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Address correspondence to Dr. Elmar Schiebel, The Paterson Institute for Cancer Research, Christie Hospital NHS Trust, Wilmslow Road, Manchester M20 4BX, United Kingdom. Tel.: 01-61-446-3783. Fax: 01-61-446-3109. E-mail: eschiebel{at}picr.man.ac.uk
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Abstract |
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Key Words: Bfa1p-Bub2p GAP; Cdc14p; MEN; polo kinase; Tem1p
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Introduction |
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The small, Ras-like GTPase Tem1p regulates MEN activity. Tem1p activation promotes its interaction with the kinase Cdc15p (Shirayama et al., 1994b; Bardin et al., 2000), which in turn activates a protein kinase complex in which the catalytic subunit Dbf2p is bound to a regulatory subunit, Mob1p (Toyn and Johnston, 1994; Luca et al., 2001; Mah et al., 2001). The polo-like kinase Cdc5p activates the MEN by inhibiting the Tem1p GTPase activating protein (GAP) complex. This bipartite GAP complex is composed of Bfa1p and Bub2p, and Cdc5p phosphorylates Bfa1p (Hoyt et al., 1991; Li, 1999). Because overproduction of CDC14 bypasses the requirement of all MEN proteins, it is thought that Cdc14p activation/release is the ultimate target of the MEN cascade (Jaspersen et al., 1998; Visintin et al., 1998).
In addition to regulation by the Bfa1pBub2p GAP complex, Tem1p activity is also modulated by the putative GDP/GTP exchange factor (GEF) Lte1p (Shirayama et al., 1994a). Tem1p forms a complex with the Bfa1pBub2p GAP on the spindle pole body (SPB), which leads the spindle into the bud (Pereira et al., 2000, 2001). The GEF Lte1p is retained at a distinct location on the cortex of the bud (Bardin et al., 2000; Pereira et al., 2000). It has therefore been proposed that SPB-associated Bfa1pBub2p GAP inactivates Tem1p until the SPB and spindle enter the bud in anaphase. This coupling of mitotic exit with nuclear migration prevents premature mitotic exit in mutants with defects in spindle orientation and has now been termed "the spindle position checkpoint."
Two recent results indicate that additional mechanisms regulate MEN activity. First, BFA1 and BUB2 only become essential for survival when nuclear migration is delayed (Bardin et al., 2000; Bloecher et al., 2000; Pereira et al., 2000). Second, deletion of LTE1 does not affect the timing of mitotic exit at 30°C (unpublished data) or 37°C (Adames et al., 2001).
The fission yeast Schizosaccharomyces pombe controls septum formation during cytokinesis through the activity of the septum initiation network (SIN) (Balasubramanian et al., 2000). The SIN is similar to the MEN in composition. However, in contrast to Cdc14p, the fission yeast homologue Clp1p/Flp1p is not essential and associates not only with the nucleolus but also with the SPB. Clp1p/Flp1p is released from the nucleolus very early in mitosis in a SIN-independent manner (Cueille et al., 2001; Trautmann et al., 2001). Furthermore, Clp1p/Flp1p does not regulate anaphase cyclin destruction and the accumulation of a Sic1p equivalent. Instead, Clp1p/Flp1p delays Cdk activation at the G2M transition and is part of a cytokinesis checkpoint that arrests cells in G2 when cytokinesis is blocked (Cueille et al., 2001; Trautmann et al., 2001). The human Cdc14p homologue, hCdc14a, localizes to the centrosome but not the nucleolus and dephosphorylates hCdh1 (Bembenek and Yu, 2001). Whether the seemingly different regulatory and functional aspects of Cdc14p, hCdc14a, and Clp1p/Flp1p have a common basis is an important question.
