1/cullin/F-box (SCF) ligase recognizes an inhibitor of cyclin-dependent kinase, sic1, and controls the G1S transition (Krek, 1998; Willems et al., 1999). A second ubiquitin ligase, the anaphase-promoting complex/cyclosome (APC), controls both the separation of sister chromatids and the exit from mitosis by ubiquitinating multiple substrates, such as anaphase inhibitors and mitotic cyclin B (Fang et al., 1999). SCF and APC each contains a ring finger subunit (Roc1 and APC11, respectively). It has been shown that both Roc1 and APC11 auto-ubiquitinate and therefore have intrinsic ubiquitin ligase activity, although the recognition of substrates requires additional subunits (Ohta et al., 1999; Seol et al., 1999; Gmachl et al., 2000; Leverson et al., 2000). Furthermore, the RF domain in each protein is critical for ligase activity, because point mutations in RF domains abolish the ligase activity.
The checkpoint protein Chfr also contains a ring finger domain (Scolnick and Halazonetis, 2000), raising the possibility that Chfr may function as a ubiquitin ligase. We show here that, like Roc1 and APC11, Chfr auto-ubiquitinates and has intrinsic ubiquitin ligase activity. The RF domain is both necessary and sufficient for the auto-ubiquitination activity in vitro. Chfr also acts as a ubiquitin ligase in vivo, and MycChfr immunopurified from HEK293T cells promotes the formation of high molecular weight ubiquitin conjugates. We characterized the specificity of Chfr toward ubiquitin-conjugating enzymes, and found that both Ubc4 and Ubc5, but not UbcH7 and UbcH10, function with the Chfr ligase. Furthermore, we developed a cell-free system to analyze the biological function of Chfr ligase. When added to Xenopus extracts, recombinant Chfr delays the activation of the Cdc2 kinase during the G2 to M transition; this delay is caused by a prolonged inhibitory phosphorylation of tyrosine 15 on Cdc2. The ubiquitin-dependent degradation by active Chfr ligase is required for the mitotic delay. The target of the Chfr ligase is the Polo-like kinase 1 (Plk1), and ubiquitination and degradation of Plk1 delays mitotic entry. Thus, Chfr represents a novel ubiquitin ligase involved in cell cycle regulation, and our biochemical analysis of the Chfr function in Xenopus extracts provides a molecular mechanism for Chfr-mediated checkpoint control at the G2 to M transition.
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Results |
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It has been shown recently that several ring fingercontaining ubiquitin ligases can auto-ubiquitinate (Joazeiro et al., 1999; Joazeiro and Weissman, 2000). To determine whether the Chfr protein has intrinsic ubiquitin ligase activity, we examined the ability of recombinant Chfr to auto-ubiquitinate. The Chfr protein was expressed in Sf9 cells and purified to homogeneity (Fig. 1 B). When the Chfr protein was incubated with radioactive ubiquitin in the presence of the recombinant E1 and Ubc4, ubiquitin conjugates were efficiently formed (Fig. 1 C). These conjugates were resistant to reduction by DTT, suggesting that ubiquitin is conjugated through an isopeptide bond rather than a thioester bond. The formation of these ubiquitin conjugates was dependent on E1, Ubc4, and Chfr; omitting any one of these proteins resulted in no DTT-resistant conjugates (unpublished data). At a high Chfr concentration (top), we detected a series of ubiquitinated protein ladders, the fastest migrating species having a mobility very close to that of the recombinant Chfr, but different from that of E1 and Ubc4 (unpublished data). We conclude that these radioactive bands represent covalent conjugates between Chfr and labeled ubiquitin, and that Chfr acts as a ubiquitin ligase to auto-ubiquitinate in the presence of E1 and Ubc4. Similarly, we detected formation of ChfrUb conjugates with the Chfr protein expressed in Escherichia coli (unpublished data), thereby excluding the possibility that a contaminating protein from Sf9 cells may function as a ubiquitin ligase for Chfr.
