InteractiveFly: GeneBrief

Archipelago: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References


Gene name - archipelago

Synonyms -

Cytological map position - 64A11--12

Function - signaling

Keywords - component of SCF ubiquitin ligase complex, eye, oogenesis, growth

Symbol - ago

FlyBase ID: FBgn0041171

Genetic map position - 3L

Classification - F-box and WD repeat protein

Cellular location - cytoplasmic



NCBI links: Precomputed BLAST | Entrez Gene
BIOLOGICAL OVERVIEW

The Myc oncoprotein is an important regulator of cellular growth in metazoan organisms. Its levels and activity are tightly controlled in vivo by a variety of mechanisms. In normal cells, Myc protein is rapidly degraded, but the mechanism of its degradation is not well understood. Genetic and biochemical evidence is presented that Archipelago (Ago), the F box component of an SCF-ubiquitin ligase and the Drosophila ortholog of a human tumor suppressor, negatively regulates the levels and activity of Drosophila Myc (dMyc) protein in vivo. Mutations in archipelago (ago) result in strongly elevated dMyc protein levels and increased tissue growth. Genetic interactions indicate that ago antagonizes dMyc function during development. Archipelago binds dMyc and regulates its stability, and the ability of Ago to bind dMyc in vitro correlates with its ability to inhibit dMyc accumulation in vivo. These data indicate that archipelago is an important inhibitor of dMyc in developing tissues. Because archipelago can also regulate Cyclin E levels and Notch activity, these results indicate how a single F box protein can be responsible for the degradation of key components of multiple pathways that control growth and cell cycle progression (Moberg, 2004).

myc genes encode basic-helix-loop-helix-zipper (bHLHZ) domain transcription factors that dimerize with Max family proteins to promote cell growth and proliferation in metazoan organisms. The Myc-Max complex is implicated in the transcriptional regulation of many genes that are required for cell growth and metabolism; such genes include those for translation initiation factors and ribosomal components. The role of Myc in promoting growth is likely to contribute to its role as an oncoprotein in a wide variety of human tumor types. myc overexpression also promotes tumorigenesis in mice and zebrafish, indicating that the oncogenic properties of myc genes are conserved in other organisms (Moberg, 2004 and references therein).

Deregulation of mammalian Myc in cancer occurs by a variety of mechanisms. In some cancers, notably lymphomas, mutations found within the Myc protein have been shown to increase its stability (Salghetti, 1999; Gregory, 2000). Myc protein is normally turned over rapidly in vivo and in cultured cells has a half-life of 20-30 min, and several studies have shown that Myc protein is subject to ubiquitin-dependent proteasomal degradation (Grandori, 2000). Ubiquitination of Myc in turn appears to be regulated by phosphorylation at two distinct sites in the protein's amino-terminal portion, Threonine 58 (Thr58) and Serine 62 (Ser62). Evidence suggests that MAP kinase mediates phosphorylation of Ser62 and that this stabilizes c-Myc. Phospho-Ser62 may be required for subsequent phosphorylation of Thr58 by glycogen-synthase kinase 3 (GSK3: Drosophila homolog, Shaggy), which promotes the ubiquitination and degradation of c-Myc (Gregory, 2003). Significantly, Thr58Ile is the most common c-Myc mutation in Burkitt's lymphoma and is known to stabilize Myc considerably (Salghetti, 1999; Gregory, 2000). These observations suggest that phosphorylation of Thr58 by GSK3 generates a motif that facilitates the interaction of Myc with a ubiquitin ligase that restricts Myc levels and activity in vivo. Currently, the identity of the ubiquitin ligase that promotes Myc degradation has not been firmly established in any organism (Moberg, 2004 and references therein).

The Drosophila F box protein Archipelago (Ago) has been implicated in the degradation of Drosophila Myc (dMyc). Ago binds dMyc, and impairment of Ago function in vivo stabilizes dMyc, resulting in markedly elevated Myc levels, and promotes cell growth. Recent evidence indicates that the Fbw7/hCDC4 tumor suppressor protein, which is the human ortholog of Ago, also inhibits c-Myc accumulation by promoting its degradation (Welcker, 2004). Because Ago proteins also regulate Cyclin E levels (Moberg, 2001; Strohmaier, 2001; Koepp, 2001), and Notch pathway activity (Moberg, unpublished observations and reviewed in Justice, 2002), these findings suggest a mechanism by which the levels of Cyclin E and dMyc and the activity of the Notch pathway can be coordinately regulated by a shared degradation pathway (Moberg, 2004).

Thus Ago, which functions as the substrate-specificity subunit of an SCFAgo ubiquitin ligase, regulates the levels of the growth-promoting transcription factor dMyc in developing Drosophila tissues. This regulation appears to occur via a posttranscriptional mechanism that involves a direct Ago-dMyc interaction that modulates dMyc stability. dMyc accumulates in ago mutant cells and likely contributes to their increased growth (Moberg, 2004).

The WD repeat domain of Ago interacts with Cyclin E (Moberg, 2001; Strohmaier, 2001; Koepp, 2001), and it also binds dMyc. The optimal binding site for the WD domain of S. cerevisiae Cdc4, the yeast ortholog of Ago, has been determined to be I/L-I/L/P-pT-P-P, in which the central threonine residue is phosphorylated (Nash, 2001). Human Cyclin E, Drosophila Cyclin E, and human c-Myc all have a single, well-conserved version of this site, whose central feature is an L-L-T-P-P motif. dMyc contains seven copies of a degenerate version of this site, in which the central threonine is often replaced by a serine, and many of the flanking residues deviate from those in the consensus sequence. Importantly, these putative sites do retain a conserved S/T residue at position +4. The equivalent +4 serine in human Cyclin E (S384) has been shown to be required for the ubiquitination of Cyclin E (Welcker, 2003) and may therefore represent an important feature of the putative Ago binding motif. The presence of multiple Ago binding sites in dMyc versus the single well-conserved site in c-Myc might indicate that although both proteins are targeted for degradation by orthologous F box proteins, the kinetics of degradation of the two Myc proteins may be different (Moberg, 2004).

The array of apparently suboptimal sites in dMyc resembles the situation in S. cerevisiae Sic1, in which nine low-affinity sites are able to cooperatively mediate a stable interaction with Cdc4 (Welcker, 2003; Orlicky, 2003). Indeed, as is the case with Sic1, mutating a single putative phosphorylation site in dMyc does not alter its Ago binding properties. In contrast, for human Cyclin E and c-Myc, the predicted Ago interaction site lies within a domain previously shown to be required for their ubiquitination and degradation (Salghetti, 1999; Gregory, 2000; Won, 1996; Clurman, 1996). Furthermore, missense mutations of the central threonine in the Ago interaction motif are the most frequent c-Myc mutations in Burkitt's lymphoma and stabilize c-Myc in cells, suggesting that Ago-dependent degradation of c-Myc is perturbed in these cancers (Moberg, 2004).

ago mutant cells grow more quickly than their wild-type neighbors, but they maintain their normal size by an apparent acceleration of the cell cycle. This differs considerably from the phenotype elicited by overexpression of either dMyc or Cyclin E. Increased expression of dMyc results in increased growth that manifests as an increase in cell size without any change in the duration of the cell cycle. dMyc also promotes S phase entry, possibly as a consequence of the increased growth. Increased expression of Cyclin E has no effect on growth but promotes S phase entry. It also results in, at best, a modest acceleration of the cell cycle. Thus, the cell cycle acceleration observed in ago mutant cells is not easily explained by the elevated level of either dMyc or Cyclin E. Both dMyc and Cyclin E promote S phase entry but maintain the normal duration of the cell cycle by apparently lengthening the S and G2 phases, respectively. Thus, it seems likely that ago loss also affects a regulatory protein that promotes the G2-M transition. Such a regulator could either be a direct substrate of SCFAgo or may be regulated indirectly (Moberg, 2004).

Interestingly, both Ago targets identified to date, Cyclin E and dMyc, are required for imaginal-disc growth. Signaling via the Notch receptor is increased in ago clones, as assessed by the activity of a reporter gene fused to the Enhancer of split mβ promoter. Notch signaling has been shown to promote imaginal-disc growth at least in part by a non-cell-autonomous pathway. Because cyclin E, dMyc, and Notch all participate in tissue growth via increases in cell number and/or cell mass, Ago may represent a way to coordinately regulate these pathways by a common degradation mechanism. Thus, increased Ago levels would be expected to impair tissue growth, and decreased levels would facilitate tissue growth, via multiple pathways. Because ago transcription is patterned in the eye imaginal disc (Moberg, 2001), ago may function to link patterning signals with the activity of these growth-promoting pathways (Moberg, 2004).

The ability of ago to regulate multiple pathways that function in growing cells has implications for understanding the role of its human ortholog (Fbw7/hCDC4) as a tumor suppressor gene. Mutations in Fbw7/hCDC4 have been identified in cancer cell lines (Moberg, 2001; Strohmaier, 2001), and more recently, mutations have been identified in Fbw7/hCDC4 in endometrial and colorectal tumors (Spruck, 2002: Rajagopalan, 2004). These tumors are likely to have elevated levels of Cyclin E. In light of the data presented here, they are predicted to have high levels of the oncoprotein c-Myc and increased Notch activity, which has also been implicated in human cancers. Thus, the neoplastic phenotype of these tumors may reflect the additive effect of activating all of these pathways that are normally inhibited by Ago (Moberg, 2004).


REGULATION

Notch-dependent expression of the archipelago ubiquitin ligase subunit in the Drosophila eye

archipelago (ago)/Fbw7 encodes a conserved protein that functions as the substrate-receptor component of a polyubiquitin ligase that suppresses tissue growth in flies and tumorigenesis in vertebrates. Ago/Fbw7 targets multiple proteins for degradation, including the G1-S regulator Cyclin E and the oncoprotein dMyc/c-Myc. Despite prominent roles in growth control, little is known about the signals that regulate Ago/Fbw7 abundance in developing tissues. This study used the Drosophila eye as a model to identify developmental signals that regulate ago expression. It was found that expression of ago mRNA and protein is induced by passage of the morphogenetic furrow (MF), and the hedgehog (hh) and Notch (N) pathways were identified as elements of this inductive mechanism. Cells mutant for N pathway components, or hh-defective cells that express reduced levels of the Notch ligand Delta, fail to upregulate ago transcription in the region of the MF; reciprocally, ectopic N activation in eye discs induces expression of ago mRNA. A fragment of the ago promoter that contains consensus binding sites for the N pathway transcription factor Su(H) is bound by Su(H) and confers N-inducibility in cultured cells. The failure to upregulate ago in N pathway mutant cells correlates with accumulation of the SCF-Ago target Cyclin E in the area of the MF, and this is rescued by re-expression of ago. These data suggest a model in which N acts through ago to restrict levels of the pro-mitotic factor Cyclin E. This N-Ago-Cyclin E link represents a significant new cell cycle regulatory mechanism in the developing eye (Nicholson, 2011).

The ago gene and its vertebrate ortholog Fbw7 have well established roles in controlling cell division, cell growth and apoptosis in developing tissues, yet only a few studies have investigated pathways that regulate ago activity in cells. It is known that dimerization of Fbw7 enhances its ability to degrade Cyclin E, and that ago and Fbw7 are both transcriptionally induced by p53/dp53 via a checkpoint pathway that responds to either energy starvation. This study shows that the N and hh pathways are necessary for the proper regulation of Ago levels in the developing Drosophila eye, specifically by increasing ago transcription in the region within and immediately behind the MF. This effect correlates with the presence of Su(H) binding sites in the ago promoter, and can be enhanced by co-expression of the N-terminal activation domain of Da. DaN can bind to Su(H) and drive elevated expression of the N-target E(spl)m8, and the requirement for da in Ago expression at the MF suggests that a similar mechanism might occur here. Interestingly, mutating the six putative Su(H) sites in the ago2kb-luc reporter does not completely abolish transactivation by NICD and DaN, suggesting that they have a secondary effect on ago transcription that is independent of the Su(H) sites. Finally, it was found that the defect in the MF-associated pulse of ago expression in N pathway-defective cells results in hyperaccumulation of the SCF-Ago target protein Cyclin E, indicating that this novel transcriptional link between N and ago is an important mechanism through which N regulates the G1-S phase transition in eye disc cells (Nicholson, 2011).

Although these data shed light on the initial inductive phase of Ago expression at the MF, additional mechanisms must operate immediately posterior to the MF to refine the pattern of Ago expression. The regular pattern of Ago expression posterior to the MF appears to overlap with gaps in Ato expression between the R8 equivalence groups, arguing that these expression patterns might share some regulatory inputs. Interestingly, N is required both for induction of Ato within MF cells and for restriction of Ato expression in cells immediately posterior to the MF via a lateral inhibition mechanism. Thus, although Ago does not display precisely the same pattern as that of Ato restriction posterior to the MF (e.g. expression only in the presumptive R8), it seems possible that N might play a similar dual activator/inhibitor role upstream of ago. The effect of the Nts allele on Ago patterning behind the MF supports such a model. Moreover, since Fbw7 can target mammalian NICDs for proteasomal degradation, the pattern of Ago expression posterior to the MF might also reflect a requirement for SCF-Ago to inhibit NICD activity in differentiating neurons. In support of this type of feedback model, expression of the N pathway reporter E(spl)mß-CD2 is elevated in ago mutant cells posterior to the MF, indicating that Ago might also regulate N activity. As overall N protein levels are not obviously altered in ago mutant eye cells, as assessed by both indirect immunofluorescence and immunoblotting with the C17.9C6 antibody that recognizes the cytoplasmic tail of the N receptor, it remains to be established whether or not this potential feedback loop involves changes in N protein turnover (Nicholson, 2011).

In addition to the potential for a cell-autonomous feedback mechanism between Ago and N, ago expression is subject to N pathway-mediated, non-cell-autonomous effects at the MF. Expression of ago>Gal4 is rescued in Dl,Ser mutant cells by adjacent wild-type cells, and Su(H) mutant cells appear to be able to upregulate ago expression in adjacent wild-type cells, probably via increased Dl expression. Alleles of other N pathway components might thus be expected to exhibit effects on ago expression at clonal boundaries. However, expression of Ago in Psn mutant clones that span the MF is not obviously rescued by adjacent wild-type cells, and, reciprocally, these cells do not induce higher levels of Ago in adjacent wild-type cells, indicating that the cross-border induction of ago transcription might be restricted to alleles of genes required for repression of Dl (Nicholson, 2011).

Depending on the developmental context, N can be either pro- or anti-mitotic. Exactly how N fulfils these roles is not fully understood. The finding that ago is regulated by N, and that N appears to act through ago to control Cyclin E levels in a subset of eye disc cells, provide a novel link between N and the core cell cycle machinery. This link could explain certain cell cycle phenotypes described in N mutant disc cells. For example, N-deficient cells in the second mitotic wave (SMW) hyperaccumulate Cyclin E but also simultaneously fail to enter the SMW properly. Su(H) mutant cells also accumulate Cyclin E, which is consistent with a role for the N pathway in promoting ago expression. The latter pro-mitotic effect of N has been attributed to a requirement for N for relief of Rbf-mediated repression of dE2f1 activity, but the former role of the N pathway in antagonizing Cyclin E levels is not well understood. Based on data presented in this study that place the N pathway upstream of ago, it is proposed that this phenotype is due to defective turnover of Cyclin E resulting from insufficient ago expression. The identity of the N target that promotes SMW entry has remained controversial. Interestingly, high Cyclin E activity can downregulate levels of the pro-S-phase transcription factor dE2f1 in the developing wing and eye. Thus, N-induced ago transcription may promote Cyclin E turnover following S-phase, but might also ensure that cells are only able to enter the SMW with an appropriate amount of Cyclin E present (Nicholson, 2011).