Here, we show that Cdc14p is initially released from the nucleolus at the beginning of anaphase (for summary see Fig. 9). This release occurs without the function of the MEN components Cdc15p, Dbf2p, and Tem1p. Cdc14p then associates with SPBs through the Bfa1pBub2p complex and facilitates MEN activation. In a second step, at the end of anaphase, Cdc14p dephosphorylates Bfa1p and thereby reactivates the Bfa1pBub2p GAP to shut down the MEN. Thus, Cdc14p shares characteristics with the human and S. pombe homologues, and its affinity for the Bfa1p- and Tem1p-like proteins may indicate a common function of Cdc14 proteins at SPBs and centrosomes.
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Results |
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We then investigated whether Tem1p alone was required for Cdc14p binding to SPBs. The essential requirement of TEM1 can be bypassed by expression of CDC15 from a 2µ plasmid (Shirayama et al., 1994b). As Bfa1p and Bub2p are still found on the bud-proximal SPB of tem1 CDC15-2µ anaphase cells, these cells can be used to directly address the role of Tem1p in the localization of Cdc14p (Pereira et al., 2000). In contrast to
bfa1 cells, Cdc14p localization was unaffected by the absence of Tem1p (Fig. 1, C and D). Thus, the reduced SPB binding of Cdc14p in
bfa1 cells likely results from the absence of the Bfa1pBub2p complex rather than the lack of Tem1p. We conclude that the Bfa1pBub2p complex facilitates the binding of Cdc14p to the SPB in the bud.
Cdc14p interacts with Bfa1p and Tem1p
We then asked whether Cdc14p could physically associate with Bfa1p, Bub2p, or Tem1p by testing for interactions in the yeast two-hybrid system. The Cdc14p interactor Net1p was used as a positive control (Shou et al., 1999). Cdc14p interacted with both Tem1p and Bfa1p. Cdc14p interacted most strongly with Tem1p, then Bfa1p, and then Net1p (Fig. 2 A, columns 1, 3, and 6). Interaction of Cdc14p with Bfa1p was mediated through the NH2-terminal domain of Bfa1p (columns 3 and 4) and not the COOH-terminal portion (column 5). Cdc14p did not give a signal with Bub2p (column 2). In conclusion, Cdc14p interacts with both Tem1p and Bfa1p in the yeast two-hybrid system.
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Because a crude yeast lysate was used as a source for Bfa1p, Bub2p, and Tem1p in the previous experiment, it was not clear whether the interaction with Cdc14p was direct or indirect. Therefore, all proteins were expressed in E. coli, purified, and mixed to test the ability of isolated proteins to physically associate. 6HisTem1p interacted with GSTCdc14p but not GST (Fig. 2 C, lanes 3 and 4). Anti-GST antibodies revealed that GST and GSTCdc14p were present in about equal amounts (lanes 1 and 2). Moreover, 6HisCdc14p also bound to GSTBfa1p1394 (lane 11) but not to GSTBfa1p199 (lane 12), GSTBub2p (lane 10), or GST (lane 9). In this experiment, similar amounts of GSTBub2p, GSTBfa1p1394, and GSTBfa1p199 (lanes 68) were on the beads, whereas GST (lane 5) was approximately twofold higher. Thus, Cdc14p binds directly to Bfa1p and Tem1p but not to Bub2p, suggesting that the interaction of Cdc14p to the Bfa1pBub2p complex (Fig. 2 B) is mediated by Bfa1p.
Cdc14p dephosphorylates Bfa1p
Because Bfa1p is a phosphoprotein (Lee et al., 2001) that interacts with the phosphatase Cdc14p (Fig. 2), we asked whether Cdc14p dephosphorylates Bfa1p. We first investigated whether Cdc14p dephosphorylates Bfa1p in vivo. For this experiment, cells deleted in the anaphase-promoting complex subunit CDC26 were shifted to 37°C, causing a metaphase arrest, a cell cycle stage where Bfa1p is phosphorylated (Zachariae et al., 1998). Cdc14p of metaphase-arrested cells is entrapped in the nucleolus and is therefore inactive (Visintin et al., 1999). Overexpression of CDC14 from the Gal1 promoter allows the accumulation of active Cdc14p in the cytoplasm. If Bfa1p is a Cdc14p substrate, then exposure to this excess of Cdc14p should dephosphorylate the Bfa1p of cdc26 cells. The phosphatase-dead CDC14C283A was used as a control for the specificity of the reaction.
