InteractiveFly: GeneBrief

Cullin 1: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References


Gene name - Cullin 1

Synonyms - lin-19-like

Cytological map position - 43F1--2

Function - protein degradation, scaffold protein within the SCF complex

Keywords - protein degradation

Symbol - Cul1

FlyBase ID: FBgn0015509

Genetic map position -

Classification - cullin

Cellular location - cytoplasmic and nuclear



NCBI links: Precomputed BLAST | Entrez Gene

Recent literature
Cho, B., Pierre-Louis, G., Sagner, A., Eaton, S. and Axelrod, J. D. (2015). Clustering and negative feedback by endocytosis in planar cell polarity signaling is modulated by ubiquitinylation of Prickle. PLoS Genet 11: e1005259. PubMed ID: 25996914
Summary:
The core components of the planar cell polarity (PCP) signaling system, including both transmembrane and peripheral membrane associated proteins, form asymmetric complexes that bridge apical intercellular junctions. While these can assemble in either orientation, coordinated cell polarization requires the enrichment of complexes of a given orientation at specific junctions. This might occur by both positive and negative feedback between oppositely oriented complexes, and requires the peripheral membrane associated PCP components. However, the molecular mechanisms underlying feedback are not understood. This study found that the E3 ubiquitin ligase complex Cullin1(Cul1)/SkpA/Supernumerary limbs(Slimb) regulates the stability of one of the peripheral membrane components, Prickle (Pk). Excess Pk disrupts PCP feedback and prevents asymmetry. Pk was found to participate in negative feedback by mediating internalization of PCP complexes containing the transmembrane components Van Gogh (Vang) and Flamingo (Fmi), and that internalization is activated by oppositely oriented complexes within clusters. Pk also participates in positive feedback through an unknown mechanism promoting clustering. these results therefore identify a molecular mechanism underlying generation of asymmetry in PCP signaling.
Suisse, A., He, D., Legent, K. and Treisman, J. E. (2017). COP9 signalosome subunits protect Capicua from MAP kinase-dependent and independent mechanisms of degradation. Development [Epub ahead of print]. PubMed ID: 28619822
Summary:
The COP9 signalosome removes Nedd8 modifications from the Cullin subunits of ubiquitin ligase complexes, reducing their activity. This study shows that mutations in the Drosophila COP9 signalosome subunit 1b (CSN1b) gene increase the activity of ubiquitin ligases that contain Cullin 1. Analysis of CSN1b mutant phenotypes revealed a requirement for the COP9 signalosome to prevent ectopic expression of Epidermal growth factor receptor (EGFR) target genes. It does so by protecting Capicua, a transcriptional repressor of EGFR target genes, from EGFR pathway-dependent ubiquitination by a Cullin 1/SKP1-related A/Archipelago E3 ligase and subsequent proteasomal degradation. The CSN1b subunit also maintains basal Capicua levels by protecting it from a separate mechanism of degradation that is independent of EGFR signaling. As a suppressor of tumor growth and metastasis, Capicua may be an important target of the COP9 signalosome in cancer.
BIOLOGICAL OVERVIEW

Cullins are the major components of a series of multimeric ubiquitin ligases that control the degradation of a broad range of proteins. The ubiquitin-like protein, Nedd8, covalently modifies members of the Cullin family. Nedd8 modifies Cullin 1 (Cul1, also known as Lin-19-like or simply Lin-19) in Drosophila. In mutants of Drosophila Nedd8 and Cul1, levels of the signal transduction effectors, Cubitus interruptus (Ci) and Armadillo (Arm), and the cell cycle regulator, Cyclin E (CycE), are unusually high, suggesting that the Cul1-based multimeric SCF ubiquitin ligase complex requires Nedd8 modification for the degradation processes of Ci, Arm, and CycE in vivo. Two distinct degradation mechanisms modulating Ci stability in the developing eye disc are separated by the morphogenetic furrow (MF) in which retinal differentiation is initiated. In cells anterior to the MF, Ci proteolytic processing promoted by PKA requires the activity of the Nedd8-modified Cul1-based SCFSlimb complex. In posterior cells, Ci degradation is controlled by a mechanism that requires the activity of Cul3, another member of the Cullin family. This posterior Ci degradation mechanism, which partially requires Nedd8 modification, is activated by Hedgehog (Hh) signaling and is PKA-independent (Ou, 2002).

Ubiquitin-mediated protein degradation mechanisms control the stability of various proteins that are essential for cellular function. Nedd8 is a ubiquitin-like small protein modifier. The Nedd8 conjugation process, called neddylation, is similar to ubiquitination. Neddylation utilizes the E1 activating-enzyme complex composed of two subunits, APP-BP1 and UBA3, and the E2 conjugating-enzyme, UBC12 (Yeh, 2000). The only known substrates of neddylation are Cullin family proteins -- Cul1, Cul2, Cul3, Cul4A, Cul4B, and Cul5 -- which have been shown to be modified by Nedd8 in mammalian cells. Cullins directly interact with Roc1, a Ring finger protein, and the Cullin-Roc1 complex comprises the core module of a series of ubiquitin E3 ligases, which confer substrate specificity and therefore regulate the degradation process. Among Cullins, many studies focused on Cul1, an essential component of the SCF complex which functions as ubiquitin E3 ligase. The SCF complex consists of core subunits: Skp1, Cul1/Cdc53, Roc1/Hrt/Rbx1, and a substrate-recognition F-box protein. Cul1 functions as a scaffold protein within the SCF complex; the N-terminal domain of Cul1 interacts with the adaptor protein Skp1 that links with the F-box protein, and the C-terminal domain interacts with Roc1 and the ubiquitin E2 enzyme (Ou, 2002 and references therein).

In vitro, neddylation of Cul1 is required for ubiquitination of IkappaBalpha and p27Kip1 (Morimoto, 2000; Podust, 2000; Read, 2000). In addition, neddylation enhances E2-ubiquitin recruitment to SCF. In fission yeast, Nedd8 is essential for the SCF-mediated degradation of Rum-1, a cyclin-dependent kinase inhibitor. In Arabidopsis thaliana, the Nedd8 pathway is required for SCF-mediated Auxin response. In mice deficient for UBA3, a subunit of the E1 enzyme in neddylation, embryonic development is aberrant, with accumulation of two putative SCF substrates, ß-catenin and cyclin E (Ou, 2002 and references therein).

In the SCF complex, F-box proteins convey substrate specificity by direct interaction with substrates for degradation. Many F-box proteins have been characterized in metazoans, and increasing numbers of specific targets for F-box proteins are being found (Deshaies, 1999). Among them, the Drosophila F-box protein Slimb and its mammalian homolog ß-TrCP are well characterized for their target specificity. The specific targets for Slimb/ß-TrCP are pIkappaBa in the Dorsal/NFkappaB pathway, Arm/ß-catenin in the Wg/Wnt pathway, and Ci/Gli in the Hedgehog (Hh) pathway (Ou, 2002 and references therein).

The Hh pathway controls growth and pattern formation in many developmental processes in both vertebrates and invertebrates. The Hh signal is transmitted through a receptor complex consisting of Patched (Ptc) and Smoothened (Smo). In the absence of Hh, Ptc inhibits Smo activity, and the effector Cubitus interruptus (Ci) is phosphorylated by PKA, leading to the proteolysis of Ci, which is converted into Ci75 with the C terminus truncated. Ci75 functions as a transcriptional repressor in the Hh signaling pathway. Upon binding to Ptc, Hh relieves Smo from its repression state. Activated Smo mediates signaling to prohibit proteolytic processing of Ci. The intact full-length Ci (CiFL) functions as a transcriptional activator for expression of target genes of the Hh pathway (Ou, 2002).

In Drosophila, Hh signaling functions in patterning the A/P compartments in developing tissues such as embryonic segments and wing and leg imaginal discs. In development of the eye imaginal disc, Hh signaling is a major driving force of the retinal differentiation wave, the morphogenetic furrow (MF), which is caused by transient constriction in cell apical surface. The MF progresses anteriorly from the posterior margin of the eye disc during the third instar larval and early pupal stages. Anterior to the advancing MF, cells are proliferating, whereas posterior cells differentiate sequentially into photoreceptor, cone, or pigment cells. Transduction of Hh signaling in the MF is revealed by the accumulation of CiFL, which activates expression of target genes such as dpp and atonal in the MF. The induced MF cells soon differentiate and produce Hh proteins for further induction of more anterior cells, thus making the MF move forward (Ou, 2002).

The effect of neddylation on a broad spectrum of E3 ligases remains largely unknown. To investigate the role of neddylation in protein degradation control during developmental processes, Nedd8 and Cul1 mutants were identified and analyzed in Drosophila. The results suggest that neddylation is required for Cul1-mediated protein downregulation of the signaling pathway effectors Ci and Armadillo (Arm) and the cell cycle regulator CycE. Using the developing eye disc as a model system to study the regulation of CiFL stability, it was found that there is mechanistic difference in controlling CiFL stability between anterior and posterior cells separated by the MF. Whereas the Cul1-based SCFSlimb complex controls CiFL stability in anterior cells, a Cul3-dependent protein degradation mechanism controls CiFL stability in posterior cells. The differences between these two protein degradation mechanisms were further investigated (Ou, 2002).

