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Skeletal muscle stem cells (MuSCs), the major source for skeletal muscle regeneration in vertebrates, are in a state of cell cycle arrest in adult skeletal muscles. Prior evidence suggests that embryonic muscle progenitors proliferate and differentiate to form myofibres and also self-renew, implying that MuSCs, derived from these cells, acquire quiescence later during development. Depletion of Dicer in adult MuSCs promoted their exit from quiescence, suggesting microRNAs are involved in the maintenance of quiescence. Here we identified miR-195 and miR-497 that induce cell cycle arrest by targeting cell cycle genes, Cdc25 and Ccnd . Reduced expression of MyoD in juvenile MuSCs, as a result of overexpressed miR-195/497 or attenuated Cdc25/Ccnd, revealed an intimate link between quiescence and suppression of myogenesis in MuSCs. Transplantation of cultured MuSCs treated with miR-195/497 contributed more efficiently to regenerating muscles of dystrophin-deficient mice, indicating the potential utility of miR-195/497 for stem cell therapies. Skeletal muscle stem cells are in a state of cell cycle arrest in adult skeletal muscles and are stimulated to proliferate and differentiate in response to injury or pathology. Here the authors identify two microRNAs, miR-195 and miR-497, which induce cell cycle arrest in the stem cells and suppress myogenesis.

Precise monitoring of DNA replication and chromosome segregation ensures that there is accurate transmission of genetic information from a cell to its daughters. Eukaryotic cells have developed a complex network of checkpoint pathways that sense DNA lesions and defects in chromosome segregation, spindle assembly and the centrosome cycle, leading to an inhibition of cell‐cycle progression for the time required to remove the defect and thus preventing genomic instability. The activation of checkpoints that are responsive to DNA damage or incomplete DNA replication ultimately results in the inhibition of cyclin‐dependent kinases. This review focuses on our understanding of the biochemical mechanisms that specifically inactivate Cdc25 (cell division cycle 25) phosphatases to achieve this. The evidence for links between checkpoint deregulation and oncogenesis is discussed.

Rhodanese domains are ubiquitous structural modules occurring in the three major evolutionary phyla. They are found as tandem repeats, with the C‐terminal domain hosting the properly structured active‐site Cys residue, as single domain proteins or in combination with distinct protein domains. An increasing number of reports indicate that rhodanese modules are versatile sulfur carriers that have adapted their function to fulfill the need for reactive sulfane sulfur in distinct metabolic and regulatory pathways. Recent investigations have shown that rhodanese domains are also structurally related to the catalytic subunit of Cdc25 phosphatase enzymes and that the two enzyme families are likely to share a common evolutionary origin. In this review, the rhodanese/Cdc25 phosphatase superfamily is analyzed. Although the identification of their biological substrates has thus far proven elusive, the emerging picture points to a role for the amino‐acid composition of the active‐site loop in substrate recognition/specificity. Furthermore, the frequently observed association of catalytically inactive rhodanese modules with other protein domains suggests a distinct regulatory role for these inactive domains, possibly in connection with signaling.

Mammalian target of rapamycin (mTOR) is a serine/threonine kinase that regulates a variety of cellular functions such as growth, proliferation and autophagy. In a variety of cancer cells, overactivation of mTOR has been reported. In addition, mTOR inhibitors, such as rapamycin and its derivatives, are being evaluated in clinical trials as anticancer drugs. However, no active mutants of mTOR have been identified in human cancer. Here, we report that two different point mutations, S2215Y and R2505P, identified in human cancer genome database confer constitutive activation of mTOR signaling even under nutrient starvation conditions. S2215Y was identified in large intestine adenocarcinoma whereas R2505P was identified in renal cell carcinoma. mTOR complex 1 prepared from cells expressing the mutant mTOR after nutrient starvation still retains the activity to phosphorylate 4E-BP1 in vitro . The cells expressing the mTOR mutant show increased percentage of S-phase cells and exhibit resistance to cell size decrease by amino-acid starvation. The activated mutants are still sensitive to rapamycin. However, they show increased resistance to 1-butanol. Our study points to the idea that mTOR activating mutations can be identified in a wide range of human cancer.