cdc26 BFA13HA,
cdc26 BFA13HA Gal1CDC14, and
cdc26 BFA13HA Gal1CDC14C283A cells were arrested in metaphase by incubation at 37°C. In all cell types, Bfa1p was similarly phosphorylated, as indicated by the accumulation of the slower migrating Bfa1p phosphoform (Fig. 3 A, lanes 2, 6, and 10; Bfa1p-P). Overexpression of Gal1CDC14 and Gal1CDC14C283A was then induced by the addition of galactose (t = 0). Bfa1p3HA phosphorylation was not affected in BFA13HA
cdc26 and BFA13HA
cdc26 Gal1CDC14C283A cells (Fig. 3 A, lanes 25 and 1013). However, in Gal1CDC14 cells, most Bfa1p3HA became dephosphorylated within 2 h of Gal1 induction (Fig. 3 A, lanes 69). Immunoblotting with anti-Cdc14p antibodies revealed that Cdc14p and Cdc14pC283A were expressed at similar levels (unpublished data). Moreover, we monitored the levels of Clb2p and the Cdc14p substrate Sic1p to ensure that overexpression of CDC14 did not induce mitotic exit of the
cdc26 cells. In all cell types, Clb2p stayed high and Sic1p did not accumulate, indicating that
cdc26 remained arrested in mitosis during the course of the experiment (Fig. 3 A). We concluded that Cdc14p dephosphorylates Bfa1p in vivo.
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Cdc14p binds to SPBs in early anaphase yet only dephosphorylates Bfa1p at mitotic exit
To gain a better insight into the relationship between Bfa1p dephosphorylation by Cdc14p and the association of Cdc14p with the SPB, we compared the timing of these two events. To enable the quantification of Cdc14p SPB binding, we again used a strain in which CDC14 was fused to YFP and the SPB marker SPC42 was fused to CFP. We generated a highly synchronized yeast culture that immediately progressed into anaphase by arresting Gal1CDC20 CDC14YFP SPC42CFP cells in metaphase through depletion of Cdc20p and subsequently resupplying Cdc20p by the addition of galactose (Shirayama et al., 1999). Depletion of Cdc20p arrested Gal1CDC20 CDC14YFP SPC42CFP cells in metaphase with phosphorylated Bfa1p (Fig. 4 A, compare lane 3 [metaphase arrest] with lanes 1 and 2 [nonphosphorylated Bfa1p]). However, the slowest migrating Bfa1p form in Gal1CDC20 arrested cells still migrated faster than the slowest migrating Bfa1p band from cdc15-1 arrested late anaphase cells (Fig. 4 A, lanes 3 and 10). Bfa1p must therefore be subject to additional phosphorylation in anaphase before full activation of the MEN (Lee et al., 2001). This additional modification became particularly apparent when the metaphase block of Gal1CDC20 cells was released. Upon induction of Cdc20p, cells entered anaphase, as indicated by the appearance of binucleated cells (Fig. 4 A, bottom). At least one additional phosphoform of Bfa1p appeared at the beginning of anaphase whose migration corresponded to Bfa1p of anaphase-arrested cdc15-1 cells (Fig. 4 A, lanes 6, 7, and 10). Phosphorylation of Bfa1p then sharply decreased at the end of anaphase as Clb2p was degraded and Sic1p accumulated (lanes 8 and 9). Because degradation of Clb2p and accumulation of Sic1p are indicators of mitotic exit (Schwab et al., 1997), we conclude that Bfa1p becomes dephosphorylated as cells exit mitosis.
