string

EVOLUTIONARY HOMOLOGS (part 2/2)

Cdc25 and checkpoint controls

Checkpoints maintain the order of cell-cycle events. At G2/M, a checkpoint blocks mitosis in response to damaged or unreplicated DNA. There are significant differences in the checkpoint responses to damaged DNA and unreplicated DNA, although many of the same genes are involved in both responses. To identify new genes that function specifically in the DNA replication checkpoint pathway, a search was carried out for high-copy suppressors of overproducer of Cdc25p [OPcdc25(+)], which lacks a DNA replication checkpoint. Two classes of suppressors were isolated. One class includes a new gene encoding a putative DEAD box helicase, suppressor of uncontrolled mitosis [sum3(+)]. This gene negatively regulates the cell-cycle response to stress when overexpressed and restores the checkpoint response by a mechanism that is independent of Cdc2p tyrosine phosphorylation. The second class includes chk1(+) (Drosophila homolog: Grapes) and the two Schizosaccharomyces pombe 14-3-3 genes, rad24(+) and rad25(+), which appear to suppress the checkpoint defect by inhibiting Cdc25p. rad24Delta mutants are defective in the checkpoint response to the DNA replication inhibitor hydroxyurea at 37 degrees: cds1Delta rad24Delta mutants, like cds1Delta chk1Delta mutants, are entirely checkpoint deficient at 29 degrees. These results suggest that chk1(+) and rad24(+) may function redundantly with cds1(+) in the checkpoint response to unreplicated DNA (Forbes, 1999).

The protein kinase Chk1 is required for cell cycle arrest in response to DNA damage. The 14-3-3 proteins Rad24 and Rad25 physically interact with Chk1 in fission yeast. Association of Chk1 with 14-3-3 proteins is stimulated in response to DNA damage. DNA damage results in phosphorylation of Chk1 and the 14-3-3 proteins bind preferentially to the phosphorylated form. Genetic analysis has independently implicated both Rad24 and Rad25 in the DNA-damage checkpoint pathway. It is suggested that DNA damage-dependent association of phosphorylated Chk1 with 14-3-3 proteins mediates an important step along the DNA-damage checkpoint pathway, perhaps by directing Chk1 to a particular substrate or to a particular location within the cell. An additional role for 14-3-3 proteins in the DNA-damage checkpoint has been suggested based on the observation that human Chk1 can phosphorylate Cdc25C in vitro creating a 14-3-3 binding site. These results suggest that in fission yeast the interaction between the 14-3-3 proteins and Cdc25 does not require Chk1 function and is unaffected by DNA damage, in sharp contrast to the interaction between the 14-3-3 proteins and Chk1 (Chen, 1999).

The DNA replication checkpoint couples the onset of mitosis with the completion of S phase. It is clear that in the fission yeast S. pombe, operation of this checkpoint requires maintenance of the inhibitory tyrosyl phosphorylation of cdc2. Cdc25 phosphatase induces mitosis by dephosphorylating tyrosine 15 of cdc2, and also accumulates to very high levels in cells arrested in S-phase. This shows that mechanisms which modulate the abundance of cdc25 are unconnected to the DNA replication checkpoint. Cdc25 activity increases approximately 10-fold during transit through M phase. Cdc25 is activated by phosphorylations that are dependent on cdc2 activity in vivo and is suppressed in cells arrested in G1 and S. However, cdc25 is more highly modified and appeared to be somewhat more active in S than in G1. This finding might be connected to the fact that progression from G1 to S increases the likelihood that constitutive cdc25 overproduction will cause inappropriate mitosis (Kovelman, 1996).