Cdc25C (cell division cycle 25C) phosphatase triggers entry into mitosis in the cell cycle by dephosphorylating cyclin B-Cdk1. Cdc25C exhibits basal phosphatase activity during interphase and then becomes activated at the G 2 /M transition after hyperphosphorylation on multiple sites and dissociation from 14-3-3. Although the role of Cdc25C in mitosis has been extensively studied, its function in interphase remains elusive. Here, we show that during interphase Cdc25C suppresses apoptosis signal-regulating kinase 1 (ASK1), a member of mitogen-activated protein (MAP) kinase kinase kinase family that mediates apoptosis. Cdc25C phosphatase dephosphorylates phospho-Thr-838 in the activation loop of ASK1 in vitro and in interphase cells. In addition, knockdown of Cdc25C increases the activity of ASK1 and ASK1 downstream targets in interphase cells, and overexpression of Cdc25C inhibits ASK1-mediated apoptosis, suggesting that Cdc25C binds to and negatively regulates ASK1. Furthermore, we showed that ASK1 kinase activity correlated with Cdc25C activation during mitotic arrest and enhanced ASK1 activity in the presence of activated Cdc25C resulted from the weak association between ASK1 and Cdc25C. In cells synchronized in mitosis following nocodazole treatment, phosphorylation of Thr-838 in the activation loop of ASK1 increased. Compared with hypophosphorylated Cdc25C, which exhibited basal phosphatase activity in interphase, hyperphosphorylated Cdc25C exhibited enhanced phosphatase activity during mitotic arrest, but had significantly reduced affinity to ASK1, suggesting that enhanced ASK1 activity in mitosis was due to reduced binding of hyperphosphorylated Cdc25C to ASK1. These findings suggest that Cdc25C negatively regulates proapoptotic ASK1 in a cell cycle-dependent manner and may play a role in G 2 /M checkpoint-mediated apoptosis.

The 14-3-3 proteins are a family of conserved regulatory molecules expressed in all eukaryotic cells. A striking feature of the 14-3-3 proteins is their ability to bind a multitude of functionally diverse signaling proteins, including kinases, phosphatases, and transmembrane receptors. This plethora of interacting proteins allows 14-3-3 to play important roles in a wide range of vital regulatory processes, such as mitogenic signal transduction, apoptotic cell death, and cell cycle control. In this review, we examine the structural basis for 14-3-3-ligand interactions, proposed functions of 14-3-3 in various signaling pathways, and emerging views of mechanisms that regulate 14-3-3 actions.

When exposed to ionizing radiation (IR), eukaryotic cells activate checkpoint pathways to delay the progression of the cell cycle 1 , 2 , 3 . Defects in the IR-induced S-phase checkpoint cause ‘radioresistant DNA synthesis’, a phenomenon that has been identified in cancer-prone patients suffering from ataxia-telangiectasia, a disease caused by mutations in the ATM gene 4 , 5 , 6 . The Cdc25A phosphatase 7 activates the cyclin-dependent kinase 2 (Cdk2) needed for DNA synthesis 8 , 9 , but becomes degraded in response to DNA damage 10 or stalled replication 11 . Here we report a functional link between ATM, the checkpoint signalling kinase Chk2/Cds1 (Chk2) 12 and Cdc25A, and implicate this mechanism in controlling the S-phase checkpoint. We show that 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. We also show that tumour-associated Chk2 alleles 13 cannot bind or phosphorylate Cdc25A, and that 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 tumour suppressor, and identify the ATM–Chk2–Cdc25A–Cdk2 pathway as a genomic integrity checkpoint that prevents radioresistant DNA synthesis.