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Inactivation of the MEN by Bfa1pBub2p at the end of mitosis
The previous experiment indicated that Bfa1p became phosphorylated at the beginning of anaphase and dephosphorylated by Cdc14p during mitotic exit. Phosphorylation of Bfa1p by Cdc5p inactivates, at least partially, the Bfa1pBub2p GAP complex (Hu et al., 2001) (unpublished data). Thus, in a simple model, dephosphorylation of Bfa1p by Cdc14p could reactivate the Bfa1pBub2p complex. As a consequence, active Bfa1pBub2p would change Tem1p into its GDP-bound, inactive state and the MEN would be inactivated at the end of mitosis. If this is the case, dephosphorylation of Bfa1p should coincide with MEN inactivation, and cells lacking the Bfa1pBub2p complex should delay Tem1p inhibition at the end of mitosis. To test these predictions, we sought a simple way to monitor Bfa1pBub2p activity. It is established that the localization of the kinase Dbf2p to SPBs is dependent upon Tem1p activation (Fesquet et al., 1999; Visintin and Amon, 2001). Thus, when the absence of the Bfa1pBub2p complex promotes premature MEN activation, Dbf2p kinase binds prematurely to SPBs. Monitoring Dbf2p association with the SPB will therefore give a read out of Tem1p activity. However, the Dbf2pYFP SPB signal is very weak (unpublished data), making it difficult to analyze. As an alternative, we investigated whether Mob1p, which forms a tight complex with Dbf2p (Komarnitsky et al., 1998), can be used as a marker for Bfa1pBub2p activity. We monitored Mob1p SPB localization in wild-type and bfa1 cells. In
-factorsynchronized CDC14CFP MOB1YFP cells, Mob1pYFP became associated with SPBs at the beginning of anaphase (Fig. 5 A, 2, and D, t = 60). In contrast, in
bfa1 cells, Mob1pYFP was already associated with the SPBs of metaphase cells (Fig. 5 B, 1, and E, t = 60). This behavior is identical to that of Dbf2p and shows that the association of Mob1pYFP with the SPBs is a good indicator of Bfa1pBub2p activity.
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In wild-type cells, Mob1p is displaced from SPBs at the same time as mitotic exit (Fig. 5 D). If Bfa1p dephosphorylation by Cdc14p is the only mechanism that triggers Mob1pYFP dissociation from SPBs at the end of anaphase, Mob1pYFP should stay at SPBs in cells lacking the Bfa1pBub2p complex. Indeed, Mob1pYFP remained associated with SPBs in bfa1 cells after the reentry of Cdc14p into the nucleolus indicated that cells had completed mitotic exit (Fig. 5 B, 35, and E, t = 105135). Together, the correlation between Bfa1p dephosphorylation and Mob1p SPB localization and the Bfa1pBub2p-dependent displacement of Mob1p from SPBs at the end of mitosis suggest that the Bfa1pBub2p complex becomes reactivated with mitotic exit through the dephosphorylation of Bfa1p by Cdc14p.
