In this study, we generated a mutant of SpCdc25 that is severely impaired in its ability to bind to the fission yeast 14-3-3 proteins (Rad 24 and Rad 25). When expressed in fission yeast, this mutant Cdc25 protein localized almost exclusively to the nucleus, in contrast to wild-type Cdc25, which localized to both the cytoplasm and the nucleus. Inhibition of Crm1-mediated nuclear export resulted in the nuclear accumulation of wild-type Cdc25, indicating that wild-type Cdc25 normally shuttles between the nucleus and the cytoplasm. Overproduction of Rad 24 caused wild-type Cdc25 to localize exclusively to the cytoplasm, whereas nuclear localization of the 14-3-3 binding mutant was not altered upon Rad 24 overproduction. Finally, cells expressing the 14-3-3 binding mutant exhibited defective G2/M checkpoint responses. Taken together, these results suggest that 14-3-3 binding regulates the intracellular compartmentalization of Cdc25 and establish that 14-3-3 binding to Cdc25 is required for fission yeast cells to arrest cell cycle progression if damaged or incompletely replicated DNA is detected.
To identify residues in Cdc25 that mediate 14-3-3 binding upon phosphorylation, we first identified residues in Cdc25 that are phosphorylated by the Cds1 protein kinase in vitro. We previously demonstrated that both the Cds1 and Chk1 protein kinases phosphorylate Cdc25 in vitro at 14-3-3 binding sites but identified only three of the sites at that time (62
). Cds1 was used in this study because it is more active than Chk1 when overproduced in insect cells. In total, we identified 12 phosphorylation sites and then characterized a mutant of Cdc25 with 9 of these residues mutated to alanine (denoted Cdc25-9A) because this mutant was severely impaired in its ability to bind 14-3-3 proteins. Importantly, Cdc25-9A fully complemented the cdc25-22
temperature-sensitive allele at the nonpermissive temperature, demonstrating that the nine mutations do not grossly affect the function of Cdc25. Human and Xenopus
Cdc25C each have a single phosphorylation site that mediates 14-3-3 binding, and in both cases mutation of this residue ablates 14-3-3 binding (26
). In contrast, fission yeast Cdc25 has several phosphorylation sites, and it is not known how many actually contribute to 14-3-3 binding. The 14-3-3 binding sites in human and Xenopus
Cdc25C match well with the consensus 14-3-3 binding sequence (40
), while those in fission yeast Cdc25 match less well (Fig. C). Throughout the course of this study, we analyzed several combinations of mutations in SpCdc25 and never found a single mutation that completely eliminated 14-3-3 binding. This indicates that fission yeast Cdc25 has more than one 14-3-3 binding site. Given that 14-3-3 is a dimer with the capability of binding two phosphorylation sites simultaneously (60
), it is possible that in fission yeast, two lower-affinity residues function together to facilitate high-affinity 14-3-3 binding.
By tagging wild-type and mutant forms of Cdc25 with GFP, we observed the subcellular localization of Cdc25 in living cells (Fig. ). In most cases, GFP-Cdc25 was observed to be evenly distributed throughout the cell. However, some cells showed enhanced nuclear fluorescence while others exhibited a more pronounced cytoplasmic fluorescence. These results are consistent with those reported by Lopez-Girona et al. (31
). Nuclear accumulation of Cdc25-WT was observed when cells were treated with LMB. This indicates that Cdc25 normally shuttles between the nucleus and the cytoplasm. Cdc25-WT was excluded from the nucleus upon Rad 24 overproduction, indicating that Rad 24 binding contributes to the nuclear exclusion of Cdc25. Further evidence in favor of 14-3-3 proteins regulating the nuclear exclusion of Cdc25 comes from the analysis of the two Cdc25 mutants. Cdc25-3A and Cdc25-9A were distinguishable in terms of 14-3-3 binding, nuclear localization, and responses to Rad 24 overproduction. The Cdc25-9A mutant, which was severely impaired in 14-3-3 binding, localized to the nucleus, and neither Rad 24 overproduction nor DNA damage affected its nuclear location. The Cdc25-3A mutant, which was partially defective in 14-3-3 binding, showed more nuclear localization than Cdc25-WT but less than Cdc25-9A did, and, unlike Cdc25-9A, Cdc25-3A was partially excluded from the nucleus upon Rad 24 overproduction. Given that 14-3-3 is competent to bind Cdc25, the simplest interpretation of our data is that 14-3-3 binding either reduces the rate of nuclear import of Cdc25 or enhances its rate of nuclear export.