Interestingly, the reduced Ago expression in N pathway mutants has no discernible effect on the levels of dMyc. One logical explanation for this is that the threshold of SCF-Ago activity required to degrade dMyc is lower than that required for Cyclin E. Such a mechanism would imply that the N-ago link selectively regulates some SCF-Ago targets but not others; alternatively, N might have a second role in this pathway by protecting dMyc from SCF-Ago activity, although there is no clear evidence of such a role (Nicholson, 2011).

The role for N upstream of Ago and Cyclin E could theoretically be significant in mediating N effects in tissues outside of the eye, and it will thus be important to examine whether N is required for optimal ago expression in other developing tissues. However, available data suggest that the N-ago link might be fairly context specific and not generalized to N signaling in all cell types. First, the pattern of Ago protein in developing larval discs (including the eye) does not generally mirror that of N pathway activity in each organ. Other signals, present at the MF but not elsewhere, must thus cooperate with N to induce ago within the MF. The proneural transcription factor Da is a clear candidate to fulfill this role: Da expression peaks in the MF and is required for the pulse of Ago protein expression at the MF. Thus, it seems likely that N signals synergize with Da, and perhaps with additional unidentified factors, to pattern ago expression. Given the role that dp53 plays in ago induction and in the control of Cyclin E protein under conditions of metabolic stress, it will be interesting to determine how pathways involving N, da and dp53 interact on the ago promoter under various stresses and developmental conditions. Furthermore, it will important to determine whether additional signaling and checkpoint mechanisms, especially those that interact functionally with the N pathway, act through the ago promoter to pattern Cyclin E levels in developing tissues (Nicholson, 2011).

Protein Interactions

To identify an SCFAgo ubiquitin ligase substrate that could explain the accelerated growth of ago mutant cells, two different interaction screens were conducted by using the Ago F box/WD domain. By mass spectrometric analysis of proteins that coprecipitate with Ago, peptides derived from a number of different SCF components were identified, including Cullins and Skp proteins. At a lower frequency, peptides derived were also recovered from putative SCFAgo substrates, including the Drosophila ortholog of the Myc transcription factor (dMyc). In addition to multiple SCF components, a single clone of dMyc was also recovered in a yeast two-hybrid screen for proteins that physically interact with the F box/WD repeat region of Ago (Moberg, 2004).

dMyc was identified as a candidate Ago binding protein, so whether the ability of ago to regulate dMyc involves a direct interaction between Ago and dMyc was examined. In protein extracts from Drosophila S2 cells transfected with epitope-tagged versions of Ago and dMyc (HAAgo and FLAGdMyc), FLAGdMyc was readily detected in anti-HA immunoprecipitates, and in the reciprocal procedure, HAAgo was readily detected in anti-FLAG immunoprecipitates. These experiments indicate that Ago and dMyc interact physically in S2 cells. Significantly, two mutant versions of Ago, Ago1 and Ago3, that correspond to mutations that deregulate dMyc levels and increase growth in vivo, are dramatically impaired in their ability to interact with dMyc in cells despite being expressed at approximately the same level as wild-type Ago protein. Thus, as is the case with the other known SCFAgo substrate, Cyclin E (Moberg, 2001), the ability of Archipelago to bind dMyc protein correlates with its ability to downregulate dMyc levels in vivo (Moberg, 2004).

Coexpression of dMyc also seems to promote Ago accumulation in cells. This increase seems more evident in forms of Ago that bind strongly to dMyc and does not appear to be a general effect of dMyc on all coexpressed proteins. However, the precise mechanism underlying this effect has not been established. It may involve direct dMyc-Ago binding, but it may also be an indirect consequence of Myc's ability to regulate cell metabolism and translation rates (Moberg, 2004).

The Drosophila ubiquitin-specific protease Puffyeye regulates dMyc-mediated growth

The essential and highly conserved role of Myc in organismal growth and development is dependent on the control of Myc protein abundance. It is now well established that Myc levels are in part regulated by ubiquitin-dependent proteasomal degradation. Using a genetic screen for modifiers of Drosophila Myc (dMyc)-induced growth, this study identified and characterized a ubiquitin-specific protease (USP), Puffyeye (Puf), as a novel regulator of dMyc levels and function in vivo. puf genetically and physically interacts with dMyc and the ubiquitin ligase archipelago (ago) to modulate a dMyc-dependent cell growth phenotype, and varying Puf levels in both the eye and wing phenocopies the effects of altered dMyc abundance. Puf containing point mutations within its USP enzymatic domain failed to alter dMyc levels and displayed no detectable phenotype, indicating the importance of deubiquitylating activity for Puf function. dMyc induces Ago, indicating that dMyc triggers a negative-feedback pathway that is modulated by Puf. In addition to its effects on dMyc, Puf regulates both Ago and its cell cycle substrate Cyclin E. Therefore, Puf influences cell growth by controlling the stability of key regulatory proteins (Li, 2013).

The mammalian Myc gene family, comprising Myc, Mycn and Mycl, is known to be crucial for growth and development. Myc proteins control multiple cellular processes, including cell growth, proliferation, metabolism and apoptosis, and deregulation of Myc plays an important role in oncogenesis. Non-mammalian Myc has been most intensively studied in Drosophila where the absolute requirement for Drosophila Myc function during development has been demonstrated by the fact that dMyc-null mutants die at an early larval stage (Li, 2013).

Myc transcript and protein abundance are subject to regulation at multiple levels ranging from transcriptional control by numerous mitogenic signaling pathways to extensive post-translational modifications. Of particular interest, given the relatively short half-life of Myc proteins, is the post-translational modification of Myc by the ubiquitin system. Protein ubiquitylation is a fundamental and versatile post-translational modification that controls multiple cellular events by marking proteins as substrates for either degradation or non-degradative processing. In mammals, distinct ubiquitin E3 ligase complexes, including Skp2 and Fbw7, have been reported to influence Myc protein stability and activity (Li, 2013).

The Drosophila ortholog of Fbw7, Archipelago (Ago) is the only ligase identified thus far as involved in proteasome-mediated ubiquitin-dependent turnover of dMyc proteins (Moberg, 2004). Ago mutant alleles were first identified in a genetic screen for regulators of tissue growth in the eye, where it was initially shown to bind and regulate Cyclin E (CycE) levels. Later work demonstrated that Ago also physically interacts with dMyc, and controls dMyc stability and biological function (Moberg, 2004). Unlike c-Myc, which was shown to have a single Myc BoxI phosphodegron associated with Fbw7 binding, several domains containing putative Ago-interacting motifs were shown in dMyc to mediate Casein kinase 1 (CK1)α-, CK1ε- and GSK3β-dependent protein degradation. Although their link to Ago function has not been precisely established, it is clear that GSK3β plays a key role in Ago-mediated dMyc ubiquitylation and degradation (Li, 2013 and references therein).

Protein ubiquitylation is a reversible process in which removal of ubiquitin chains is mediated by deubiquitylating enzymes (DUBs), and the role of DUBs in controlling various cellular processes has attracted considerable interest. DUBs are classified into five subfamilies based on their deubiquitylating domain. Ubiquitin-specific proteases (USPs), which constitute the largest DUB subfamily, share a structurally conserved USP domain of ~350 to 450 amino acids. The USP domain is the catalytic core that mediates the cleavage of ubiquitin conjugates, whereas domains required for protein-protein interaction and substrate specificity are located within N and/or C termini of the USP protein.Although several ubiquitin E3 ligases have been implicated in modulating c-Myc stability, only one deubiquitylating enzyme, USP28, has been demonstrated to catalyze the deubiquitylation of Myc in mammals. Thus far, no deubiquitylating enzyme has been identified that modulates dMyc function or antagonizes Ago-mediated dMyc degradation. Of the 41 predicted Drosophila DUBs, 21 are predicted to have a mammalian USP ortholog. Interestingly, Drosophila does not encode an USP28 ortholog, suggesting that a distinct USP may be responsible for reversing dMyc ubiquitylation in Drosophila. This study reports the identification and characterization of Puffyeye (Puf), a Drosophila USP that antagonizes Ago function and interacts genetically and physically with dMyc. Evidence is presented that Puf regulates dMyc activity at the level of cell and organ growth (Li, 2013).

Although a great deal has been learned recently concerning ubiquitin ligases that interact with Myc proteins, to date only one DUB has been reported that targets Myc. This study has employed a genetic screen based on the rough eye phenotype induced by dMyc overexpression in the eye (GMM) in Drosophila. This screen led to the identification of a USP-type DUB, which was named Puffyeye (Puf; CG9754), as a novel regulator of dMyc function in vivo. Reduced puf expression suppresses, whereas puf overexpression augments, the GMM phenotype. This phenotype is largely an effect of cell overgrowth, yet overgrowth in the eye due to cyclin D/Cdk4 was not influenced by altered Puf abundance. Moreover, knockdown of four other USPs had no effect on the GMM phenotype. This suggests that puf possesses specificity for dMyc-induced growth in the eye. Indeed, puf itself induced a dose-dependent rough eye phenotype, displaying augmented ommatidial size that can be modulated by altering dMyc levels. In the wing disc, dMyc and Puf also were found to collaborate in cell growth. It was also found that Puf is essential for normal development, consistent with a crucial role for Puf in cell growth (Li, 2013).

dMyc levels markedly increase in cells in which puf is overexpressed, whereas dMyc levels are decreased in Puf hypomorphic mutants. These changes in dMyc are predominantly post-translational. This is consistent with the finding that Puf overexpression results in a dramatic increase in dMyc protein stability. Importantly, all of the biological effects of Puf, as well as its effects on dMyc abundance and turnover, are abolished by point mutations in the highly conserved Puf USP catalytic domain. It is surmised that Puf stabilizes Myc through its function as a deubiquitylating enzyme that antagonizes the activity of the Ago ubiquitin ligase, previously shown to target Myc for ubiquitylation and degradation (Moberg, 2004). Importantly, increased Puf exacerbates, and decreased Puf suppresses, the effect of Ago heterozygotes in enhancing the GMM phenotype. The notion that Puf and Ago act as antagonists receives further support from the findings that Puf protein physically associates with both dMyc and Ago in vivo. Interactions between DUBs and their antagonistic E3 ligases, as well as their substrates have been reported previously. The ability of both the Puf short and long isoforms to modify the dMyc-mediated eye phenotype, and stabilize dMyc and Ago proteins in an ubiquitylation domain-dependent manner suggests that domain(s) required for Puf to interact with dMyc or Ago are located in a region N-terminal to the core catalytic domain (Li, 2013).

It was also found that Puf stabilizes CycE, another known Ago substrate, suggesting that Puf antagonizes Ago function in regulating other targets that are crucial for cell cycle control. Indeed, flies homozygous for puf and ago double mutations do not survive, raising the possibility that, in addition to regulating common substrates, they each possess unique targets, as shown for other ubiquitin ligases and DUBs. Notch would be another potential candidate for Puf activity (Moberg, 2004); however, no significant effect of Puf on Notch levels was found in wing discs. In mammalian cells, the ubiquitin-specific protease USP28 was demonstrated to regulate the turnover of c-Myc by binding and antagonizing the activity of Fbw7α, the vertebrate ortholog of Ago. However, Puf and USP28 are not homologs: they appear to be two very distinct USPs in terms of their overall size and amino acid sequence similarity in both their core enzymatic domains and the protein sequence as a whole. The closest mammalian homolog of Puf is USP34 (3546aa). Puf and USP34 are highly homologous in their core catalytic domains (67% identity; 80% similarity) with the catalytic triad conserved, whereas the overall similarity between the two proteins is ~52% (~37% identity) (Li, 2013).

Previous studies have shown that multiple signaling pathways regulate Ago and Fbw7 expression and activity. This study found that Ago levels are increased upon dMyc, as well as upon Puf overexpression. Although the mechanisms by which dMyc and Puf regulate Ago expression are unclear, dMyc-dependent Ago expression may provide a mechanism for dMyc autoregulation, whereas Puf may stabilize Ago by deubiquitylating it. Indeed, Fbw7 has been shown to be regulated through ubiquitylation. A similar type of dynamic relationship has been reported for the ubiquitin ligase Mdm2 and deubiquitylase HAUSP/USP7 in regulating the stability and function of the tumor suppressor p53. Taken together, these data suggest that Ago and Puf represent a regulatory node that controls degradation of Myc and CycE, and very likely other growth control factors. Further studies will be required to identify additional substrates of Puf and to understand the physiological importance of Puf-mediated regulation of protein degradation in Drosophila (Li, 2013).

Drosophila Minus is required for cell proliferation and influences Cyclin E turnover

Turnover of cyclins plays a major role in oscillatory cyclin-dependent kinase (Cdk) activity and control of cell cycle progression. This study analyses a novel cell cycle regulator, called minus, which influences Cyclin E turnover in Drosophila . minus mutants produce defects in cell proliferation, some of which are attributable to persistence of Cyclin E. Minus protein can interact physically with Cyclin E and the SCF Archipelago/Fbw7/Cdc4 ubiquitin-ligase complex. Minus does not affect dMyc, another known SCFAgo substrate in Drosophila . It is proposed that Minus contributes to cell cycle regulation in part by selectively controlling turnover of Cyclin E (Szuplewski, 2009).

Progression through the cell cycle requires periodic activation of cyclin-dependent kinases (Cdks). Oscillation in Cdk activity is achieved in part through cyclical synthesis and controlled degradation of cyclins, the regulatory subunits of the Cdks. Cyclin E is an evolutionarily conserved nuclear cyclin that controls G1/S transition and S-phase progression in animal cells, predominantly by activating Cdk2. CycE and cdk2 are essential genes in Drosophila . Cyclin E acts as the limiting factor for G1-S-phase transition. Cyclin E turnover is important for cell cycle progression and is regulated by a conserved ubiquitin-ligase complex, called SCF. The SCF complex is built on an elongated scaffold protein, Cullin-1, which recruits the substrate recognition module consisting of Skp1 and an F-box protein, as well as a ring domain-containing ubiquitin-ligase module. Substrate selectivity is mediated by the F-box subunit, in part through recognition of phosphorylated motifs on substrate proteins. The Drosophila F-box protein encoded by archipelago (ago) is the ortholog of Fbw7/Cdc4. Scf-Ago promotes degradation of CycE, dMyc, and Trachealess (Szuplewski, 2009).

This study reports the characterization of the classical Drosophila mutant minus. Minus protein can interact physically with Cyclin E and the SCF-Ago complex. Cells lacking Minus fail to degrade CycE, resulting in persistence of CycE. minus mutants show defects in cell proliferation, attributable in part to excess CycE activity, reflecting that normal regulation of CycE turnover is essential for normal cell proliferation during Drosophila development. It is proposed that Minus acts as a cell cycle regulator by selectively controlling CycE turnover (Szuplewski, 2009).