Partial release of Cdc14p from the nucleolus is independent of Cdc15p, Dbf2p, and Tem1p
The experiments in the previous section established that in CDC14CFP MOB1YFP cells, Cdc14p was released from the nucleolus and Mob1p bound to SPBs at the same time (Fig. 5 D). We did not observe any cells (n > 200) in which Mob1p was already at SPBs while Cdc14p was still in the nucleolus. In fact, 5% of early anaphase cells showed Cdc14p, but not Mob1p, at SPBs (unpublished data). This observation suggests that Cdc14p dissociates from Net1p shortly before Tem1p activation. In turn, this implies that MEN activity is not essential for the initial release of Cdc14p from the nucleolus. If this is the case, then the initial release of Cdc14p from the nucleolus should still occur in cells where the MEN is defective. To test this possibility, the resident nucleolar protein Net1p, which forms a complex with Cdc14p (Shou et al., 1999; Visintin et al., 1999), was fused to CFP in wild-type, cdc5-10, cdc15-1, and dbf2-2 cells that contained CDC14YFP. We then monitored the localization of Cdc14pYFP and Net1pCFP as cells recovered from an
-factor block at 37°C, the restrictive temperature for these MEN mutants. In all cell types, Cdc14pYFP and Net1pCFP showed 100% colocalization in G1, S, and G2 cells. In wild-type cells, Cdc14p was released from the nucleolus in early anaphase. Partial release of Cdc14pYFP (Fig. 6 A, PR, and B, green line) was followed by the complete release of the nucleolar Cdc14pYFP (Fig. 6 A, R, and B, red line). In cdc15-1 and dbf2-2 cells, we saw a partial release of Cdc14p from the nucleolus in early anaphase (Fig. 6, A and B). This release had similar kinetics to wild-type cells. In contrast to wild-type cells, complete liberation of nucleolar Cdc14p was very rare in cdc15-1 and dbf2-2 cells. Most cells of cdc5-10 behaved differently, i.e., they failed to show any degree of Cdc14p release (Fig. 6 B). As has been previously reported, cdc15-1, cdc5-10, and dbf2-2 cells all eventually arrested in telophase with Cdc14p in the nucleolus (Shou et al., 1999; Visintin et al., 1999) (Fig. 6 B, t = 140).
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Our data suggest that the Cdc14p released from the nucleolus in early anaphase is performing a distinct function from the Cdc14p liberated at the end of mitosis. If this is the case, then this early anaphase population of Cdc14p should not affect phosphorylation of Bfa1p, because the kinase responsible for phosphorylating it at this time, Cdc5p, is active until the end of anaphase (Shirayama et al., 1998). To test this possibility, we compared the accumulation of phosphorylated Bfa1p in -factorsynchronized wild-type and cdc15-1 cells. Phosphorylated Bfa1p appeared with similar kinetics in both cell types (Fig. 6 C). Thus, the MEN-independent release of Cdc14p did not affect Bfa1p phosphorylation.
Cdc14p binds transiently to SPBs in an MEN-independent manner
We then asked whether the partial nucleolar release of Cdc14p in cdc15-1 and dbf2-2 cells was coordinated with the acquisition of affinity for the SPBs. Cells of cdc5-10, in which Cdc14p remains in the nucleolus, were used as controls. -Factorsynchronized wild-type, cdc5-10, cdc15-1, and dbf2-2 cells with CDC14YFP SPC42CFP were followed at 37°C. No Cdc14pYFP SPB signal was observed from interphase to early mitosis in any of the four cell types. Localization of Cdc14pYFP at SPBs in wild-type cells peaked at 120 min after the release of the
-factor block (Fig. 7 A, a, and B). This corresponded to the timing of the complete release of Cdc14p from the nucleolus in anaphase (Fig. 6 B). In early anaphase, Cdc14pYFP was also found at SPBs in cdc15-1 (Fig. 7 A, b) and dbf2-2 cells (Fig. 7 A, c). However, the maximal SPB association was reached 15 min before the peak of wild-type cells (Fig. 7 B). The peak of Cdc14p at SPBs of the MEN mutants coincided with the maximal partial release of Cdc14p from the nucleoli of cdc15-1 and dbf2-2 cells (Fig. 6 B). Identical behavior was seen when the MEN was inactivated by Tem1p depletion from a Gal1UPL-TEM1 CDC14YFP SPC42CFP strain (unpublished data). In contrast to these MEN-deficient mutants, Cdc14pYFP was virtually absent from the anaphase SPBs of cdc5-10 cells (Fig. 7 A, d, and B). At later time points, when cdc15-1, cdc5-10, and dbf2-2 cells were arrested in late anaphase, no Cdc14p SPB staining was observed (Fig. 2 B, t = 130150). We concluded that both the release of Cdc14p from the nucleolus and its association with the SPB in early anaphase are independent of MEN activation.