This work has shown that in anterior cells of developing discs, CiFL proteolytic processing requires the activity of the Nedd8-modified, Cul1-based SCFSlimb complex. This CiFL proteolytic processing is inhibited by Smo signaling and promoted by PKA phosphorylation on CiFL. The mechanism by which CiFL is proteolyzed from CiFL to Ci75 is not clear. It is proposed that Nedd8 modifies and activates SCFSlimb for Ci ubiquitination and then proteolysis, as evidenced by Cul1 modification by Nedd8 and CiFL accumulation in Nedd8, Cul1, and slimb mutants. Consistently, proteolysis of CiFL depends on 26S proteasome activity. However, ubiquitinated Ci is not detected in cells treated with 26S proteasome inhibitors (Ou, 2002).

In the Hh signaling pathway, it is not clear how Smo signaling prevents CiFL from proteolysis. According to double mutant analysis, Nedd8 could be downstream or parallel to Smo and PKA signaling. Thus, it is possible that Hh signaling prevents CiFL from proteolysis through downregulating the level of Nedd8-modified Cul1. However, no change in the level of Nedd8-modified Cul1 could be detected in cell extracts prepared from the eye discs with ectopic Hh expression. It is therefore inferred that Hh may affect CiFL proteolysis through a Nedd8-independent mechanism (Ou, 2002).

Two modes of Ci downregulation in Drosophila eye development are proposed. In the undifferentiated cells anterior to the MF, Ci is phosphorylated by PKA constantly and processed by an SCFSlimb-dependent mechanism to generate the repressor form of Ci75. Upon binding to Hh, cells in the MF transduce Smo signaling to prevent this proteolytic processing. Thus, the transcriptional activator CiFL is preserved for activation of downstream genes in the MF (Ou, 2002).

In the posterior cells that are undergoing differentiation, a novel mechanism controls Ci degradation. Mutant analyses suggest that this mechanism is comprised of Smo signaling, Nedd8 modification, and Cul3 activity. The effect of Smo signaling in promoting Ci degradation in the posterior cells is in contrast to its effect on the anterior cells, in which Smo signaling prohibits CiFL processing. In addition to Smo signaling, Nedd8 modification activity also participates in this posterior Ci degradation. Further Cul1 mutant analysis suggests that Cullin proteins other than Cul1 are likely involved in this posterior degradation mechanism. This hypothesis has led to the identification of Cul3 as one candidate functioning in Ci degradation. More surprisingly, Cul3 activity is very restricted; Cul3 controls Ci degradation in the posterior, but not anterior, cells of the eye disc. CiFL accumulation may have an impact on proper differentiation of the posterior cells. In Cul3 mutants, cone cell differentiation is affected, probably due to the accumulation of CiFL (Ou, 2002).

Furthermore, the Ci degradation process is also distinct in posterior cells; Ci degradation is independent of PKA phosphorylation and proteolytic processing to the short form Ci-75. Based on these results, it is proposed that Smo signaling, acting in concert with the Nedd8 pathway, activates a Cul3-based ubiquitin ligase to degrade Ci in a PKA-independent mechanism in posterior cells of the eye disc (Ou, 2002).

It is not clear how Nedd8 modifies Cul3 in flies. Strong genetic interaction is observed between Nedd8 and Cul3 during eye and antennal development, suggesting that Nedd8 may also regulate Cul3. However, depletion of Nedd8 activity affects only posterior cells abutting the MF, in contrast to depletion of Cul3 activity, which increases the CiFL level in all posterior clones, indicating that some Cul3 activity is Nedd8-independent. It is possible that a basal Cul3 activity for Ci degradation is further enhanced by Nedd8 modification near the MF in which accumulated Ci may require efficient degradation for cells to enter proper differentiation (Ou, 2002).

Different protein-protein interactions may result in a switch between two Ci degradation mechanisms in eye discs. Ci is known to interact with Cos2, Fu, and Su(fu) to comprise a protein complex that promotes Ci degradation. Cos2, a motor-like protein with a kinesin motif, is required for tethering Ci in the cytosolic compartment and Ci proteolytic processing in the Drosophila developing wing. Similarly, Fu, a serine/threonine kinase, is also required for Ci processing. However, in Su(fu) mutants, levels of both long and short forms of Ci are reduced, suggesting that Su(fu) plays an additional role in Ci protein stability. Interestingly, the role of Su(fu) in controlling Ci stability seems modulated by Hh signaling. The results in this study indicate that, in contrast to the effect of Hh signaling in the anterior cells, Hh signaling downregulates the Ci level in the posterior cells of the eye disc. It is possible that the Ci protein complex is modulated by the sweep of the MF, and this change requires Hh signaling to expose Ci to the Cul3-based protein degradation machinery. Alternatively, additional factors may be activated by the sweeping of the MF and be required for Hh signaling to induce Cul3 activity that leads to constitutive Ci degradation (Ou, 2002).

Similar to Ci, CycE is degraded by two different mechanisms in mammalian cells. The Cul1-based SCF complex recognizes the phosphorylated form of Cdk2-bound CycE for ubiquitination (Dealy, 1999; Skowyra, 1999; Koepp, 2001; Strohmaier, 2001; Yeh, 2001), and Cul3 targets unbound CycE for ubiquitination (Singer, 1999) in a process that is independent of protein phosphorylation (Singer, 1999). ß-catenin is also degraded by two different mechanisms in mammalian cells (Polakis, 2001). One mechanism involves the SCFßTrCP complex (ßTrCP is the mammalian homolog of Slimb) that recognizes phosphorylated ß-catenin. The other mechanism involves the Ebi complex comprised of Ebi, Skp1, SIP, and Siah-1, which targets ß-catenin in a phosphorylation-independent manner (Ou, 2002).

In vertebrates, the three Ci-related proteins Gli1, Gli2, and Gli3 transduce Hh signaling in different developmental processes. Ectopic expression of the Gli proteins in Drosophila has shown that Gli2 and Gli3, but not Gli1, are proteolyzed to generate repressor forms. Although the proteolytic cleavage of Gli3 is under the regulation of Hh signaling, Gli2 proteolysis is independent of Hh. Consistently, proteolytic processing of Gli3, but not Gli1, has been observed in mouse embryos. In cultured cells, Gli3 processing is dependent on Hh signaling and PKA activity, in contrast to Gli1 and Gli2. Apparently, the Gli proteins are controlled by different protein downregulation mechanisms. It will be interesting to investigate whether Nedd8, Cul1, Cul3, and perhaps other Cullins are differentially involved in protein degradation of the Gli proteins (Ou, 2002 and references therein).


REGULATION

Protein Interactions

Cul-1 is an SCF core subunit

Many proteins are targeted to proteasome degradation by a family of E3 ubiquitin ligases, termed SCF complexes, that link substrate proteins to an E2 ubiquitin-conjugating enzyme. SCFs are composed of three core proteins-Skp1, Cdc53/Cull, Rbx1/Hrt1-and a substrate specific F-box protein. The closest homologs to the human components of the SCF(betaTrCP) complex and the E2 ubiquitin-conjugating enzyme UbcH5 have been identified in Drosophila. Putative Drosophila SCF core subunits skpA and Rbx1 both interact directly with Cu11 and the F-box protein Slimb. The direct interaction of UbcH5 related protein UbcD1 with Cul1 and Slimb is also reported. In addition, a functional complementation test performed on a Saccharomyces cerevisiae Hrt1p-deficient mutant shows that Drosophila Rbx1 is able to restore the yeast cells viability. These results suggest that Rbx1, SkpA, Cullin1, and Slimb proteins are components of a Drosophila SCF complex that functions in combination with the ubiquitin conjugating enzyme UbcD1 (Bocca, 2001).

The effect of the Nedd8 pathway on CiFL processing

Nedd8, the ubiquitin-like protein that covalently modifies members of the Cullin family, is highly conserved from yeast to mammals. Several Nedd8 alleles have been identifed in Drosophila, including two null alleles Nedd8AN015 and Nedd8AN024. The Nedd8 null mutants were growth-arrested in the first-instar larval stage and died within several days without further growth. Mutant clones were generated to analyze Nedd8 loss-of-function phenotypes; in the adult flies very few Nedd8AN015 mutant cells are identified, while in control experiments, large Nedd8+ clones are frequently recovered. Nedd8 mutant clones of small size, however, are present in the developing discs, suggesting that Nedd8 mutant cells are defective in proliferation and survival (Ou, 2002).

To study the relationship between Nedd8 and the F-box protein Slimb-mediated protein degradation, the protein stability for substrates of Slimb was studied in Nedd8 mutant cells. Nedd8 mutant cells in developing wing discs accumulate high levels of full-length Ci (CiFL) and Arm proteins, phenotypes identical to those observed in the slimb mutants. In Drosophila embryonic development, the signaling pathway mediated by the NFkappaB homolog Dorsal is required for patterning the dorsoventral identity. Accumulation of pIkappaBa/Cactus inhibits Dorsal activation, leading to repression of the downstream target gene, twist, an effect that has been observed in slimb mutants. twist expression was examined in embryos laid by Nedd8AN015/Nedd8203 females in which Nedd8203 is a hypomorphic allele. In such embryos, the twist expression domain is reduced along the dorsoventral axis and often found missing in many cells, revealing a requirement for Nedd8 in Dorsal signaling (Ou, 2002).