Flies homozygous mutant for a spontaneous mutation in the minus gene mi1 showed small body size, small bristles, and delayed completion of pupal development. minus alleles were also isolated in a screen for female sterility, and the minus gene has been mapped to the cytogenetic interval 59E on the right arm of the second chromosome. To isolate new minus alleles, P-element insertions in 59E were screened for failure to complement the mi1 bristle phenotype. Flies carrying the l(2)SH0818 P-element insertion in trans to mi1 showed a small bristle phenotype, milder than that produced by the combination of mi1 in trans to a deletion. The stronger mutant combination was also female sterile. Thus, l(2)SH0818 appears to have reduced minus activity. l(2)SH0818 is semilethal, but rare homozygous survivors showed small body size and small bristles. These phenotypes were confirmed to be due to the P-element insertion, since flies from which the P-element was precisely excised were homozygous viable and normal in size. Animals homozygous for a null allele of minus also showed reduced body size in larval and pupal stages (Szuplewski, 2009).

The l(2)SH0818 P-element is inserted in the 5' untranslated region (UTR) of the annotated gene CG5360. Two other transposons inserted in this 5' UTR, EY01258 and l(2)k06908, also produced weak bristle phenotypes in trans to mi1, suggesting that they are weak minus alleles. mi1 was isolated as a spontaneous mutation, which can be caused by transposon insertions. It was not possible to amplify DNA spanning the second intron of CG5360 from mi1 homozygous animals by PCR, consistent with the possibility that an insertion of DNA disrupts the CG5360 transcription unit (other parts of the gene amplified normally). An additional mi allele was generated by imprecise excision of the viable P-element insertion EY01258. miDeltaEY22 is a deletion that removes the two first exons and part of the third exon of CG5360. miDeltaEY22 produced phenotypes equivalent to those of a larger deletion that completely removes the gene, and so behaves genetically like a null allele. Homozygous miDeltaEY22 mutants died mainly during early larval stages. The remaining mutants showed a developmental delay and reduced growth rate. After 5 d, the largest mutant larvae were much smaller than comparably aged control larvae. By 11 d, the surviving mutants that had pupated were also small (Szuplewski, 2009).

Minus protein lacks domains of known function, but was identified as a CycE-interacting protein in a genome-wide yeast two-hybrid screen. Ten cyclin-binding sites are predicted using the Eukaryotic Linear Motif server. Some of the predicted cyclin-binding sites are conserved in other insects—Anopheles gambiae, Tribolium castaneum, and Apis mellifera—but none resides in a region of sequence conservation sufficient to permit identification of an orthologous protein outside of the insects. The interaction between Minus and CycE was confirmed in vitro using GST pull-down assays. The Drosophila Cyclin E gene encodes two proteins that differ in their N termini. A GST-Minus fusion protein was able to bind CycE-I from lysates of S2 cells transfected to express Myc-tagged CycE-I. Similar results were obtained with Myc-tagged Drosophila CycE-II protein and with Myc-tagged human CycE isoform 1 (CCNE1) (Szuplewski, 2009).

This study provides evidence that efficient Cyclin E turnover is essential for normal cell proliferation and endoreplication during Drosophila development. Endoreplicative growth and more typical proliferative diploid cell cycles are impaired as a consequence of the elevated Cyclin E levels in minus mutants. Attempts to suppress the cell proliferation phenotype by reducing CycE activity were only partially successful, perhaps due to incomplete compensation for elevated CycE levels. It is also possible that Minus has other targets, in addition to CycE (Szuplewski, 2009).

Minus was identified in a screen for female sterility. Minus' role in Cyclin E turnover suggests a possible link to the encore mutant. encore encodes a protein of unknown function that has been proposed to promote CycE degradation by localizing the SCF complex to a germline-specific cytoplasmic structure called the fusome. In mammals, different Fbw7/Cdc4 isoforms can target different Myc functions in distinct subcellular compartments. Interestingly, the Drosophila Fbw7/hCdc4 protein Archipelago exists in only one isoform, limiting the possibility for isoform-specific subfunctions. Use of accessory proteins, such as Minus, may be another means to confer target specificity on the core Archipelago/SCF complex (Szuplewski, 2009).

At present no Minus ortholog has been detected outside the insects. But, it is noted that Minus binds to human CCNE1 and influences its expression, much as it does with Drosophila CycE. Although this does not constitute evidence for the existence of a mammalian protein with a function analogous to Minus, it is compatible with this possibility. A vertebrate protein having the motifs required for Minus function but in a different number or arrangement might not be readily identified unless these short motifs were embedded in more extensive blocks of sequence similarity. In view of the importance of Cyclin E turnover in cancer, a functional equivalent of Minus might be a good candidate for a tumor suppressor (Szuplewski, 2009).

The Drosophila F-box protein Archipelago controls levels of the Trachealess transcription factor in the embryonic tracheal system

The archipelago gene (ago) encodes the F-box specificity subunit of an SCF(skp-cullin-f box) ubiquitin ligase that inhibits cell proliferation in Drosophila and suppresses tumorigenesis in mammals. ago limits mitotic activity by targeting cell cycle and cell growth proteins for ubiquitin-dependent degradation, but the diverse developmental roles of other F-box proteins suggests that it is likely to have additional protein targets. This study shows that ago is required for the post-mitotic shaping of the Drosophila embryonic tracheal system, and that it acts in this tissue by targeting the Trachealess (Trh) protein, a conserved bHLH-PAS transcription factor. ago restricts Trh levels in vivo and antagonizes transcription of the breathless FGF receptor, a known target of Trh in the tracheal system. At a molecular level, the Ago protein binds Trh and is required for proteasome-dependent elimination of Trh in response to expression of the Dysfusion (Dys) protein. ago mutations that elevate Trh levels in vivo are defective in binding forms of Trh found in Dysfusion-positive cells. These data identify a novel function for the ago ubiquitin-ligase in tracheal morphogenesis via Trh and its target breathless, and suggest that ago has distinct functions in mitotic and post-mitotic cells that influence its role in development and disease (Mortimer, 2007).

The biological properties of individual F-box proteins are to a large degree determined by their repertoire of target proteins. In the case of the Drosophila Ago F-box protein, failure to degrade these targets promotes excess proliferation of imaginal disc cells. This observation has led to the identification of Cyclin E and Myc proteins as ago targets. However the broad pattern of Ago expression in the embryo suggests that it might regulate distinct processes and targets in other cell types. In view of the rapidly growing body of work showing that inactivation of human ago/Fbw7 is a common event in a variety of cancers (e.g., Malyukova, 2007; O'Neil, 2007; Thompson, 2007), identification of these targets may provide important insight into the biology of cancers lacking ago function (Mortimer, 2007).

This study shows that Drosophila Ago is required for the post-mitotic morphogenesis of the embryonic tracheal system and that this requirement is due, at least in part, to the ability of Ago to bind directly to a previously unrecognized target, the Trh transcription factor, and stimulate its proteasomal degradation. This ago degradation mechanism appears to fulfill different regulatory roles in different populations of tracheal cells. In non-fusion tracheal cells, ago is required to limit overall levels of Trh, which is normally expressed at moderate levels throughout the tracheal system. In tracheal fusion cells the ago degradation mechanism appears to be strongly potentiated by an unidentified signal generated by Dys, such that Trh is completely eliminated from Dys-expressing cells. At a genetic level, the dependence of ago tracheal phenotypes on trh gene dosage argues that that elevated Trh levels are primarily responsible for branching defects that occur in ago zygotic mutant embryos. In support of this, persistent Trh expression is also observed in ago mutant fusion cells in other tracheal branches. This novel role for Ago in tracheal development is supported by the independent finding that homozygosity for a genomic deletion containing the ago locus is associated with cell migration defects in embryonic tracheal metameres (Mortimer, 2007).

Many important developmental events are controlled by multiple mechanisms that collaborate to regulate a key step in the process. This somewhat redundant control insulates the process from defects in any single pathway, such that major defects only occur when multiple control mechanisms are blocked. The observation that the effect of ago mutations on Trh and btl levels is completely penetrant, but the resulting morphological defects are not, suggests that another pathway acts redundantly to ago to control tracheal development. The strong, dominant enhancement of the ago phenotype by a mutation in the abnormal wing disc (awd) gene (Dammai, 2003) fits very well into a model in which multiple pathways are responsible for the precisely timed down-regulation of the Breathless/FGF pathway: ago attenuates btl transcription by degrading Trh, awd lowers levels of Btl protein on the cell surface by promoting its endocytic internalization (Dammai, 2003), and other pathways act independently to control expression of the FGF ligand branchless in non-tracheal cells. Thus, the incomplete penetrance of the ago phenotype is not indicative of an insignificant role for the gene in tracheal development, but rather may indicate that the tracheal system uses multiple mechanisms to redundantly control a key step in its development (Mortimer, 2007).

The Ago WD repeat region binds Cyclin E and dMyc, and the current work demonstrates that it also binds Trh. Broadly, the Ago-Trh interaction is quite similar to interactions with Cyclin E and dMyc: it is required for the down-regulation of substrate levels in vivo, and its disruption elevates levels of substrate that then drive downstream phenotypes. For substrates like Myc, site-specific phosphorylation generates a motif that binds to the Ago WD-region and stimulates rapid, SCF-mediated protein turnover of the target protein (reviewed in Minella, 2005). In contrast, the data in this study suggest that Trh can physically interact with Ago in two distinct configurations: one that does not require an intact WD-domain and a second WD-dependent mode of binding. The observation that the Ago1 allele can participate in the first complex but not the second and is defective in Trh regulation in vivo, suggests that like other Ago targets, WD-dependent binding is associated with rapid Trh turnover. Expression of Dys appears to shift the balance in favor of this second mode of binding. Combined with the genetic and phenotypic data implicating ago as an in vivo regulator of Trh activity, these molecular data support a model in which Ago can bind to Trh in the absence of Dys and inefficiently stimulate Trh turnover by a WD-dependent mechanism. This inefficient mechanism may be responsible for the fairly mild increase in Trh levels observed in all ago mutant dorsal trunk cells. However, in the presence of Dys, the efficiency of Trh turnover in dorsal trunk fusion cells is enhanced to the degree that the entire pool of Trh is rapidly eliminated. Interestingly, the correlate of this hypothesis - that ectopic expression of dys in non-fusion cells should be sufficient to trigger down-regulation of endogenous Trh - was confirmed in a recent study (Jiang, 2006; Mortimer, 2007).

The nature of the Dys-generated signal responsible for this effect is not currently known. Precedent with other Ago targets suggests that it may involve Trh phosphorylation. Recent work on the mammalian ago ortholog Fbw7 has shown that interactions with substrates can also be modulated by interaction with accessory factors (Punga, 2006), or by conformational changes in the substrate driven by the isomerization of proline residues within the Ago/Fbw7 binding motif (van Drogen, 2006). Proline isomerization has been implicated in the degradation of mammalian c-Myc, but such mechanisms are not currently known to play a role in the degradation of either Myc or bHLH-PAS proteins in Drosophila . An important goal of future studies will be to determine if any of these types of mechanisms are involved in Dys-induced Trh degradation in tracheal cells (Mortimer, 2007).

The requirement for ago in tracheal cells suggests that the consequences of ago loss vary considerably depending on the proliferative state of the cells, their location within the organism, and their developmental stage. ago mutant clones in the mitotically active larval eye disc show no evidence of excessive Trh levels or deregulated Btl/FGF signaling and conversely, ago zygotic mutant trachea do not display 'extra cell' defects similar to those observed in the eye. The origins of this tissue specificity are currently not clear, although it might simply reflect the differential expression patterns of Ago targets in various mitotic and post-mitotic cell populations. There is currently no evidence that the mammalian Trh homologs NPAS-1 and NPAS-3 are degraded by an Ago/Fbw7 dependent mechanism in mammalian cells. However the finding that Fbw7 knock-out mouse embryos display defects in vascular development (Tetzlaff, 2004; Tsunematsu, 2004) seems to indicate that the Fbw7 ligase may also target proteins involved in tubular morphogenesis, and intriguingly both NPAS1 and NPAS3 have been linked either to this process (Levesque, 2007) or to the transcriptional control of FGFR genes (Pieper, 2005). It has been suggested that the Fbw7 vascular defects arise due to Notch misregulation. However since FGF signaling is known to control branching morphogenesis of the mammalian vasculature and lung, the data presented in this study raise the possibility that vascular phenotypes in Fbw7 knock-out mouse embryos may also reflect the deregulation of developmental pathways that control branching morphogenesis via mammalian homologs of trh and btl (Mortimer, 2007).

Regulation of Drosophila embryonic tracheogenesis by dVHL and hypoxia

The tracheal system of Drosophila is an interconnected network of gas-filled epithelial tubes that develops during embryogenesis and functions as the main gas-exchange organ in the larva. Larval tracheal cells respond to hypoxia by activating a program of branching and growth driven by HIF-1α/sima-dependent expression of the breathless (btl) FGF receptor. By contrast, the ability of the developing embryonic tracheal system to respond to hypoxia and integrate hard-wired branching programs with sima-driven tracheal remodeling is not well understood. This study shows that embryonic tracheal cells utilize the conserved ubiquitin ligase (von Hippel-Lindau) (dVHL) to control the HIF-1 α/sima hypoxia response pathway, and two distinct phases of tracheal development with differing hypoxia sensitivities and outcomes were identified: a relatively hypoxia-resistant 'early' phase during which Sima activity conflicts with normal branching and stunts migration, and a relatively hypoxia-sensitive 'late' phase during which the tracheal system uses the dVHL/sima/btl pathway to drive increased branching and growth. Mutations in the archipelago (ago) gene, which antagonizes btl transcription, re-sensitize early embryos to hypoxia, indicating that their relative resistance can be reversed by elevating activity of the btl promoter. These findings reveal a second type of tracheal hypoxic response in which Sima activation conflicts with developmental tracheogenesis, and identify the dVHL and ago ubiquitin ligases as key determinants of hypoxia sensitivity in tracheal cells. The identification of an early stage of tracheal development that is vulnerable to hypoxia is an important addition to models of the invertebrate hypoxic response (Mortimer, 2008).

The development and survival of an organism are dependent on its ability to adapt to changing environmental conditions. Responses to some environmental changes, for example in nutrient availability, temperature, or oxygen concentration, involve alterations in patterns of gene expression that allow the organism to survive periods of environmental stress. In metazoan cells, the cellular response to reduced oxygen is mediated primarily by the HIF (hypoxia inducible factor) family of transcription factors, which are heterodimers composed of α and β subunits belonging to the bHLH Per-ARNT-Sim (bHLH-PAS) protein family. The HIF-1 αβ heterodimer is the primary oxygen-responsive HIF in mammalian cells and binds to a specific DNA sequence termed hypoxia response element (HRE) present in the promoters of target genes involved in energy metabolism, angiogenesis, erythropoiesis, and autophagy. HIF-1 activity is inhibited under normoxic conditions by two hydroxylase enzymes that use dioxygen as a substrate for catalysis to hydroxylate specific proline or aspartate residues in the HIF-1α subunit. These modifications limit HIF-1 activity by either reducing HIF-1α levels or inhibiting its ability to activate HRE-containing target promoters. One of these inhibitory mechanisms involves the 2-oxoglutarate/Fe(II)-dependent HIF-1 prolyl hydroxylase (HPH), which attaches a hydroxyl group onto each of two conserved proline residues in the oxygen-dependent degradation domain (ODD) of mammalian HIF-1α. These modifications create a binding site in the HIF-1α ODD for the Von Hippel-Lindau (VHL) protein, the substrate adaptor component of a ubiquitin ligase that subsequently polyubiquitinates HIF-1α and targets it for degradation by the proteasome. This degradation mechanism operates constitutively in normoxia and is epistatic to otherwise wide spread expression of HIF-1α mRNA. HIF-1α protein is also modified by a second oxygen-dependent hydroxylase termed Factor Inhibiting HIF (FIH) that hydroxylates an asparagine residue in the HIF-1α C-terminal activation domain. This blocks interaction with the CBP/p300 transcriptional co-factor and thus further restricts expression of HIF-1 responsive genes. These parallel O2-dependent hydroxylation mechanisms by HPH and FIH ensure that HIF-1α levels and activity remain low in normoxic conditions. However as oxygen levels become limiting in the cellular environment, rates of hydroxylation decline and HIF-1α is rapidly stabilized in a form that dimerizes with HIF-1β, translocates to the nucleus, and promotes transcription of HRE-containing target genes (Mortimer, 2008).