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The interaction of Cdc14p with Bfa1p at SPBs at the beginning of anaphase (Fig. 4) raises the possibility that Cdc14p activates the MEN through inactivation of the Bfa1pBub2p GAP complex. If this is the case, we would expect that the Bfa1pBub2p GAP remains active when Cdc14p function is impaired. This failure to inactivate Bfa1pBub2p should be reflected in the SPB localization of Mob1p, which, as we demonstrated, can be used as a marker for Bfa1pBub2p activity (Fig. 5). To understand how failure to inactivate the Bfa1pBub2p complex affects Mob1p SPB localization in anaphase, we first studied Mob1pGFP in -factorsynchronized cdc5-10 cells. In wild-type cells, Cdc5p phosphorylated Bfa1p with anaphase onset and this inactivated, at least partially, the Bfa1pBub2p complex (Hu et al., 2001; unpublished data) (Fig. 8 C, lane 2). In contrast, cdc5-10 cells failed to hyperphosphorylate Bfa1p (Fig. 8 C, lane 6) and, therefore, arrested in anaphase with an active Bfa1pBub2p complex (unpublished data). In
88% of anaphase cdc5-10 cells, Mob1pGFP was only seen on the SPB in the mother cell body, the cell body with the mating projection, whereas in wild-type anaphase cells, both SPBs carried a Mob1pGFP signal (Luca et al., 2001) (Fig. 8, A and B). Because in cdc5-10 cells Bfa1p and Bub2p were only associated with the SPB in the bud (unpublished data), active Bfa1pBub2p complex may prevent binding of Mob1pGFP to this SPB. Indeed, Mob1pGFP was at both SPBs in 96% of the double cdc5-10
bub2 mutant cells (Fig. 8, A and B), where the Bfa1pBub2p complex is absent from SPBs (unpublished data). We conclude that the presence of active Bfa1pBub2p complex on the bud-proximal SPB blocked Mob1p association to this SPB.
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Discussion |
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Cdc14p activates the MEN in early anaphase
Our results show that Cdc14p is partially released from the nucleolus in early anaphase independently of the MEN components Cdc15p, Dbf2p, and Tem1p (Fig. 9 I). Evidence for an MEN-independent step in releasing the nucleolar Cdc14p has already been obtained while studying Cdc14p in net1-1 cells (Shou et al., 1999). In addition, during revision of our manuscript, a paper by Stegmeier et al. (2002) also reported that Cdc14p is partially released from the nucleolus in cells with an inactive MEN. These authors showed that separase, Cdc5p, the kinetochore protein Slk19p, and Spo12p all have a role in the control of Cdc14p localization during early anaphase. Consistent with this, we observed that Cdc14p remained largely in the nucleolus in cdc5-10 cells.
What is the function of the fraction of Cdc14p that is released from the nucleolus in early anaphase in an MEN-independent manner? In cdc14-2 cells, the Bfa1pBub2p complex was still partially active, as assessed by Mob1p localization, despite phosphorylation of Bfa1p by Cdc5p. This suggests a role of Cdc14p in Bfa1pBub2p inactivation, resulting in the conversion of the GTPase Tem1p into its GTP-bound active form and thereby the activation of the MEN. Thus, phosphorylation of Bfa1p by Cdc5p at anaphase onset may not be sufficient to fully inactivate the Bfa1pBub2p GAP. A similar conclusion has been reached based on the observation that a constitutively active BFA1-11A does not significantly delay mitotic exit (Hu et al., 2001).
