[Cell Cycle 5:18, 2172-2173, 15 September 2006]; ©2006 Landes Bioscience
Redox Control of Cell Cycle Progression via Cdc25 Phosphatase (Mih1p) in S. cerevisiae Letter to the Editor
INTRODUCTION
*Correspondence to: Johannes Rudolph; Departments of Biochemistry and Chemistry, Duke University Medical Center; Durham, North Carolina 27710 USA; Tel.: 919.668.6188; Fax: 919.613.8642; Email:
[email protected]
ABBREVIATIONS ROS Cdk/Cyclins
reactive oxygen species cyclin-dependent kinases
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MATERIALS AND METHODS
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ACKNOWLEDGEMENTS
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Cdc25 phosphatase, Mih1p, redox regulation, protein tyrosine phosphatase, yeast, oxidative stress, redox, reactive oxygen species
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KEY WORDS
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Previously published online as a Cell Cycle E-publication: http://www.landesbioscience.com/journals/cc/abstract.php?id=3252
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Original manuscript submitted: 06/13/06 Manuscript accepted: 07/31/06
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Departments of Biochemistry and Chemistry, Duke University Medical Center; Durham, North Carolina USA
Cdc25 phosphatases are key regulators of the eukaryotic cell cycle through their activation of the cyclin-dependent protein kinases (Cdk/Cyclins).1,2 Additionally the Cdc25 phosphatases, by their phosphorylation and subsequent degradation or sequestration, play an important role in integrating the signals of checkpoint control.3 Like other members of the protein tyrosine phosphatase family, Cdc25s are also directly and reversibly inhibited by oxidation.4,5 The initial oxidation product, the sulfenic acid of the active site cysteine, is rapidly and effectively trapped as an intramolecular disulfide by a so-called backdoor cysteine. This backdoor cysteine is conserved in all known Cdc25s as part of the Cdc25-homology domain A (CH2-A).6 Because the backdoor cysteine is located deep within the protein, the active site loop undergoes a major rearrangement during oxidation. The resulting intramolecular disulfide is buried and substrate binding and phosphatase activity is precluded.7 A new pocket next to the active site emerges to allow for ready access by thioredoxin, an efficient and physiologically relevant reductant for the Cdc25 phosphatases. In the yeast S. cerevisiae, the Cdc25 phosphatase homolog is Mih1p, which in the absence of one of two negative regulators (Hsl1p or Hsl7p) of the opposing kinase (Swe1p), is essential for normal growth due to Swe1p-mediated hyper-phosphorylation of the Cdk/Cyclins.8 This block in cell cycle progression is readily observable under the microscope as hyphal cell growth. Herein we use S. cerevisiae as a model organism to investigate the in vivo importance of Mih1p oxidation and the role of the backdoor cysteine (Cys270).
RIB
Divya Seth Johannes Rudolph*
Hydrogen peroxide and menadione were obtained from Sigma and solutions of each were prepared fresh daily. Anti-myc antibody was purchased from Cell Signaling. Parent strain JMY1289 with the genotype GAL:MIH1:TRP1 hsl1\∆\ 1:URA3 was obtained from D. Lew (Department of Pharmacology and Cancer Biology, Duke University Medical Center) and has the same phenotype as the analogous strain JMY1290.8 JMY1289 was transformed with an integrating plasmid containing myc-tagged mih1 under the control of its own genomic promoter, thus preserving the copy of mih1 under control of the gal promoter. This transformation was performed with wild type or the C426S (human cdc25B) equivalent of mih1(C270S). The expression of Mih1p in these transformants was confirmed by Western blotting using the Myc antibody. Yeast cultures were grown overnight at 30˚C in YPGal (10 g/L yeast extract, 20 g/L peptone, 20 g/L galactose) media and were streaked onto YPGlu (10 g/L yeast extract, 20 g/L peptone, 20 g/L glucose) plates or diluted into YPGlu media. Sterile filters (Whatman #1) were overlayed on the plates and the yeast were allowed to grow for 24–48 h at 30˚C until small colonies were visible. Oxidative stress was induced by addition of 10 µL of menadione (2.5 mM) or hydrogen peroxide (1–5 mM) to the Whatman filters. Following growth at 30˚C in liquid culture monitored by OD600 or incubation overnight on plates, cell morphology was scored by light microscopy (400x).