Evidence suggests that invertebrate homologs of HIF-1 are also regulated in response to changes in oxygen availability. In the fruit fly Drosophila melanogaster, the HPH homolog fatiga (fga) has been shown to genetically antagonize the HIF-1α homolog similar (sima) during development. The Drosophila VHL homolog dVHL has also been shown to be capable of binding to human HIF-1α and stimulating its proteasomal turnover in vitro. In addition, the Drosophila genome encodes a well-characterized HIF-1β homolog tango (tgo), and two potential FIH homologs (CG13902 and CG10133; Berkeley Drosophila Genome Project) that have yet to be analyzed functionally. Spatiotemporal analysis of sima activation using sima-dependent hypoxia-reporter transgenes has shown that exposure to an acute hypoxic stress induces Sima most strongly in cells of the larval and embryonic tracheal system, while induction of reporter activity in other tissues requires more chronic exposure to low oxygen. The larval tracheal system is composed of an interconnected network of polarized, epithelial tubes that duct gases through the organism. As the trachea acts as the primary gas-exchange organ in the larva, it is thus a logical site of hypoxia sensitivity. During larval stages, specific cells within the tracheal system called 'terminal cells' respond to hypoxia by initiating new branching and growth that results in the extension of fine, unicellular, gas-filled tubes toward hypoxic tissues in a manner somewhat analogous to mammalian angiogenesis . Studies have shown that sima and its upstream antagonist fga function within terminal cells to regulate this process. sima is necessary for terminal cell branching in hypoxia and its ectopic activation, by either transgenic overexpression or loss of fga, is sufficient to induce excess branching even in normoxia. These phenotypes have been linked to the ability of sima to promote expression of the breathless (btl) gene, which encodes an FGF receptor that is activated by the branchless (bnl) FGF ligand. This receptor/ligand pair is known to act via a downstream MAP-kinase signaling cascade to promote cell motility and tubular morphogenesis in a variety of systems. Excessive activation of this pathway within tracheal cells by transgenic expression of btl is sufficient to drive excess branching. Reciprocally, misexpression of the bnl ligand in certain peripheral tissues is sufficient to attract excess terminal cell branching. Indeed production of secreted factors such as Bnl may be a significant part of the physiologic mechanism by which hypoxic cells attract new tracheal growth. Sima-driven induction of btl in conditions of hypoxia thus allows larval terminal cells to enter what has been termed an 'active searching' mode in which they are hyper-sensitized to signals emanating from nearby hypoxic non-tracheal cells (Mortimer, 2008 and references therein).

The role of the btl/bnl pathway in tracheal development is not restricted to hypoxia-induced branching of larval terminal cells. It also plays a critical, earlier role in the initial development of the embryonic tracheal system from the tracheal placodes, groups of post-mitotic ectodermal cells distributed along either side of the embryo that undergo a process of invagination, polarization, directed migration, and fusion to create a network of primary and secondary tracheal branches . btl and bnl are each required for this process via a mechanism in which restricted expression of bnl in cells outside the tracheal placode represents a directional cue for the migration of btl-expressing cells within the placode. Accordingly, btl expression is normally highest in pre-migratory and migratory embryonic fusion cells. In contrast to the larval hypoxic response, sima does not appear to be required for morphogenesis of the embryonic tracheal system. Rather, developmentally programmed signals in the embryo dictate a stereotyped pattern of btl and bnl expression that leads to a similarly stereotyped pattern of primary and secondary tracheal branches. The btl/bnl pathway thus responds to developmental signals to drive a fixed pattern of branching in the embryo, while in the subsequent larval stage it responds to hypoxia-dependent sima activity to facilitate the homeostatic growth of larval terminal cells and tracheal remodeling (Mortimer, 2008 and references therein).

Under normal circumstances, developing Drosophila tissues do not begin to experience hypoxia until the first larval stage, when organismal growth and movement begin to consume more oxygen than can be provided by passive diffusion alone. As a consequence, the first hypoxic challenge normally occurs after the btl/bnl-dependent elaboration of the primary and secondary embryonic branches is complete. Thus, the ability of the larval tracheal system to drive new branching and remodeling via sima and btl represents the response of a developed 'mature' tracheal system to reduced oxygen availability. By contrast the effect of hypoxia on embryonic tracheal development, which requires tight spatiotemporal control of Btl signaling to pattern the tracheal network, is not as well understood. Given that the trachea does not function as a gas-exchange organ until after fluid is cleared from the tubes at embryonic stage 17, it may be that the transcriptional response of embryonic tracheal cells to hypoxia leads to mainly metabolic changes rather than to a btl-driven program of tubulogenesis and remodeling. However, if the embryonic tracheal system does utilize the sima pathway to induce hypoxia-dependent changes in btl gene transcription, then hypoxic exposure of embryos might be predicted to produce a situation of competing developmental and homeostatic inputs that converge on the btl/bnl pathway. The ability of tracheal cells to integrate such signals may then determine whether or not the embryonic tracheal system is able to adapt to oxygen stress, or whether embryonic tracheal development represents a sensitive period during which the organism's ability to respond to changes in oxygen levels is inherently limited by a pre-programmed pattern of developmental gene expression (Mortimer, 2008).

This study shows that the embryonic tracheal system utilizes the dVHL/sima pathway to respond to hypoxia, but that the type and severity of resulting phenotypes depend on the developmental stage of exposure. Hypoxic challenge while embryonic tracheal cells are responding to developmentally programmed btl/bnl migration signals disrupts tracheal development and results in fragmented and unfused tracheal metameres. In contrast, hypoxic challenge at a somewhat later embryonic stage after fusion is complete results in overgrowth of the primary tracheal branches and the production of extra secondary branches. Interestingly, it was found that the threshold of hypoxia required to induce tracheal phenotypes in the early embryo is higher than that required to induce excess branching phenotypes in later embryonic stages, indicating that tracheal patterning events in the embryo are relatively resistant to hypoxia. Genetic analysis indicates that both types of hypoxic tracheal phenotypes -- stunting and overgrowth -- require sima and can be phenocopied in normoxia by reducing expression of the HIF-1α ubiquitin ligase gene dVHL specifically within tracheal cells. Moreover, it was found that reduced dVHL expression in the larval trachea leads to excess terminal cell branching in a manner quite similar to that observed in fga mutants. Molecular and genetic data indicate that excess btl transcription is a major cause of hypoxia-induced tracheal phenotypes. Consistent with this, mutations in the archipelago (ago) gene, which antagonizes btl transcription in tracheal fusion cells, synergize strongly with dVHL inactivation to disrupt tracheal migration and branching. Interestingly, ago mutations also lower the threshold of hypoxia required to elicit tracheal phenotypes in the 'early' embryo, suggesting that the relative activity of the btl promoter can affect hypoxic sensitivity. These findings show that the dVHL/sima pathway plays an important role in tracheal development, and identify two distinct phases of embryonic development that show different phenotypic outcomes of activating this pathway: an early phase during which sima activity conflicts with developmental control of tracheal branching and migration, and a later phase during which the tracheal system uses the dVHL/sima/btl pathway to adapt to hypoxia by increasing its future capacity to deliver oxygen to target tissues (Mortimer, 2008).

Hypoxia-induced remodeling of tracheal terminal cells represents the response of a developed larval tracheal system to reduced levels of O2 in the environment. By contrast, the response of the developing embryonic tracheal system to systemic hypoxia has not been as well characterized. In light of the observation that embryonic tracheal cells display hypoxia-induced activation of a Sima-reporter) and that sima promotes btl expression in larval tracheal cells, embryonic exposure to hypoxia may thus produce a situation in which hard-wired btl/bnl patterning signals in the embryo come into conflict with the type of sima/btl-driven plasticity of tracheal cell branching seen in the larva. This study examined the effect of hypoxia on embryonic tracheal branching and migration. It was found that hypoxia has dramatic effects on the patterns of morphogenesis of the primary and secondary tracheal branches. Surprisingly, varying the timing and severity of hypoxic challenge is able to shift the outcome from severely stunted tracheal branching to excess branch number and enhanced branch growth. Genetic and molecular data indicate that both classes of phenotypes, stunting and overgrowth, involve regulation of sima activity and btl transcription by dVHL, and that the effects of hypoxia on tracheal development can be mimicked in normoxia by tracheal-specific knockdown of dVHL. This observation confirms a central role for dVHL in restricting the hypoxic response in vivo, and identifies a role for dVHL as a required inhibitor of sima and btl during normal tracheogenesis (Mortimer, 2008).

Since Trh and Sima/HIF-1α share a similar consensus DNA binding site, it is likely that the tracheal phenotypes elicited by either hypoxia or dVHL knockdown are to some degree the product of a combined 'Trh/Sima-like' transcriptional activity in tracheal cells. This conclusion is supported both by the general phenotypic similarity (i.e. migration and overgrowth defects) between hypoxia/dVHL knockdown and trh overexpression, by the modest ability of trh alleles to suppress dVHLi phenotypes, and by the previously demonstrated overlap of transcriptional activity between Trh and human HIF-1α. Indeed, Trh is well-established as a required activator of developmental btl expression. However, because the excess Btl activity that occurs in hypoxia or in the absence of dVHL occurs independently of a change in Trh expression, it thus appears to be mediated largely by increased sima activity (Mortimer, 2008).

This analysis suggests that there are two distinct developmental 'windows' of embryogenesis during which hypoxia has opposite effects on tracheal branching. The first corresponds to a period immediately before and during primary branch migration that is relatively insensitive to hypoxia. Embryos in this stage show a minimal response to 1% O2, but show a nearly complete arrest of migration in 0.5% O2. Interestingly, a prior study found that similarly staged embryos (stage 11) respond to complete anoxia by prolonged developmental arrest, from which they can emerge and resume normal development. These somewhat paradoxical results -- that acute hypoxia is more detrimental to development than chronic anoxia -- might be explained by the observation that chronic exposure to low O2 induces Sima activity throughout the embryo while acute exposure activates Sima only in tracheal cells. The former scenario may result in coordinated developmental and metabolic arrest throughout the organism, while in the latter scenario developmental patterns of gene expression in non-tracheal cells may proceed such that tracheal cells emerging from an 'early' hypoxic response find an embryonic environment in which developmentally hard-wired migratory signals emanating from non-tracheal cells have ceased (Mortimer, 2008).

The second type of tracheal response occurs during a later 'window' of embryogenesis after btl/bnl-driven primary and secondary branch migration and fusion are largely complete. It involves sinuous overgrowth of the primary and secondary branches, and duplication of secondary branches. As in the 'early' response, 'late' hypoxic phenotypes are controlled by the dVHL/sima pathway, yet unlike the 'early' response, these phenotypes occur at high penetrance even at 1% O2. Thus the 'late' embryonic tracheal system is relatively sensitized to hypoxia and responds with increased branching in a manner similar to larval terminal cells. Indeed, much as larval branching increases with decreasing O2 levels, it was observed that dorsal trunk growth in the late embryo is graded to the degree of hypoxia. The mechanism underlying the differential sensitivity of the 'early' and 'late' tracheal system may be quite complex. However, it was found that tracheogenesis can be sensitized to hypoxia by reducing activity of ago, a ubiquitin ligase component that restricts btl transcription in tracheal cells via its role in degrading the Trh transcription factor. Increasing transcriptional input on the btl promoter thus appears to sensitize 'early' tracheal cells to hypoxia. As Sima also controls btl transcription, one explanation of the difference in sensitivity between different embryonic stages may thus lie in differences in the activation state of the btl promoter. If so then the activity of the endogenous btl regulatory network may be an important determinant of the threshold of hypoxia required to elicit changes in tracheal architecture (Mortimer, 2008).

An organism can have its hypoxic response triggered in two ways, either by systemic exposure of the whole organism to a reduced O2 environment or by localized hypoxia produced by increased O2 consumption in metabolically active tissues. Data from this study and others suggests there may be distinctions between these two triggers. Exposing larvae or embryos to a systemic pulse of hypoxia results in a 'btl-centric' response specifically in tracheal cells. Outside of an 'early' vulnerable period which corresponds to embryonic branch migration and fusion, elevated Btl activity in embryonic tracheal cells promotes branch duplications and overgrowth similar to that seen in larvae. By contrast, tracheal growth induced by localized hypoxia in the larva has been suggested to involve a 'bnl-centric' model in which the hypoxic tissue secretes Bnl and recruits new tracheal branching. Whether this type of mechanism operates in embryos, or whether embryos ever experience localized hypoxia in non-tracheal cells, has not been established (Mortimer, 2008).

tHE data indicate that dVHL is a central player in the hypoxic response pathway in embryonic and larval tracheal cells. A prior study found that injection of dVHL dsRNA into syncytial embryos disrupted normal tracheogenesis, but was technically limited in its ability to conduct a detailed analysis of dVHL function in development and homeostasis. The current study found that dVHL knockdown specifically in tracheal cells mimics the effect of systemic hypoxia on embryonic tracheal architecture and larval terminal cell branching. dVHL knockdown thus phenocopies loss of the HPH gene fga, which normally functions to target Sima to the dVHL ubiquitin ligase in normoxia. Moreover, all phenotypes that result from reduced dVHL expression can be rescued by reducing sima activity, suggesting that Sima is the major target of dVHL in the tracheal system. These data support a model in which dVHL, fga, and sima function as part of a conserved VHL/HPH/HIF-1α pathway to control tracheal morphogenesis in embryos and larvae. The btl receptor appears to be an important target of this pathway in embryonic (this study) and larval tracheal cells. Knockdown of dVHL elevates btl transcription in embryonic placodes and tracheal branches, and removal of a copy of the gene effectively suppresses dVHL tracheal phenotypes. Reciprocally, overexpression of wild type btl in embryonic tracheal cells can produce migration defects and sinuous overgrowth, while expression of a constitutively active btl chimera (btlλ) also leads to primary branch stunting and duplication of secondary branches. Interestingly, pupal lethality associated with tracheal-specific knockdown of dVHL is not sensitive to the dose of btl, but is dependent on sima. Thus the dVHL/sima pathway may have btl independent effects on tracheal cells in later stages of development (Mortimer, 2008).