Whether Nedd8 affects the protein level of CycE was examined. Levels of CycE are regulated by the F-box protein, Archipelago (Ago; Moberg, 2001). CycE accumulates in Nedd8 mutant cells in the eye disc. These results suggest that Nedd8 might affect the stability of a broad range of proteins through F-box proteins in flies (Ou, 2002).

The Drosophila eye imaginal disc is an excellent model system for developmental study. Cells are undifferentiated and dividing randomly anterior to the MF, and cells posterior to the MF are differentiating into different types of cells. Thus, Nedd8 phenotypes can be observed in cells of different differentiation states in a single eye disc. The Hh pathway is the major signaling pathway in eye development, and the protein level of its effector Ci is tightly regulated in Drosophila. These studies focused on how Nedd8 regulates the CiFL level in the Hh pathway and the effects of Ci upregulation on eye development. It was found that in the Nedd8 clones that located anterior to the MF, CiFL accumulates to a level identical to that in the MF cells that transduce the Hh signaling pathway. Accumulation of CiFL also exists in posterior mutant cells that locate proximally but not distally to the MF. CiFL accumulation in Nedd8 mutant cells is not caused by an increase in the ci transcription level, because expression of ci-lacZ that recapitulates endogenous ci expression remains constant in Nedd8 mutant cells, indicating that posttranscriptional defects resulted in CiFL accumulation (Ou, 2002).

Elevated CiFL levels causes anterior Nedd8 mutant cells to adopt MF fate precociously. Nedd8 mutant cells are constricted on their apical surface, as revealed by the intensified phalloidin staining, and express the Hh-target gene, dpp, as detected by the expression of dpp-lacZ reporter gene. Furthermore, the early photoreceptor marker, Atonal, is induced. These phenotypes are observed only in mutant cells abutting the MF anteriorly, suggesting that accumulated CiFL in Nedd8 mutant cells is able to respond to Hh signaling (Ou, 2002).

CiFL accumulation in Nedd8 cells results from a defect in the machinery controlling CiFL protein processing. Ci protein processing is known to depend on the phosphorylation status of CiFL by PKA. The level of CiFL is downregulated when PKA is constitutively activated by the expression of its catalytic subunit. Therefore, the functional relationship between PKA activity and Nedd8 modification was examined. When the UAS-C* transgene was driven by eq-GAL4 for misexpression in the equator region of the eye disc, as visualized by the coexpressed GFP, the level of CiFL in the equator region was reduced, consistent with the observations that PKA phosphorylates Ci and promotes Ci proteolysis. Nedd8 mutant clones were then generated in the equator region where PKA is constitutively activated. In Nedd8 clones that overlap the eq-GAL4 expression domain, CiFL accumulates to a high level, identical to the level in the Nedd8 clone located externally to the eq-GAL4 expression domain. These results indicate that CiFL downregulation by PKA activity requires Nedd8 activity, and the effect of the Nedd8 pathway on CiFL processing is unlikely to be mediated through modulation of PKA activity (Ou, 2002).

Ci downregulation in the posterior cells of the eye disc requires Smo signaling and Nedd8 modification activity; CiFL degradation is mediated by a Cul3-dependent mechanism

The finding that CiFL accumulates in posterior smo3 clones indicates that Smo signaling contributes to the downregulation of CiFL in the posterior cells of the eye disc. This effect is in contrast to the smo role in the MF, where smo is required for CiFL activation. CiFL accumulation was also observed in the posterior Nedd8 mutant clones located proximally to the MF. In the smo3 Nedd8 double mutant clones, the level of CiFL is further enhanced, even in clones located distally to the MF, whereas no CiFL accumulation is detected in Nedd8 or smo3 clones, suggesting that Nedd8 and Smo function partially redundantly to downregulate Ci stability in the posterior cells of the eye disc (Ou, 2002).

The involvement of Nedd8 in controlling CiFL levels in the posterior cells of the eye disc suggests that Cullin proteins other than Cul1 may be involved in the posterior mechanism to control Ci stability. Among the mammalian Cullin family, Cul3 shares with the Cul1-based SCF complex the substrate CycE (Singer, 1999). To test whether Cul3 affects CiFL degradation in the eye disc, the available Drosophila Cul3 mutants were analyzed. CiFL accumulates in Cul3 mutant clones located posterior to the MF, with a higher level in nondifferentiating cells that surround differentiating photoreceptor clusters. In contrast, no CiFL accumulation is detected in anterior Cul3 mutant clones, indicating that Cul3 controls CiFL protein stability only in the posterior cells of the eye disc. Ci accumulation in posterior Cul3 mutant cells is controlled at the posttranscriptional level because ci expression is normal, as revealed by in situ hybridization. These results show that the CiFL degradation machinery in the posterior cells of the eye disc requires a Cul-3-mediated degradation mechanism. Ci accumulation is also detected in Cul3 mutant cells located in the A/P boundary of the wing disc. The level of Arm in Cul3 mutant clones in wing discs and the level of CycE in Cul3 mutant clones in eye discs remain constant, suggesting that Cul3 activity is specific to Ci (Ou, 2002).

In contrast to the Cul1-based SCFSlimb complex that controls CiFL processing only in the anterior cells of the eye disc, the Cul3-mediated Ci degradation mechanism is specific to the posterior cells. These specific activities in controlling Ci protein stability are not caused by differential gene expression of Cul1 and Cul3 in the eye disc. Ubiquitous mRNA expression patterns of both Cul1 and Cul3, and ubiquitous Cul1 protein expression are found all along the eye disc, suggesting that control of specificity is mediated by mechanisms other than regulation of Cul1 and Cul3 expression (Ou, 2002).

PKA phosphorylation promotes CiFL processing, and plays a role in the Hh signaling pathway for Ci activation. The requirement of PKA in CiFL degradation in the posterior cells of the eye disc was examined; CiFL downregulation is not regulated by PKA activity. Proteolytic processing of CiFL to the short form Ci75 is not a prerequisite for complete degradation in the posterior cells, in contrast to the proteolytic processing of the phosphorylated CiFL to the short form Ci75 in the anterior cells. To sum up, the results suggest that in the posterior cells of the eye disc, CiFL is degraded constitutively, and this degradation process is independent of PKA phosphorylation (Ou, 2002).

The COP9 signalosome promotes degradation of cyclin E during early Drosophila oogenesis

The COP9 signalosome (CSN) is an eight-subunit complex that regulates multiple signaling and cell cycle pathways. The CSN has been linked to the degradation of Cyclin E, which promotes the G1-S transition in the cell cycle and then is rapidly degraded by the ubiquitin-proteasome pathway. Using CSN4 and CSN5/Jab1 mutants, it has been shown that the CSN acts during Drosophila oogenesis to remove Nedd8 from Cullin1, a subunit of the SCF ubiquitin ligase. Overexpression of Cyclin E causes similar defects as mutations in CSN or SCFAgo subunits (see Archipelago) -- extra divisions or, in contrast, cell cycle arrest and polyploidy. Because the phenotypes are so similar and because CSN and Cyclin E mutations reciprocally suppress each other, Cyclin E appears to be the major target of the CSN during early oogenesis. Genetic interactions among CSN, SCF, and proteasome subunits further confirm CSN involvement in ubiquitin-mediated Cyclin E degradation (Doronkin, 2003).

The effect of the CSN on the activity of the SCF complex has been controversial. Although Nedd8 modification of Cullin1 stimulates SCF activity, the opposite process, deneddylation, has also been shown to be important for SCF function and cell cycle progression. For example, point mutations in the JAMM domain of the S. cerevisiae CSN5 homolog Rri abolish its deneddylation activity and enhance the growth defect shown by ts alleles of SCF genes. These results have led to the proposal that repeated cycles of neddylation and deneddylation are required for the sustained activity of the SCF. However, a recent gain-of-function analysis suggests that deneddylation by the CSN inhibits degradation of the SCF target p27kip1 (Doronkin, 2003).

The results of this study strongly support the idea that deneddylation of Cullin1 by the CSN is necessary for activity of the SCF complex. CSN mutations have the same, not opposite, effects on oogenesis as do Nedd8, cullin1, or ago mutations. CSN5 and CSN4 mutations also interact dominantly with cullin1 and ago mutations, further suggesting that the CSN works along with the SCF to promote Cyclin E degradation. These requirements for the CSN appear to demand its deneddylase activity, because the CSN5quo2 mutation, with a single amino acid substitution in the metalloprotease domain, behaves similarly to a CSN5 null (Doronkin, 2003).

Cycles of neddylation and deneddylation might control the association of an F box protein with an E3 ubiquitin ligase core complex or the association of a ubiquitin-loaded E2-conjugating enzyme with the E3 complex. Neddylation might also affect Cullin1 stability as suggested by the Cullin1 accumulation that is seen in CSN5 mutants and its reduction in Nedd8 mutants (Doronkin, 2003).