Rad 24 reportedly contains an NES, and it has been proposed that Rad 24 contributes an “attachable” NES to mediate the nuclear export of Cdc25 (31
). The sequence comprising the putative NES consists of the sequence STLIMQLLRDNLT LW (amino acids 219 to 233). A mutant of Rad 24 containing alanine in place of isoleucine 222 and leucine 226 [Rad 24(I222A, L226A)] is reported to be more nuclear than wild-type Rad 24 and to be unable to deplete Cdc25 from the nucleus upon irradiation (31
). We confirmed that Rad 24(I222A, L226A) was more nuclear than wild-type Rad 24 and that overexpression of wild-type Rad 24 but not Rad24(I222A, L226A) resulted in the nuclear exclusion of Cdc25 (Fig. and data not shown). Furthermore, cell elongation was noted upon overexpression of wild-type but not mutant Rad24 (data not shown). These results are consistent with the NES hypothesis, which states that 14-3-3 contributes an NES to export Cdc25 from the nucleus. However, we found that Rad 24(I222A, L226A) was severely impaired in its ability to bind to Cdc25 both in vitro and in vivo (Fig. ). Furthermore, fluorescence polarization experiments with a fluorescein-labelled phospho-Raf peptide as ligand demonstrated that Rad24(I222A, L226A) had a Kd
at least 30 times higher than wild-type Rad24 (data not shown). Thus, the phenotypes described for the Rad24(I222A, L226A) mutant by Lopez-Girona et al. (31
) could easily be accounted for by the failure of the Rad 24 mutant to efficiently bind to Cdc25 or possibly to other target proteins. Interestingly, Xenopus
Cdc25 contains its own NES, and in this case 14-3-3 binding appears to regulate the rate of nuclear import of Cdc25 as opposed to its nuclear export (24
). Thus, it is possible that 14-3-3 binding perturbs the nuclear import of fission yeast Cdc25 rather than actually promoting its nuclear export by providing an attachable NES or that the same sequence in Rad 24 has dual functions.
Upon completion of DNA synthesis or after recovery from DNA damage, Cdc25 must accumulate in the nucleus to promote mitotic entry. This could be accomplished by either reduced phosphorylation or enhanced dephosphorylation of Cdc25 followed by loss of 14-3-3 binding. Differential subcellular localization provides a level of regulation that potentially explains how phosphorylation of Cdc25 and subsequent 14-3-3 binding function to prevent premature activation of Cdc2 in response to G2
/M checkpoint activation. However, in the absence of checkpoint activation, fission yeast cells expressing Cdc25-9A were indistinguishable from those expressing Cdc25-WT, even though Cdc25-9A was constitutively nuclear. Given that fission yeast Cdc2 has been reported to be nuclear (1
), these results indicate that nuclear colocalization of Cdc25 with Cdc2 is not sufficient to advance cells into mitosis from S phase during a normal cell cycle. Thus, the importance of 14-3-3/Cdc25 interactions in coupling S-phase completion to M-phase entry during a normal cell cycle is unclear due to the apparent lack of any phenotype in cells expressing Cdc25-9A.