EFFECTS OF MUTATION

Archipelago regulates Cyclin E levels in Drosophila and is mutated in human cancer cell lines

During Drosophila development and mammalian embryogenesis, exit from the cell cycle is contingent on tightly controlled downregulation of the activity of Cyclin E-Cdk2 complexes that normally promote the transition from G1 to S phase. Although protein degradation has a crucial role in downregulating levels of Cyclin E, many of the proteins that function in degradation of Cyclin E have not been identified. In a screen for Drosophila mutants that display increased cell proliferation, archipelago, a gene encoding a protein with an F-box and seven tandem WD (tryptophan-aspartic acid) repeats, has been identified. archipelago mutant cells have persistently elevated levels of Cyclin E protein without increased levels of cyclin E RNA. They are under-represented in G1 fractions and continue to proliferate when their wild-type neighbors become quiescent. The Archipelago protein binds directly to Cyclin E and probably targets it for ubiquitin-mediated degradation. A highly conserved human homolog is present and is mutated in four cancer cell lines including three of ten derived from ovarian carcinomas. These findings implicate archipelago in developmentally regulated degradation of Cyclin E and potentially in the pathogenesis of human cancers (Moberg, 2002).

To identify genes that restrict cell numbers and tissue growth during development, a genetic screen was conducted to identify recessive mutations that give homozygous mutant cells even a subtle proliferative advantage over their wild-type neighbors. Clones of homozygous mutant tissue were generated in the eyes of heterozygous flies and their size was compared with the wild-type twin spots generated from the same recombination events. Flies were retained in which there was more mutant than wild-type tissue. Of more than 23 loci identified in the screen, multiple alleles were obtained of homologs of several known human tumor-suppressor genes, including TSC1, TSC2 and PTEN. A previously unknown locus, represented by a lethal complementation group consisting of three alleles, was named archipelago (Moberg, 2002).

Compared with an unmutagenized control, adult eyes mosaic for mutations in ago were composed mostly of mutant tissue. Within ago mutant clones, most ommatidial clusters lacked the wild-type complement of photoreceptor cells, and the distance between adjacent photoreceptor clusters was increased. Staining of apical cell profiles during pupal eye development show that the enlarged interommatidial spaces in ago mutant clones contain excess cells. TUNEL revealed no significant decrease in the extent of cell death in ago mutant clones. Moreover, co-expression of the baculovirus p35 protein, which blocks caspase-dependent cell death, resulted in a further increase in the number of interommatidial cells. These data suggest that loss of ago leads to increased cell proliferation that is partially offset by apoptosis (Moberg, 2002).

Because ago mutant cells proliferate more than wild-type cells, it seemed likely that ago mutations would result in increased levels of a positive regulator of the cell cycle. Clones of homozygous mutant tissue were generated in eye imaginal discs and then examined for changes in cyclin levels. In wild-type third-instar discs, Cyclin E is expressed at varying levels and is unpatterned in cells anterior to the morphogenetic furrow; this lack of pattern is thought to correlate with expression at specific stages of the cell cycle in cells that are proliferating asynchronously. A strong stripe of expression can be found immediately posterior to the furrow; this condition corresponds to cells of the second mitotic wave. Cells posterior to the second mitotic wave express low levels of Cyclin E. In clones of ago mutant tissue anterior to the furrow, almost all cells express high levels of Cyclin E. Clones posterior to the furrow display mild elevations in Cyclin E levels. In contrast to the increase in Cyclin E protein, no alteration in the expression pattern of cyclin E RNA is observed in discs that contain many large ago mutant clones. In wild-type discs, ago mRNA is also expressed throughout the disc, but is expressed at particularly high levels in the morphogenetic furrow. In contrast to the results obtained with Cyclin E, the levels of Cyclin B, Cyclin A and the SCF substrates Cubitus interruptus, Armadillo and Tramtrack are not appreciably elevated in ago mutant clones in the larval imaginal disc, nor are the levels of the putative substrates Dacapo and the intracellular domain of Notch (Moberg, 2002).

Because Cyclin E promotes S-phase entry, an increase in the level of Cyclin E can perturb regulation of the cell cycle. To examine the proliferative properties of ago mutant cells, ago clones were generated in the wing disc of third-instar larvae and their DNA content was compared with that of wild-type cells from the same imaginal discs. In mutant clones, a smaller fraction of cells (21.4%) is found in G1 when compared with wild-type cells (36.8%). The proportion of cells found in S phase and in G2/M is increased. These alterations are extremely similar to those elicited by the overexpression of Cyclin E (Moberg, 2002).

The effect of ago mutations on the patterns of cell proliferation in vivo was also examined. Cells anterior to the morphogenetic furrow in the larval eye disc proliferate asynchronously. It is therefore difficult to visualize differences in rates of cell proliferation in mutant clones at this stage of eye development. In contrast, very few cells proliferate in the wild-type pupal retina. The bristle precursor cell is the only mitotically active cell type detected during this stage; it divides twice during the mid-pupal phase to generate the four cells of the 'bristle complex'. Levels of Cyclin E rapidly decrease after these divisions. In ago mutant clones, elevated levels of Cyclin E are detected in the four cells of the bristle complex well after the levels in the corresponding cells of adjacent wild-type ommatidia have declined. Some of these ago mutant cells also continue to incorporate 5-bromodeoxyuridine (BrdU), suggesting that they continue to cycle after the corresponding cells in adjacent wild-type tissue have exited from the cell cycle. Such additional divisions are likely to contribute to the increased number of interommatidial cells observed in the pupal retina. Thus, the persistence of Cyclin E in ago mutant cells disrupts exit from the cell cycle in a manner similar to that elicited by Cyclin E overexpression (Moberg, 2002).

The simplest explanation of the role of ago in cell cycle control is that Ago binds to Cyclin E and targets it for ubiquitin-mediated degradation. Genetic and physical interactions between Ago and Cyclin E were therefore sought. A genetic interaction was observed between ago and the cyclin EJP allele, which reduces the levels of cyclin E transcription in the developing eye. The rough-eye phenotype of cyclin EJP flies is suppressed in flies that are also heterozygous for a mutation in ago. In addition, ago mutations dominantly suppress the small-eye phenotype produced by eyGAL4-driven overexpression of the cyclin-dependent kinase inhibitor dacapo, which has been shown to reduce Cyclin E-Cdk2 activity. Thus flies that are heterozygous for mutant alleles of ago are likely to have increased levels of Cyclin E (Moberg, 2002).

To test for a direct physical interaction between Archipelago and Cyclin E, the portion of Archipelago containing the F-box and WD repeats was expressed as a protein fused to glutathione S-transferase (GST: GST-AgoDeltaN) and its ability to bind Cyclin E protein was evaluated in lysates of S2 cells transfected with cyclin E and cdk2. Versions of GST-AgoDeltaN were generated that contained the mutations found in the ago1 and ago3 alleles. Binding was readily detected with the wild-type version of GST-AgoDeltaN and was greatly reduced with both mutant versions. Thus the ability of Archipelago to bind Cyclin E in vitro correlates with its ability to downregulate Cyclin E levels in vivo (Moberg, 2002).

These findings, together with the observation that mutations in the C. elegans genes cul1 and lin-23 (which encode a cullin and an F-box protein respectively) have increased cell divisions, highlight the importance of SCF-mediated degradation in regulating cell proliferation through Cyclin E. Because ago RNA is expressed in a dynamic pattern, these results indicate that degradation of Cyclin E is not constitutive in vivo. Dynamic expression of Ago provides another mechanism by which cyclin/cdk activity and cell proliferation can be regulated during development. Finally, impaired proteolysis of Cyclin E is implicated in the pathogenesis of human cancers (Moberg, 2002).

The Drosophila F box protein Archipelago regulates dMyc protein levels in vivo

archipelago (ago) mutations lead to overproliferation of mutant tissue in the developing Drosophila eye, and ago mutant cells express elevated levels of Cyclin E protein and are delayed in their exit from the cell cycle (Moberg, 2001). The Ago protein, as well as its human ortholog Fbw7/hCDC4, is the F box component of an SCF E3-ubiquitin ligase, and Ago binds Cyclin E and targets it for ubiquitination and subsequent degradation. The overgrowth of ago mutant tissue implies that the ago mutant cells collectively grow (i.e., accumulate mass) at an accelerated rate in comparison to wild-type cells. Because Drosophila Cyclin E has been shown to promote S phase entry but not growth, the increased growth of ago mutant cells suggests that there are other Ago substrates that promote cell growth (Moberg, 2004).

To examine the growth properties of ago mutant cells, marked pairs of ago mutant clones and wild-type sister clones (twin spots) were generated in the developing larval wing imaginal disc. ago mutant clones in the wing and the eye are consistently larger and contain more cells than their respective twin spots (labeled control). The increased cell number in ago clones indicates that ago mutant cells divide more frequently over a fixed period of time than do control cells and thus have a shortened cell cycle duration. Indeed, the calculated length of an average cell cycle in ago cells is approximately 15% shorter than in control cells. However, flow-cytometric analysis indicates that ago cells are not decreased in size. These observations indicate that ago cells coordinately accelerate rates of cell growth and cell division such that normal cell size is maintained (Moberg, 2004).

Because dMyc has been shown to promote growth in imaginal-disc cells, the role of Ago in regulating dMyc levels was examined further. Eye imaginal discs containing ago mutant clones were stained with an anti-dMyc monoclonal antibody. dMyc protein is strongly elevated in ago mutant cells, both in third-larval-instar eye discs and in early pupal-phase eye discs. Increased dMyc staining is observed in ago cells found throughout the larval eye disc and antennal discs. In the pupal eye disc, dMyc levels are especially high in the clusters of nuclei of the bristle cell complex. Immunoblot analysis also indicates that dMyc levels are highly elevated in extracts of ago mutant discs. In this experiment, the twinspots carry two copies of a strong Minute mutation [M(3)] that dramatically impairs their growth such that more than 95% of the disc cells are ago mutant. The dMyc protein detected in ago/M(3) discs also appears to have reduced mobility in SDS-PAGE, suggesting that in the absence of Ago, dMyc accumulates in a modified form. Significantly, overexpression of cyclin E in larval eye discs did not detectably alter dMyc levels, suggesting that accumulation of dMyc protein in ago mutant cells is not an indirect effect of the concurrent deregulation of Cyclin E levels (Moberg, 2004).

To determine if dMyc-dependent transcription is also deregulated in ago cells, expression of a dMyc target gene was examined in ago/M(3) and FRT80B discs. In three independent RNA preparations from equal numbers of staged discs, ago mutant discs were observed to contain approximately twice as much total RNA as control discs. Since ago/M(3) and FRT80B discs are approximately the same size and contain similar levels of total protein, this indicates that ago mutant cells contain more RNA than control cells. The increase in the amount of RNA appears to result from a disproportionate increase in rRNA in ago/M(3) discs. As a result, when equal amounts (4 μg) of total RNA are analyzed by Northern blotting, a control RNA (β-Tubulin 56D mRNA) is less abundant in the ago/M(3) sample compared to FRT80B. In contrast, the level of RNA of the dMyc target gene pitchoune is increased. If this change were normalized to the levels of β-Tubulin 56D RNA, this would represent an approximately 3-fold increase of pitchoune RNA in ago/M(3) discs relative to the wild-type. These findings provide evidence for increased expression of a putative dMyc target gene in ago mutant cells (Moberg, 2004).

To begin to examine how ago normally functions to inhibit dMyc levels, in situ analysis of dMyc mRNA on FRT80B and ago/M(3) larval eye discs was performed. In control discs, an anti-sense dMyc RNA probe detects dMyc expression at low levels throughout the eye disc, with a stronger stripe immediately posterior to the morphogenetic furrow, whereas a sense dMyc RNA probe produces no discernable staining in wild-type discs. The pattern and level of dMyc expression is unchanged in ago/M(3) discs. It is possible that subtle increases in dMyc mRNA levels are below the limits of the detection techniques used in this study, but it is clear that the anti-sense dMyc RNA probe easily detects increased dMyc transcripts in pGMR-Gal4;UAS-dMyc eye discs. These data suggest that ago inhibits dMyc accumulation largely through a posttranscriptional mechanism (Moberg, 2004).

Because Ago and dMyc proteins interact, whether perturbing Ago function in S2 cells could modulate dMyc levels and stability was examined. A putative dominant-negative form of Ago that lacks the F box domain (AgoΔF) was constructed. AgoΔF is predicted to bind target proteins via an intact WD repeat domain but to be unable to recruit them into SCFAgo. Expression of AgoΔF is thus predicted to stabilize SCFAgo targets. Coexpression of dMyc and AgoΔF in cells increases the amount of Ago-dMyc complex recovered in coimmunoprecipitation experiments and increases the overall levels of dMyc in these cells. To determine whether AgoΔF expression alters dMyc stability, dMyc levels were assayed in the presence or absence of coexpressed AgoΔF protein after treatment with the translation inhibitor cycloheximide (CHX) . When dMyc is expressed alone, its levels decline rapidly after CHX treatment, indicating that dMyc is normally quite unstable. In contrast, dMyc coexpressed with AgoΔF persists longer in cells after CHX addition, indicating that dMyc is more stable when SCFAgo activity is reduced. In support of this, treatment of cells with double-stranded ago RNA (dsRNA) or with the proteasome inhibitor MG132 was found to increase the amount of transfected dMyc detected in cells. These data indicate that dMyc is degraded via the proteasome in vivo in a manner similar to mammalian c-Myc and that, as the substrate specificity component of an SCFAgo ubiquitin-ligase, Ago is likely to participate in this process (Moberg, 2004).

In addition to Ago, one or more of the 23 other F box proteins encoded by the Drosophila genome might also play a role in regulating dMyc levels in vivo. Of particular note are the Drosophila F box proteins Slmb and CG9772. The Slmb protein, encoded by the supernumerary limbs (slmb) gene, is the Drosophila protein most similar to Ago within the F box and WD repeats. The gene CG9772 may encode the Drosophila ortholog of the human F box protein Skp2 (54% similarity and 31% identity between CG9772-PA and Skp2 across their length), which has recently been shown to regulate c-Myc levels in a transformed mammalian cell line (Moberg, 2004).

To assess the relative roles of these F box family members in regulating endogenous dMyc levels, double-stranded RNA interference (dsRNAi) was used in S2 cells to reduce the RNA levels of ago, slmb, and CG9772. Reducing ago function in S2 cells by ago dsRNAi results in the stabilization and accumulation of dMyc protein. In contrast, despite significant reduction in the levels of slmb and CG9772 RNAs, there is no discernible change in dMyc levels. Whether the CG9772-PA protein (a CG9772 isoform containing the complete F box and leucine-rich repeat domains) could bind dMyc in a manner similar to Skp2, its putative human ortholog, was also tested. Ago and CG9772 accumulate to similar levels in the absence of coexpressed dMyc. However, CG9772-PA displayed very little dMyc binding activity compared to Ago. Although these data do not rule out a role for other F box proteins in regulating dMyc levels and/or activity, they do indicate that at least in S2 cells, and among the Ago, Slmb, and CG9772 proteins, only Ago is able to bind to dMyc and regulate its stability (Moberg, 2004).