Conjugation of Nedd8 to cullins may regulate not only their activity, but also their subcellular distribution. Shuttling between the nucleus and cytoplasm has been proposed as a regulatory mechanism for E3 ubiquitin ligases when the target protein is ubiquitinated in the nucleus. The results showing that in CSN mutants, Nedd8-modified Cullin1 accumulates in the cytoplasm suggest that neddylation may be one way to regulate shuttling. Neddylation might favor nuclear export of Cullin1, and nuclear CSN would be required to remove Nedd8 and prevent export. Alternatively, neddylation might prevent Cullin1 nuclear import, and recycling of SCF into the nucleus would require cytoplasmic CSN. On either model, the CSN would be an important regulator of SCF activity. For example, modulation of SCF nuclear shuttling might affect the timing of Cyclin E degradation and entry into S phase of the cell cycle (Doronkin, 2003).

Mutations in Drosophila ago, the C. elegans gene cul1, or the F box-encoding lin23 have been shown to cause increased cell proliferation, suggesting a critical role for SCF in regulating cell divisions. Extra cell division is found to be a frequent phenotype produced by mutations in CSN5, CSN4, cullin1, ago, or by overexpression of Cyclin E. However, SCF and CSN mutations have also been shown to cause the opposite effect on the cell cycle. In null mutant clones of cullin1 or Nedd8, cell proliferation in Drosophila eye discs is arrested. Similarly, loss of CSN5, CSN4, Cullin1, or ago inhibits and finally stops cell proliferation and often leads to enlarged nuclei. The abundance of Cyclin E and giant polyploid nuclei are also present in mice that are mutant for cul1 (Doronkin, 2003).

Elevated levels of Cyclin E that may give cells a proliferative advantage are found in many human tumors. In many of these tumors the Cyclin E gene itself is amplified. However, among breast and ovarian cancer cell lines that overexpress Cyclin E protein without amplification, several lines have mutations in hCDC4, the human homolog of archipelago, suggesting that SCF[hcdc4] acts to suppress tumor formation. The results suggest that the CSN might have a similar effect (Doronkin, 2003).

In summary, these genetic and functional relationships between the CSN, the SCF, and the proteasome link these complexes in the regulation of Cyclin E degradation during normal development. When either the CSN or SCF are disrupted, the periodic degradation of Cyclin E is prevented, and cell cycle deregulation ensues (Doronkin, 2003).

Targeted disruption of Drosophila Roc1b reveals functional differences in the Roc subunit of Cullin-dependent E3 ubiquitin ligases

Cullin-dependent ubiquitin ligases regulate a variety of cellular and developmental processes by recruiting specific proteins for ubiquitin-mediated degradation. Cullin proteins form a scaffold for two functional modules: a catalytic module comprised of a small RING domain protein Roc1/Rbx1 and a ubiquitin-conjugating enzyme (E2), and a substrate recruitment module containing one or more proteins that bind to and bring the substrate in proximity to the catalytic module. This study presents evidence that the three Drosophila Roc proteins are not functionally equivalent. Mutation of Roc1a causes lethality that cannot be rescued by expression of Roc1b or Roc2 by using the Roc1a promoter. Roc1a mutant cells hyperaccumulate Cubitus interruptus, a transcription factor that mediates Hedgehog signaling. This phenotype is not rescued by expression of Roc2 and only partially by expression of Roc1b. Targeted disruption of Roc1b causes male sterility that is partially rescued by expression of Roc1a by using the Roc1b promoter, but not by similar expression of Roc2. These data indicate that Roc proteins play nonredundant roles during development. Coimmunoprecipitation followed by Western or mass spectrometric analysis indicate that the three Roc proteins preferentially bind certain Cullins, providing a possible explanation for the distinct biological activities of each Drosophila Roc/Rbx (Donaldson, 2004).

One possible explanation for the inability of a given Roc protein to rescue the phenotype of a different Roc mutant is that each Roc protein may form a unique set of E3 ubiquitin ligase complexes by preferentially interacting with different Cullin family members. To test this, coimmunoprecipitation experiments were performed with Roc1agrf::FLAG-Roc transgenes. Lysates from control, nontransgenic (w1118) embryos or embryos expressing each of the FLAG-Roc transgenes were incubated with anti-FLAG-agarose and immunocomplexes were analyzed by Western blotting or mass spectrometry. Western analysis with a CUL-1 antibody showed that CUL-1 efficiently coprecipitates with FLAG-Roc1a. Relatively little, but still above-background, amounts of CUL-1 was present in immunocomplexes from FLAG-Roc1b or FLAG-Roc2 lysates. This result shows that whereas Roc1a, Roc1b, and Roc2 are each able to bind to CUL-1 when expressed from the Roc1a promoter, Roc1a does so much more efficiently. Immunocomplexes were analyzed from each of the FLAG-Roc transgenic lysates by mass spectrometry. Proteins from a Coomassie-stained polyacrylamide gel that migrated with the predicted molecular weight of the Cullins and that were present in one or more of the transgenic lines but absent from wild-type, nontransgenic lysate were excised and identified by tandem mass spectrometry. Using this approach, CUL-1 and CUL-2 was identified in Roc1a immunocomplexes, CUL-3 in Roc1b immunocomplexes, and CUL-5 in Roc2 immunocomplexes. Because weaker Cullin-Roc interactions may not permit the precipitation of enough Cullin protein to be visible on a Coomassie-stained gel, this technique does not rule out any particular Cullin-Roc interactions. However, the data do suggest that there is a preference for the formation of certain Cullin-Roc complexes (Donaldson, 2004).

The results indicate that there are significant differences in the biological roles of the three Drosophila Roc proteins and that these differences are not simply the result of distinct expression patterns during development. In all of the experimental paradigms, Roc1a and Roc1b could partially, but not completely, substitute for one another, whereas Roc2 showed no ability to substitute for either Roc1 paralogue. Results of coimmunoprecipitation experiments suggest that these differences are due to preferential interactions between Roc and Cullin family members. For example, CUL-1 seems to interact most strongly with Roc1a, suggesting that a majority of SCF (i.e., CUL-1) targets require Roc1a. However, it cannot be ruled out that Roc1b or Roc2 function within the context of an SCF complex, since both showed weak interactions with CUL-1. Indeed, Roc1b seems to be capable of participating in SCF-mediated ubiquitylation, because it was able to rescue the aberrant accumulation of Ci, a bona fide SCF target, when overexpressed (Donaldson, 2004).

Because the Drosophila Roc proteins share between 40% and 60% overall sequence identity, it is somewhat surprising that a higher degree of complementation was not observed in rescue assays. Most of the conservation is within the C-terminal 67 residues, which contains the catalytic RING domain. Roc1a and Roc1b share 76% identity and 88% similarity in this domain, whereas Roc1a and Roc2 are 45% identical and 59% similar. In the N-terminal regions, the sequence identity/similarity is lower (38%/50% between Roc1a and Roc1b; 41%/57% between Roc1a and Roc2). Deletion of the Rbx1 (Roc1) N-terminus prevents interaction with CUL-1 in 293T cells. The crystal structure of the SCF complex shows that the association between Rbx1 and the C-terminal portion of the CUL-1 protein (termed the Cullin homology domain or CHD) consists of two parts. First, the RING domain of Rbx1 packs into a V-shaped groove formed by the alpha/B and WH-B domains of CUL-1. Second, the Rbx1 N terminus threads into CUL-1 and makes a five-stranded intermolecular ß-sheet (four strands provided by CUL-1 and one by Rbx1). This intermolecular ß-sheet seems to provide the primary mechanism of Rbx1 recruitment. Together, these data implicate the N terminus of the Drosophila Roc proteins as the region responsible for mediating the differential binding to Cullins (Donaldson, 2004).

This study used the powerful genetic techniques of the fruit fly to assess how the RING domain subunit contributes to the function of Cullin-dependent ubiquitin ligases. The Drosophila Roc proteins have nonredundant roles during development, and these differences may be mediated by the formation of specific Cullin-Roc ligase complexes. The results are consistent with studies of mammalian Roc proteins showing that although both Rbx1 and mammalian Roc2 can associate with all Cullin proteins, these interactions, as well as the associated ligase activities of the different complexes, seem to show certain preferences. Because each Cullin family member may use a distinct mechanism to target nonoverlapping sets of proteins for ubiquitylation, preferential Cullin binding provides a sufficient, if not the only, explanation for the functional differences among the three Drosophila Roc proteins. Further experiments are needed identify which complexes exist in vivo and to determine exactly what mediates these specific Cullin-Roc interactions (Donaldson, 2004).