At a high Chfr concentration (800 µg/ml; Fig. 1 C, top), we detected a ladder of ubiquitinated Chfr. Interestingly, at a lower Chfr concentration (20 µg/ml; Fig. 1 C, bottom), the majority of ubiquitin conjugates formed had such high molecular weights that they failed to enter the stacking gel. Only a minor portion of the conjugates was trapped between the stacking gel and the separating gel. No mono- or oligo-ubiquitin conjugates were detected. The different modes of Chfr reactivity were likely due to the relative ratio of Chfr to Ubc4 in these reactions, because the concentration of Ubc4 used in both panels was 10 µg/ml, a concentration twofold higher than the molar concentration of Chfr used in the bottom panel, but 20-fold lower than that of Chfr in the top panel.
The ring finger domain in Chfr is required for its ligase activity
We next examined whether the ring finger domain is required for the ubiquitin ligase activity of the Chfr protein. The domain structure of Chfr is illustrated in Fig. 2 A (Scolnick and Halazonetis, 2000). We constructed three deletion mutants, ChfrF13, which consist of various regions of the protein. All three fragments were expressed as soluble proteins in E. coli (Fig. 2 B). When the lysates of E. coli expressing mutant proteins were incubated with radioactive ubiquitin in the presence of E1 and Ubc4, we found that all three fragments efficiently promoted auto-ubiquitination (Fig. 2 C). Similarly, purified recombinant fragments also ubiquitinated themselves (Fig. 2 D). The smallest construct used in this experiment, glutathione-S-transferase (GST)ChfrF3, has less than 40 amino acids NH2-terminal to the RF domain and less than 15 amino acids COOH-terminal to the RF domain. We conclude that the RF domain and its adjacent regions are sufficient for the auto-ubiquitination activity of the Chfr protein. Interestingly, kinetic studies indicate that the FHA domain at the NH2 terminus of the protein actually reduces the ubiquitination activity of the Chfr protein (Fig. 2 D, compare ChfrF1 with ChfrF2), suggesting a potential role of the FHA domain in regulation of the ligase activity.
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The Chfr ligase acts with Ubc4 and Ubc5, but not with UbcH7 and UbcH10
We determined the specificity of the ubiquitin-conjugating enzymes for the Chfr ligase. An immunofluorescence study of the GFPChfr fusion protein indicated that Chfr is a nuclear protein (unpublished data). There are several ubiquitin-conjugating enzymes that have been implicated in nuclear functions; these include Ubc4, Ubc5A, Ubc5B, and UbcH10 (Jentsch et al., 1990; Hershko and Ciechanover, 1998). We tested whether these E2s act with Chfr in the ubiquitination reaction. We also included UbcH7 as a negative control in our analysis, because UbcH7 is involved in ubiquitinating ER-associated proteins.
Ubc4, Ubc5A, Ubc5B, UbcH10, and UbcH7 were expressed as recombinant proteins in E. coli and purified to homogeneity (unpublished data). When incubated with recombinant E1 and radioactive ubiquitin, all these E2s formed conjugates with ubiquitin that were only detectable by nonreducing SDS-PAGE (Fig. 4 A), suggesting that these conjugates were formed through thioester bonds. Thus, the recombinant E2s analyzed here are all active.
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Chfr inhibits the activation of MPF
Chfr delays entry into mitosis when the checkpoint pathway is activated by mitotic stress, induced with taxol or nocodazole (Scolnick and Halazonetis, 2000). We developed a cell-free system to investigate how the Chfr pathway delays the cell cycle progression.
Entry into mitosis can be monitored in Xenopus extracts (Murray and Kirschner, 1989; Solomon et al., 1990). Addition of 90 cyclin B, a mutant cyclin B with the first 90 amino acids truncated, to interphase extracts efficiently activated the Cdc2 kinase activity and drove extracts into mitosis (Fig. 5 A, top). However, the addition of wild-type Chfr protein reduced the rate of Cdc2 activation and delayed entry into mitosis (Fig. 5, A and B). This inhibition of Cdc2 kinase activity required an active ligase, because the two inactive mutants, ChfrI306A and ChfrW332A, both failed to delay mitotic entry. Thus, Cdc2 is a key target for the Chfr pathway to control the entry into mitosis.