Entry into mitosis occurs after activation of Cdk1, resulting in chromosome condensation in the nucleus and centrosome separation, as well as increased microtubule nucleation activity in the cytoplasm. The active cyclin-B1–Cdk1 complex first appears at the centrosome, suggesting that the centrosome may facilitate the activation of mitotic regulators required for the commitment of cells to mitosis. However, the signalling pathways involved in controlling the initial activation of Cdk1 at the centrosome remain largely unknown. Here, we show that human Chk1 kinase localizes to interphase, but not mitotic, centrosomes. Chemical inhibition of Chk1 resulted in premature centrosome separation and activation of centrosome-associated Cdk1. Forced immobilization of kinase-inactive Chk1 to centrosomes also resulted in premature Cdk1 activation. Conversely, under such conditions wild-type Chk1 impaired activation of centrosome-associated Cdk1, thereby resulting in DNA endoreplication and centrosome amplification. Activation of centrosomal Cdk1 in late prophase seemed to be mediated by cytoplasmic Cdc25B, whose activity is controlled by centrosome-associated Chk1. These results suggest that centrosome-associated Chk1 shields centrosomal Cdk1 from unscheduled activation by cytoplasmic Cdc25B, thereby contributing to proper timing of the initial steps of cell division, including mitotic spindle formation.

In a wide variety of animal species, oocyte maturation is arrested temporarily at prophase of meiosis I (ref. 1 ). Resumption of meiosis requires activation of cyclin-dependent kinase-1 (CDK1, p34cdc2), one component of maturation-promoting factor (MPF) 2 , 3 . The dual specificity phosphatases Cdc25a, Cdc25b and Cdc25c are activators of cyclin-dependent kinases 4 , 5 , 6 , 7 , 8 ; consequently, they are postulated to regulate cell-cycle progression in meiosis and mitosis as well as the DNA-damage response 9 , 10 , 11 , 12 . We generated Cdc25b -deficient ( Cdc25b −/− ) mice and found that they are viable. As compared with wildtype cells, fibroblasts from Cdc25b −/− mice grew vigorously in culture and arrested normally in response to DNA damage. Female Cdc25b −/− mice were sterile, and Cdc25b −/− oocytes remained arrested at prophase with low MPF activity. Microinjection of wildtype Cdc25b mRNA into Cdc25b −/− oocytes caused activation of MPF and resumption of meiosis. Thus, Cdc25b −/− female mice are sterile because of permanent meiotic arrest resulting from the inability to activate MPF. Cdc25b is therefore essential for meiotic resumption in female mice. Mice lacking Cdc25b provide the first genetic model for studying the mechanisms regulating prophase arrest in vertebrates.

The CDC25 protein phosphatases drive cell cycle advancement by activating cyclin-dependent protein kinases (CDKs). Humans and mice encode three family members denoted CDC25A, -B and -C and genes encoding these family members can be disrupted individually with minimal phenotypic consequences in adult mice. However, adult mice globally deleted for all three phosphatases die within one week after Cdc25 disruption. A severe loss of absorptive villi due to a failure of crypt epithelial cells to proliferate was observed in the small intestines of these mice. Because the Cdc25s were globally deleted, the small intestinal phenotype and loss of animal viability could not be solely attributed to an intrinsic defect in the inability of small intestinal stem and progenitor cells to divide. Here, we report the consequences of deleting different combinations of Cdc25s specifically in intestinal epithelial cells. The phenotypes arising in these mice were then compared with those arising in mice globally deleted for the Cdc25s and in mice treated with irinotecan, a chemotherapeutic agent commonly used to treat colorectal cancer. We report that the phenotypes arising in mice globally deleted for the Cdc25s are due to the failure of small intestinal stem and progenitor cells to proliferate and that blocking cell division by inhibiting the cell cycle engine (through Cdc25 loss) versus by inducing DNA damage (via irinotecan) provokes a markedly different response of small intestinal epithelial cells. Finally, we demonstrate that CDC25A and CDC25B but not CDC25C compensate for each other to maintain the proliferative capacity of intestinal epithelial stem and progenitor cells.