Cdc25A Regulation: To Destroy or Not to Destroy, Is That the Only ...

[Cell Cycle 2:5, 455-457; September/October 2003]; ©2003 Landes Bioscience

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Cdc25A Regulation To Destroy or Not To Destroy—Is That the Only Question?

Previously published online as a Cell Cycle E-publication at: http://www.landesbioscience.com/journals/cc/tocnew25.php?volume=2&issue=5

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Cdc25A, cell cycle, checkpoints, stress, phosphorylation, Chk1, Chk2, p38MAPK

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Received 07/14/03; Accepted 07/16/03

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*Correspondence to: Helen Piwnica-Worms; Howard Hughes Medical Institute and Departments of Cell Biology and Physiology and Internal Medicine; Washington University School of Medicine; 660 South Euclid Avenue, Box 8228; St. Louis, Missouri 63110-1093 USA; Tel.: 314.362.6812; Fax: 314.362.3709; Email: [email protected]

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Howard Hughes Medical Institute and Departments of Cell Biology and Physiology and Internal Medicine; Washington University School of Medicine; 660 South Euclid Avenue, Box 8228; St. Louis, Missouri USA

Cdc25A is a member of the Cdc25 family of dual-specificity protein phosphatases. This family of protein phosphatases function as positive regulators of the cell division cycle by activating cyclin-dependent protein kinases (CDKs). In mammals, there are three family members, Cdc25A, Cdc25B, and Cdc25C. Whereas Cdc25B and Cdc25C are primarily important in regulating the G2- to M-phase transition, Cdc25A regulates both early (G1/S) and late (G2/M) cell-cycle transitions (for review see ref. 1). Additionally, the Cdc25 family members are key targets of negative regulation by various checkpoint and stress pathways. The absence of checkpoint control results in genomic instability and can lead to uncontrolled cell growth and possibly the development of cancer. Indeed, a subset of aggressive human cancers have been found to display increased levels of Cdc25A or B expression.2-6 Several recent reports have investigated the regulation of Cdc25A stability, during an unperturbed cell cycle and in response to various forms of cellular stress (Fig. 1). It is quite clear that Cdc25A levels are tightly regulated- Cdc25A levels remain low throughout interphase but rise during mitosis and Cdc25A is rapidly destroyed when cells are exposed to agents that impede DNA replication (UV light and hydroxyurea), induce double-strand DNA breaks [ionizing radiation (IR)] or induce osmotic stress.7-14 Cdc25A is ubiquitinated and degraded by the 26S proteosome in a phosphorylationdependent manner.7,10,11,15 Thus, identifying Cdc25A phosphorylation sites and the kinases that phosphorylate these sites is critical to understanding how the cell division cycle is regulated during a normal cell cycle and how the cell division cycle is interrupted during periods of stress and possibly in cancer cells. Serine 123 was the first site reported to regulate Cdc25A stability. Phosphorylation of Cdc25A on serine 123 was reported to be IR-inducible and the Chk2 protein kinase was proposed to be the S123 kinase.16 Subsequent studies demonstrated that Cdc25A is phosphorylated on S123 throughout interphase even in the absence of IR, and that the Chk1 protein kinase is a key regulator of Cdc25A.14 Chk1 functions to keep Cdc25A levels low throughout interphase and Chk1 is required for Cdc25A to be destroyed in the presence of DNA damage.12-14 Chk1 also promotes the degradation of Cdc25A in Xenopus.17,18 The contribution made by Chk2 to the proteolysis of Cdc25A following IR-treatment is still under debate. Recent studies indicate that Cdc25A degradation requires phosphorylation of sites in addition to S123. Sorenson and colleagues identified three Chk1-dependent sites (serines 178, 278, and 292) that cooperate with S123 to regulate Cdc25A stability both in the presence and absence of cellular stress.12 A report in this issue of Cell Cycle by Goloudina and colleagues, along with a recent report by Hassepass and colleagues, identified another Chk1-dependent site, serine 75 that also regulates Cdc25A stability.9 In Xenopus, Cdc25A is phosphorylated on serine 73 (equivalent of S75 in human Cdc25A) and this modification is required for Cdc25A degradation at the mid-blastula transition.18 However, in Xenopus, phosphorylation of S73 appears to be catalyzed by a kinase distinct from Chk1. Hassepass and colleagues reported that Chk1-dependent phosphorylation of Cdc25A on serine 75 was required for UV-mediated Cdc25A degradation.9 The inability to destroy a mutant of Cdc25A containing alanine in place of serine at position 75 resulted in failure to inhibit cyclin E/Cdk2 kinase activity following UV-treatment. The authors proposed that this pathway could be important in regulating cell cycle arrest following UV-treatment, but this was not tested experimentally. Goloudina and colleagues (this issue) report that Cdc25A is phosphorylated on S75 by Chk1 following UV-treatment and, in addition, by p38 MAPK in response to osmotic stress. However, Goloudina and colleagues failed to observe checkpoint bypass when Cdc25A proteolysis was disrupted by mutation of key phosphorylation sites. Both studies highlight the importance of S75- rather than S123phosphorylation in regulating Cdc25A destruction. It is not clear why in one case four sites (serines 123, 178, 278, and 292) had to be