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Chfr is a ubiquitin ligase and addition of an active ligase to extracts could have a nonspecific effect on the entry into mitosis if there is a specific requirement for ubiquitin-dependent proteolysis in the G2 to M transition. For example, active Chfr ligase may nonspecifically interfere with physiological ubiquitin-dependent proteolysis by depleting the ubiquitin pool or by competing for proteasome degradation in extracts. To establish whether there is a requirement for ubiquitin-dependent proteolysis at the G2 to M transition, we examined the effect of methylated ubiquitin (methyl-ubiquitin) and N-acetyl-Leu-Leu-norleucinal peptide (LLnL) on entry into mitosis. Methyl-ubiquitin interferes with the formation of poly-ubiquitin conjugates required for degradation and LLnL is an inhibitor of proteasomes. Neither methyl-ubiquitin nor LLnL delayed the mitotic entry, as measured by the kinetics of activation of the Cdc2 kinase (Fig. 6 A, left). The amount of methyl-ubiquitin and LLnL used in these experiments, when added to mitotic extracts, reduced the rate of cyclin degradation (Fig. 6 B), suggesting that both methyl-ubiquitin and LLnL interfere with ubiquitin-dependent proteolysis pathways in Xenopus extracts. We conclude that there is no requirement for ubiquitin-dependent proteolysis at the G2 to M transition under normal physiological conditions.
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Chfr prolongs the phosphorylated state of Tyr15 in Cdc2 at the G2 to M transition
We investigated how the Cdc2 kinase activity is regulated by Chfr. When recombinant Chfr was incubated with interphase extracts and 90 cyclin B, the Chfr pathway delayed the removal of inhibitory phosphorylation on Cdc2, as analyzed by an antibody specific to the tyrosine 15phosphorylated Cdc2 (Fig. 7 A). 25 min after addition of
90 cyclin B, Cdc2 from both the control extracts and the Chfr-treated extracts had significant levels of phosphorylated tyrosine. Interestingly, by 35 min, the level of phosphorylated tyrosine was greatly reduced in control extracts, correlating well with the activation of the Cdc2 kinase (Fig. 7 A). On the other hand, the level of phosphorylated tyrosine in Chfr-treated extracts remained very high, consistent with the observation that the Cdc2 kinase was not activated until 45 min after the addition of
90 cyclin B (Fig. 7 A). We conclude that Chfr delays mitotic entry by prolonging the phosphorylated state of Cdc2.
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We observed a more pronounced delay of mitotic entry in Xenopus cycling extracts. Control extracts entered mitosis twice in the first 2 h, first at 70 min and then at 120 min, as analyzed by the activity of the Cdc2 kinase and the phosphorylation states of Cdc25C and Wee1 (Fig. 7 B). However, the entry into mitosis in Chfr-treated extracts was delayed by more than one cell cycle; such extracts did not enter mitosis until 130 min. This delay for the G2 to M transition correlates well with the delayed phosphorylation of Cdc25C and Wee1 (Fig. 7 B). It is likely that the shorter delay observed in extracts treated with 90 cyclin B reflects the high concentration of
90 cyclin B used in these experiments, and that cycling extracts more closely reflect the physiological process in vivo.