In response to low doses of ultraviolet (UV) radiation, cells undergo a G2 delay. The G2 delay results in the accumulation of inactive forms of cyclin B1/cdc2 and both the G2 and mitotic complexes of cyclin A/cdk. This appears to be through a block in the cdc25-dependent activation of these complexes. The expression and localization of cyclin A and cyclin B1/cdk complexes are similar in UV-induced G2 delay and normal early G2 phase cells. Cdc25B and cdc25C also accumulate to normal G2 levels in UV irradiated cells, but the mitotic phosphorylation associated with increased activity of both cdc25B and cdc25C is absent. The cdc25B accumulates in the nucleus of UV irradiated cells and in normal G2 phase cells. Thus the block in cyclin B/cdc2 activation is in part due to the physical separation of cyclin B/cdc2, localized in the cytoplasm, from the cdc25B and cdc25C phosphatases localized in the nucleus. The data positions the UV-induced G2 checkpoint at either the S/G2 transition or early G2 phase, prior to the activation of cyclin A/cdk2 (Gabrielli, 1997b).

Arrest of the cell cycle at the G2 checkpoint, induced by DNA damage, requires inhibitory phosphorylation of the kinase Cdc2 in both fission yeast and human cells. The kinase Wee1 and the phosphatase Cdc25, which regulate Cdc2 phosphorylation, were evaluated as targets of Chk1, a kinase essential for the checkpoint. Fission yeast cdc2-3w Deltacdc25 cells, which express activated Cdc2 and lack Cdc25, are responsive to Wee1 but insensitive to Chk1 and irradiation. Expression of large amounts of Chk1 produces the same phenotype as does the loss of the cdc25 gene in cdc2-3w cells. Cdc25 associates with Chk1 in vivo and is phosphorylated when copurified in Chk1 complexes. These findings identify Cdc25, but not Wee1, as a target of the DNA damage checkpoint (Furnari, 1997).

A single double-stranded DNA (dsDNA) break will cause yeast cells to arrest in G2/M at the DNA damage checkpoint. If the dsDNA break cannot be repaired, cells will eventually override (that is, adapt to) this checkpoint, even though the damage that elicited the arrest is still present. Two adaptation-defective mutants have been identified that remain permanently arrested as large-budded cells when faced with an irreparable dsDNA break in a nonessential chromosome. This adaptation-defective phenotype is entirely relieved by deletion of RAD9, a gene required for the G2/M DNA damage checkpoint arrest. One mutation resides in CDC5, which encodes a polo-like kinase, whereas a second, less penetrant, adaptation-defective mutant is affected at the CKB2 locus, which encodes a nonessential specificity subunit of casein kinase II (see Drosophila CKII) . It is likely that Cdc5p promotes checkpoint adaptation by inhibiting or bypassing the checkpoint pathway. The Cdc5p polo-like kinase has a role in activating Cdc25C, a conserved tyrosine phosphatase that removes an inhibitory phosphate on Cdc2. It may be that CKII acts to inhibit some part of the cell cycle arrest machinery that is not essential for maintaining the checkpoint arrest but is extremely important for maintaining viability during arrest (Toczyski, 1997).

In response to DNA damage, mammalian cells prevent cell cycle progression through the control of critical cell cycle regulators. A human gene has been identified that encodes the protein Chk1, a homolog of the Schizosaccharomyces pombe Chk1 protein kinase, which is required for the DNA damage checkpoint. Human Chk1 protein is modified in response to DNA damage. In vitro Chk1 binds to and phosphorylates the dual-specificity protein phosphatases Cdc25A, Cdc25B, and Cdc25C, which control cell cycle transitions by dephosphorylating cyclin-dependent kinases. Chk1 phosphorylates Cdc25C on serine-216. Serine-216 phosphorylation creates a binding site for 14-3-3 protein and inhibits function of the phosphatase. These results suggest a model whereby in response to DNA damage, Chk1 phosphorylates and inhibits Cdc25C, thus preventing activation of the Cdc2-cyclin B complex and mitotic entry (Sanchez, 1997).