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Kristen E. Neely Helen Piwnica-Worms*

www.landesbioscience.com

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Figure 1. Serine 75 in command of Cdc25A destruction. Cdc25A positively regulates the cell division cycle by activating cyclin-dependent protein kinases (Cdks). Cdc25A is phosphorylated on several residues in vivo and phosphorylation targets Cdc25A for ubiquitin-mediated proteolysis. The Chk1 protein kinase is a major regulator of Cdc25A in vivo – it serves to keep Cdc25A levels low throughout interphase. In addition, the integrity of the Chk1/Cdc25A regulatory pathway is essential for cells to delay cell cycle progression in response to agents that stall DNA replication (such as hydroxyurea (HU) and UV light) and induce double strand DNA breaks (i.e. gamma irradiation (IR)). p38 MAPK targets Cdc25A for destruction under conditions of osmotic stress. The phosphorylation of several residues, including serines 75, 123, 178, 278, and 292, has been proposed to regulate Cdc25A stability. Studies in this issue of Cell Cycle by Goloudina and colleagues indicate that serine 75-phosphorylation is perhaps the most important for regulating Cdc25A stability.

mutated to impair Cdc25A proteolysis12 whereas in the recent studies by Hassepass et al. and Goloudina et al. only a single site (S75) had to be mutated. For the field to fully understand the individual contributions made by these phosphorylation sites to the regulation of Cdc25A, it will be important to determine if mutation of S75 indirectly effects phosphorylation at the four serines reported by Sorenson et al. and vice versa. The generation of phosphospecific antibodies could help to address this possibility. A striking finding by Goloudina and colleagues (this issue), was that stabilization of Cdc25A by mutation of serine 75 to alanine (S75A) or serines 75 and 123 to alanines (S75/123A) was not enough to overcome the UV- or IR-induced S phase checkpoint. Yet other studies have shown that overproduction of Cdc25A can accelerate cell cycle transitions and cause checkpoint bypass.10,11,16,19-21 Was there enough residual proteolysis of the phosphorylation-site mutants to prevent Cdc25A levels from reaching the threshold levels seen, for example, when Chk1 protein levels are lowered by siRNA-treatment or when Chk1 kinase activity is inhibited with drugs?12-14,22-24 Goloudina et al. conclude from their study that regulation of Cdc25A stability must cooperate with other regulatory pathways to contribute to an effective S-phase checkpoint. If this is the case, what might these pathways be? Is targeting Cdc25A for ubiquitin-mediated proteolysis only one way in which Chk1 negatively regulates Cdc25A? Does Chk1 regulate additional pathways that cooperate with the Chk1/Cdc25A pathway to regulate the DNA damaged induced S-phase checkpoint? Destruction of Cdc25A is likely to be just the tip of the iceberg. 456