Consistent with the observed physical interaction between the Ago and dMyc proteins, it was found that an ago mutation could modify dMyc mutant phenotypes. The dMyc allele diminutive1 (dm1) is a hypomorphic viable mutation caused by a gypsy element insertion into the first intron of the dMyc genomic locus. dm1 homozygous females and dm1 hemizygous males are smaller than wild-type flies, and the females are sterile. dm1 flies that are heterozygous for ago (dm1;ago1/+) are larger than dm1 flies. Quantitation of this effect shows that heterozygosity for a mutation in ago increases dm1 female body length by approximately 12%. The wings of these dm1;ago1/+ adults are also approximately 15% larger than those of dm1 adults. To determine whether these effects are due to an increase in cell number, cell size, or both, wing hair density was determined in the relevant genotypes. Because each cell in the wing generates a single hair, hair density varies inversely with cell size. The hair density in dm1, dm1;ago/+, and wild-type wings is the same, indicating that the cells are of comparable size. Thus, dm1 wings are small because they contain fewer cells, and a 2-fold reduction in wild-type ago gene dosage increases the size of these mutant wings by increasing the number of normally sized cells. However, ago mutations do not rescue size defects associated with the dMyc alleles dmP0 and dmP1. These are stronger loss-of-function mutations than dm1 and reduce organism size by reducing cell size, with little effect on cell number. dm1 may therefore represent a weaker dMyc allele whose body-size phenotype remains sensitive to ago gene dosage (Moberg, 2004).

In addition to restoring cell number in the dm1 mutant wings, reducing ago function can also ameliorate the female fertility defect of dMyc mutant animals. Unlike dm1;FRT80B/+ females, dm1;ago1/+ females lay eggs that can give rise to viable larvae. In addition, dm1/dmPG45;ago/+ females are approximately 10-fold more fertile than dm1/dmPG45;FRT80B/+ females, which normally show a 2%-3% egg hatching rate. These data indicate that, in addition to modifying dMyc organ and organism size phenotypes, ago is also an antagonist of dMyc in the female germline (Moberg, 2004).

Consistent with a role for Ago in inhibiting dMyc, ago expression retards organ growth. Expression of an N-terminally truncated ago cDNA, that contains an intact F box domain and WD repeat region, in the posterior compartment of the wing decreases posterior compartment size by approximately 35%. Hair density measurements indicate that cells in the ago-expressing compartment are approximately 32% smaller than controls, suggesting that the reduction in compartment size is largely an effect of reduced cell size. Thus, overproduction of an N-terminally truncated version of Ago in the developing wing mimics the effect of dMyc mutations and inhibits growth (Moberg, 2004).

GMCs in an embryo overexpressing Cyclin E, or in an embryo mutant for archipelago, undergo self-renewing asymmetric divisions.

In the Drosophila CNS, neuroblasts undergo self-renewing asymmetric divisions, whereas their progeny, ganglion mother cells (GMCs), divide asymmetrically to generate terminal postmitotic neurons. It is not known whether GMCs have the potential to undergo self-renewing asymmetric divisions. It is also not known how precursor cells undergo self-renewing asymmetric divisions. Maintaining high levels of Mitimere or Nubbin, two POU proteins, in a GMC causes it to undergo self-renewing asymmetric divisions. These asymmetric divisions are due to upregulation of Cyclin E in late GMC and its unequal distribution between two daughter cells. GMCs in an embryo overexpressing Cyclin E, or in an embryo mutant for archipelago, also undergo self-renewing asymmetric divisions. Although the GMC self-renewal is independent of inscuteable and numb, the fate of the differentiating daughter is inscuteable and numb-dependent. These results reveal that regulation of Cyclin E levels, and asymmetric distribution of Cyclin E and other determinants, confer self-renewing asymmetric division potential to precursor cells, and thus define a pathway that regulates such divisions. These results add to understanding of maintenance and loss of pluripotential stem cell identity (Bhat, 2004).

Maintenance of a self-renewing fate can be viewed as a state where activities of certain genes maintain that state. Once the activity of such genes is switched off, the cells become committed to a differentiation pathway. The results reported in this study indeed support this type of mechanism. That POU genes might be a class of genes that maintain a self-renewing capacity is indicated by the fact that the Oct4 POU gene (Pou5f1 -- Mouse Genome Informatics), which is expressed in pluripotent stem cells of the mouse early embryo, is turned off when these cells begin to differentiate (Rosner, 1990). Similarly, SCIP is expressed in the progenitors of oligodendrocytes, but it is downregulated when these cells are induced to differentiate (Collarini, 1992). The current results provide direct evidence that these genes can induce a cell that is committed to a differentiation pathway to acquire a self-renewing capability in a lineage specific manner. Moreover, studies undertaken in the past several years using the Drosophila nervous system as a paradigm have revealed how asymmetry can be generated during cell division to produce two distinct postmitotic cells. However, there is very little information on how an asymmetric self-renewing division pattern is determined. In this paper, results are presented that provide insight into this particular process. (Bhat, 2004).

The strongest evidence that a GMC-1 undergoes a self-renewing type of asymmetric division in embryos overexpressing miti/nub or CycE, and in embryos mutant for ago, comes from the presence of hemisegments with two sibs and one RP2. There are two ways the second sib cell can be generated: (1) a self-renewed GMC-1 generates another sib when it divides, and (2) some other cell is transformed into a sib. The following set of evidence indicates the former scenario: (1) the second sib cell always appears later in development, i.e. at ~8.5 hours of age (as opposed to in wild type where the GMC-1 terminally divides by ~7.5 hours of age into an RP2 and a sib); (2) the dynamics of Eve expression itself in the sib -- expression of eve is switched off in a sib during the asymmetric division of GMC-1 and there is no de novo synthesis of Eve thereafter. If a postmitotic cell from an Eve-negative lineage transforms into a sib, it would be negative for Eve and would not be detected. The development of the other Eve-positive neuronal lineages is normal in these embryos, thus it is unlikely that a cell from those Eve-positive lineages is transformed into a sib. (3) The Eve and Spectrin staining of UAS-nub; ftz-GAL4 embryos provides more direct evidence for the self-renewal of GMC-1. In ~8. 5-hour-old UAS-nub; ftz GAL4 embryos, the larger GMC-1 (this Eve-positive cell is Zfh1 negative, indicating that it is indeed a GMC-1) can be observed undergoing asymmetric cytokinesis for the second time. From the heat-shock induction experiments of nub or miti mutant embryos, it can be argued that higher levels of these proteins in the parental NB4-2 cause later born GMCs to adopt a GMC-1 fate. However, the GMC-1 self-renewing phenotype observed following targeted expression of nub using the ftz-GAL driver makes this scenario unlikely. (4) The results obtained with the mitiP; insc and mitiP; nb double mutant embryos (P referring to prolonged expression), and the mis-localization of Insc in GMC-1 of these embryos, are also consistent with this conclusion. (Bhat, 2004).

These results indicate that the level, timing and duration of presence of Miti or Nub proteins determine the dynamics of the GMC-1 division pattern. For example, the asymmetric divisions (which generate the 3-cell phenotypes) and the symmetric divisions (which generate the 4-cell phenotype) were observed when the transgenes were induced for 20-25 minutes. However, the multiple cell-phenotype was observed only when the transgenes were induced for 90 minutes. Once the induction was stopped and the levels returned to normal, the two GMC-1s appeared to exit from the cell cycle to generate postmitotic cells. Similarly, when the transgene was induced with ftz-GAL4, only the 3-cell phenotypes, and not the 4-cell or multi-cell phenotypes were observed. Thus, the following picture emerges from these results. Although high levels of Miti and Nub proteins are required for the specification of GMC-1 identity, their level must be downregulated in order for the GMC-1 to divide asymmetrically into postmitotic RP2 and sib. Maintaining a high level of these proteins in GMC-1 commits that cell to adopt a self-renewing stem cell type of division pattern. The results described here also show that Miti and Nub prevent GMC-1 from exiting the cell cycle by upregulation of CycE (Bhat, 2004).

The results clearly show that upregulation of CycE in late GMC-1 is the cause for the adoption of a self-renewing asymmetric division pattern. In other words, presence of high levels of CycE in late GMC-1 and its unequal distribution to one of the two daughter cells prevents this cell from exiting the cell cycle. Since this daughter cell still maintains the GMC-1 identity and has sufficient CycE to divide again, a further asymmetric division(s) is ensured. The cell that has lower amounts of CycE becomes committed to a differentiation pathway (RP2 or sib) (Bhat, 2004).

What lines of evidence support this conclusion? (1) In contrast with wild type, there is a significant amount of CycE present in a late GMC-1 in embryos overexpressing miti or nub. This CycE preferentially segregates to one of the two daughters of that GMC-1, usually the larger cell. When miti or nub genes are overexpressed only briefly, the level of CycE is downregulated after just one additional round of division, whereas with prolonged induction, the level is maintained at high levels in one or two cells of the multi-cell cluster for a prolonged duration of time (Bhat, 2004).

(2) Upregulation of CycE in a late GMC-1 is also observed in embryos mutant for ago, which is known to regulate CycE levels. In ago mutants, the two daughter cells of such a GMC-1 have unequal CycE levels accompanied by a self-renewing asymmetric division phenotype. The CycE is always downregulated after one additional GMC-1 division, which is consistent with the finding that the self-renewal occurs only once in these embryos. Since penetrance in ago mutants is partial, and CycE is downregulated in this lineage after just one additional division, there must be additional factors that mediate the downregulation of CycE in this lineage (Bhat, 2004).

(3) Embryos expressing high levels of CycE from a CycE transgene exhibit the same GMC-1 phenotypes as embryos expressing high levels of Miti or Nub. Thus, these results indicate that upregulation of CycE alone is sufficient for the GMC-1 to adopt a self-renewing type of division pattern. Finally, mitiP phenotypes are found to be dependent on CycE. That is, no multi-cell clusters were observed in mitiP; CycE double mutant embryos (Bhat, 2004).

In wild type, the downregulation of CycE in GMCs appears to occur through switching off CycE transcription and degradation of the protein by factors such as Ago. At what level does Miti or Nub regulate CycE? Since POU genes are thought to be transcriptional activators, they can regulate transcription of CycE either directly or indirectly. However, this does not seem to be the case since expressing high levels of miti does not have a discernible effect on the levels of CycE mRNA in GMC-1, as assessed by whole-mount RNA in situ hybridization. In addition, the putative promoter/enhancer region of CycE gene does not contain any consensus POU protein-binding sites. Therefore, it seems likely that Miti and Nub regulate factors that are involved in the degradation of CycE in late GMC-1 (Bhat, 2004).

The question arises as to how only one cell has a high level of CycE. There are several ways this can happen. There might be an asymmetric degradation of CycE. This scenario seems unlikely since there is only one of two daughter cells with high levels of CycE in ago mutants. Given that Ago downregulates CycE via a protein degradation mechanism, if there was an asymmetric degradation, in those hemisegments where the levels of CycE was elevated in GMC-1, it would initially be expected that both the daughter cells would have high CycE levels. However, this was not the case. An asymmetric transcription of the CycE gene also seems unlikely since the transcription of CycE ceases prior to GMC-1 division, as judged by whole-mount RNA in situ hybridization. The most likely possibility is that CycE is unequally distributed between the two daughter cells of GMC-1. The unequal distribution of CycE could be a passive process due to the size difference between daughter cells, especially in the GMC-1-->RP2/sib lineage. Moreover, no cytoplasmic crescent of CycE was observed during mitosis. By contrast, it could also be an active process. For instance, the size difference between an aCC and a pCC (or between a GMC1-1a and an aCC) is very small, and the fact that GMC1-1a undergoes a self-renewing asymmetric division suggests that the segregation of CycE may not be entirely a passive process (Bhat, 2004).

Finally, the results indicate that while a GMC that does not normally express Miti or Nub is insensitive to its ectopic expression (e.g., GMC1-1a of NB1-1; this GMC produces an aCC/pCC pair of neurons), a brief induction of CycE in the same GMC causes it to undergo self-renewing asymmetric division. Therefore, CycE can confer a stem cell type of division potential to more than one GMC. Another important conclusion one can draw from this result is that the segregation of CycE may be an active process. In the case of GMC1-->RP2/sib lineage, the cytokinesis of GMC-1 is asymmetric, and the size difference between an RP2 and a sib is significant. Thus, CycE can be asymmetrically segregated because of this size difference. However, the size difference between an aCC and a pCC (or between a GMC1-1a and an aCC) is very small, and the fact that GMC1-1a undergoes a self-renewing asymmetric division suggests that the segregation of CycE may not be entirely a passive process. It is possible that the difference between the levels of CycE needed to retain a cell within the cell cycle and the levels that do not support maintaining the cell within the cell cycle are quite small. Thus, even a minor change in the amount that a cell receives during division might be sufficient to make a difference. Thus, the segregation of CycE can still be a passive process. Nonetheless, these results reveal how a cell can adopt a self-renewing asymmetric division potential through CycE. (Bhat, 2004).

CycE and Ago are part of a mechanism that converts a normal cell into a cancer cell. In ago mutants, CycE protein is not degraded and a number of cancer cell lines carry a mutation in ago. The current results showing that these genes are also involved in a stem cell type of division suggest a commonality between stem cells and cancer cells. These results also provide a molecular mechanism of how self-renewing asymmetric divisions are possible (Bhat, 2004).

Ago/hCdc4 (Archipelago) is crucial for endocycle progression in the follicular epithelium

The Notch signaling pathway controls the follicle cell mitotic-to-endocycle transition in Drosophila oogenesis by stopping the mitotic cycle and promoting the endocycle. To understand how the Notch pathway coordinates this process, a functional analysis was performed of genes whose transcription is responsive to the Notch pathway at this transition. These genes include String, the G2/M regulator Cdc25 phosphatase; Hec/CdhFzr, a regulator of the APC ubiquitination complex and Dacapo, an inhibitor of the CyclinE/CDK complex. Notch activity leads to downregulation of String and Dacapo, and activation of Fzr. All three genes are independently responsive to Notch. In addition, CdhFzr, an essential gene for endocycles, is sufficient to stop mitotic cycle and promote precocious endocycles when expressed prematurely during mitotic stages. In contrast, overexpression of the growth controller Myc does not induce premature endocycles but accelerates the kinetics of normal endocycles. F-box/WD40-domain protein Ago/hCdc4 (Archipelago), a SCF-regulator is dispensable for mitosis, but crucial for endocycle progression in follicle epithelium. CycE oscillation remains critical for endocycling; continuous high level of CycE expression blocks the cell cycle in G2. The regulation of CycE levels is achieved by the function of Ago that presumably binds to auto-phosphorylated CycE and directs it to SCF-complex degradation: high levels of CycE and no endocycling is observed in ago-clones. The results support a model in which Notch activity executes the mitotic-to-endocycle switch by regulating all three major cell cycle transitions. Repression of String blocks the M-phase, activation of Fzr allows G1 progression, and repression of Dacapo assures entry into the S-phase. This study provides a comprehensive picture of the logic that external signaling pathways may use to control cell cycle transitions by the coordinated regulation of the cell cycle (Shcherbata, 2004).

The exit from mitosis and/or progression through G1 requires the inactivation of cyclin-dependent kinases, mediated by the APC/C-dependent destruction of cyclins. APC/C is regulated by multiple mechanisms, such as phosphorylation and by spindle checkpoints. Key factors for APC/C function and regulation are the WD proteins Cdc20 and Hec1/Cdh. These proteins seem to bind directly to substrates and recruit them to the APC/C core complex. Importantly, Cdc20 and Hec1/Cdh bind and activate APC/C in a sequential manner during mitosis. APC/C-Cdc20 is activated at the metaphase/anaphase transition, and gets replaced by APC/C-Hec1/Cdh in telophase. This second complex remains active in the subsequent G1 phase. In Drosophila the homolog of Hec1/Cdh, Fzr, also induces the APC/C-complex-dependent proteolysis of CycA and B and is required for the G1-phase progression. Fzr is required for cyclin removal during G1 when the embryonic epidermal cell or follicle epithelial proliferation stops and the cells enter endocycles. Premature Hec1/CdhFzr transcription in follicle cells is sufficient to block mitosis and initiate precocious endocycling. This suggests that Fzr is a powerful player in the mitotic-to-endocycle switch, yet regulation of other components is also required for the efficiency of this process. Regulators of G1-S transition, such as Dacapo/CIP/KIP, which also turns out to be a Notch-regulated component, possibly abort premature attempts by follicle cells to enter the endocycle (Shcherbata, 2004).