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This research was supported by NIH grant R01 GM61822
RESULTS AND DISCUSSION
Yeast strain JMY1289 serves as an ideal model to test the in vivo significance of oxidation of Mih1p (Cdc25) and the role of the backdoor cysteine in this process.8 JMY1289 does not grow normally on standard glucose media because it lacks both hsl1, a negative regulator of swe1, and wild type mih1. This combination of knockouts stalls normal cell cycle progression because of highly phosphorylated and therefore inactivated Cdk/Cyclins, and as a result the cells grow hyphally. As expected, this cell growth defect can be 2172
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2006; Vol. 5 Issue 18
Cdc25 (Mih1p) and Redox Regulation in S. cerevisiae
complemented with a wild type copy of mih1 that is under the control of its own promoter. Strain survival in the absence of complementation is ensured in galactose media because of the presence of a wild type copy of mih1 under the control of the gal promoter. Following transformation of JMY1289 with an integrating plasmid bearing the backdoor mutation (C270S, Mih1p numbering; C426S, human Cdc25B numbering), we selected several transformants expressing wild type levels of Mih1p and tested them for cell viability on glucose-containing plates. As for wild type, the backdoor mutant yields rapidly growing colonies wherein the cells are round and appear to divide normally (Fig. 1). In contrast, the colonies from the parent strain lacking Mih1p appear more slowly and their cells exhibit hyphal growth, with long clusters of incompletely divided cells (Fig. 1). Interestingly, while the backdoor mutant cells grown in roller tubes in liquid media on glucose were normal, those grown in shaker flasks at 100 rpm showed a slightly elongated and clumpy phenotype compared to wild type cells (not shown). This delay or abnormality in cell division indicates the high sensitivity of Mih1p to mild oxidative stress. We next tested the response of the backdoor mutant towards induced oxidative stress. Freshly growing colonies on glucosecontaining plates were exposed to nontoxic concentrations of menadione or hydrogen peroxide, the latter applied repeatedly because of its intrinsic instability and/or rapid degradation by catalase and cellular reductants. Closest to the filter, both wild type and the C270S mutant did not grow. The cells nearest the filters that grew were inspected by microscopy. The hyphal morphology of cells near the oxidants demonstrated a concentration-dependent effect for the backdoor mutant strain, but not for the wild type strain (Fig. 1). As a control, the backdoor mutant strain grown in galactose wherein the wild type copy of Mih1p is also expressed does not have this hyphal phenotype even upon oxidative stress, indicating additionally the C270S is not a dominant negative (data not shown). We conclude from these results that Mih1p is highly susceptible to oxidation in vivo, as we have previously demonstrated for Cdc25B in vitro.5 Note that we have previously shown that wild type and the backdoor mutants of Cdc25s have equivalent phosphatase activity and are equally susceptible to oxidation and inactivation.5 It is only the subsequent events that are different, namely protective disulfide formation for wild type and further oxidation to an irreversibly inactivated form for the backdoor mutant. We expect the same for Mih1p, and thus the backdoor mutant yeast strain serves as readout for oxidation events occurring at the active site in a cellular environment. In this strain, any oxidation that occurs is irreversible, leading to a block in cell cycle progression and conversion to hyphal growth. As is evident from Figure 1, mildly oxidative conditions are sufficient to induce this nondominant phenotype in the backdoor mutant strain. Additionally, growth in liquid culture with even moderate aeration is sufficient to induce hyphal growth. We therefore presume that Mih1p in the wild type strain subjected to these same oxidation conditions undergoes successive cycles of oxidation, intramolecular disulfide bond formation, and re-reduction by thioredoxin, thereby allowing normal growth even in the presence of oxidative stress. Left unanswered is the more difficult question of whether oxidation of Mih1p plays a direct role in the transient delay of the cell cycle in response to mild oxidative stress or other signals that generate an ROS response (e. g. signaling via growth factors). That is, does the cell cycle stall briefly under oxidative stress due to inactivation of Cdc25, to be quickly retriggered once reducing conditions are restored? Because of the complexity of cell cycle regulation and the www.landesbioscience.com
Figure 1. JMY1289 parent strain and JMY1289 transformed with Mih1p and Mih1pC270S were grown overnight in YPGal liquid media and then streaked onto YPGlu plates to test for their viability on glucose containing media. Following incubation at 30˚C to allow the growth of colonies, the phenotype of the cells was visualized by microscopy (400x). Treatment with menadione and hydrogen peroxide was performed as described in the Materials and Methods and the results shown are representative of these conditions. Milder effects were seen using lower concentrations of menadione and hydrogen peroxide (not shown). Expression of Mih1p was confirmed by Western blotting using the c-myc antibody, as shown for representative cultures.
numerous other intracellular responses to oxidative stress not necessarily dependent on Mih1p, this is a difficult question to address conclusively. Despite this uncertainty, we reiterate that the dramatically different responses of the wild type and backdoor mutant strains differing by only one amino acid following treatment by oxidants emphasizes the importance of the backdoor cysteine for the function of the Cdc25 phosphatases. That is, at a minimum, Cys270 serves an essential protective role to prevent the irreversible oxidation of Mih1p and the backdoor cysteine, which is conserved in all known Cdc25s, can be considered essential to the normal function of this phosphatase. References 1. 2. 3. 4. 5. 6. 7. 8.
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Nilsson I, et al. Prog Cell Cycle Res 2000; 4:107-14. Kristjánsdóttir K, et al. Chem and Biol 2004; 11:1043-51. Bartek J, et al. Cancer Cell 2003; 3:421-9. Savitsky PA, et al. J Biol Chem 2002; 277:20535-40. Sohn J, et al. Biochemistry 2003; 42:10060-70. Keyse SM, et al. Trends Biochem Sci 1993; 18:377-8. Buhrman G, et al. Biochemistry 2005; 44:5307-16. McMillan JN, et al. Molec Cell Biol 1999; 19:6929-39.
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