References 1. Draetta G, Eckstein J. Cdc25 protein phosphatases in cell proliferation. Biochem Biophys Acta 1997; 1332:M53-63. 2. Cangi MG, Cukor B, Soung P, Signoretto S, Moreira G, Ranashinge M, Cady B, Pagano M, Loda M. Role of the Cdc25A phosphatase in human breast cancer. J Clin Invest 2000; 106:753-61. 3. Dixon D, Moyana T, King MJ. Elevated expression of the cdc25A protein phosphatase in colon cancer. Exp Cell Res 1998; 240:236-43. 4. Galaktionov K, Lee AK, Eckstein J, Draetta G, Meckler J, Loda M, Beach D. CDC25 phosphatases as potential human oncogenes. Science 1995; 269:1575-7. 5. Hernandez S, Hernandez L, Bea S, Pinyol M, Nayach I, Bellosillo B, Nadal A, Ferrer A, Fernandez PL, Montserrat E, Cardesa A, Campo E. Cdc25A and the splicing variant cdc25B2, but not cdc25B1, -B3 or -C, are over-expressed in aggressive human nonHodgkin's lymphomas. Int J Cancer 2000; 89:148-52. 6. Wu W, Fan Y-H, Kemp BL, Walsh G, Mao L. Overexpression of cdc25A and cdc25B Is Frequent in Primary Non-Small Cell Lung Cancer but Is Not Associated with Overexpression of c-myc1. Cancer Res 1998; 58:4082-5. 7. Bernardi R, Lieberman DA, Hoffman B. Cdc25A stability is controlled by the ubiquitinproteasome pathway during cell cycle progression and terminal differentiation. Oncogene 2000; 19:2447-54. 8. Goloudina A, Yamaguchi H, Chervyakova DB, Appella E, Fornace AJ, Bulavin DV. Regulation of human Cdc25A stability by serine 75 phosphorylation is not sufficient to activate a S-phase checkpoint. Cell Cycle 2003; 2: (this issue). 9. Hassepass I, Voit R, Hoffmann I. Phosphorylation at serine-75 is required for UV-mediated degradation of human Cdc25A phosphatase at the S-phase checkpoint. J Biol Chem 2003; In Press. 10. Mailand N, Falck J, Lukas C, Syljuasen RG, Welcker M, Bartek J, Lukas J. Rapid destruction of human Cdc25A in response to DNA damage. Science 2000; 288: 1425-9. 11. Molinari M, Mercurio C, Dominguez J, Goubin F, Draetta GF. Human Cdc25A inactivation in response to S phase inhibition and its role in preventing premature mitosis. EMBO Rep 2000; 1:71-9. 12. Sorensen CS, Syluasen RG, Falck J, Schroeder T, Ronnstrand L, Khanna KK, Zhou B-B, Bartek J, Lukas J. Chk1 regulates the S phase checkpoint by coupling the physiological turnover and ionizing radiation-induced accelerated proteolysis of Cdc25A. Cancer Cell 2003; 3:247-58.

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13. Xiao Z, Chen Z, Gunasekera AH, Sowin TJ, Rosenberg SH, Fesik S, Zhang H. Chk1 Mediates S and G2 Arrests through Cdc25A Degradation in Response to DNA-damaging Agents. J Biol Chem 2003; 278:21767-73. 14. Zhao H, Watkins JL, Piwnica-Worms H. Disruption of the checkpoint kinase 1/cell division cycle 25A pathway abrogates ionizing radiation-induced S and G2 checkpoints. Proc Natl Acad Sci USA 2002; 99: 14795-800. 15. Donzelli M, Squatrito M, Ganoth D, Hershko A, Pagano M, Draetta GF. Dual mode of degradation of Cdc25 A phosphatase. EMBO J 2002; 21:4875-84. 16. Falck J, Mailand N, Syljuasen RG, Bartek J, Lukas J. The ATM-Chk2-Cdc25A checkpoint pathway guards against radioresistant DNA synthesis. Nature 2001; 410:842-7. 17. Kim SH, Chuan L, Maller JL. A maternal form of the phosphatase Cdc25A regulates early embryonic cell cycles in Xenopus laevis. Dev Biol 1999; 212:381-91. 18. Shimuta K, Nakajo N, Uto K, Hayano Y, Okazaki K, Sagata N. Chk1 is activated transiently and targets Cdc25A for degradation at the Xenopus midblastula transition. EMBO J 2002; 21:3694-703. 19. Blomberg I, Hoffman I. Ectopic expression of Cdc25A accelerates the G1/S transition and leads to premature activation of cyclin E-and cyclin A-dependent kinases. Mol Cell Biol 1999; 19:6183-94. 20. Mailand N, Podtelejnikov AV, Groth A, Mann M, Bartek J, Lukas J. Regulation of G2/M events by Cdc25A through phosphorylation-dependent modulation of its stability. EMBO J 2002; 21:5911-20. 21. Sexl V, Diehl J, Sherr C, Ashmun R, Beach D, Roussel MF. A rate limiting function of Cdc25A for S phase entry inversely correlates with tyrosine dephosphorylation of Cdk2. Oncogene 1999; 18:573-82. 22. Busby EC, Leistritz DF, Abraham RT, Karnitz LM, Sarkaria JN. The radiosensitizing agent 7-hydroxystaurosporine (UCN-01) inhibits the DNA damage checkpoint kinase hChk1. Cancer Res 2000; 60:2108-212. 23. Graves PR, Yu L, Schwarz JK, Gales J, Sausville EA, O'Connor PM, Piwnica-Worms H. The Chk1 protein kinase and the Cdc25C regulatory pathway are targets of the anticancer agent UCN-01. J Biol Chem 2000; 275:5600-5. 24. Jackson JR, Gilmartin A, Imburgia C, Winkler JD, Marshall LA, Roshak A. An indolocarbazole inhibitor of human checkpoint kinase (Chk1) abrogates cell cycle arrest caused by DNA damage. Cancer Res 2000; 60:566-72.

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