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Gene name - roughex > Synonyms - Cytological map position - 5D1--5D3 Function - regulator of cyclins Keywords - eye, testis, cell cycle |
Symbol - rux FlyBase ID: FBgn0003302 Genetic map position - 1-15.0.
Cellular location - cytoplasmic and nuclear
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The Drosophila compound eye is well suited to the study of cell cycle regulation. The eye develops from a columnar epithelium called the eye imaginal disc. During the third and final larval stage in Drosophila development, a wave of differentiation sweeps across the eye disc, from posterior to anterior. The front of this wave is marked by a depression in the disc epithelium, the morphogenetic furrow (MF). Ahead of the MF, cells are unpatterned and undifferentiated; they progress through the cell cycle asynchronously. All cells become synchronized in G1 beginning just anterior to the MF, such that cells at the anterior edge of the MF are in early G1. Cells in G2 ahead of the furrow are driven through mitosis by a burst of string expression in a band of cells just anterior to the MF under the control of the patterning gene hedgehog. String drives G2 cells through mitosis and into G1 (Heberlein, 1995).
Cells proceeding throgh G1 are found more posterior (relative to the MF). Cells emerging from the posterior edge of the MF either become postmitotic, without entering S phase, and differentiate into neurons, or they up-regulate Cyclin E before entering a final synchronous S phase. As cells transit S and G2, Cyclin A and CycB proteins accumulate. G1 cells are prevented from entering S phase by the product of the roughex locus. In roughex null mutants, cells enter S phase precociously in early G1. By preventing early accumulation of Cyclin A, Roughex delays premature entry into S phase, one part of the cell cycle driven by Cyclin A. In addition, Cyclin E promotes down-regulation of Roughex, allowing accumulation of Cyclin A for its function in S and G2 (Thomas, 1997 and references).
The R8 neuron is likely to be the first cell fate to be established within the MF, and several lines of evidence suggest that cellular interactions play a role in restricting to one per cluster the number of R8 cells that form within the MF. Atonal acts as the proneural gene for photoreceptor neurons. Notch is required for restriction of atonal expression to a single R8 precursor. Rough functions to downregulate atonal in cells not selected for R8 fate. Boss is subsequently expressed in R8 cells and is required for the R7 photoreceptor fate (Dokucu, 1996 and KrĀmer, 1991).
Mutation of roughex perturbs cell fate determination. Many rux mutant clusters contain multiple boss-expressing cells. In some of these clusters, R8 cells are missing. There is also a reduction in the number of cells expressing bar and Seven-up. This may be due to errors in cell fate determination. Alternatively, the reduced number of cells expressing these markers may reflect cell death. Extensive cell death is seen in rux mutants beginning with the MF and extending to the posterior edge of the disc. In rux mutant discs, neuronal differentiation is delayed by approximately 6 hours of development (Thomas, 1994).
It is found thations in ras1 and Star enhance rux phenotypes. In rux mutations, the length of G1 may be reduced, making cellular interactions more sensitive to a reduction in the level of intercellular signaling molecules. A more intriguing notion is that the establishment of G1 itself may be promoted by intercellular signals. For example, Ras1 and Star may act in a signaling pathway to activate Rux function, or Rux may be part of a signaling cascade that negatively regulates cell cycle progression in the MF. MF movement may actually be driven by a signaling cascade that, among other processes, regulates cell cycle synchronization. hedgehog is thought to function as a secreted diffusible signal to induce progression of the MF (Thomas, 1994 and references).
The key to understandinge fuction of roughex comes from an analysis of the interactions of rux with cell cycle proteins. Rux acts as a negative regulator of Cyclin A. In screens for dominant suppressors of the roughex rough-eye phenotype, three regulators of cyclin dependent kinase (Cdk) activity were identified: cycA, string and Regulator of cyclin A1 (Rca1) (Thomas, 1994 and Dong, 1997). Because Stg activates CycA-Cdk complexes in vitro and Rca1 encodes an upstream positive regulator of cycA (Dong, 1997), a test was made as to whether rux suppresses entry in S phase by preventing ectopic activation (directly or indirectly) of a CycA-Cdk complex in the G1 domain within the MF. Consistent with this interpretation, overexpression of cycA mimics the rux mutant phenotype, showing extensive induction of S-phase cells just anterior to and within the MF. Coexpression of rux results in suppression of the ectopic S phases induced by cycA in all discs assayed. Therefore, ectopic CycA expression can drive G1 cells into S phase, and coexpression of Rux inhibits this phenotype. It is concluded that rux acts as a negative regulator of CycA. This is the first demonstration of a role for CycA in regulation of G1 or S phase in Drosophila (Thomas, 1997).