Mouse Cul1 overexpression provides evidence indicating a role for an SCFSlmb complex in elimination of E2F during S phase in Drosophila

Cul1 is a core component of the evolutionarily conserved SCF-type ubiquitin ligases that target specific proteins for destruction. SCF action contributes to cell cycle progression but few of the key targets of its action have been identified. Expression of the mouse Cul1 (mCul1) in the larval wing disc of Drosophila has a dominant negative effect. It reduces, but does not eliminate, the function of SCF complexes, promotes accumulation of Cubitus interruptus (a target of SCF action), triggers apoptosis, and causes a small wing phenotype. A screen for mutations that dominantly modify this phenotype showed effective suppression upon reduction of E2F function, suggesting that compromised downregulation of E2F contributes to the phenotype. Partial inactivation of Cul1 delays the abrupt loss of E2F immunofluorescence beyond its normal point of downregulation at the onset of S phase. Additional screens showed that mild reduction in function of the F-box encoding gene slimb enhances the mCul1 overexpression phenotype. Cell cycle modulation of E2F levels is virtually absent in slimb mutant cells in which slimb function is severely reduced. This implicates Slimb, a known targeting subunit of SCF, in E2F downregulation. In addition, Slimb and E2F interacted in vitro in a phosphorylation-dependent manner. Thus the G1/S transcription factor E2F is an SCFSlmb target in Drosophila. These results argue that the SCFSlmb ubiquitin ligase directs E2F destruction in S phase (Hériché, 2003).

This study provides evidence indicating a role for an SCFSlmb complex in elimination of E2F during S phase in Drosophila. Mouse Cul1 overexpression has a dominant negative effect on SCF function in the wing imaginal disc resulting in apoptotic cell death leading to a small wing phenotype in adult flies. Thus, SCF activity normally suppresses cell death. The cell death that occurs upon reduction of Cul1 function is suppressed when E2F function is reduced by mutation. This genetic interaction might be due to action of E2F and Cul1 in parallel pathways, one suppressing and one enhancing cell death, or they might act in opposite directions in the same pathway. Because SCF complexes are involved in controlling protein degradation, the hypothesis is favored that Cul1 promotes the destruction of E2F and that the inappropriate persistence of E2F leads to cell death. Consistent with this idea, E2F overproduction can induce apoptosis. Furthermore, cell cycle specific destruction of E2F is suggested by the finding that E2F protein becomes undetectable in the synchronized S phase cells of the morphogenetic furrow of the eye imaginal disc. Double labelling analysis extended this finding to the asynchronously cycling cells of the wing imaginal disc and demonstrated that E2F is rapidly degraded prior to significant DNA replication. In contrast, it was found that E2F is detected in a fraction of S phase cells when SCF function is reduced by mCul1. This shows that normal E2F downregulation is delayed or slowed by reduction in SCF function (Hériché, 2003).

Although the dominant negative action of mCul1 in Drosophila was unanticipated, it offers a fortuitously convenient tool for the analysis of SCF function. The Drosophila Cul1 mutant is less useful because maternal contributions of Cul1 are so large that homozygous mutant animals develop to pupariation. Additionally, unlike the spatially restricted expression of mCul1, the strong Cul1 mutant alleles result in lethality. The stage and generality of the defects make analysis of the Cul1 mutant phenotype particularly difficult. In contrast, the induced expression of mCul1 in the wing disc does not compromise viability and it gives a distinctive graded phenotype well suited for the study of genetic modification. While the basis for the dominant negative effect of mCul1 remains unknown, it is important to recognize that inactivation of the endogenous Cul1 is incomplete so that a reduced level of Cul1 activity persists. One likely explanation for the negative effect is that mCul1 overexpression leads to sequestration of some limiting SCF components into weakly active complexes. This hypothesis is consistent with the fact that mCul1 retains some positive function in flies as revealed by its ability to prolong survival of dCul1 mutant flies (J.-K. Hériché and P.H. O'Farrell, unpublished data) (Hériché, 2003).

The essentially complete suppression of the mCul1 phenotype by reduction of the genetic dose of E2F suggests a remarkable level of specificity, in which, among the many targets of Cul1 action, the destruction of E2F appears to be particularly important. Similarly, limitations of Cul1 function in other organisms also uncovered the disproportionate importance of particular substrates. For example, Cul1 mutations in mice result in the accumulation of the SCF substrate cyclin E but not of p27, another well characterized SCF substrate. Among all of the substrates targeted for degradation by SCF in S. cerevisiae, it is the failure to degrade Sic1 that underlies the G1 arrest in cdc53 mutants. Thus, different substrates of the SCF appear to have particularly high dependence on SCF activity in different biological contexts. The experimental context using mCul1 expression in the wing disc is thought to be particularly effective in exposing the involvement of SCF in E2F destruction (Hériché, 2003).

Since SCF complexes function in conjunction with a variety of F-box proteins that act as specificity factors, mutations in individual F-box proteins ought to affect particular subsets of SCF substrates. In an extensive screen for loci that modify the reduction of function phenotype for Cul1, the gene encoding the F-box protein Slimb as a modifier was identified, but no contributions of other F-box encoding genes to the phenotype were detected. This implicates Slimb in the action of SCF on E2F. Analysis of E2F levels reveals that cell cycle oscillations in E2F levels were absent when Slimb function was severely reduced. Note that the severity of this E2F destruction phenotype in comparison to the mild defect in cell cycle programming of E2F destruction upon mCul1 expression is entirely consistent with the fact that the slimb mutant gives a stronger loss of function than the reduction of function imposed by mCul1 overexpression. The absence of cell cycle oscillation in E2F presence in the slimb mutant suggests that SCFSlmb is responsible for targeting E2F for S phase destruction. If this destruction is the consequence of direct action of SCFSlmb on E2F, the F-box protein would be expected to interact with E2F. This prediction was confirmed by pull-down experiments. Furthermore, it is demonstrated that the interaction between Slimb and E2F is dependent on phosphorylation, as expected for the interaction of F-box proteins with their substrates. The S phase specificity of E2F destruction is regulated by a targeting phosphorylation event, but this level of regulation has yet to be investigated (Hériché, 2003).

Since E2F is a positive regulator of the G1/S transition, it is not clear why S phase destruction of E2F is required. However, it is noted that slimb mutant cells do not replicate DNA normally and the replication defect is correlated with an increase in E2F in individual cells. This observation is consistent with observations indicating that E2F can limit DNA replication both in mammalian cells and in flies. This enigmatic feature of E2F regulation suggests that its continued presence has a negative effect on DNA replication and indicates that there is still much to learn about E2F roles in cell cycle regulation (Hériché, 2003).

SCFSlmb appears to influence other cell cycle events, perhaps by actions on other substrates, or perhaps as a result of events that are secondary to its influence on E2F destruction. For example, a slimb allele shows defects in centrosome duplication control, and slimb null cells undergo apoptosis. Both effects can be explained by a failure to inactivate E2F, since E2F promotes centrosome duplication in mammalian cells and E2F can induce apoptosis. A link between Slimb function and the RB/E2F pathway of cell proliferation control is also suggested by the genetic interaction between the C. elegans slimb ortholog lin-23 and the RBF ortholog lin-35 in which lin-35 function limits the severity of the loss of lin-23 . Since RBF is an important modifier of E2F activity, perhaps this interaction is a reflection of the action of both Lin-23 and Lin-35 on E2F. However, in the Drosophila system there was no modification of the mCul1 overexpression phenotype by mutations in either the RBF or DP gene, two known partners of E2F. This result suggests that these factors are not limiting factors in this situation or that E2F acts independently of RBF and DP. The latter hypothesis is the most likely explanation for the lack of an RBF interaction for the following reason. The E2F/RBF complex has to be disrupted for E2F to drive cells into S phase and E2F degradation occurs after the G1/S phase transition, which only happens after E2F has been released from its association with RBF. According to this view, it would be predicted that the mCul1-induced phenotype should be specifically sensitive to factors impinging on the elimination of E2F activity during S phase, and factors involved in the unknown processes by which persistence of E2F has negative outcome. Consequently, this experimental system may provide an avenue for the genetic dissection of this mysterious facet of E2F function (Hériché, 2003).

Thus, to explore potential roles for Cul1 in cell cycle control in D. melanogaster, mouse Cul1 (mCul1) was overexpressed in the wing imaginal disc. This overexpression has a dominant negative effect leading to a reduction in SCF function. The resulting small wing phenotype was used in a modifier screen to identify mutations in cell cycle genes capable of dominantly modifying the mCul1-induced phenotype. E2F loss-of-function mutations were the strongest modifiers since they completely suppress the phenotype. The reduction in SCF function associated with mCul1 overexpression also correlates with a failure to downregulate E2F normally in S phase. Additionally, mutations in slimb that reduce the function of the F-box protein Slimb enhance the phenotype and lead to persistence of E2F in S phase cells. Finally, this F-box protein interacts with E2F in vitro. These results indicate that an SCFSlmb complex is involved in regulating E2F activity during S phase (Hériché, 2003).

Encore facilitates SCF-Ubiquitin-proteasome-dependent proteolysis during Drosophila oogenesis: Encore physically interacts with cyclin E and Cul1

In Drosophila, egg development starts at the anterior tip of the ovary, in the germarium, where the germline stem cells divide to produce a cystoblast and a self-renewing stem cell. Each cystoblast undergoes four mitotic divisions with incomplete cytokinesis. The resulting 16 cells of each egg chamber are connected by intercellular bridges called ring canals. Exit from the cell cycle at the end of these four mitotic divisions requires the downregulation of Cyclin/Cdk activity. In the ovary of Drosophila, Encore activity is necessary in the germline to exit this division program (Ohlmeyer, 2003).