Chfr ubiquitinates Polo-like kinase 1 (Plk1)
To identify the substrate of the Chfr ligases, we analyzed the ability of Chfr to ubiquitinate regulators of Cdc2 in Xenopus extracts. The addition of recombinant Chfr to interphase extracts did not change the stability of in vitro translated Cdc25C and Wee1 (Fig. 8 A), consistent with the Western blot analysis of the endogenous Cdc25C and Wee1 (Fig. 7). Thus, neither Cdc25C nor Wee1 is the substrate of Chfr. We next tested the stability of Plk1, a positive regulator for Cdc25C and a negative regulator for Wee1 at the G2 to M transition (King et al., 1994). When incubated with interphase extracts, in vitrotranslated Plk1 was efficiently ubiquitinated in a Chfr-dependent manner (Fig. 8 A). Indeed, Plk1 is a substrate of Chfr. In an in vitro reconstituted reaction, Plk1, but not Cdc25C and Wee1, was quantitatively ubiquitinated in the presence of purified recombinant E1, Ubc4, and Chfr (Fig. 8 B).
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The extent of ubiquitination of endogenous Plx1 is limited in interphase extracts. We noticed that Xenopus extracts had a limited capacity for ubiquitination and that efficient ubiquitination of cell cycle regulators required exogenously added recombinant ubiquitin protein. For example, complete degradation of mitotic cyclin B by the APC pathway requires the addition of ubiquitin to mitotic extracts (Fang et al., 1998a,b). In the presence of recombinant ubiquitin, endogenous Plx1 was quantitatively ubiquitinated by Chfr and subsequently degraded by proteasomes (Fig. 8 E). Complete ubiquitination and degradation of Plx1 prevented the phosphorylation of Cdc25C and Wee1 in Chfr-treated extracts, such that Cdc2 remained phosphorylated and its kinase activity was not activated, even after extended incubation with 90 cyclin B (Fig. 8 E). In the presence of Chfr, even the residual level of Cdc2 kinase activity detectable in interphase extracts was downregulated. We conclude that Chfr ubiquitinates and downregulates Plx1, which, in turn, simultaneously regulates Cdc25C and Wee1 activities, leading to prolonged phosphorylation of Cdc2 and a delay in entry into mitosis.
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Discussion |
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The checkpoint protein Chfr is a ubiquitin ligase
Chfr is a ubiquitin ligase based on the following observations. First, the Chfr protein has a ring finger domain common to a growing number of ubiquitin ligases (Jackson et al., 2000; Joazeiro and Weissman, 2000; Scolnick and Halazonetis, 2000). Sequence alignment of the Chfr ring finger domain with those from APC11 and c-Cbl indicates that they are highly homologous. The cocrystal structure of c-Cbl and UbcH7 have been solved recently and the ring finger domain in c-Cbl has been shown to directly bind to UbcH7 (Zheng et al., 2000), indicating an important role of the ring finger domain in the ubiquitin ligase activity. Residues in c-Cbl involved in interacting with UbcH7 are partly conserved in Chfr; these residues include Ile306, Cys328, Trp332, and Pro340 (amino acid numbers derived from the Chfr protein). Second, recombinant Chfr auto-ubiquitinates in an E1- and E2-dependent manner. Auto-ubiquitination is a hallmark of ring fingercontaining ligases (Joazeiro et al., 1999; Huang et al., 2000; Suzuki et al., 2001). Indeed, both c-Cbl and APC11 have auto-ubiquitination activities in the absence of substrates (Joazeiro et al., 1999; Gmachl et al., 2000; Leverson et al., 2000). We showed here that the ring finger domain in Chfr is both necessary and sufficient for the auto-ubiquitination activity. Third, when expressed in mammalian tissue culture cells, the Chfr immunoprecipitate also contains a ubiquitinated protein(s) and promotes the formation of high molecular weight ubiquitin conjugates, indicating that the Chfr immunoprecipitate has a ubiquitin ligase activity. Again, the ring finger domain in Chfr is essential for the ligase activity in vivo, because point mutations of conserved residues in this domain abolish the ubiquitin ligase activity. Fourth, Plk1 is ubiquitinated in Xenopus extracts in a Chfr-dependent manner, and in a purified, reconstituted system, Chfr directly ubiquitinates Plk1. Thus, Chfr by itself is sufficient to ubiquitinate the substrate.