Striking defects in the level and subcellular distribution of CycA are observed in cells ectopically expressing high levels of Rux. In the eye, dividing cells lose their connection to the basal lamina and mitotic nuclei are found at the extreme apical surface of the disc epithelium. In wild type cells, CycA accumulates in the cytoplasm of cells with basally located nuclei. As nuclei rise apically on entry into mitosis, CycA localizes transiently to the nucleus and then disappears. In CycA overexpressors, no cytoplasmic CycA staining is detected in apical focal planes. Instead, CycA accumulates transiently in basally located nuclei of cells behind the MF. These cells do not show features of cells that are entering mitosis. It is thought that Rux controls CycA levels by promoting its nuclear localization and thereby its rapid degradation. Since Rux does not physically associate with CycA, exactly how Rux regulates CycA levels and subcellular distribution remains unknown (Thomas, 1997).
Rux can be shown to physically interact with CycE, but does not inhibit the kinase activity of Drosophila CycE-Cdk complexes. Also, Rux overexpression fails to inhibit CycE-induced S phase. Therefore, rux does not appear to inhibit CycE-dependent processes in vivo. Conversely, CycE inhibits Rux accumulation. Rux rapidly disappears from cycling cells at the posterior edge of the MF where the level of CycE increases. It can be shown that overexpression of CycE, but not of CycA, inhibits Rux protein accumulation. These results suggest that CycE targets Rux for destruction in cells that re-enter S phase behind the MF. Therefore, during normal eye development, accumulation of CycE in late G1 cells may down-regulate Rux protein. In turn, this facilitates the accumulation of CycA, which is required in subsequent cell cycle stages (Thomas, 1997).
The control of CycA activity in cells in the MF shows striking similarity to the control of G2 cyclins during G1 in yeast. Degradation of the G2 cyclin, CLB2, in S. cerevisiae continues in early G1 prior to Start (the initiation of DNA synthesis). In addition, overexpression of CLB2 protein in G1 drives cells into S phase, and the CLB degradation pathway is inhibited by G1 cyclins in late G1 (Amon, 1994).
It is concluded that cell cycle arrest in G1 at the onset of patterning in the Drosophila eye is mediated by roughex. Rux inhibits entry into S phase by preventing accumulation of CycA protein during G1, and CycE promotes down-regulation of Rux, allowing accumulation of CycA for its function in S and G2 (Thomas, 1997). The next several years should see renewed efforts to unravel the role of CycA in the G1-S transition in Drosophila. These future analyses should reveal the pathway by which Roughex regulates CycA.
Differentiation in the developing Drosophila eye requires synchronization of cells in the G1 phase of the cell cycle. The roughex gene product plays a key role in this synchronization by negatively regulating cyclin A protein levels in G1. Coexpressed Roughex and cyclin A physically interact in vivo. Roughex is a nuclear protein, while cyclin A has previous been shown to be exclusively cytoplasmic during interphase in the embryo. In contrast, in interphase cells in the eye imaginal disc, cyclin A has been shown to be present in both the nucleus and the cytoplasm. In the presence of ectopic Roughex, cyclin A becomes strictly nuclear and is later degraded. Nuclear targeting of both Roughex and cyclin A under these conditions is dependent on a C-terminal nuclear localization signal in Roughex. Disruption of this signal results in cytoplasmic localization of both Roughex and cyclin A, confirming a physical interaction between these molecules. Cyclin A interacts with both Cdc2 and Cdc2c, the Drosophila Cdk2 homolog, and Roughex inhibits the histone H1 kinase activities of both cyclin A-Cdc2 and cyclin A-Cdc2c complexes in whole-cell extracts. Two-hybrid experiments have suggested that the inhibition of kinase activity by Roughex results from competition with the cyclin-dependent kinase subunit for binding to cyclin A. These findings suggest that Roughex can influence the intracellular distribution of cyclin A and define Roughex as a distinct and specialized cell cycle inhibitor for cyclin A-dependent kinase activity (Avedisov, 2000).