Chk1 kinase, a DNA damage/replication G2 checkpoint kinase, has recently been shown to phosphorylate and inhibit Cdc25C, a Cdc2 Tyr-15 phosphatase, thereby directly linking the G2 checkpoint to negative regulation of Cdc2. Immature Xenopus oocytes are arrested naturally at the first meiotic prophase (prophase I) or the late G2 phase, with sustained Cdc2 Tyr-15 phosphorylation. A Xenopus homolog of Chk1 has been cloned, its developmental expression determined, and its possible role in prophase I arrest of oocytes examined. Xenopus Chk1 protein is expressed at approximately constant levels throughout oocyte maturation and early embryogenesis. Overexpression of wild-type Chk1 in oocytes prevents the release from prophase I arrest by progesterone. Conversely, specific inhibition of endogenous Chk1 either by overexpression of a dominant-negative Chk1 mutant or by injection of a neutralizing anti-Chk1 antibody facilitates prophase I release by progesterone. Moreover, when ectopically expressed in oocytes, a Chk1-nonphosphorylatable Cdc25C mutant alone can induce prophase I release much more efficiently than wild-type Cdc25C; if endogenous Chk1 function is inhibited, however, even wild-type Cdc25C can induce the release very efficiently. These results suggest strongly that Chk1 is involved in physiological prophase I arrest of Xenopus oocytes via the direct phosphorylation and inhibition of Cdc25C. Chk1 might function either as a G2 checkpoint kinase or as an ordinary cell cycle regulator in prophase-I-arrested oocytes (Nakajo, 1999).

Chk1, a nuclear DNA damage/replication G2 checkpoint kinase, phosphorylates Cdc25 and causes its nuclear exclusion in yeast and mammalian cells, thereby arresting the cell at the G2 phase until DNA repair/replication is completed. Chk1 is also involved, at least in part, in the natural G2 arrest of immature Xenopus oocytes, but it is unknown how Chk1 inhibits Cdc25 function and undergoes regulation during oocyte maturation. By using enucleated oocytes, it has been shown that Chk1 inhibits Cdc25 function in the cytoplasm of G2-arrested oocytes and that Cdc25 is activated exclusively in the cytoplasm of maturing oocytes. Moreover, Chk1 activity is not appreciably altered during maturation, being maintained at basal levels; the C-terminal truncation mutants of Chk1 have very high kinase activities, strong abilities to inhibit maturation, and altered subcellular localization in oocytes. These results suggest that the Chk1/Cdc25 pathway is involved cytoplasmically in G2 arrest of Xenopus oocytes, but moderately and independent of the G2 checkpoint, and that the C-terminal region of Chk1 negatively regulates its kinase activity and also determines its subcellular localization. Based on these results, Chk1 (with the basal activity) may function as an ordinary regulator of Cdc25 in oocytes (and in other cell types) and Chk1 might be hyperactivated in response to the G2 checkpoint via its dramatic conformational change (Oe, 2001).

In Xenopus embryos, cell cycle elongation and degradation of Cdc25A (a Cdk2 Tyr15 phosphatase) occur naturally at the midblastula transition (MBT), at which time a physiological DNA replication checkpoint is thought to be activated by the exponentially increased nucleo-cytoplasmic ratio. The checkpoint kinase Chk1, but not Cds1 (Drosophila homolog: loki), is activated transiently at the MBT in a maternal/zygotic gene product-regulated manner and is essential for cell cycle elongation and Cdc25A degradation at this transition. A constitutively active form of Chk1 can phosphorylate Cdc25A in vitro and can target it rapidly for degradation in pre-MBT embryos. Intriguingly, for this degradation, however, Cdc25A also requires a prior Chk1-independent phosphorylation at Ser73. Ectopically expressed human Cdc25A can be degraded in the same way as Xenopus Cdc25A. Finally, Cdc25A degradation at the MBT is a prerequisite for cell viability at later stages. Thus, the physiological replication checkpoint is activated transiently at the MBT by developmental cues, and activated Chk1, only together with an unknown kinase, targets Cdc25A for degradation to ensure later development (Shimuta, 2002).