How Cdc14p regulates the MEN in early anaphase is not fully understood. However, given that Cdc14p interacts with Bfa1p and Tem1p and that Cdc14p is required to fully activate the MEN, it is possible that Cdc14p acts through Bfa1p and Tem1p. Considering that Cdc14p does not dephosphorylate Bfa1p before mitotic exit and the observation that phosphatase-independent functions of Cdc14p exist (Shirayama et al., 1996), inactivation of the Bfa1pBub2p complex or activation of Tem1p in early anaphase may not require the phosphatase activity of Cdc14p. Cdc14p binding to Bfa1p or Tem1p may activate the MEN by decreasing the GAP activity of the Bfa1pBub2p complex or by facilitating the exchange of GDP to GTP of Tem1p. Dephosphorylation of the Tem1p binding kinase Cdc15p in early anaphase by Cdc14p probably also contributes to MEN activation (Jaspersen and Morgan, 2000; Stegmeier et al., 2002).
Cdc14p inactivates the MEN at the end of anaphase through Bfa1p dephosphorylation
Although Cdc14p probably activates the MEN in early anaphase (as discussed above), it seems to have the opposite function when cells exit mitosis (Fig. 9 II). We show that Bfa1p is dephosphorylated by the phosphatase Cdc14p as cells exit mitosis. Because phosphorylation of Bfa1p by Cdc5p inactivates the Bfa1pBub2p GAP (Hu et al., 2001; unpublished data), it is likely that dephosphorylation of Bfa1p by Cdc14p reactivates the Bfa1pBub2p GAP complex in telophase/G1. Evidence for the reactivation of the Bfa1pBub2p complex with mitotic exit was obtained through studying the SPB localization of the MEN protein Mob1p. In wild-type cells, inactivation of the MEN coincided with Bfa1p dephosphorylation at the end of mitosis. Furthermore, in cells lacking the Bfa1pBub2p complex, Tem1p remained active even when cells entered G1. This suggests a Bfa1pBub2p-dependent inactivation of the MEN when wild-type cells exit mitosis. Taken together, these observations support a model in which the dephosphorylation of Bfa1p by Cdc14p at the end of mitosis activates the Bfa1pBub2p complex and thereby inhibits the MEN until the renewed phosphorylation of Bfa1p by Cdc5p during the next mitosis.
Cdc14p is part of a positive feedback loop
We propose the order of events outlined in Fig. 9. Cdc14p is released from the nucleolus in early anaphase in an MEN-independent manner and binds immediately to SPBs (Fig. 9 I). Our data are consistent with a model in which this fraction of Cdc14p activates the MEN in early anaphase together with the polo-like kinase Cdc5p (Hu et al., 2001; Lee et al., 2001; unpublished data). Thus, the partially released Cdc14p stimulates the MEN, which in turn promotes the further release of Cdc14p or prevents its nucleolar reuptake (Fig. 9 I). The latter has been suggested for SIN regulation of Clp1p/Flp1p (Cueille et al., 2001; Trautmann et al., 2001). This scenario creates a positive feedback loop that is essential to produce an efficient wave of cytoplasmic Cdc14p, which is probably required to switch the balance toward the dephosphorylation of proteins and promote exit from mitosis. Only this massive liberation of Cdc14p is sufficient to dephosphorylate Bfa1p and thereby reactivate the Bfa1pBub2p GAP in late anaphase, a step that will subsequently inhibit the MEN (Fig. 9 II). Cdc14p also dephosphorylates Hct1p, Sic1p, and Swi5p, which then trigger inactivation of CdkClb (Visintin et al., 1998). Thus, Cdc14p first activates the MEN in early anaphase, and when sufficient Cdc14p is released from the nucleolus, it triggers mitotic exit and inactivates the MEN through the dephosphorylation of Bfa1p. This dual role of Cdc14p will restrict MEN activity to a short period, sufficient for mitotic exit.
Do Cdc14p-like proteins fulfill a common function at SPBs?
