Gene name - roughex
Cytological map position - 5D1--5D3
Function - regulator of cyclins
Symbol - rux
FlyBase ID: FBgn0003302
Genetic map position - 1-15.0.
Classification - novel protein
Cellular location - cytoplasmic and nuclear
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 through 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 Krmer, 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 that mutations 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 understanding the function 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 G1 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).
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).
Rux is a nuclear protein both in SL2 cells and in eye imaginal discs. In Drosophila embryos, CycA is cytoplasmic during those stages of interphase when it can be detected (late S phase and G2). A different pattern of localization has been found in eye discs where CycA, as in higher eukaryotes, is also present in the nuclei of S- and G2-phase cells. A similar distribution of Drosophila CDKs in S-phase cells has been seen in the developing eye using anti-PSTAIR antibodies, indicating that active CycA-Cdk complexes may be present in both cellular compartments. As a consequence, it is suggested that some of the activities associated with CycA-dependent kinase complexes are likely to be regulated at the level of subcellular distribution. In support of this hypothesis, eye discs expressing the RuxDeltaNLS construct show an expansion in the domain of S-phase cells behind the MF, as compared with a similar domain in control discs, consistent with an increase in the length of S phase. This observation suggests that the subcellular localization of CycA is important for S-phase progression and is blocked by expression of the RuxDeltaNLS mutant protein but not by expression of wild-type Rux (Avedisov, 2000).
How does Rux function to reduce CycA levels in G1? It is suggested that CycA normally exists in an equilibrium between nuclear and cytoplasmic fractions. In support of this notion, CycA expressed from a heat-inducible promoter in a GMR-Rux background is predominantly cytoplasmic immediately after heat shock and gradually becomes localized to the nucleus when the heat shock is removed. It is suggested that in G1 cells in the MF, the level of endogenous CycA protein is very low as a consequence of the abrupt destruction of mitotic cyclins just prior to G1 arrest in the MF. In contrast, Rux is stable in these G1 cells but is absent in cells that are actively cycling. Thus, relatively high levels of Rux in G1 can shift the CycA subcellular distribution by binding to and effectively targeting CycA protein to the nucleus. Rux may then inhibit CycA-dependent kinase activity by preventing or disrupting the CycA-CDK interaction. Nuclear CycA is also targeted for destruction by binding with Rux, although proteolysis of CycA is apparently not required for inactivation of CycA-dependent functions. When cells reenter S phase behind the MF, Rux levels decline and CycA reaccumulates for its S/G2 functions. This model implies that the level of Rux relative to that of CycA must be significantly higher in G1 (where inhibition of CycA occurs) than in S phase (where Rux levels are reduced) (Avedisov, 2000).
Rux 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).
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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