Thus Chk1, but not Cds1, is essential for cell cycle elongation or the physiological DNA replication checkpoint at the MBT and for cell viability shortly after the MBT in Xenopus. Chk1 is also essential for normal cell proliferation at the MBT in Drosophila and at the blastocyst stage in mice. In these organisms, however, it is not known whether Chk1 undergoes any regulation at the relevant stages of development. The results in Xenopus do show, however, that Chk1 undergoes ATR-mediated phosphorylation (and hence activation) weakly and transiently at the MBT or, more accurately, during the maternal/zygotic transition. This result, which is the first direct demonstration of a physiological Chk1 activation in metazoan development, would imply that replication checkpoint signaling in early embryos is induced, rather than constitutive, and intrinsically weak. Notably, however, very low basal levels of Chk1 modification was consistently observed even in post-MZT embryos, which could imply that constitutive replication checkpoint signaling occurs at low basal levels in normal cells. At present, it is not known whether such basal signaling is essential for later development, although, in Drosophila, ATR or Chk1 has been shown to be non-essential for post-embryonic development (Shimuta, 2002).

DNA damage activates a cell-cycle checkpoint that prevents mitosis while DNA repair is under way. The protein Chk1 enforces this checkpoint by phosphorylating the mitotic inducer Cdc25. Phosphorylation of Cdc25 by Chk1 creates a binding site in Cdc25 for 14-3-3 proteins, but it is not known how 14-3-3 proteins regulate Cdc25. Rad24 is a 14-3-3 protein that is important in the DNA-damage checkpoint in fission yeast. Rad24 controls the intracellular distribution of Cdc25. Elimination of Rad24 causes nuclear accumulation of Cdc25. Activation of the DNA-damage checkpoint causes the net nuclear export of Cdc25 by a process that requires Chk1, Rad24 and nuclear-export machinery. Mutation of a putative nuclear-export signal in Rad24 impairs the nuclear exclusion of Rad24, the damage-induced nuclear export of Cdc25 and the damage checkpoint. Thus, Rad24 appears to function as an attachable nuclear-export signal that enhances the nuclear export of Cdc25 in response to DNA damage (Lopez-Girona, 1999).

Binding of 14-3-3 proteins near the nuclear localization sequence of Xenopus Cdc25 suppresses its ability to induce entry into mitosis. Results reported here are based on studies of intracellular localization of green fluorescent protein (GFP)-tagged wild-type Cdc25 or a mutant (S287A) that cannot bind 14-3-3 proteins. Upon coexpression with Myc-14-3-3epsilon, GFP-Cdc25-WT is predominantly cytoplasmic, whereas GFP-Cdc25-S287A is exclusively nuclear. Leptomycin B, an inhibitor of nuclear export, elicits a prompt redistribution of GFP-Cdc25-WT to the nucleus. Mutagenesis experiments demonstrate that Cdc25 contains multiple nuclear export sequences. These studies indicate that the binding of 14-3-3 proteins and nuclear export regulate the intracellular localization of Cdc25 (Kumagai, 1999).

In human cells, the mitosis-inducing kinase Cdc2 is inhibited by phosphorylation on Thr14 and Tyr15. Disruption of these phosphorylation sites abrogates checkpoint-mediated regulation of Cdc2 and renders cells highly sensitive to agents that damage DNA. Phosphorylation of these sites is controlled by the opposing activities of the Wee1/Myt1 kinases and the Cdc25 phosphatase. The regulation of these enzymes is therefore likely to be crucial for the operation of the G2-M DNA-damage checkpoint. The activity of Cdc25 decreases following exposure to ionizing radiation. The irradiation-induced decrease in Cdc25 activity is suppressed by wortmannin, an inhibitor of phosphatidylinositol 3-kinases, and is dependent on the function of the gene that is mutated in ataxia telangiectasia. Two human kinases have been identified that phosphorylate and inactivate Cdc25 in vitro. One is the previously characterized Chk1 kinase. The second is novel and is homologous to the Cds1/Rad53 family of checkpoint kinases in yeast. Human Cds1 is activated in response to DNA damage. These results suggest that, in human cells, the DNA-damage checkpoint involves direct inactivation of Cdc25 catalyzed by Cds1 and/or Chk1 (Blasina, 1999)

Human Cdc25C is a dual-specificity protein phosphatase that controls entry into mitosis by dephosphorylating the protein kinase Cdc2. Throughout interphase, but not in mitosis, Cdc25C is phosphorylated on serine-216 and binds to members of the highly conserved and ubiquitously expressed family of 14-3-3 proteins (see Drosophila Leonardo). A mutation preventing phosphorylation of serine-216 abrogates 14-3-3 binding. Conditional overexpression of this mutant perturbs mitotic timing and allows cells to escape the G2 checkpoint arrest induced by either unreplicated DNA or radiation-induced damage. Chk1, a fission yeast kinase involved in the DNA damage checkpoint response, phosphorylates Cdc25C in vitro on serine-216. These results indicate that serine-216 phosphorylation and 14-3-3 binding negatively regulate Cdc25C and identify Cdc25C as a potential target of checkpoint control in human cells (Peng, 1997).

