The tumor suppressor protein p53 mediates stress-induced growth arrest or apoptosis and plays a major role in safeguarding genome integrity. In response to DNA damage, p53 can be modified at multiple sites by phosphorylation and acetylation. We report on the characterization of p53 C-terminal phosphorylation by CHK1 and CHK2, two serine/ threonine (Ser/Thr) protein kinases, previously implicated in the phosphorylation of the p53 N terminus. Using tryptic phosphopeptide mapping, we have identified six additional CHK1 and CHK2 sites residing in the final 100 amino acids of p53. Phosphorylation of at least three of these sites, Ser366, Ser378, and Thr387, was induced by DNA damage, and the induction at Ser366 and Thr387 was abrogated by small interfering RNA targeting chk1 and chk2. Furthermore, mutation of these phosphorylation sites has a different impact on p53 C-terminal acetylation and on the activation of p53-targeted promoters. Our results demonstrate a possible interplay between p53 C-terminal phosphorylation and acetylation, and they provide an additional mechanism for the control of the activity of p53 by CHK1 and CHK2. INTRODUCTIONThe protein p53 is often referred to as a "tumor suppressor," because it is frequently mutated in Ͼ50% of human cancers (Olivier et al., 2002). In response to stress, p53 undergoes extensive posttranslational modification, including phosphorylation and acetylation (Appella and Anderson, 2001;Brooks and Gu, 2003; Xu, 2003). Consequently, p53 is stabilized and activated (Vousden, 2002). As a transcription factor, p53 is composed of an N-terminal activation domain, a central specific DNA binding domain, and a C-terminal tetramerization domain, followed by a regulatory domain rich in basic amino acids (Ko and Prives, 1996). On activation, p53 binds to an array of gene promoters, some of which, such as p21/waf1, are responsible for stress-induced cell cycle arrest, whereas others, such as bax and puma, are responsible for driving cells into apoptosis (Vousden and Lu, 2002;Harms et al., 2004).Phosphorylation of p53 mostly occurs in the N-terminal activation domain at the Ser6, Ser9, Ser15, Thr18, Ser20, Ser33, Ser37, Ser46, Thr55, and Thr81 residues, with some phosphorylation occurring in the C-terminal linker and basic regions at Ser315, Ser371, Ser376, Ser378, and Ser392. Phosphorylation on most of these sites is induced by DNA damage, with some, such as Thr55 and Ser376, being repressed upon genotoxic stress (Appella and Anderson, 2001;Holmberg et al., 2002). How these individual residues contribute to p53 stabilization and activation is still not fully understood, and, at times, has been the subject of debate.Many protein kinases have been implicated in phosphorylating p53 (Holmberg et al., 2002). Notably, phosphorylation at Ser15 by ATM/ATR, either directly or through CHK1/ CHK2, or at Ser20 by CHK1/CHK2 has been shown to alleviate the inhibition or degradation of p53 by Mdm2, leading to p53 stabilization and activation (Shieh et al., 1997(Shieh et al., , 2000Banin et al., 1998;...
Proper regulation of cell cycle progression is pivotal for maintaining genome stability. In a search for DNA damage-inducible, CHK1-modulated genes, we have identified BTG3 (B-cell translocation gene 3) as a direct p53 target. The p53 transcription factor binds to a consensus sequence located in intron 2 of the gene both in vitro and in vivo, and depletion of p53 by small interfering RNA (siRNA) abolishes DNA damage-induced expression of the gene. Furthermore, ablation of BTG3 by siRNA in cancer cells results in accelerated exit from the DNA damage-induced G2/M block. In vitro, BTG3 binds to and inhibits E2F1 through an N-terminal domain including the conserved box A. Deletion of the interaction domain in BTG3 abrogates not only its growth suppression activity, but also its repression on E2F1-mediated transactivation. We also present evidence that by disrupting the DNA binding activity of E2F1, BTG3 participates in the regulation of E2F1 target gene expression. Therefore, our studies have revealed a previously unidentified pathway through which the activity of E2F1 may be guarded by activated p53.
CHK2/hCds1 plays important roles in the DNA damage-induced cell cycle checkpoint by phosphorylating several important targets, such as Cdc25 and p53. To obtain a better understanding of the CHK2 signaling pathway, we have carried out a yeast two-hybrid screen to search for potential CHK2-interacting proteins. Here, we report the identification of the mitotic checkpoint kinase, TTK/hMps1, as a novel CHK2-interacting protein. TTK/hMps1 directly phosphorylates CHK2 on Thr-68 in vitro. Expression of a TTK kinase-dead mutant, TTK D647A , interferes with the G 2 /M arrest induced by either ionizing radiation or UV light. Interestingly, induction of CHK2 Thr-68 phosphorylation and of several downstream events, such as cyclin B1 accumulation and Cdc2 Tyr-15 phosphorylation, is also affected. Furthermore, ablation of TTK expression using small interfering RNA results not only in reduced CHK2 Thr-68 phosphorylation, but also in impaired growth arrest. Our results are consistent with a model in which TTK functions upstream from CHK2 in response to DNA damage and suggest possible cross-talk between the spindle assembly checkpoint and the DNA damage checkpoint.
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