In encore mutant germaria, Cyclin A persists longer than in wild type. In addition, Cyclin E expression is not downregulated after the fourth mitosis and accumulates in a polyubiquitinated form. Mutations in genes coding for components of the ubiquitin-protease pathway such as cul1, UbcD2 and effete enhance the extra division phenotype of encore. Encore physically interacts with the proteasome, Cul1 and Cyclin E. The association of three factors, Cul1, phosphorylated Cyclin E, and the proteasome 19S-RP subunit S1, with the fusome is affected in encore mutant germaria. It is proposed that in encore mutant germaria the proteolysis machinery is less efficient and, in addition, reduced association of Cul1 and S1 with the fusome may compromise Cyclin E destruction and consequently promote an extra round of mitosis (Ohlmeyer, 2003).

Overexpression or loss-of-function mutations in a third group of genes such as Cyclin A, Cyclin B, Cyclin E and mutations in the gene encoding the E2 Ubiquitin conjugating enzyme UbcD1 lead to the production of cysts with 32 or 8 cells. These genes do not affect fusome integrity and thus timing and spatial characteristics of cell division appear to be intact. The encore gene belongs to this group of genes: its product is necessary for exit from mitosis. Loss of Encore activity results in egg chambers containing 32 rather than 16 cells. Mutations in the encore gene produce additional phenotypes, which show differential temperature sensitivity. encore mutant females raised at 18°C produce egg chambers with 16 cells, but they give rise to ventralized eggs. The extra cell division phenotype is only observed when encore mutant females are raised at high temperatures (25°-29°C). The encore gene encodes a 200 kDa protein with no homolog of a defined biochemical function. The mechanism by which Encore promotes exit from the cell cycle after four germline mitoses has been investigated (Ohlmeyer, 2003).

Cell cycle progression is controlled by a series of cell cycle dependent kinases (Cdk). Cdk activity is carefully regulated by the levels of the Cyclin subunits, by Cdk inhibitors (CKI) and by post-translational modification of the Cdk subunit through both activating and inactivating phosphorylation. Transition from G1 to S phase depends on Cdk2/Cyclin E activity, and on the timely destruction of the Cdk2/Cyclin E inhibitor p27. The Drosophila p27 homologue, Dacapo, is required for exit from the cell cycle in the embryo and eye imaginal disc. In addition, exit from the cell cycle requires destruction of the cyclins by the ubiquitin-proteasome system (UPS). The addition of ubiquitin requires three different activities; the ubiquitin activating enzyme (E1), the ubiquitin conjugating enzyme (E2) and the ubiquitin ligase enzyme (E3). The ubiquitinated protein bound to E3 is presented to the proteasome, isopeptidase activities in the 19S-recognition particle (RP) of the proteasome cleave the ubiquitin tail, the protein is unfolded and finally destroyed by the proteasome 20S-core particle (CP) (Ohlmeyer, 2003).

There are two E3 enzyme complexes that regulate the cell cycle progression. The first, the APC/cyclosome, regulates progression from G2 to M phase transition. The second, the SCF complex regulates the G1 to S phase transition. The SCF complex is composed of Skp/Cullin/Rbx1 and F-box proteins and controls substrate ubiquitination via an interaction between the F-box component and the phosphorylated target protein. In Drosophila and mammalian systems, mutations in the Cul3 and Ago genes have been shown to cause the accumulation of Cyclin E, entry to S-phase and doubling of cell number. Thus, proper regulation of the destruction machinery is important for maintaining normal levels of Cyclin E and assuring proper cell cycle progression (Ohlmeyer, 2003).

The work presented in this study demonstrates that the encore gene product associates with the SCF-ubiquitin-proteasome system and is required for proper exit from germline mitosis. The failure to downregulate Cyclin E after four cell divisions in conjunction with an accumulation of Cyclin A protein provide the conditions to promote an extra cell division. Encore can bind to Cul1, Cyclin E-Ub(n) and the proteasome. Cul1 and the proteasome 19S-RP subunit S1 are associated with the fusome and these associations are very much attenuated in encore mutant ovaries. It is proposed that as a direct consequence, Cyclin E is not degraded properly, its activity is misregulated and the cyst undergoes one extra cell division (Ohlmeyer, 2003).

This study shows that the fusome is a regulator of cell division during early oogenesis. Some of the functions ascribed to the fusome are to synchronize cyst mitosis and to provide the scaffold for the transport system necessary for oocyte determination. Limiting the number of cell divisions in the germarium could be achieved by regulating the association of proteins such as the cyclins and/or other cell cycle regulators with the fusome. The expression pattern of Cyclin A, Cul1, P-Cyclin E and 19S-S1 proteins in the germarium supports the idea that the fusome plays an important role in the regulation of mitosis. Indeed, Cyclin A association with the fusome is transient and occurs only during cyst division. In encore mutant germaria, Cyclin A remains associated with the fusome after cell division has stopped. Cul1 localization to the fusome suggests that the rest of the SCF complex also associates with the fusome and that substrate ubiquitination may happen at the fusome. The SCF component Cul1 is mainly associated with the fusome in the wild-type germaria. In encore mutant germaria, Cul1 localization to the fusome is very poor, leading to a proposal that this may be one reason why Cyclin E is not degraded properly. This also suggests that the degradation of Cyclin E and perhaps of other proteins degraded by the SCF-UPS may occur at the fusome. The association of P-Cyclin E supports this idea. The localization of P-Cyclin E in the wild type seems to be dynamic, consistent with the idea that the phosphorylated substrate is localized to the fusome, and then rapidly degraded via the SCF-UPS. In encore mutant germaria, the poor localization of Cul1 may result in an inefficient assembly of SCF complexes at the fusome. P-Cyclin E is localized to the fusome, but its degradation is compromised and as a result a consistent expression of P-Cyclin E is observed at the fusome. The partial association of the proteasome 19S-RP subunit S1 to the fusome supports the idea that proteolysis may occur at the fusome. The proteasome 19S-RP would recognize the polyubiquitinated substrate and recruit the rest of the proteasome to the fusome (Ohlmeyer, 2003).

The results suggest that Encore can associate with the SCF ubiquitin-proteasome system machinery and assists with the degradation of Cyclin E and perhaps other SCF substrates. Since the mutant Encore protein can still interact with SCF-UPS components, the mutant protein may form complexes but these might be inactive and/or the mutant protein poisons the degradation machinery. Consistent with such a hypothesis, the encore extra cell division phenotype is milder in hemizygous versus homozygous females at 25°C (Hawkins, 1996). Encore is required for the proper localization of Cul1, P-Cyclin E, S1 and presumably the rest of the proteolysis complex to the fusome. This localization may be more crucial at 29°C, whereas at lower temperatures a less efficient degradation system may have enough time for normal cell cycle regulation. encore mutations do not affect the 20S-Core Particle activity as measured by the rate of degradation of a fluorogenic peptide. It is not known whether Encore retains Cul1 at the fusome or whether Encore directly or indirectly modifies Cul1 in order to promote its localization at the fusome. Cul1 is known to be modified by the addition of Nedd8; however, Cul1 seems to be equally neddylated in encore and wild-type ovary extracts (Ohlmeyer, 2003).

In summary, the results suggest that the Encore protein assists with proper cell cycle progression in the Drosophila germarium by ensuring that Cul1 and the proteolysis machinery is localized at the mitosis coordination center, the fusome (Ohlmeyer, 2003).

A novel F-box protein is required for caspase activation during cellular remodeling in Drosophila

Terminal differentiation of male germ cells in Drosophila and mammals requires extensive cytoarchitectural remodeling, the elimination of many organelles, and a large reduction in cell volume. The associated process, termed spermatid individualization, is facilitated by the apoptotic machinery, including caspases, but does not result in cell death. From a screen for genes defective in caspase activation in this system, a novel F-box protein, which was termed Nutcracker, was isolated that is strictly required for caspase activation and sperm differentiation. Nutcracker interacts through its F-box domain with members of a Cullin-1-based ubiquitin ligase complex (SCF): Cullin-1 and SkpA. This ubiquitin ligase does not regulate the stability of the caspase inhibitors DIAP1 and DIAP2, but physically binds Bruce, a BIR-containing giant protein involved in apoptosis regulation. Furthermore, nutcracker mutants disrupt proteasome activity without affecting their distribution. These findings define a new SCF complex required for caspase activation during sperm differentiation and highlight the role of regulated proteolysis during this process (Bader, 2010).

Most F-box proteins also possess another protein-interaction domain, usually comprising WD40 or LRR motifs, that is responsible for binding the ubiquitylation substrate. Nutcracker belongs to the class of F-box proteins that do not contain a known protein-protein interaction domain, and differs topologically from most F-box proteins in that its F-box domain is at the very C-terminus (Kirk, 2008). Sequence alignments with several F-box-only proteins revealed that Nutcracker shares some limited amino acid similarity with the mammalian FBXO7 protein, which also contains the F-box domain at the C-terminus. Although the sequence conservation is limited primarily to the F-box domain, it is possible that these two proteins share functional properties, as do other proteins that are conserved only within limited regions. For example, the C. elegans p53 protein displays less than 20% overall primary sequence similarity to the human protein, mostly in the active sites, but has been demonstrated to function in related cellular processes. Since FBXO7 has been shown to regulate the stability of cIAP1 (BIRC2) (Chang, 2006), it is possible that these two E3 ligases have a conserved function in caspase regulation (Bader, 2010).