The data suggest that a component regulating growth and thereby the kinetics of G1/S transition in follicle cell endocycles is the Myc oncogene instead and independent of CycD. In mammals c-Myc controls the decision to divide or not to divide and thereby functions as a crucial mediator of signals that determine organ and body size. Interestingly, overexpression of Myc in follicle cells does not affect the mitotic cycles but induces, instead, extra endocycles. Because the timing for entering and exit from the endocycles has not changed, however, increased ploidy is observed; therefore, it is suggested that the rate of endocycles is increased because of the overexpression of Myc. This finding is in accordance with recent loss-of-function analysis on myc in follicle cells, suggesting that myc mutant follicle cells can make the transition from mitosis to the endocycle, but that they can only very inefficiently support the endocycle. Therefore, both loss-of-function and overexpression experiments suggest that Myc is an essential component for the proper rate of endocycles in follicle cells (Shcherbata, 2004).

In addition to Myc and Cyclin D, Cyclin E also plays an important role in the regulation of the G1/S-transition. Cyclin E binds to and activates the cyclin-dependent kinase Cdk2, and thereby promotes the transition from G1 to S. Oscillation of Cyclin E activity is a mechanism responsible for the timely inactivation of this G1 cyclin/Cdk complex and an arrest in cell proliferation. The oscillation of Cyclin E level is controlled partly by a SCF-ubiquitin-dependent proteolysis. Fluctuations of Cyclin E are critical for multiple rounds of endocycles. Cyclin E is critical for endocycles in follicle cells as well, and this analysis shows that the CycE level is controlled by an SCF-regulator, F-box protein, Ago/hCdc4/Fbw7. Fbw7 (Ago) associates specifically with phosphorylated Cyclin E, and catalyzes Cyclin E ubiquitination in vitro. Depletion of Ago leads to accumulation and stabilization of Cyclin E in vivo in human and D. melanogaster. This leads to increased mitosis in certain mammalian and Drosophila cell types. In addition, ago loss-of-function clones in the germ line will cause extra mitotic divisions or, in contrast, cell cycle arrest and polyploidy. However, increased Cyclin E levels observed in ago loss-of-function mutant clones do not affect the mitotic cycles in follicle cells but do halt the transition to endocycles that normally occurs at stage 6 (Shcherbata, 2004).

Why is the function of Ago/hCdc4/Fbw7 critical to endocycles but not to mitotic cycles in follicle epithelial cells? A potential answer might reside in Dacapo, a CIP/KIP-type inhibitor of Cyclin E/Cdk2 complexes that is regulated in the mitotic to endocycle transition by activation of Notch pathway. dacapo is downregulated at mitotic-to-endocycle transition because of Notch activation and ectopic expression of dacapo represses endocycle progression. It is plausible that during mitotic phases Ago and Dacapo share a redundant role for regulating the Cyclin E activity level, however, dacapo is downregulated by Notch pathway at the time of mitotic-to-endocycle transition and at that point Ago gains the critical role of sole regulator of Cyclin E protein activity level. However, downregulation of Dacapo does not readily explain the reduction of CycE levels observed in mitotic-to-endocycle transition. Elevation of CycE protein level is detected in response to Dacapo overexpression, pointing out that this CKI may stabilize CycE in an inactive form. One possibility therefore is that less CycE protein is observed after the Dacapo downregulation because Dacapo is no longer stabilizing it (Shcherbata, 2004).

Why is Dacapo downregulated at the time of endocycle transition? Expression of Dacapo is important for proper cell cycle regulation. For example, during vertebrate development, members of the CIP/KIP family of CKIs are often upregulated as cells exit the mitotic cycle and begin to terminally differentiate. Also, reduced expression of p27Kip1 is frequently shown to correlate with a poor prognosis in various cancers, and in the absence of p21, DNA-damaged cells arrest in a G2-like state, but then undergo additional S-phases without intervening normal mitoses. They thereby acquire grossly deformed, polyploid nuclei and subsequently die through apoptosis. Also, p21 elimination causes centriole overduplication and polyploidy in human hematopoietic cells. In the Drosophila germ line Dap is differentially regulated in the nurse cells versus the oocyte. High Dap levels in the oocyte are critical to the maintenance of the prophase I meiotic arrest and ultimately to later events of oocyte differentiation, and in the nurse cells the oscillations of Dap drive the endocycle. In contrast to all these examples, in endocycling follicle cells reduction of p21/Dacapo is a requirement for normal endocycle progression. Similarly, in a megakaryocytic cell line, differentiation is correlated with a downregulation of p27. It is proposed that the downregulation of Dacapo is a reasonable strategy to bypass the G1/S transition and to enter endocycling when mitosis is not completed, however, how these endocycling cells escape possible centrosome amplification and apoptosis that could be consequences of the lack of Dacapo/p21-activity is not clear. This diversity in the processes, that allow cells to exit from mitotic cell cycle, is generating or representing regulatory multiplicity that might be reflected in the ways eukaryotic cells acquire tumor formation capacity (Shcherbata, 2004).


EVOLUTIONARY HOMOLOGS

Cdc4 function in yeast

In eukaryotes, the ubiquitin-proteasome system plays a major role in selective protein breakdown for cellular regulation. A new essential component of this degradation machinery has been discovered. The Saccharomyces cerevisiae protein Cic1 attaches to 26S proteasomes playing a crucial role in substrate specificity for proteasomal destruction. Whereas degradation of short-lived test proteins is not affected, cic1 mutants stabilize the F-box proteins Cdc4 and Grr1, which are substrate recognition subunits of the SCF complex. Cic1 interacts in vitro and in vivo with Cdc4, suggesting a function as a new kind of substrate recruiting factor or adaptor associated with the proteasome (Jager, 2001).

The Cdc6 DNA replication initiation factor is targeted for ubiquitin-mediated proteolysis by the E3 ubiquitin ligase SCF(CDC4) from the end of G1phase until mitosis in the budding yeast Saccharomyces cerevisiae. A dominant-negative CDC6 mutant is described that, when overexpressed, arrests the cell cycle by inhibiting cyclin-dependent kinases (CDKs) and, thus, prevents passage through mitosis. This mutant protein inhibits CDKs more efficiently than wild-type Cdc6, in part because it is completely refractory to SCF(CDC4)-mediated proteolysis late in the cell cycle and consequently accumulates to high levels. The mutation responsible for this phenotype destroys a putative CDK phosphorylation site near the middle of the Cdc6 primary amino acid sequence. This site lies within a novel Cdc4-interacting domain distinct from a Cdc4-interacting site identified previously near the N-terminus of the protein. Both sites can target Cdc6 for proteolysis in late G1/early S phase while only the newly identified site can target Cdc6 for proteolysis during mitosis (Perkins, 2001).

Degradation of Saccharomyces cerevisiae G(1) cyclins Cln1 and Cln2 is mediated by the ubiquitin-proteasome pathway and involves the SCF E3 ubiquitin-ligase complex containing the F-box protein Grr1 [SCF(Grr1)]. The domain of Cln2 that confers instability has been identified and the signals in Cln2 that result in binding to Grr1 and rapid degradation are describe. Mutants of Cln2 that lack a cluster of four Cdc28 consensus phosphorylation sites are highly stabilized and fail to interact with Grr1 in vivo. Since one of the phosphorylation sites lies within the Cln2 PEST motif, a sequence rich in proline, aspartate or glutamate, serine, and threonine residues found in many unstable proteins, various Cln2 C-terminal domains containing combinations of the PEST and the phosphoacceptor motifs were fused to stable reporter proteins. Fusion of the Cln2 domain to a stabilized form of the cyclin-dependent kinase inhibitor Sic1 (Delta N-Sic1), a substrate of SCF(Cdc4), results in degradation in a phosphorylation-dependent manner. Fusion of Cln2 degradation domains to Delta N-Sic1 switches degradation of Sic1 from SCF(Cdc4) to SCF(Grr1). Delta N-Sic1 fused with a Cln2 domain containing the PEST motif and four phosphorylation sites binds to Grr1 and is unstable and ubiquitinated in vivo. Interestingly, the phosphoacceptor domain of Cln2 binds to Grr1 but is not ubiquitinated and is stable. In summary, a small transferable domain in Cln2 has been identified that can redirect a stabilized SCF(Cdc4) target for SCF(Grr1)-mediated degradation by the ubiquitin-proteasome pathway (Berset, 2002).

The S. cerevisiae SCF(Cdc4) is a prototype of RING-type SCF E3s, which recruit substrates for polyubiquitination by the Cdc34 ubiquitin-conjugating enzyme. Current models propose that Cdc34 ubiquitinates the substrate while remaining bound to the RING domain. In contrast, it was found that the formation of a ubiquitin thiol ester regulates the Cdc34/SCF(Cdc4) binding equilibrium by increasing the dissociation rate constant, with only a minor effect on the association rate. By using a F72VCdc34 mutant with increased affinity for the RING domain, it was demonstrated that release of ubiquitin-charged Cdc34-S - Ub from the RING is essential for ubiquitination of the SCF(Cdc4)-bound substrate Sic1. Release of ubiquitin-charged E2 from E3 prior to ubiquitin transfer is a previously unrecognized step in ubiquitination, which can explain both the modification of multiple lysines on the recruited substrate and the extension of polyubiquitin chains (Deffenbaugh, 2003).

Ubiquitin ligases direct the transfer of ubiquitin onto substrate proteins and thus target the substrate for proteasome-dependent degradation. SCF complexes constitute a family of ubiquitin ligases composed of a common core of components and a variable component called an F-box protein that defines substrate specificity. Distinct SCF complexes, defined by a particular F-box protein, target different substrate proteins for degradation. Although a few have been identified to be involved in important biological pathways, such as the cell division cycle and coordinating cellular responses to changes in environmental conditions, the role of the overwhelming majority of F-box proteins is not clear. Creating inhibitors that will block the in vivo activities of specific SCF ubiquitin ligases may provide identification of substrates of these uncharacterized F-box proteins. Using Saccharomyces cerevisiae as a model system, it has been demonstrated that overproduction of polypeptides corresponding to the amino terminus of the F-box proteins Cdc4p and Met30p results in specific inhibition of their SCF complexes. Analyses of mutant amino-terminal alleles demonstrate that the interaction of these polypeptides with their full-length counterparts is an important step in the inhibitory process. These results suggest a common means to inhibit specific SCF complexes in vivo (Dixon, 2003).

The CDK inhibitor Sic1 must be phosphorylated on at least six sites in order to allow its recognition by the SCF ubiquitin ligase subunit Cdc4. However, because Cdc4 appears to have only a single phospho-epitope binding site, the apparent cooperative dependence on the number of phosphorylation sites in Sic1 cannot be accounted for by traditional thermodynamic models of cooperativity. A general kinetic model has been developed that predicts an unexpected multiplicative increase in affinity as a function of ligand sites. This effect, termed allovalency, derives from a high local concentration of interaction sites moving independently of each other. Modeling of this interaction by a first exit time approach indicates that the probability of ligand rebinding increases exponentially with the number of sites. This type of interaction is relatively immune to loss of any one site and may be easily tuned to any given threshold by adjusting the properties of individual sites. The allovalency model suggests that a previously undescribed mechanism may underlie certain cooperative interactions. The widespread occurrence of flexible polyvalent ligands in biological systems suggests that this principle may be broadly applicable (Klein, 2003).

Cell cycle progression depends on precise elimination of cyclins and cyclin-dependent kinase (CDK) inhibitors by the ubiquitin system. Elimination of the CDK inhibitor Sic1 by the SCFCdc4 ubiquitin ligase at the onset of S phase requires phosphorylation of Sic1 on at least six of its nine Cdc4-phosphodegron (CPD) sites. A 2.7 Å X-ray crystal structure of a Skp1-Cdc4 complex bound to a high-affinity CPD phosphopeptide from human cyclin E reveals a core CPD motif, Leu-Leu-pThr-Pro, bound to an eight-bladed WD40 propeller domain in Cdc4. The low affinity of each CPD motif in Sic1 reflects structural discordance with one or more elements of the Cdc4 binding site. Reengineering of Cdc4 to reduce selection against Sic1 sequences allows ubiquitination of lower phosphorylated forms of Sic1. These features account for the observed phosphorylation threshold in Sic1 recognition and suggest an equilibrium binding mode between a single receptor site in Cdc4 and multiple low-affinity CPD sites in Sic1 (Orlicky, 2003).

The ubiquitin-dependent targeting of proteins to the proteasome is an essential mechanism for regulating eukaryotic protein stability. The minimal signal for the degradation of the S phase CDK inhibitor Sic1 has been defined. Of 20 lysines scattered throughout Sic1, 6 N-terminal lysines serve as major ubiquitination sites. Sic1 lacking these lysines (K0N) is stable in vivo, but readdition of any one restores turnover. Nevertheless, ubiquitin chains attached at different N-terminal lysines specify degradation in vitro at markedly different rates. Moreover, although K0N can be ubiquitinated by SCF(Cdc4)/Cdc34 in vitro in the absence (but not in the presence) of S-CDK, it is degraded slowly. These results reveal that a single multiubiquitin chain can sustain a physiological turnover rate, but that chain position plays an unexpectedly significant role in the rate of proteasomal proteolysis (Petroski, 2003).

Two multiprotein E3 (ubiquitin-protein ligase) ubiquitin ligases, the SCF (Skp1-Cullin-1-F-box) and the APC/C (anaphase promoting complex/cyclosome), are vital in ensuring the temporal order of the cell cycle. Particularly, timely destruction of cyclins via these two E3s is essential for down-regulation of cyclin-dependent kinase. In general, G1 and S phase cyclins are ubiquitylated by the SCF, whereas ubiquitylation of mitotic cyclins is catalyzed by the APC/C. Fission yeast S phase cyclin Cig2 is ubiquitylated and degraded via both the SCF and the APC/C. Cig2 instability during G2 and M phase is dependent upon the SCF complex, whereas the APC/C is responsible for Cig2 destruction during anaphase and G1, thereby ensuring a spike pattern of Cig2 levels, peaking only at S phase. Two F-box/WD proteins Pop1 and Pop2, homologs of budding yeast Cdc4 and human Fbw7, are responsible for Cig2 instability. Pop1 binds Cig2 in vivo. An in vitro binding assay shows that 93 internal amino acid residues comprising a part of the cyclin box are necessary and sufficient for this binding. Cig2 phosphorylation is also required for interaction with Pop1. Transcriptional oscillation of cig2+ requires Pop1 and Pop2 function. SCF(Pop1/Pop2) therefore regulates Cig2 levels in a dual manner, transcriptionally and post-translationally. These results also highlight a collaborative action of the APC/C and the SCF toward the common substrate Cig2. This type of composite degradation control may be more general as the regulatory mechanism in other complex systems (Yamano, 2004).