Although genetic and immunohistochemical experiments indicate that Rux prevents CycA accumulation in early G1 in the developing Drosophila eye, an understanding of the mechanism by which Rux functions to reduce CycA protein levels has been unclear. Using two in vivo techniques, two-hybrid analysis and coimmunoprecipitation, it has been shown that Rux and CycA interact in both Drosophila and mammalian cells. Although the possibility that other as yet unidentified proteins mediate the interaction between Rux and CycA cannot be ruled out, analysis of Rux point mutations as well as in vitro experiments suggest that the interaction is direct. Binding of Rux to CycA both in vitro and in vivo is eliminated by a mutation in a motif, RXL, which has been shown in mammalian cells to mediate binding of a variety of proteins to CycA, including p107, p130, and the CKIs p21 and p27. In Rux, a single amino acid substitution in this motif is sufficient to eliminate CycA interaction in both the two-hybrid assay and Drosophila cultured cells. These data provide strong evidence that Leu-31 is part of a CycA-binding site that contains the same minimal consensus sequence seen in mammalian cell cycle inhibitors (Avedisov, 2000).
Although in vitro experiments indicate that Leu-31 is necessary for CycA binding, the phenotype resulting from overexpression of the Rux[L31A] mutant in the eye is unexpectedly complicated. In the presence of the mutant protein, CycA still localizes to the nucleus, both in the eye disc and in SL2 cells. It is possible that, although Leu-31 is critical for binding to CycA in cultured cells and in vitro, residual binding occurs via one or both of the remaining two RXL sites in the protein. However, Rux mutant proteins in which all three RXL sites are eliminated still display nuclear localization of CycA in SL2 cells. This result suggests that Rux is not directly involved in CycA nuclear import. CycA protein is stabilized in Rux[L31A] relative to expression of wild-type Rux, indicating that binding to Rux via Leu-31 may be required for degradation of CycA. Finally, mitosis does not occur in eye discs expressing Rux[L31A], a phenotype also seen in nondegradable CycA mutant proteins lacking a destruction box. However, in contrast to cells expressing nondegradable CycA mutant proteins, which arrest in metaphase, cells expressing Rux[L31A] arrest prior to chromosome condensation (Avedisov, 2000).
The simplest explanation of these data, taken together, is that the Rux[L31A] mutant protein displays residual binding to CycA in vivo. Because the Rux[L31A]
mutant protein is stable in cells that reenter the cell cycle behind the MF whereas wild-type Rux is degraded, the Rux[L31A] mutant protein is expressed to much
higher levels in these S-phase cells than is the wild-type protein. In addition, mutation of a second RXL motif in Rux (at position 248) showed a reduction in
CycA binding in the mammalian two-hybrid system, suggesting that this second RXL site also participates in binding. It is possible that this weak residual binding
coupled with the stabilization of the mutant protein in S/G2 cells leads to disruption of mitotic CycA-Cdk complexes and the observed G2 arrest. Indeed,
fly transformant lines in which Rux[L31A] is expressed at lower levels than in the line analyzed in this study display a completely wild-type phenotype,
indicating that extremely high levels of expression of the mutant protein are required to detect these mitotic effects (Avedisov, 2000).
P>The Rux-CycA interaction occurs via a motif similar to that of characterized CKIs. However, unlike other CKIs, which typically bind both cyclin and CDK subunits,
Rux does not interact with either Drosophila CDK in the two-hybrid assay. In addition, coimmunoprecipitation of CDKs with Rux and CycA
from SL2 cells expressing all three proteins is not observed. Instead, two-hybrid data indicate that Rux competes with CDKs for binding to CycA. Rux may do
this by reducing the stability of CycA-CDK complexes or, alternatively, by preventing CDKs from binding to CycA. This conclusion is conditioned by the finding that
low levels of added Rux cause a modest stimulation of CycA-CDK interaction, suggesting that the associations between these proteins may be more complex than
has been suggested by a simple competition model (Avedisov, 2000).
<>In addition to the expected interaction between CycA and the G2 CDK Cdc2, an interaction between CycA and the G1 Cdk2 homolog Cdc2c was detected.