How is the ligase activity toward itself and the substrates differentially regulated? There are at least two possible mechanisms for this regulation. First, the auto-ubiquitination activity may be separable from the ligase activity toward substrates, with self-ubiquitination and ubiquitination of substrates being mutually exclusive. An example of a ligase with such a mechanism is the MDM2 protein, a ring fingercontaining ligase for the tumor suppressor p53. MDM2 ubiquitinates itself as well as p53 (Fang et al., 2000). The ligase is modified in the ring finger domain by SUMO-1, a ubiquitin-like protein. Modification by SUMO-1 prevents self-ubiquitination and increases its ligase activity toward p53 (Buschmann et al., 2000). On the other hand, a reduction of the SUMO-1 modification destabilizes MDM2 and increases the level of p53. A second possibility is that Chfr may ubiquitinate itself and its substrates at the same time, and ubiquitinated Chfr and substrates are degraded by proteasomes. Such a mechanism exists for the c-Cbl ligase. C-Cbl mediates the ubiquitination of itself and c-Src. Ubiquitination of both proteins requires Src kinase activity and tyrosine phosphorylation of c-Cbl by Src (Yokouchi et al., 2001), suggesting that substrate ubiquitination and auto-ubiquitination are interdependent. The Xenopus extract system should allow us to determine the regulatory mechanism of Chfr self-ubiquitination versus ubiquitination of Plk1.
Chfr delays entry into mitosis by inhibiting the activation of Cdc2
The Chfr-mediated checkpoint controls entry into mitosis (Scolnick and Halazonetis, 2000). Normal primary cells and tumor cell lines with wild-type Chfr exhibit a delay in entry into mitosis when centrosome separation is inhibited by nocodazole or taxol. On the other hand, tumor cell lines that lack the functional Chfr protein enter mitosis without such a delay in the presence of mitotic stress. Importantly, ectopic expression of Chfr in tumor cell lines restores the delay, indicating that the Chfr protein is responsible for the delay in entry into mitosis. Similarly, ectopic expression of the active Chfr protein in Xenopus extracts is likely to mimic a checkpoint-activated state, and our cell-free system allows us to investigate the molecular pathway that Chfr regulates. It is interesting to note that the Chfr-minus cells seem to have a normal G2 to M transition in the absence of mitotic stress (Scolnick and Halazonetis, 2000). Thus, the Chfr protein is only required for a checkpoint mechanism that controls the kinetics of mitotic entry in the presence of mitotic stress. This is consistent with our observation that there is no requirement for ubiquitin-dependent proteolysis at the normal G2 to M transition. Although we have not examined the function of endogenous Chfr protein in Xenopus extracts due to the lack of a high-quality anti-Chfr antibody for immunodepletion experiments, the endogenous Chfr, if expressed in Xenopus embryonic extracts, is unlikely to be required for entry into mitosis in the absence of the checkpoint signal.
When added to Xenopus extracts, the Chfr ligase delays entry into mitosis with a prolonged phosphorylation of tyrosine 15 in the Cdc2 kinase. In cycling extracts, the Chfr protein delays the activation of Cdc2 by more than one cell cycle. Given that the inactive Chfr mutants have no effect on mitotic entry, and the inhibition of ubiquitin-dependent protein degradation rescues the delay phenotype, the effect of Chfr on mitotic entry is specific and requires ubiquitin-dependent protein degradation.
It is interesting to note that in mammalian tissue culture cells, the Chfr pathway causes a delay in entry into mitosis without a detectable delay in the activation of the Cdc2 kinase (Scolnick and Halazonetis, 2000). It is possible that the temporal delay in activation of Cdc2 kinase found in Xenopus extracts could have escaped from detection in the mammalian system due to a large time interval (2 h) between time points used in the original analysis of the Cdc2 kinase activity. This discrepancy between Xenopus extracts and mammalian tissue culture cells may also result from difficulties in achieving an optimal synchronization in mammalian cells (Scolnick and Halazonetis, 2000). In addition, we found that constitutive expression of Chfr in mammalian cells is detrimental to cell proliferation and the expression level of the exogenously introduced Chfr was quickly diminished in stable Chfr cell lines (unpublished data). On the other hand, it has been reported that the Chfr checkpoint temporarily arrests mammalian cells at prophase with an intact nuclear envelope and uncondensed chromosomes (Scolnick and Halazonetis, 2000), consistent with a low level of the Cdc2 kinase activity.