Effects of hCDC4 mutation

Aneuploidy, an abnormal chromosome number, has been recognized as a hallmark of human cancer for nearly a century; however, the mechanisms responsible for this abnormality have remained elusive. This study reports the identification of mutations in hCDC4 (also known as Fbw7 or Archipelago) in both human colorectal cancers and their precursor lesions. Genetic inactivation of hCDC4, by means of targeted disruption of the gene in karyotypically stable colorectal cancer cells, results in a striking phenotype associated with micronuclei and chromosomal instability. This phenotype can be traced to a defect in the execution of metaphase and subsequent transmission of chromosomes, and is dependent on cyclin E, a protein that is regulated by hCDC4. These data suggest that chromosomal instability is caused by specific genetic alterations in a large fraction of human cancers and can occur before malignant conversion (Rajagopalan, 2004).

Degradation of c-Myc by the ubiquitin-proteasome pathway

The human proto-oncogene c-myc encodes a highly unstable transcription factor that promotes cell proliferation. Although the extreme instability of Myc plays an important role in preventing its accumulation in normal cells, little is known about how Myc is targeted for rapid destruction. Mechanisms regulating the stability of Myc have been investigated. Myc is destroyed by ubiquitin-mediated proteolysis, and two elements are defined in Myc that oppositely regulate its stability: a transcriptional activation domain that promotes Myc destruction, and a region required for association with the POZ domain protein Miz-1 that stabilizes Myc. Myc is stabilized by cancer-associated and transforming mutations within its transcriptional activation domain. These data reveal a complex network of interactions regulating Myc destruction, and imply that enhanced protein stability contributes to oncogenic transformation by mutant Myc proteins (Salghetti, 1999).

The c-Myc oncoprotein is a transcription factor that is a critical regulator of cellular proliferation. Deregulated expression of c-Myc is associated with many human cancers, including Burkitt's lymphoma. The c-Myc protein is normally degraded very rapidly with a half-life of 20 to 30 min. Proteolysis of c-Myc in vivo is mediated by the ubiquitin-proteasome pathway. Inhibition of proteasome activity blocks c-Myc degradation, and c-Myc is a substrate for ubiquitination in vivo. Furthermore, an increase in c-Myc stability occurs in mitotic cells and is associated with inhibited c-Myc ubiquitination. Deletion analysis was used to identify regions of the c-Myc protein that are required for rapid proteolysis. A centrally located PEST sequence, amino acids 226 to 270, is necessary for rapid c-Myc degradation, but not for ubiquitination. Also, N-terminal sequences, located within the first 158 amino acids of c-Myc, are necessary for both efficient c-Myc ubiquitination and subsequent degradation. c-Myc is significantly stabilized (two- to sixfold) in many Burkitt's lymphoma-derived cell lines, suggesting that aberrant c-Myc proteolysis may play a role in the pathogenesis of Burkitt's lymphoma. Finally, mutation of Thr-58, a major phosphorylation site in c-Myc and a mutational hot spot in Burkitt's lymphoma, increases c-Myc stability; however, mutation of c-Myc is not essential for stabilization in Burkitt's lymphoma cells (Gregory, 2000).

The c-Myc protein is a transcription factor that is a central regulator of cell growth and proliferation. Thr-58 is a major phosphorylation site in c-Myc and is a mutational hotspot in Burkitt's and other aggressive human lymphomas, indicating that Thr-58 phosphorylation restricts the oncogenic potential of c-Myc. Mutation of Thr-58 is also associated with increased c-Myc protein stability. Here it is shown that inhibition of glycogen synthase kinase-3 (GSK-3) activity with lithium increases c-Myc stability and inhibits phosphorylation of c-Myc specifically at Thr-58 in vivo. Conversely, overexpression of GSK-3 alpha or GSK-3 beta enhances Thr-58 phosphorylation and ubiquitination of c-Myc. Together, these observations suggest that phosphorylation of Thr-58 mediated by GSK-3 facilitates c-Myc rapid proteolysis by the ubiquitin pathway. Furthermore, GSK-3 binds c-Myc in vivo and in vitro and GSK-3 colocalizes with c-Myc in the nucleus, strongly arguing that GSK-3 is the c-Myc Thr-58 kinase. c-MycS, which lacks the N-terminal 100 amino acids of c-Myc, is unable to bind GSK-3; however, mutation of Ser-62, the priming phosphorylation site necessary for Thr-58 phosphorylation, does not disrupt GSK-3 binding. Finally, Thr-58 phosphorylation is shown to alter the subnuclear localization of c-Myc, enhancing its localization to discrete nuclear bodies together with GSK-3 (Gregory, 2003).

Myc proteins regulate cell growth and division and are implicated in a wide range of human cancers. Fbw7, a component of the SCF(Fbw7) ubiquitin ligase and a tumor suppressor, promotes proteasome-dependent c-Myc turnover in vivo and c-Myc ubiquitination in vitro. Phosphorylation of c-Myc on threonine-58 (T58) by glycogen synthase kinase 3 regulates the binding of Fbw7 to c-Myc as well as Fbw7-mediated c-Myc degradation and ubiquitination. T58 is the most frequent site of c-myc mutations in lymphoma cells, and these findings suggest that c-Myc activation is one of the key oncogenic consequences of Fbw7 loss in cancer. Because Fbw7 mediates the degradation of cyclin E, Notch, and c-Jun, as well as c-Myc, the loss of Fbw7 is likely to elicit profound effects on cell proliferation during tumorigenesis (Welcker, 2004).

Degradation of cyclin E by the ubiquitin-proteasome pathway

Cyclin E binds and activates the cyclin-dependent kinase Cdk2 and catalyzes the transition from the G1 phase to the S phase of the cell cycle. The amount of cyclin E protein present in the cell is tightly controlled by ubiquitin-mediated proteolysis. The ubiquitin ligase responsible for cyclin E ubiquitination has been identifed as SCFFbw7; it is functionally conserved in yeast, flies, and mammals. Fbw7 associates specifically with phosphorylated cyclin E, and SCFFbw7 catalyzes cyclin E ubiquitination in vitro. Depletion of Fbw7 leads to accumulation and stabilization of cyclin E in vivo in human and Drosophila melanogaster cells. Multiple F-box proteins contribute to cyclin E stability in yeast, suggesting an overlap in SCF E3 ligase specificity that allows combinatorial control of cyclin E degradation (Koepp, 2001).

SCF ubiquitin ligases target phosphorylated substrates for ubiquitin-dependent proteolysis by means of adapter subunits called F-box proteins. The F-box protein Cdc4 captures phosphorylated forms of the cyclin-dependent kinase inhibitor Sic1 for ubiquitination in late G1 phase, an event necessary for the onset of DNA replication. The WD40 repeat domain of Cdc4 binds with high affinity to a consensus phosphopeptide motif (the Cdc4 phospho-degron, CPD), yet Sic1 itself has many sub-optimal CPD motifs that act in concert to mediate Cdc4 binding. The weak CPD sites in Sic1 establish a phosphorylation threshold that delays degradation in vivo, and thereby establishes a minimal G1 phase period needed to ensure proper DNA replication. Multisite phosphorylation may be a more general mechanism to set thresholds in regulated protein-protein interactions (Nash, 2001).

Cyclin E, one of the activators of the cyclin-dependent kinase Cdk2, is expressed near the G1-S phase transition and is thought to be critical for the initiation of DNA replication and other S-phase functions. Accumulation of cyclin E at the G1-S boundary is achieved by periodic transcription coupled with regulated proteolysis linked to autophosphorylation of cyclin E. The proper timing and amplitude of cyclin E expression seem to be important, because elevated levels of cyclin E have been associated with a variety of malignancies and constitutive expression of cyclin E leads to genomic instability. Turnover of phosphorylated cyclin E depends on an SCF-type protein-ubiquitin ligase that contains the human homolog of yeast Cdc4, which is an F-box protein containing repeated sequences of WD40 (a unit containing about 40 residues with tryptophan (W) and aspartic acid (D) at defined positions). The gene encoding hCdc4 was found to be mutated in a cell line derived from breast cancer that expressed extremely high levels of cyclin E (Strohmaier, 2001).

Ubiquitin-proteasome pathway shuts off Notch signaling

The Notch signaling pathway is essential in many cell fate decisions in invertebrates as well as in vertebrates. After ligand binding, a two-step proteolytic cleavage releases the intracellular part of the receptor which translocates to the nucleus and acts as a transcriptional activator. Although Notch-induced transcription of genes has been reported extensively, its endogenous nuclear form has been seldom visualized. The nuclear intracellular domain of Notch1 is stabilized by proteasome inhibitors and is a substrate for polyubiquitination in vitro. SEL-10, an F-box protein of the Cdc4 family, was isolated in a genetic screen for Lin12/Notch-negative regulators in Caenorhabditis elegans. Human and murine counterparts of SEL-10 were isolated and the role was investigated of a dominant-negative form of this protein, deleted of the F-box, on Notch1 stability and activity. This molecule can stabilize intracellular Notch1 and enhance its transcriptional activity but has no effect on inactive membrane-anchored forms of the receptor. SEL-10 specifically interacts with nuclear forms of Notch1 and this interaction requires a phosphorylation event. Taken together, these data suggest that SEL-10 is involved in shutting off Notch signaling by ubiquitin-proteasome-mediated degradation of the active transcriptional factor after a nuclear phosphorylation event (Gupta-Rossi, 2001).

Mammalian Fbw7 (also known as Sel-10, hCdc4, or hAgo) is the F-box protein component of an SCF (Skp1-Cul1-F-box protein-Rbx1)-type ubiquitin ligase, and the mouse Fbw7 is expressed prominently in the endothelial cell lineage of embryos. Mice deficient in Fbw7 were generated: the embryos died in utero at embryonic day 10.5-11.5, manifesting marked abnormalities in vascular development. Vascular remodeling was impaired in the brain and yolk sac, and the major trunk veins were not formed. In vitro para-aortic splanchnopleural explant cultures from Fbw7-/- embryos also manifested an impairment of vascular network formation. Notch4, which is the product of the proto-oncogene Int3 and an endothelial cell-specific mammalian isoform of Notch, accumulate in Fbw7-/- embryos, resulting in an increased expression of Hey1, which encodes a transcriptional repressor that acts downstream of Notch signaling and is implicated in vascular development. Expression of Notch1, -2, or -3 or of cyclin E was unaffected in Fbw7-/- embryos. Mammalian Fbw7 thus appears to play an indispensable role in negative regulation of the Notch4-Hey1 pathway and is required for vascular development (Tsunematsu, 2004).

Ubiquitin-proteasome pathway and Presenilin

Mutations in the human presenilin genes (PS1 or PS2) have been linked to autosomal dominant, early onset Alzheimer's disease (AD). Presenilins, probably as an essential part of gamma-secretase, modulate gamma-cleavage of the amyloid protein precursor (APP) to the amyloid beta-peptide (Abeta). Mutations in sel-12, a Caenorhabditis elegans presenilin homolog, cause a defect in egg laying that can be suppressed by loss of function mutations in a second gene, SEL-10. SEL-10 protein is a homolog of yeast Cdc4, a member of the SCF (Skp1-Cdc53/CUL1-F-box protein) E2-E3 ubiquitin ligase family. Human SEL-10 interacts with PS1 and enhances PS1 ubiquitination, thus altering cellular levels of unprocessed PS1 and its N- and C-terminal fragments. Co-transfection of sel-10 and APP cDNAs in HEK293 cells leads to an alteration in the metabolism of APP and to an increase in the production of amyloid beta-peptide, the principal component of amyloid plaque in Alzheimer's disease (Li, 2002).

hCDC4 and cancer

Cyclin-dependent kinase 2 activated by cyclin E is involved in the initiation of DNA replication and other S phase functions. Consistent with this role, cyclin E protein accumulates at the G1-S phase transition and declines during early S phase. This profile of expression is the result of periodic transcription and ubiquitin-mediated proteolysis directed by SCF(hCdc4). However, in many types of human tumors cyclin E protein is elevated and deregulated relative to the cell cycle by an unknown mechanism. The F-box protein hCdc4 that targets cyclin E to the SCF (Skp1-Cull-F-box) protein ubiquitin ligase is mutated in at least 16% of human endometrial tumors. Mutations were found either in the substrate-binding domain of the protein or at the amino terminus, suggesting a critical role for the region of hCdc4 upstream of the F-box. hCDC4 gene mutations are accompanied by loss of heterozygosity and correlate with aggressive disease. The hCDC4 gene is localized to chromosome region 4q32, which is deleted in over 30% of human tumors. These results show that the hCDC4 gene is mutated in primary human tumors and suggests that it may function as a tumor suppressor in the genesis of many human cancers (Spruck, 2002).

Cyclin E overexpression occurs in a subset of endometrial carcinomas (ECs), but the molecular mechanisms underlying this alteration remain to be established. The present study has analysed amplification of the cyclin E gene (CCNE) and mutation in hCDC4, the gene coding for the F-box protein, which tags phosphorylated cyclin E for proteosomal degradation, to ascertain whether these alterations might be responsible for cyclin E overexpression in ECs. Cyclin E and p53 expression was studied by immunohistochemistry in eight atypical endometrial hyperplasias (AEHs), 51 endometrioid endometrial carcinomas (EECs), and 22 non-endometrioid endometrial carcinomas (NEECs). CCNE amplification was analysed by fluorescence in situ hybridization (FISH). Mutations in exons 2-11 of the hCDC4 gene were screened by PCR-SSCP-sequencing. Finally, the polymorphic marker D4S1610 was used to assess loss of heterozygosity (LOH) in the hCDC4 gene. Cyclin E overexpression was found in 26/81 (32%) cases and was associated with the histological type of the lesion, since it was not found in any AEHs but was present in 27% of EECs and 54.5% of NEECs (p=0.035). Cyclin E overexpression was associated with histological grade (p=0.011) and p53 immunostaining in EECs (p=0.033). CCNE amplification was found in 6 of 37 (16%) ECs examined. There was a significant association between CCNE amplification and the histological type of the lesion, since five (83%) of the six cases with amplification were NEECs (p=0.008). One EEC harbored an hCDC4 mutation -- a CGA to CAA (Arg/Gln) change at codon 479. In addition, D4S1610 LOH was found in 7 of 23 (30%) informative cases analysed, but no correlation with cyclin E overexpression was found. However, the tumor with hCDC4 mutation also showed LOH. This is the first study demonstrating that cyclin E overexpression is associated with gene amplification in ECs, these alterations being more frequent in NEECs. Although hCDC4 exhibits a low mutation frequency in ECs overexpressing cyclin E, it seems to function as a tumor suppressor gene that is involved in endometrial carcinogenesis (Cassia, 2003).


REFERENCES

Search PubMed for articles about Drosophila Archipelago

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Deffenbaugh, A. E., et al. (2003). Release of ubiquitin-charged Cdc34-S - Ub from the RING domain is essential for ubiquitination of the SCF(Cdc4)-bound substrate Sic1. Cell 114(5): 611-22. 13678584

Dixon, C., et al. (2003). Overproduction of polypeptides corresponding to the amino terminus of the F-box proteins Cdc4p and Met30p inhibits ubiquitin ligase activities of their SCF complexes. Eukaryot. Cell 2(1): 123-33. 12582129

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Gregory, M. A., Qi, Y. and Hann, S. R. (2003). Phosphorylation by glycogen synthase kinase-3 controls c-myc proteolysis and subnuclear localization. J. Biol. Chem. 278: 51606-51612. 14563837

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Biological Overview

date revised: 10 June 2012

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