Previous experiments using stage 11 Drosophila embryos have detected coimmunoprecipitation of only Cdc2 with CycA. Stage 11 corresponds roughly to
embryonic cell cycle 16, which consists of a regulated G2 phase with no apparent G1. It is possible that CycA-Cdc2c complexes are normally present in S
phase at such low levels that they cannot be detected at this stage of embryonic development. Human CycA associates with Cdk1 in G2 and with Cdk2 in S phase. These data suggest that the same may happen during larval cell divisions in Drosophila melanogaster. If such an interaction occurs, the activity of this complex
may also be a target for regulation by Rux (Avedisov, 2000).
Rx contains four consensus phosphorylation sites for CDKs, and Rux itself is a good substrate for phosphorylation by both CycE-Cdk2 and CycA-Cdc2 activities
immunoprecipitated from Drosophila embryos and SL2 cells. Phosphorylation of these sites is not required for binding to CycA. The effect of ectopic Rux expression on CycA localization and stability in eye imaginal tissue can be overcome by overexpression of
CycE, suggesting that Rux itself may be a target for CycE-dependent kinase activity. In both yeast and mammalian cells, phosphorylation of CKIs in G1 is
absolutely required for their destruction by ubiquitin-mediated proteolysis. The sequence defined in this paper as a CycA-binding site overlaps a region
predicted to be important for ubiquitin-mediated degradation, suggesting that CycA may compete with the ubiquitination apparatus for binding to Rux. Indeed, the
Rux[L31A] mutant protein, in which this motif is disrupted, shows increased stability in cells that reenter S phase behind the MF. It remains to be seen, however,
whether Rux is phosphorylated and/or ubiquitinated in vivo. Experiments to address the role of CycE in inhibiting Rux function are in progress (Avedisov, 2000).
The cyclin-dependent kinase 1 (Cdk1)-cyclin B (CycB) complex plays critical roles in cell-cycle regulation. Before Drosophila male meiosis, CycB is exported from the nucleus to the cytoplasm via the nuclear porin 62kD (Nup62) subcomplex of the nuclear pore complex. When this export is inhibited, Cdk1 is not activated, and meiosis does not initiate. This study investigated the mechanism that controls the cellular localization and activation of Cdk1. Cdk1-CycB continuously shuttled into and out of the nucleus before meiosis. Overexpression of CycB, but not that of CycB with nuclear localization signal sequences, rescued reduced cytoplasmic CycB and inhibition of meiosis in Nup62-silenced cells. Full-scale Cdk1 activation occurred in the nucleus shortly after its rapid nuclear entry. Cdk1-dependent centrosome separation did not occur in Nup62-silenced cells, whereas Cdk1 interacted with Cdk-activating kinase and Twine/Cdc25C in the nuclei of Nup62-silenced cells, suggesting the involvement of another suppression mechanism. Silencing of roughex rescued Cdk1 inhibition and initiated meiosis. Nuclear export of Cdk1 ensured its escape from inhibition by a cyclin-dependent kinase inhibitor. The complex re-entered the nucleus via importin β at the onset of meiosis. A model is proposed regarding the dynamics and activation mechanism of Cdk1-CycB to initiate male meiosis (Yamazoe, 2023).
A conserved molecular mechanism that controls the initiation of cell division in eukaryotes involves the activation of cyclin-dependent kinase 1 (Cdk1), which serves as a master regulator of the M phase of mitosis and meiosis. In eukaryotes, the following three conditions are indispensable for activating this protein kinase: complex formation with its regulatory subunit, cyclin B (CycB); phosphorylation of Thr161 of Cdk1; and removal of phosphate groups from Thr14 and Tyr15, both of which are involved in the negative regulation of the kinase phosphorylated by Wee1/Myt1. Cdk1 is activated at the onset of the M phase via dephosphorylation of Thr14 and Tyr15 by cell division cycle 25 (Cdc25) orthologues. Thr161 of Cdk1 also needs to be phosphorylated by Cdk- activating kinase (CAK). In addition to Cdk1 modification, another type of inhibitors known as Cdk inhibitors (CKIs), such as p21, play an important role in controlling the cell cycle. CKIs were originally identified as negative factors that bind to suppress Cdk activity at the G1/S phase and also affect CycB-Cdk1 during the G2/M transition . These inhibitors need to be released from Cdk1 before the onset of the M phase. From later stages of the G2 phase towards the beginning of the M phase, Cdk1 activity depends on an increase in the expression of CycB. In vitro assays using animal oocyte extracts have revealed that Cdks are activated progressively. A small population of the CycB-Cdk1 complex is first activated by a trigger Consequently, the balance between Cdc25 and Wee1/Myt1 activities is shifted so that Cdc25 activity becomes predominant (Yamazoe, 2023).