Plk1 is ubiquitinated by Chfr
The direct target of the Chfr pathway is Plk1. In vitrotranslated Plk1 is ubiquitinated, in a Chfr-dependent manner, both in Xenopus interphase extracts as well as in a purified system reconstituted with recombinant proteins. On the other hand, neither Cdc25C nor Wee1 is ubiquitinated in both assays, demonstrating the specificity of the Chfr pathway. The endogenous Plx1 is also ubiquitinated in Chfr-treated Xenopus extracts, and this ubiquitination correlates with the delay in entry into mitosis. Indeed, if the efficiency of the Chfr pathway is augmented by recombinant ubiquitin, endogenous Plx1 is quantitatively ubiquitinated and the Chfr-treated extracts are arrested at G2 with minimal Cdc2 kinase activity. We expect that Xenopus extracts reflect certain aspects of Plk1 regulation in mammalian somatic cells. The expression of Plk1 is regulated in mammalian cells; Plk1 begins to accumulate in G2 and its level peaks in mitosis (Fang et al., 1998a). Activation of the Chfr pathway by mitotic stress leads to ubiquitination and degradation of Plk1, and blocks entry into mitosis. Once mitotic stress is relieved and defects are repaired, the Chfr ligase is inactivated and de novo synthesis of Plk1 drives cells into mitosis.
Plk1 regulates both the Wee1 kinase and the Cdc25C phosphatase, which in turn control the Cdc2 kinase activity at the G2 to M transition (Russell and Nurse, 1986; Gould and Nurse, 1989; Ferrell et al., 1991; Gautier et al., 1991; Kumagai and Dunphy, 1991; Osmani et al., 1991; Kumagai and Dunphy, 1992). Wee1 mediates the inhibitory phosphorylation of Tyr15 and Thr14 on Cdc2, whereas Cdc25C dephosphorylates and activates Cdc2. Cdc25C and Wee1 themselves are regulated by complex molecular circuits in the cell cycle (King et al., 1994). The NH2-terminal regulatory domain in Cdc25C is phosphorylated by Plk1 and Cdc2/cyclin B, and phosphorylation enhances the phosphatase activity of Cdc25C (Izumi et al., 1992; Kumagai and Dunphy, 1992; Izumi and Maller, 1993; Kumagai and Dunphy, 1996). On the other hand, Wee1 is also likely to be phosphorylated by Plk1 and Cdc2/cyclin B, but phosphorylation inhibits the kinase activity of Wee1. Thus, the activation of Cdc25C and Cdc2 and the inhibition of Wee1 at mitosis constitute an auto-activating feedback loop that allows the Cdc2 kinase to be activated in an all-or-none fashion (King et al., 1994). The initial activity of Plk1 at the G2 to M transition can determine the status of the auto-activating loop and the level of the Cdc2 kinase activity.
We reported here that Plk1 is a target for the Chfr-mediated checkpoint control. Plk1 is a kinase initially identified for its role in controlling the separation of duplicated centrosomes at the G2 to M transition (Llamazares et al., 1991; Lane and Nigg, 1996). Thus, the Chfr pathway may also delay the separation of centrosomes in the presence of microtubule drugs, thereby providing additional time for repairing defects in the centrosome structure. We speculate that the Chfr checkpoint may control multiple steps at the G2 to M transition. Indeed, in the two other ubiquitination pathways, SCF ligase and APC ligase, each has multiple substrates and controls several events in the cell cycle (Fang et al., 1999; Jackson et al., 2000). The demonstration of Chfr as a ubiquitin ligase and the identification of Plk1 and Cdc2 as targets for the Chfr pathway will allow us to begin to understand the biological function of this novel ubiquitination pathway.