Cdc25, the dual-specificity phosphatase that dephosphorylates the Cdc2-cyclin B complex at mitosis, is highly regulated during the cell cycle. In Xenopus egg extracts, Cdc25 is associated with two isoforms of the 14-3-3 protein. Cdc25 is complexed primarily with 14-3-3epsilon and to a lesser extent with 14-3-3zeta. The association of these 14-3-3 proteins with Cdc25 varies dramatically during the cell cycle: binding is high during interphase but virtually absent at mitosis. Interaction with 14-3-3 is mediated by phosphorylation of Xenopus Cdc25 at Ser-287, which resides in a consensus 14-3-3 binding site. Recombinant Cdc25 with a point mutation at this residue (Cdc25-S287A) is incapable of binding to 14-3-3. Addition of the Cdc25-S287A mutant to Xenopus egg extracts accelerates mitosis and overrides checkpoint-mediated arrests of mitotic entry due to the presence of unreplicated and damaged DNA. These findings indicate that 14-3-3 proteins act as negative regulators of Cdc25 in controlling the G2-M transition (Kumagai, 1998).

Checkpoints maintain the order and fidelity of events of the cell cycle by blocking mitosis in response to unreplicated or damaged DNA. In most species this is accomplished by preventing activation of the cell-division kinase Cdc2, which regulates entry into mitosis. The Chk1 kinase, an effector of the DNA-damage checkpoint, phosphorylates Cdc25, an activator of Cdc2. Phosphorylation of Cdc25 promotes its binding to 14-3-3 proteins, preventing it from activating Cdc2. It is proposed that a similar pathway is required for mitotic arrest in the presence of unreplicated DNA (that is, in the replication checkpoint) in fission yeast. It is shown by mutagenesis that Chk1 functions redundantly with the kinase Cds1 at the replication checkpoint and that both kinases phosphorylate Cdc25 on the same sites, which include serine residues at positions 99, 192 and 359. Mutation of these residues reduces binding of 14-3-3 proteins to Cdc25 in vitro and disrupts the replication checkpoint in vivo. It is concluded that both Cds1 and Chk1 regulate the binding of Cdc25 to 14-3-3 proteins as part of the checkpoint response to unreplicated DNA (Zeng, 1998).

Cdc2-cyclin B1 in the G2-arrested Xenopus oocyte is held inactive by phosphorylation of Cdc2 at two negative regulatory sites, Thr14 and Tyr15. Upon treatment with progesterone, these sites are dephosphorylated by the dual specificity phosphatase, Cdc25, leading to Cdc2-cyclin B1 activation. Whereas maintenance of the G2 arrest depends on preventing Cdc25-induced Cdc2 dephosphorylation, the mechanisms responsible for keeping Cdc25 in check in these cells have not yet been described. Cdc25 in the G2-arrested oocyte is bound to 14-3-3 proteins and progesterone treatment abrogates this binding. Cdc25, apparently statically localized in the cytoplasm, is actually capable of shuttling in and out of the oocyte nucleus. Binding of 14-3-3 protein markedly reduces the nuclear import rate of Cdc25, allowing nuclear export mediated by a nuclear export sequence present in the N-terminus of Cdc25 to predominate. If 14-3-3 binding to Cdc25 is prevented while nuclear export is inhibited, the coordinate nuclear accumulation of Cdc25 and Cdc2-cyclin B1 facilitates their mutual activation, thereby promoting oocyte maturation (Yang, 1999).