CycB-Cdk1 further accelerates dephosphorylation of the kinase via positive feedback loops, leading to maximal activation. In contrast, a double-negative feedback loop implemented by the inactivation of a counteracting phosphatase by Cdk1 can also contribute to Cdk1's own activation. In addition, the subcellular localization of Cdk1 and its regulatory factors and the timing of their migration to other compartments are considered critical points for mitotic entry in mammalian cells. In the G2 phase, CYCB1 is enriched in the cytoplasm but continuously shuttles into and out of the nucleus until shortly before the onset of mitosis. Mitosis is triggered by the activation of Cdk1-CycB and its translocation from the cytoplasm to the nucleus. The spatial and feedback regulation ensures a rapid and irreversible transition from interphase to mitosis (Yamazoe, 2023).
Much progress has already been made in elucidating special regulatory activities that control Cdk1 activation during the G2/M transition in mitosis. However, several issues remain to be uncovered regarding the mechanism of meiotic initiation. Meiosis is expected to be highly susceptible to spatial and temporal control of the cell cycle in cooperation with the developmental program. For example, in mouse oocytes, spatial regulation of anaphase-promoting complex (APC)/CCdh1-induced CycB degradation maintains G2 arrest of oocytes for several years. The stepwise activation of Cdk1 may, rather, play a more important role in meiosis than in the mitotic cell cycle. In Drosophila, the developmental program and cell-cycle progression in meiosis have been better studied. As the meiotic cycle in Drosophila generally constitutes a prolonged G2-like growth period, the timing of meiosis initiation is expected to be strictly regulated. However, the mechanism by which Cdk1-dependent phosphorylation is timed to occur shortly before the nuclear envelope breaks down remains to be explored. With reference to the regulatory mechanisms of mitotic initiation, a similar regulatory mechanism can be expected to initiate male meiosis in Drosophila. In contrast, several specific regulations separate from the core regulatory system take place during meiosis. For example, a Cdc25 orthologue encoded by twine plays a meiosis-specific role in activating Cdk1 before the onset of meiosis during oogenesis and spermatogenesis, whereas string is required at the initiation of mitotic events during embryogenesis and those during the development of germline stem cells and their progenitor cells. cycB mRNA is expressed at low levels in the spermatogonia during mitotic proliferation. Then, it is downregulated after the completion of mitotic divisions and re-expressed at high levels in spermatocytes during the growth phase before meiosis. In contrast, CycB protein levels in spermatocytes remain low until spermatocytes enter the G2/M transition after the appearance of mRNA. CycB translation is repressed until before the onset of male meiosis by two proteins that bind to cycB mRNA in spermatocytes. CycB accumulates in the cytoplasm prior to the initiation of chromatin condensation, remains at a high level during prophase, and then enters the nucleus at the onset of meiosis (Yamazoe, 2023).
Drosophila spermatocytes before or during meiosis I offer several advantages with respect to the investigation of cell-cycle regulation at the G2/M phase. Identifying and observing meiotic cells is easy due to the large cell size, which originates the remarkable cell growth. This facilitates the observation of the subcellular localization of specific regulatory proteins. Nevertheless, comparing the spermatocytes at similar developmental stages in different cysts is not easy. The growth phase has been classified into six stages, based on the chromatin morphology and the intracellular structure of pre-meiotic spermatocytes. Recently, the characteristic size and morphology of the nucleolus in the growth phase have allowed precise identification of the developmental stages of spermatocytes (Yamazoe, 2023).
Bases in 5' UTR - 85
Bases in 3' UTR - 221
Roughex is novel, with no homology to any other reported protein (Thomas, 1994). The interaction of a variety of proteins, including CKIs, with cyclins is mediated by RXL motifs. Rux contains three RXL motifs, starting at positions 30, 197 and 249, that could mediate the observed interaction of Rux with cyclins (Foley, 1999).
date revised: 3 June 97
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