Comparing the MEN and SIN, it becomes apparent that the regulatory aspects of the two pathways are similar. A common property of Clp1p/Flp1p and Cdc14p is their association with the SPB (Cueille et al., 2001; Trautmann et al., 2001). Although it is unclear how Clp1p/Flp1p binds to SPBs, our data indicate that the association of the budding yeast Cdc14p with the SPB is mediated through an association with Bfa1p. Cdc14p also interacts with Tem1p. It will be interesting to see whether the fission yeast Clp1p/Flp1p binds to the Bfa1pBub2p complex and Tem1p homologues, named Byr4pCdc16p and Spg1p, respectively. At least, localization of Clp1p/Flp1p with SPBs was in part dependent on Cdc16p (Cueille et al., 2001). It is important to note that Cdc14p must bind to additional SPB or SPB-associated MEN components, because colocalization of Cdc14pYFP and the SPB marker Spc42pCFP was observed in 10% of anaphase bfa1 cells in which Bub2p, Bfa1p, and Tem1p all fail to associate with the SPB (Pereira et al., 2000). Thus, additional SPB components or cell cycle regulators associated with the SPB may be the targets of Cdc14p and Clp1p/Flp1p (Bridge et al., 1998).
A further common feature is the MEN/SIN-independent release of Cdc14p/Clp1p/Flp1p from the nucleolus (Cueille et al., 2001; Trautmann et al., 2001). A third conserved mechanism is the inactivation of Byr4pCdc16p by polo kinase (Balasubramanian et al., 2000; Hu et al., 2001; Tanaka et al., 2001). The data from fission yeast are also consistent with an activation of the SIN by Clp1p/Flp1p through Byr4pCdc16p. The fission yeast cytokinesis checkpoint requires SIN activity but is impaired in the absence of Clp1p/Flp1p (Cueille et al., 2001; Trautmann et al., 2001). In addition, it was found that SPB localization of Sid1pGFP, which is dependent on an active SIN (Guertin et al., 2000), required the presence of Clp1p/Flp1p. Thus, the activation of the MEN and SIN via Bfa1pBub2p or Byr4pCdc16p may be a conserved feature of spindle poleassociated Cdc14-related proteins.
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Materials and methods |
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In vitro binding experiments
GST and GSTCdc14p fusion proteins, produced in E. coli, were incubated with glutathione-Sepharose beads as recommended by Amersham Pharmacia Biotech. Proteins bound to beads were washed with UB buffer (0.05 M Hepes, pH 7.5, 0.1 M KCl, 3 mM MgCl2, 1 mM EGTA, 1 mM DTT, 1 mM PMSF, 0.2% Triton X-100, 1 mM GTP, EDTA-free protease inhibitors cocktail tablets [Roche]). A yeast lysate of BFA19MYC BUB23HA cells was prepared using glass beads (Knop et al., 1999). In brief, yeast cells were resuspended in UB buffer and acid-washed glass beads (Sigma-Aldrich) were added. Cells were lysed using a vortex mixer until >90% of the cells were sheared. The lysate was cleared by centrifugation (5,100 g, 10 min, 4°C). The supernatant was incubated for 60 min at 4°C with the purified GST proteins bound to the glutathione-Sepharose beads. After three washes with UB buffer, the associated proteins were resuspended in HU-DTT (200 mM Tris, pH 6.8, 8 M urea, 5% SDS, 0.1 mM EDTA, 15 mg/ml DTT, bromophenol blue) and incubated for 15 min at 65°C. Samples were analyzed by immunoblotting. Alternatively, recombinant 6HisTem1p or 6HisCdc14p, produced in E. coli, were presented to Ni2+-NTA-agarose and affinity purified as recommended by the manufacturer (QIAGEN). The purified 6HisTem1p or 6HisCdc14p were incubated for 60 min at 4°C with recombinant GST, GSTCdc14p, GSTBfa1p, and GSTBub2p, produced in E. coli, and bound to glutathione-Sepharose. Subsequent washes and immunoblotting were performed as described above.
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Footnotes |
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Acknowledgments |
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The work of E. Schiebel is supported by the Cancer Research UK and by the Human Frontier Science Program.
Submitted: 18 December 2001
Revised: 18 March 2002
Accepted: 20 March 2002
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References |
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