In response to DNA damage and replication blocks, cells prevent cell cycle progression through the control of critical cell cycle regulators. Chk2 is the mammalian homolog of the Saccharomyces cerevisiae Rad53 and Schizosaccharomyces pombe Cds1 protein kinases required for the DNA damage and replication checkpoints. Chk2 is rapidly phosphorylated and activated in response to replication blocks and DNA damage; the response to DNA damage occurs in an ataxia telangiectasia mutated (ATM)-dependent manner. In vitro, Chk2 phosphorylates Cdc25C on serine-216, a site known to be involved in negative regulation of Cdc25C. This is the same site phosphorylated by the protein kinase Chk1, which suggests that, in response to DNA damage and DNA replicational stress, Chk1 and Chk2 may phosphorylate Cdc25C to prevent entry into mitosis (Matsuoka, 1998).

Checkpoint genes cause cell cycle arrest when DNA is damaged or DNA replication is blocked. Although a human homolog of Chk1 (hChk1) has recently been reported to be involved in the DNA damage checkpoint through phosphorylation of Cdc25A, B, and C, it is not known at which phase(s) of the cell cycle hChk1 functions and how hChk1 causes cell cycle arrest in response to DNA damage. In the present study, it is demonstrated that, in normal human fibroblasts (MJ90), hChk1 is expressed specifically at the S to M phase of the cell cycle at both the RNA and protein levels and that it is localized to the nucleus at this time. hChk1 activity, as determined by phosphorylation of Cdc25C, is readily detected at the S to M phase of the cell cycle, and DNA damage induced by UV or ionizing radiation does not enhance the expression of hChk1 or its activity. Furthermore, hChk1 exists in an active form at the S to M phase in fibroblasts derived from patients with ataxia telangiectasia (AT): these patients lack the functional AT mutated (ATM) gene product, suggesting that hChk1 expression is independent of functional ATM. Taken together with the findings that phosphorylation of Cdc25C on serine 216 is increased at the S to M phase, it is suggested that at this particular phase of the cell cycle, even in the absence of DNA damage, hChk1 phosphorylates Cdc25C on serine 216: this modification is considered to be a prerequisite for the G2/M checkpoint. Thus, hChk1 may play an important role in keeping Cdc25C prepared for responding to DNA damage by phosphorylating its serine residue at 216 during the S to M phase (Kaneko, 1999).

In response to DNA damage and replication blocks, cells activate pathways that arrest the cell cycle and induce the transcription of genes that facilitate repair. In mammals, ATM (ataxia telangiectasia mutated) kinase together with other checkpoint kinases are important components in this response. Rat and human homologs of Saccharomyces cerevisiae Rad 53 and Schizosaccharomyces pombe Cds1, called checkpoint kinase 2 (chk2), have been cloned. Complementation studies suggest that Chk2 can partially replace the function of the defective checkpoint kinase in the Cds1 deficient yeast strain. Chk2 is phosphorylated and activated in response to DNA damage in an ATM dependent manner. However, its activation in response to replication blocks by hydroxyurea (HU) treatment is independent of ATM. Using mass spectrometry, it was found that, similar to Chk1, Chk2 can phosphorylate serine 216 in Cdc25C, a site known to be involved in negative regulation of Cdc25C. These results suggest that Chk2 is a downstream effector of the ATM-dependent DNA damage checkpoint pathway. Activation of Chk2 might not only delay mitotic entry, but also increase the capacity of cultured cells to survive after treatment with gamma-radiation or with the topoisomerase-I inhibitor topotecan (Chaturvedi, 2000).