The ubiquitin-proteasome system is implicated in regulating caspase activity. Several studies have shown that the ubiquitylation and degradation of DIAP1 is a means of displacing it from caspases when apoptosis is favored. Also, ubiquitylation of caspases themselves contributes to their regulation by preventing a critical mass of full-length caspases from auto-activation in a normal setting. The wide variety of other ubiquitin-modifying proteins that regulate apoptosis and caspase activity, including Bruce, Morgue and Uba1, imply the existence of an elaborate regulatory network that is controlled by ubiquitylation (Bader, 2010).

In the screen isolated another ubiquitin ligase, a Cullin-3-based complex, was isolated, indicating that caspase activation in this system is tightly controlled by ubiquitin modifications. These two complexes could regulate the stability of the same substrate, as is the case for regulation of Cubitus interruptus (Ci) stability in Hedgehog signaling by both Cullin-1-based and Cullin-3-based complexes, or they might target multiple important substrates. Alternatively, the E3 ligases isolated in the screen might play non-degradative roles in controlling caspase activity. For example, mono-ubiquitylation affects the targeted localization of proteins and these ubiquitin ligases might control the proper localization of caspase regulators. Another possibility is that these E3 ligases mediate the non-classical Lys63 ubiquitin chain addition that is important for protein-protein interaction. Thus, instead of degradation, these proteins might actually control interactions between caspase regulators (Bader, 2010).

Although DIAP1 is the only Drosophila BIR-containing protein that has been shown to directly inhibit caspases in vivo, Bruce has been implicated in modifying apoptosis in several death paradigms, and mutations in its mammalian homolog cause defects associated with excess cell death. This study showed that Bruce can physically bind Nutcracker, and that this interaction is independent of the F-box domain. Therefore, Bruce might be a substrate of Nutcracker. However, it was not possible to determine the steady-state levels of Bruce in nutcracker mutants, so it is as yet unclear whether it is indeed a substrate or a complex partner. The fact that Bruce also binds to another E3 ligase isolated in the screen suggests that this protein is a common regulator of caspase activation during individualization (Bader, 2010).

nutcracker mutants cause a reduction in proteasome activity. This decreased activity does not seem to be due to proteasome mislocalization or a reduction in their numbers, suggesting that Nutcracker controls proteasome activity directly. It is possible that Nutcracker modifies proteasome regulators, which could include, for example, proteins of the regulatory particle of the proteasome. An attractive model is that Nutcracker functions through proteasomes to activate caspases. Caspase activity is tightly controlled by the ubiquitin proteasome system. Therefore, it is possible that local activation of proteasomes controls localized caspase activation (Bader, 2010).

Many questions remain regarding the non-lethal role of caspases in cellular remodeling. For instance, is it a specialized activation that is governed by dedicated proteins, and to what extent are known apoptotic regulators involved in this process? Another intriguing question is how cells tolerate a certain level of caspase activation and avoid destruction by these potentially deadly proteases. Answers to these questions will not only uncover novel caspase regulators, but might also help us to understand how diseased cells, such as cancer cells, manage to escape cell death (Bader, 2010).

insomniac and Cullin-3 regulate sleep and wakefulness in Drosophila

In a forward genetic screen in Drosophila, insomniac, a mutant that severely reduces the duration and consolidation of sleep, has been isolated. Anatomically restricted genetic manipulations indicate that insomniac functions within neurons to regulate sleep. insomniac expression does not oscillate in a circadian manner, and conversely, the circadian clock is intact in insomniac mutants, suggesting that insomniac regulates sleep by pathways distinct from the circadian clock. The protein encoded by insomniac is a member of the BTB/POZ superfamily, which includes many proteins that function as adaptors for the Cullin-3 (Cul3) ubiquitin ligase complex. It was shown that Insomniac can physically associate with Cul3, and that reduction of Cul3 activity in neurons recapitulates the insomniac phenotype. The extensive evolutionary conservation of insomniac and Cul3 suggests that protein degradation pathways may have a general role in governing the sleep and wakefulness of animals (Stavropoulos, 2011).

Emerging evidence has suggested that the sleep states of diverse animals may be regulated by conserved molecular mechanisms, although many of these mechanisms remain undefined. insomniac, a gene that governs the duration of sleep and wakefulness in Drosophila is likely to engage protein degradation pathways to regulate sleep. Both insomniac and these pathways are well conserved, suggesting that they may be employed generally to regulate sleep in animals (Stavropoulos, 2011).

In rats and Drosophila, chronic sleep deprivation leads to reduced lifespan and lethality. Mutations in Shaker, sleepless, and Hyperkinetic that strongly reduce sleep in Drosophila are also associated with decreased longevity. In each case, longevity has been assessed for classical mutants in which gene function is reduced or absent in all tissues. Two independent insomniac mutants exhibit similarly decreased longevity. However, neuronally restricted depletion of insomniac, which sharply reduces the duration of sleep, has no measurable effect on longevity, demonstrating that the two attributes can be uncoupled. Similarly, fumin mutants affecting the Drosophila dopamine transporter gene display a strong decrease in sleep but normal longevity (Stavropoulos, 2011).

These results do not contradict the notion that sleep has critical physiological functions or that sleep deprivation leads to deficits in waking performance, although they do suggest that certain disruptions of sleep can be tolerated without impacting lifespan. Reductions in sleep duration may need to exceed a certain threshold to affect longevity, and the lethality elicited by chronic sleep deprivation regimens, as well as that of especially severe sleep mutants, may reflect the reduction of sleep to extremely low levels. For mutations with more modest effects on sleep, interpretations that attribute a causal relationship between altered sleep and reduced longevity may be problematic, particularly for those genes that are broadly expressed and whose loss-of-function is likely to have numerous pathological consequences. Additional genetic manipulations that perturb sleep in increasingly specific ways are required to further assess the relationship between sleep and longevity in both Drosophila and vertebrates (Stavropoulos, 2011).

Anatomically restricted manipulations of insomniac indicate that its expression within neurons is essential for normal sleep and wakefulness. The neuronal requirement for insomniac appears to be broad, as drivers that provide panneuronal or broad neuronal expression alter sleep most strongly in depletion and rescue experiments. The possibility cannot however be excluded that insomniac regulates sleep by functioning in a smaller number of neurons that are dispersed within the brain and not effectively represented by individual drivers were assayed. In particular, an insomniac-Gal4 reporter is expressed in regions of the Drosophila brain that are implicated in regulating sleep, including the mushroom bodies and the pars intercerebralis, although driving insomniac expression in these areas individually does not rescue the sleep defect of insomniac mutants, with the exception of a weak rescue provided by the pars-intercerebralis-specific Mai301-Gal4 driver. Further manipulations of insomniac within the nervous system are necessary to understand the neuroanatomical basis by which it regulates sleep (Stavropoulos, 2011).

Several lines of evidence indicate that insomniac exerts its effects on sleep by a mechanism functionally distinct from the circadian clock. The circadian clock is intact in insomniac mutants, and insomniac expression is not regulated in a circadian fashion. Furthermore, the expression of insomniac in clock neurons is unable to restore normal sleep patterns in insomniac mutant backgrounds. Consistent with these data, daily sleep profiles indicate that the circadian control of sleep is intact in insomniac mutants. As is the case for wild-type animals, the highest probability of sleep during the dark phase is observed soon after the onset of darkness, with a decreasing sleep drive as the dark phase proceeds. The profile of sleep probability during the light phase is similarly intact. The principal alteration of sleep in insomniac animals is a reduced likelihood of sleeping throughout the day and night, consistent with the inference that insomniac may contribute to homeostatic mechanisms that regulate sleep need (Stavropoulos, 2011).

Cullins are scaffold proteins that assemble multisubunit E3 ubiquitin ligase complexes that ubiquitinate and degrade a variety of protein substrates in diverse biological contexts. The C termini of cullins interact with RING-domain ubiquitin ligases, while the N termini interact with adaptor proteins that recruit substrates for ubiquitination. Cul3 complexes utilize proteins of the BTB superfamily as their adaptor. In addition to the KCTD proteins that are known to function as Cul3 adaptors, more than half of the non-channel KCTD proteins, including the three vertebrate orthologs of Insomniac, are candidate Cul3 adaptors, as they copurify specifically with Cul3, but not with other cullins. For several of these candidate adaptors, including KCTD5 and TAG-303, the C. elegans ortholog of Insomniac, independent biochemical evidence confirms their ability to associate physically with Cul3. The finding that Insomniac is able to physically interact with Cul3 indicates that this interaction is evolutionarily conserved and supports the hypothesis that Insomniac serves as a Cul3 adaptor (Stavropoulos, 2011).

