SCFβ-TRCP links Chk1 signaling to degradation of the Cdc25A protein phosphatase

In response to DNA damage and DNA replication interference, cells activate an elaborate network of signaling pathways collectively called the DNA damage stress response pathway (Zhou and Elledge 2000). The central conduits of this network are two parallel but partially overlapping protein kinase cascades, the ATM-Chk2 and ATR-Chk1 kinase modules, that transduce the damage signal to downstream effectors involved in cell cycle control, DNA repair, and apoptosis (Zhou and Elledge 2000; Shiloh 2003). ATM primarily responds to DNA double-strand breaks, whereas ATR responds to agents that interfere with DNA replication in addition to double-strand breaks (Zou and Elledge 2003). The branches of these pathways used in arresting the cell cycle are called checkpoints. In many eukaryotes, the targets of checkpoint pathways are the cyclin-dependent kinases, Cdks, which control multiple cell cycle transitions including the G1/S transition, late origin firing during S phase, and the G2/M transition, each of which is inhibited in response to DNA damage. Cells have evolved multiple mechanisms to inhibit Cdk activity. In mammals, the Cdk inhibitor p21Cip1 is induced in response to DNA damage through activation of the p53 transcription factor, which is activated and stabilized in response to DNA damage (Giaccia and Kastan 1998; Zhou and Elledge 2000). In addition, levels of cyclin D1 rapidly decrease in response to DNA damage during G1, leading to redistribution of p21Cip1 from Cdk4 to Cdk2 (Agami and Bernards 2000). Cdks are also regulated by inhibitory phosphorylation on tyrosine, which is altered in response to DNA damage (Jin et al. 1997; Rhind et al. 1997; for review, see Takizawa and Morgan 2000; Donzelli and Draetta 2003). The regulatory mechanisms controlling Cdk phosphorylation have been extensively studied. Tyrosine phosphorylation is regulated by the opposing activities of the tyrosine kinases Wee1 and Myt1, and a group of tyrosine phosphatases known as Cdc25A, Cdc25B, Cdc25C (for review, see Morgan 1997). A direct connection between checkpoint signaling and Cdc25 was established when it was found that the checkpoint kinase Chk1, and later Chk2, could phosphorylate Cdc25C on a site relevant to its checkpoint function in vivo (Peng et al. 1996; Sanchez et al. 1996; Furnari et al. 1997; Matsuoka et al. 1998). Furthermore, these kinases were shown to phosphorylate all three Cdc25 family members, suggesting they were general targets of DNA damage stress response pathways (Sanchez et al. 1996; Matsuoka et al. 1998). Analysis of Chk regulation of Cdc25 from several systems showed that phosphorylation of Cdc25C both inhibited kinase activity (Blasina et al. 1999; Furnari et al. 1999) and maintained Cdc25C in the cytoplasm, where it cannot access Cdk/cyclin complexes efficiently (Zeng et al. 1998; Kumagai and Dunphy 1999; Lopez-Girona et al. 1999; Zeng and Piwnica-Worms 1999). However, mice lacking Cdc25C grow normally and have intact checkpoint responses (Chen et al. 2001), suggesting that other family members may play more prominent roles in Cdk regulation. A second family member implicated in the damage response is Cdc25A. Cdc25A is capable of removing inhibitory tyrosine phosphorylation from both Cdk1 and Cdk2 kinases to promote entry into and progression through S phase and mitosis (Hoffmann et al. 1994; Vigo et al. 1999; for review, see Donzelli and Draetta 2003). Cdc25A has also been shown to be a phosphorylation target of Chk kinases (Sanchez et al. 1996) and to be regulated by Chk kinases in response to DNA damage (for review, see Donzelli and Draetta 2003). In contrast to regulation of Cdc25C, Cdc25A is destroyed in response to ionizing radiation (IR) and ultraviolet (UV) light through a process involving ubiquitin-mediated proteolysis. During G1, UV treatment leads to Chk1-dependent elimination of Cdc25A (Mailand et al. 2000) and persistent Cdk2 Y15 phosphorylation. During an unperturbed S phase, Cdc25A is unstable and this instability requires Cdc25A phosphorylation by Chk1 (Falck et al. 2001; Sorensen et al. 2003). IR during S phase leads to accelerated Cdc25A phosphorylation by Chk1 with a concomitant increase in turnover. Defects in this intra-S-phase checkpoint lead to radio-resistant DNA synthesis (RDS; Falck et al. 2001; Xiao et al. 2003). Whereas depletion of Chk1 leads to an RDS phenotype, expression of a Cdk2 mutant that is resistant to inhibitory tyrosine phosphorylation overcomes IR-dependent S-phase arrest (Falck et al. 2001), implicating elimination of Cdc25A in the intra-S-phase checkpoint. Recent studies indicate that Cdc25A turnover through the ubiquitin pathway involves at least two temporally distinct components (Donzelli et al. 2002; Donzelli and Draetta 2003). During mitotic exit and early G1, Cdc25A stability is controlled by the anaphase-promoting complex in conjunction with Cdh1. During interphase, however, Cdc25A turnover is dependent on Cul1 (Donzelli et al. 2002), a central component of the SCF (Skp1/Cul1/F-box protein) ubiquitin ligase (Feldman et al. 1997; Skowyra et al. 1997). Precisely how Cul1 promotes turnover of Cdc25A is unknown. In SCF complexes, Cul1 together with the RING-H2 finger protein Rbx1 forms the core ubiquitin ligase that binds ubiquitin-conjugating enzymes (for review, see Deshaies 1999; Koepp et al. 1999). Specificity in these reactions is achieved by a substrate-binding module composed of Skp1 and a member of the F-box family of proteins. F-box proteins interact with Skp1 through the F-box motif (Bai et al. 1996) and with substrates through C-terminal protein interaction domains, including WD40 propellers (Skowyra et al. 1997; Wu et al. 2003; Orlicky et al. 2003). Frequently, association of SCF targets with the requisite F-box protein requires that the substrate be modified, typically by phosphorylation to produce a short peptide motif displaying properties of a phosphodegron (Winston et al. 1999a; Koepp et al. 2001; Nash et al. 2001; Wu et al. 2003; Orlicky et al. 2003). Here we report that constitutive and DNA-damage-induced turnover of Cdc25A occurs via the SCFβ-TRCP ubiquitin ligase. Depletion of β-TRCP by shRNA stabilizes Cdc25A, leading to inappropriately high levels of Cdk2 kinase activity characteristic of a checkpoint defect. Cdc25A ubiquitination by SCFβ-TRCP in vitro involves Chk1-dependent phosphorylation principally at S76, consistent with the requirement for Chk1 in vivo. However, Chk1-mediated phosphorylation of Cdc25A does not appear to be sufficient to generate the requisite phosphodegron for β-TRCP recruitment. We find that residues 79-84 of Cdc25A constitute a phosphodegron for recognition by β-TRCP and implicate S79 and S82 as phosphoacceptor sites in this motif. Indeed, S82 is in a sequence context (Asp-Ser-Gly-Phe) reminiscent of previously identified phosphodegrons in the β-TRCP substrates IκBα and β-catenin. We suggest that Chk1-mediated phosphorylation of S75 may promote Cdc25A turnover by facilitating the phosphorylation of the adjacent phosphodegron targeted by β-TRCP.