In this study, a new function for the Chk1 protein kinase in regulating mitotic entry is proposed (Fig. ). We demonstrate that Chk1 phosphorylates Cdc25A at S178 and T507 in vitro and that phosphorylation of these residues mediates binding of 14-3-3 protein to Cdc25A in vivo. Because phosphorylation of Cdc25A in vivo is absolutely dependent on Chk1 (57
) and because Chk1 phosphorylates Cdc25A at S178 and T507 in vitro (Fig. ), we infer that Chk1 regulates S178 and T507 phosphorylation in vivo. This conclusion is supported by the finding that phosphorylation of T507 and, to a lesser extent, S178 was impaired in Chk1-deficient cells (Fig. ). Thus, Chk1 negatively regulates entry into mitosis by preventing functional interactions between Cdc25A and the mitotic inducer Cdk1. Sorenson et al. (46
) reported that both Chk1 and Chk2 phosphorylate Cdc25A to promote its turnover. Here we show that Chk1, but not Chk2, phosphorylates Cdc25A at T507 to prevent cyclin B1/Cdk1 binding. Thus, this study uncovers a unique role for Chk1 in the negative regulation of Cdc25A.
FIG. 9. Regulation of Cdc25A by Chk1. Chk1 phosphorylates Cdc25A on several N-terminal serines to promote its ubiquitin-mediated proteolysis (11, 16, 20, 46, 57). Chk1 also phosphorylates Cdc25A on S178 and T507 to facilitate 14-3-3 binding. 14-3-3 binding to (more ...)
In general, the binding of 14-3-3 proteins to their targets is mediated by the phosphorylation of the target protein at one of two 14-3-3-binding consensus motifs. These motifs include the RSXpSXP (mode 1) and RX(Y/F)XpSXP (mode 2) sequences, where pS denotes either phosphoserine or phosphothreonine (33
). Given that each monomer of the 14-3-3 dimer can simultaneously bind distinct phosphoepitopes on a single protein (35
), it is possible that a single 14-3-3 dimer simultaneously binds to pS178 and pT507 on a single Cdc25A molecule. The sequences surrounding and inclusive of S178 (RQNpSAP) fit nicely with the mode 1 consensus, whereas sequences inclusive of and surrounding T507 (TKSRpTWA) do not fit either consensus very well. Thus, it is predicted that S178 is a higher-affinity 14-3-3 binding site and could serve a gatekeeper function, thereby increasing the affinity of 14-3-3 binding to the C terminus of Cdc25A when T507 is phosphorylated (53
). In support of S178 serving a gatekeeper function, mutation of S178 to alanine impaired the interactions between Cdc25A and 14-3-3 to a much greater extent than did mutation of T507 (Fig. and ). However, pT507 must be able to bind 14-3-3 independently of pS178, as the phenotype observed in cells expressing the S178 mutant was much less severe than that observed in cells expressing the T507 mutant.
T507 is located within a cyclin B1/Cdk1 docking site in the C terminus of Cdc25A, and 14-3-3 binding to this region appears to block Cdc25A from functionally interacting with cyclin B1/Cdk1. Sequences comprising the cyclin B1/Cdk1 docking domain are highly conserved among vertebrate Cdc25 homologues, particularly Cdc25A and Cdc25B family members (39
), and this region is disordered in the crystal structures of both Cdc25A and Cdc25B (12
). A motif within the N terminus of Cdc25A confers binding of Cdc25A to Cdk complexes containing cyclins A and E but not B1 (43
). Mutation of this domain reduced binding of Cdc25A to cyclin A and E complexes but not to cyclin B1/Cdk1. Thus, domains within the N and C termini of Cdc25A are important for facilitating interactions between Cdc25A and various cyclin/Cdks, but the C-terminal domain appears to be the major determinant for cyclin B1/Cdk1 binding.
Taken together, these data suggest that phosphorylation of Cdc25A on T507 and subsequent 14-3-3 binding impair the ability of Cdc25A to interact with cyclin B1/Cdk1. Cdc25A is phosphorylated on T507 during the G1 and S phases of the cell cycle, and reduced phosphorylation of T507 was noted during G2/M transition. This suggests that the cyclin B1/Cdk1 binding site is masked throughout much of interphase. Substitution of alanine for T507 prevented phosphorylation of Cdc25A on T507 and subsequent 14-3-3 binding at the C terminus of Cdc25A. This, in turn, is predicted to expose the cyclin B1 binding site, allowing inappropriate interactions between Cdc25A and cyclin B1/Cdk1 during S phase. This model is consistent with the observation that the single T507A and double S178A/T507A mutants were more effective in activating cyclin B1/Cdk1 both in vivo and in vitro and in promoting premature chromosome condensation and phospho-histone H3 staining during S phase.
It was recently reported that phosphorylation of S178 together with phosphorylation of serines 123, 278, and 292 regulates the turnover of Cdc25A both during interphase and in response to IR (46
). This conclusion was based on studies carried out with Cdc25A phosphorylation-site mutants. In our hands, mutation of S178 alone does not impair Cdc25A turnover (data not shown). It is unclear how phosphorylation of S178 regulates the turnover of Cdc25A, given that S178 phosphorylation facilitates the binding of 14-3-3 proteins to Cdc25A. It may be that S178 needed to be mutated to block 14-3-3 binding in order to observe the effects of subsequent mutations of S123, S278, and S292 on Cdc25A stability.
14-3-3 binding regulates several important mitotic regulators. 14-3-3 binding impairs the ability of Cdc25A to interact with cyclin B1/Cdk1 (this study), 14-3-3 binding to Cdc25B and Cdc25C regulates their intracellular trafficking (9
), and 14-3-3 regulates the enzymatic activity of Wee1 (28
). Finally, 14-3-3σ prevents the nuclear accumulation of cyclin B1/Cdk1 following DNA damage (7
). Thus, 14-3-3 binding to mitotic regulators is a commonly employed mechanism used by cells to regulate progression through an unperturbed cell cycle as well as following checkpoint activation.
Maintenance of the Chk1/Cdc25A pathway is essential for cells to progress normally through an unperturbed cell division cycle and for cells to arrest in response to checkpoint activation. Importantly, failure to regulate Cdc25A protein levels causes bypass of the IR-induced DNA damage checkpoint and the DNA replication checkpoint, resulting in decreased cell survival (2
). Thus, drugs that induce accumulation and/or inappropriate activation of Cdc25A are expected to be potent checkpoint abrogators and may prove useful in combination therapies for treating human cancers. Specific Chk1 inhibitors may be particularly useful in this regard, as they would not only lead to Cdc25A accumulation but also to activation of cyclin B1/Cdk1 in S phase to promote mitotic catastrophe.