When exposed to ionizing radiation (IR), eukaryotic cells activate checkpoint pathways to delay the progression of the cell cycle. Defects in the IR-induced S-phase checkpoint cause 'radioresistant DNA synthesis' (RDS), a phenomenon that has been identified in cancer-prone patients suffering from ataxia-telangiectasia, a disease caused by mutations in the ATM gene. The Cdc25A phosphatase activates the cyclin-dependent kinase 2 (Cdk2) needed for DNA synthesis, but becomes degraded in response to DNA damage or stalled replication. A functional link that exists between ATM, the checkpoint signaling kinase Chk2/Cds1 (Chk2) and Cdc25A, and this mechanism is implicated in controlling the S-phase checkpoint. IR-induced destruction of Cdc25A requires both ATM and the Chk2-mediated phosphorylation of Cdc25A on serine 123. An IR-induced loss of Cdc25A protein prevents dephosphorylation of Cdk2 and leads to a transient blockade of DNA replication. Tumor-associated Chk2 alleles cannot bind or phosphorylate Cdc25A, and cells expressing these Chk2 alleles, elevated Cdc25A or a Cdk2 mutant unable to undergo inhibitory phosphorylation (Cdk2AF) fail to inhibit DNA synthesis when irradiated. These results support Chk2 as a candidate tumor suppressor, and identify the ATM-Chk2-Cdc25A-Cdk2 pathway as a genomic integrity checkpoint that prevents radioresistant DNA synthesis (Falck, 2001).

Because RDS occurs in cells with a defective ATM gene, as well as through deregulation of Chk2 or Cdc25A, and because ATM activates Chk2 directly, the response of the Chk2-Cdc25A-Cdk2 pathway in lymphoblasts derived from ataxia-telangiectasia patients was compared to those isolated from normal individuals. Unlike normal lymphoblasts, irradiated ataxia-telangiectasia cells are unable to activate Chk2 and downregulate Cdc25A protein and activity. Consequently, exposure of ataxia-telangiectasia lymphoblasts to IR causes neither an increase in Cdk2 Tyr 15 phosphorylation nor an inhibition of cyclin E/Cdk2 kinase activity, which is consistent with the well-documented RDS phenotype in these cells (Falck, 2001).

The human Cdc25A phosphatase plays a pivotal role at the G1/S transition by activating cyclinE and A/Cdk2 complexes through dephosphorylation. In response to ionizing radiation (IR), Cdc25A is phosphorylated by both Chk1 and Chk2 on Ser-123. This in turn leads to ubiquitylation and rapid degradation of Cdc25A by the proteasome resulting in cell cycle arrest. In response to UV-irradiation, Cdc25A is phosphorylated at a different serine residue, Ser-75. Significantly, Cdc25A mutants carrying alanine instead of either Ser-75 or Ser-123 demonstrate that only Ser-75 mediates protein stabilization in response to UV-induced DNA damage. As a consequence, cyclinE/Cdk2 kinase activity is high. Furthermore, Cdc25A is phosphorylated by Chk1 on Ser-75 in vitro and the same site is also phosphorylated in vivo. Taken together, these data strongly suggest that phosphorylation of Cdc25A on Ser-75 by Chk1 and its subsequent degradation is required to delay cell cycle progression in response to UV-induced DNA lesions (Hassepass, 2003).

DNA-responsive checkpoints prevent cell-cycle progression following DNA damage or replication inhibition. The mitotic activator Cdc25 is suppressed by checkpoints through inhibitory phosphorylation at Ser287 (Xenopus numbering) and docking of 14-3-3. Ser287 phosphorylation is a major locus of G2/M checkpoint control, although several checkpoint-independent kinases can phosphorylate this site. Mitotic entry requires 14-3-3 removal and Ser287 dephosphorylation. DNA-responsive checkpoints also activate PP2A/B56Δ phosphatase complexes to dephosphorylate Cdc25 at a site distinct from Ser287 (T138), the phosphorylation of which is required for 14-3-3 release. However, phosphorylation of T138 is not sufficient for 14-3-3 release from Cdc25. These data suggest that creation of a 14-3-3 'sink,' consisting of phosphorylated 14-3-3 binding intermediate filament proteins, including keratins, coupled with reduced Cdc25-14-3-3 affinity, contribute to Cdc25 activation. These observations identify PP2A/B56Δ as a central checkpoint effector and suggest a mechanism for controlling 14-3-3 interactions to promote mitosis (Margolis, 2006).

back to String: Evolutionary Homologs part 1/2

Home page: The Interactive Fly © 1995, 1996 Thomas B. Brody, Ph.D.

The Interactive Fly resides on the
Society for Developmental Biology's Web server.