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Materials and methods |
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Expression and purification of recombinant proteins
Human Chfr gene was subcloned by PCR amplification into pFastBac (GIBCO BRL), expressed as a soluble protein in Sf9 cells, and purified to homogeneity. ChfrI306A and ChfrW332A mutants were constructed with the Quick Change Site-Directed Mutagenesis Kit (Stratagene), and mutant proteins were expressed and purified. Deletion mutants of Chfr were constructed by PCR amplification and mutant proteins were expressed in E. coli. ChfrF1 and ChfrF2 were purified by Ni-NTA beads (QIAGEN), and GSTChfrF3 was purified by glutathione-agarose beads (Amersham Pharmacia Biotech). Human Ubc5A, Ubc5B, and UbcH7 genes were cloned into a modified pET28a vector by PCR amplification from human fetal thymus cDNA. Recombinant proteins were expressed in E. coli and purified by Ni-NTA beads. Expression and purification of recombinant E1, Ubc4, and UbcH10 were described previously (Fang et al., 1998a, b).
Ubiquitination Assays
Ubiquitin (Sigma-Aldrich) was labeled with 125I to a specific activity of 100 µCi/µg using the chloramine T procedure. For auto-ubiquitination of recombinant Chfr in Figs. 13, reactions were performed in a total volume of 10 µl. The reaction mixture contained an energy-regenerating system, 400 µg/ml labeled ubiquitin, 20 µg/ml recombinant E1, 10 µg/ml Ubc4, and various amounts of Chfr as indicated in figure legends. Reactions were incubated at room temperature for various times, quenched with SDS sample buffer, and analyzed by reducing 12% SDS-PAGE. Gels were scanned with a PhosphorImager (Molecular Dynamics).
For ubiquitination assays with MycChfr complexes from HEK293T cells (Figs. 1 and 3), anti-Myc immunoprecipitates were purified from one jumbo dish of transfected HEK293T cells and incubated with 400 µg/ml labeled ubiquitin, 20 µg/ml recombinant E1, 300 µg/ml Ubc4, and an energy-regenerating system at room temperature for 1 h. The reactions were quenched with SDS sample buffer and analyzed by reducing 10% SDS-PAGE.
For auto-ubiquitination assays with various E2s in Fig. 4 B, Chfr at 400 µg/ml were incubated at room temperature for 5 min with 30 µg/ml labeled ubiquitin, 200 µg/ml recombinant E1, 200 µg/ml various E2s, and an energy-regenerating system. The reactions were quenched with SDS sample buffer and analyzed by reducing 10% SDS-PAGE.
For ubiquitination of Plk1, recombinant Chfr was incubated at room temperature for various times with in vitrotranslated [35S]Plk1 in the presence of E1, Ubc4, ubiquitin, and ubiquitin aldehyde, quenched with SDS sample buffer, and analyzed by SDS-PAGE. To confirm that the endogenous Plx1 is ubiquitinated in a Chfr-dependent manner, immobilized GSTUb was incubated with Xenopus interphase extracts in the presence or absence of recombinant Chfr protein. The final concentration of GSTUb in extracts was 0.85 mg/ml. Ubiquitinated products were purified by glutathione beads and immunoblotted with an anti-Plx1 antibody.
The thioester assay for E2, histone H1 kinase assay, and cyclin degradation assay were performed as described previously (Fang et al., 1998a, b).
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Footnotes |
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Acknowledgments |
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J. Wong is a recipient of the Howard Hughes pre-doctoral fellowship. G. Fang is a Searle scholar, a Kimmel scholar in Cancer Research, and a recipient of the Beckman Young Investigator Award and the Burroughs-Wellcome Career Award in Biomedical Sciences. This work was also supported by a Research Scholar grant from the American Cancer Society (RSG-01-143-01) and by grants from Concern Foundation and the Office of Technology Licensing at Stanford University.
Submitted: 3 August 2001
Revised: 30 November 2001
Accepted: 4 December 2001
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