In an earlier study, we demonstrated that reduced 14-3-3 binding to Cdc25 coupled with a deletion in mik1+ partially compromised the DNA replication checkpoint. Here we show that a more complete disruption of Cdc25/14-3-3 interactions coupled with mik1+ deletion results in an almost complete bypass of the DNA replication checkpoint. We also establish a role for 14-3-3/Cdc25 interactions in the DNA damage checkpoint, since cells expressing the 9A mutant of Cdc25, which is severely impaired in 14-3-3 binding, do not arrest cell cycle progression following DNA damage. Thus, in fission yeast, 14-3-3 binding to Cdc25 is required for cell cycle arrest in response to both DNA damage and replication blocks.
Studies conducted with a wide variety of organisms indicate that G2
/M checkpoints operate to maintain Cdc25 in a 14-3-3-bound form rather than to induce 14-3-3 binding. In humans, Cdc25C is bound to 14-3-3 proteins throughout interphase (49
). A similar situation exists in Xenopus
(that is, Cdc25 is stoichiometrically bound to 14-3-3 proteins throughout interphase), and so it is unclear how checkpoints could further stimulate the binding of 14-3-3 (26
). That checkpoints operate to maintain rather than induce 14-3-3 binding to Cdc25 is also supported by studies with fission yeast. In fission yeast, activation of the DNA replication checkpoint with HU did not alter the phosphorylation state of Cdc25, suggesting that the DNA replication checkpoint operates to maintain Cdc25 in a phosphorylated state to ensure 14-3-3 binding (62
). In addition, the amount of 14-3-3 bound to Cdc25 has not been observed to change during G2
/M checkpoint responses in fission yeast (10
In humans there are at least three protein kinases that potentially regulate the interactions between Cdc25 and 14-3-3 proteins. These kinases include C-TAK1 (43
), Chk1 (5
), and Cds1/Chk2 (5
). Elimination of Chk1 in either Xenopus
or fission yeast does not detectably alter the levels of 14-3-3 bound to Cdc25 (10
). A recently published study reported that fission yeast cells lacking both Cds1 and Chk1 still contain Cdc25 bound to 14-3-3 proteins both before and after DNA damage (10
). The authors interpreted their findings to indicate that neither Chk1 nor Cds1 function is required for Cdc25/14-3-3 interactions either before or after DNA damage. Given what we know about the interactions between the human and fission yeast Cdc25 proteins with 14-3-3, it is perhaps not unexpected that loss of Cds1 and Chk1 could affect checkpoint function without noticeably affecting overall 14-3-3/Cdc25 interactions. As indicated above, at least three kinases potentially regulate Cdc25/14-3-3 interactions in humans. C-TAK1 resides in the cytoplasm, whereas Chk1 and Cds1/Chk2 are in the nucleus. Thus, elimination of any two of the kinases, as was done by Chen et al. (10
), would not be expected to eliminate the interactions between Cdc25 and 14-3-3 in human cells. Furthermore, the studies of Lopez-Girona et al. (31
) and the findings reported here indicate that the major role of 14-3-3 binding in the case of fission yeast is to keep Cdc25 out of the nucleus. Thus, elimination of two nuclear checkpoint kinases (Chk1 and Cds1) could abrogate the checkpoint response by allowing Cdc25 to accumulate in the nucleus without affecting 14-3-3 binding to the bulk population of Cdc25 remaining in the cytoplasm. Furthermore, Lopez-Girona et al. (31
) showed that DNA damage does not cause Cdc25 to be excluded from the nucleus in the absence of Chk1, indicating that Chk1 does indeed regulate Cdc25 function in fission yeast.
In summary, our data support the hypothesis that both the DNA replication and the DNA damage checkpoints use similar mechanisms to arrest the cell cycle in fission yeast. The mechanism involves maintaining Cdc25 in a phosphorylated form that binds 14-3-3 proteins. 14-3-3 binding keeps Cdc25 out of the nucleus, away from its substrate, either by inhibiting nuclear import or by indirectly facilitating nuclear export. The net effect is to prevent Cdc25 from activating Cdc2 and thereby to delay entry into mitosis. At this time, Cds1 and Chk1 remain viable candidate kinases for mediating Cdc25/14-3-3 interactions during a G2/M checkpoint response.