The reductions in sleep elicited by neuronal depletion of Cul3, and of its activator Nedd8, show that protein degradation pathways have a vital role in regulating sleep in Drosophila. Although alternative mechanisms cannot be excluded, the simplest hypothesis consistent with the data is that Insomniac engages the Cul3 protein degradation pathway to regulate sleep. One clear implication of this hypothesis is that the increased wakefulness of insomniac and Cul3 mutants may result from the inappropriate accumulation of substrates whose degradation is normally mediated by these proteins. The results suggest that such target substrates promote wakefulness and inhibit sleep, but they do not distinguish the neuronal function of these substrates. Target substrates regulated by Insomniac and Cul3 might function in a developmental manner, for example, in the elaboration of neural circuits that regulate sleep. Indeed, Cul3 has been implicated in regulating axonal and dendritic branching. Alternatively, such substrates might actively promote waking in adult animals, such that their ongoing degradation is part of the homeostatic mechanism contributing to the regulation of sleep-wake cycles (Stavropoulos, 2011).

Knockdown of SCFSkp2 function causes double-parked accumulation in the nucleus and DNA re-replication in Drosophila plasmatocytes

In Drosophila, circulating hemocytes are derived from the cephalic mesoderm during the embryonic wave of hematopoiesis. These cells are contributed to the larva and persist through metamorphosis into the adult. To analyze this population of hemocytes, data was considered from a previously published RNAi screen in the hematopoietic niche, which suggested several members of the SCF complex play a role in lymph gland development. eater-Gal4;UAS-GFP flies were crossed to UAS-RNAi lines to knockdown the function of all known SCF complex members in a plasmatocyte-specific fashion, in order to identify which members are novel regulators of plasmatocytes. This specific SCF complex contains five core members: Lin-19-like (Cul-1), SkpA, Skp2, Roc1a and complex activator Nedd8. The complex was identified by its very distinctive large cell phenotype. Furthermore, these large cells stained for anti-P1, a plasmatocyte-specific antibody. It was also noted that the DNA in these cells appeared to be over-replicated. Gamma-tubulin and DAPI staining suggest the cells are undergoing re-replication as they had multiple centrioles and excessive DNA content. Further experimentation determined enlarged cells were BrdU-positive indicating they have progressed through S-phase. To determine how these cells become enlarged and undergo re-replication, cell cycle proteins were analyzed by immunofluorescence. This analysis identified three proteins that had altered subcellular localization in these enlarged cells: Cyclin E, Geminin and Double-parked. Previous research has shown that Double-parked must be degraded to exit S-phase, otherwise the DNA will undergo re-replication. When Double-parked was titrated from the nucleus by an excess of its inhibitor, geminin, the enlarged cells and aberrant protein localization phenotypes were partially rescued. The data in this report suggests that the SCFSkp2 complex is necessary to ubiquitinate Double-parked during plasmatocyte cell division, ensuring proper cell cycle progression and the generation of a normal population of this essential blood cell type (Kroeger, 2013).

The generation of an eaterGal4; UAS-GFP strain allowed identification the functional importance of SCF complex members for the plasmatocyte blood cell lineage by a RNAi knockdown approach. Using this technique, several genes belonging to the core SCF complex were identified that, when knocked-down, caused a very distinctive giant cell phenotype. Importantly, as eater was bing used as a driver to identify complex components, it was confirmed that these enlarged cells were plasmatocytes by anti-P1 plasmatocyte-specific antibody staining. This suggested, as proof-of-principle, that knockdown of gene function in mature plasmatocytes could elicit aberrant phenotypes dependent on the functional requirement of an essential gene/gene complex (Kroeger, 2013).

Previous research has shown that there are several Drosophila genes that may be involved in SCF complexes in order to determine specificity for a substrate. The F-box is thought to convey specificity of this complex by recruiting the substrate, however activation of the Cullin by neddylation factors also plays a role in ubiquitation of the substrate. A comprehensive list of all known and predicted complex members was used to identify the remaining members of the specific SCF complex that function in Drosophila hematopoiesis, as knockdown of only one of each of the core components caused enlarged plasmatocytes. lin19, SkpA and Roc1a likewise play a role in the hematopoietic niche, the PSC of the larval lymph gland. Knockdown of these genes caused a decrease in the number of PSC cells, as well as an increase in the size of these cells. These data, along with the findings in this current study, suggest that the SCF complex has a significant role in multiple aspects of Drosophila larval hematopoiesis (Kroeger, 2013).

Using fluorescence microscopy, it was noted that the enlarged cells caused by the SCF knockdown had a significant excess of DNA in the nuclear region. To investigate the hypothesis that DNA re-replication was occurring in plasmatocytes with the SCF complex knockdown, anti-gamma- Tubulin staining of centrioles was performed. Previously, it was shown that knockdown of Gem elicits DNA re-replication, therefore this study used it as a positive control. It was evident that the lin19 knockdown had multiple centrioles in one giant plasmatocyte, similar to plasmatocytes from the gem RNAi samples. It was also clear that the DNA had replicated many times, without any cellular division as indicated by BrdU-positive, but phospho-Histone H3-negative enlarged cells. These data support the idea that plasmatocytes from SCF knockdown animals undergo DNA re-replication, thus the SCF complex is necessary for Dup degradation. Additionally, previous research had identified a number of proteins that when misexpressed or knocked-down cause an enlarged cell phenotype with excess DNA replication. Several papers have shown that misregulation of Cyclin E can cause aberrant DNA synthesis. Research has also suggested that knockdown of Gem can cause this excessive DNA phenotype. In the current experiments, antibody staining identified that the subcellular localization of both these proteins changed between control samples and the lin19 knockdown. Importantly, Dup is necessary for DNA replication, but it must be degraded to prevent re-replication. As the main role of Gem is to inhibit Dup, and Gem was no longer found in the nucleus in the knockdown, this is suggestive that Gem had complexed with Dup, removing it from the nucleus. Conversely, Cyclin E was found in the nucleus. This is notable because Cyclin E is known to phosphorylate Dup marking it for ubiquitination, leading to its nuclear localization. It is also known that SCFSkp2 degrades Cyclin E. This is another explanation for the accumulation of Cyclin E in the nucleus of SCF knockdown hemolymph samples. These data suggest that Dup may be the target substrate for the SCF complex being studied, with a secondary target possibly being Cyclin E. Previous research in human cells has shown that SCFSkp2 regulates the degradation of Cdt1 (the homolog of Drosophila Dup)(Li, 2003). It has also been shown that the activated SCFSkp2 complex plays a role in murine hematopoiesis, by ubiquitinating proteins necessary for proper cell cycle, such as Cyclin E. There are still many questions to be answered about SCF regulation in blood cells, as some of these results are contradictory (Kroeger, 2013).

In addition to these data, protein localization in the knockdown of Cyclin E showed that Gem had been removed from the nucleus, again consistent with the notion that it was titrated away from the nucleus by binding Dup. This is plausible because the SCF complex can recognize its substrates due to phosphorylation state. Since Cyclin E was knocked-down, Dup was not properly phosphorylated, and it was not recognized as the substrate by the SCF complex, therefore never being ubiquitinated nor degraded. Furthermore, in the Cyclin E knockdown, Dup localized to the nucleus similar to its localization in the SCF knockdown. This would make it necessary for Gem to inhibit Dup, causing Gem to take on a non-nuclear localization, while Dup would have a nuclear localization, if Dup was in excess. Taken together, these lines of investigation support the hypothesis that Cyclin E is necessary to phosphorylate Dup, allowing the SCF complex to recognize and ubiquitinate it. Dup that remains in the nucleus after degradation must be bound by Gem for the cell cycle to progress properly. DNA re-replication will occur if Dup remains in the nucleus. It is highly suggestive that knockdown of Cyclin E or the SCF complex perturbs this mechanism, causing Dup accumulation in the nucleus, and the cells to re-initiate DNA replication. Furthermore, others have shown there must be a balance of Gem and Dup in the nucleus for proper progression through the cell cycle. This research shows that there is a lack of Gem and an accumulation of Dup in the nucleus, which leads to excessive DNA replication and additional centriole replication in five percent of the plasmatocyte population (Kroeger, 2013).

Although re-replication is one mechanism to explain the SCF loss-of-function phenotype, a similar non-canonical process, known as endoreplication, could also account for the over-replicative system in these cells. Endoreplication is a cycle in which cells undergo S phases that are separated only by gap phases but not an intervening mitosis. However, endoreplication is not known to occur in wild-type Drosophila plasmatocytes. Further, Drosophila plasmatocytes are most similar to mammalian macrophages, which also do not endoreplicate. Since several of the proteins studied in this paper have been implicated in re-replication with phenotypes including enlarged cells, increased DNA content, and multiple centriole replication, the hypothesis is favored that re-replication is triggered in plasmatocyte development in the absence of SCF complex activity (Kroeger, 2013).

It is intriguing that only five percent of the cells display the re-replication phenotype. One explanation is that the smaller cells have arrested. There are many intrinsic mechanisms to ensure proper cell cycle progression preventing re-replication and ultimately cancer. It is possible these enlarged cells have escaped these mechanisms, causing the cell to replicate their DNA many times without going through mitosis, while the smaller cells arrest, to prevent this phenotype. It is also possible that only five percent of these cells are going through cell division during the time the RNAi is functionally knocking down the gene. Previous research has suggested that during mid-to-late third instar larval stages, only one to two percent of cells are going through mitosis at a given time. eaterGal4 is activated during second instar, however there is likely a latent period between activation of Gal4 and protein knockdown by the RNAi. This is consistent with only five percent of cells having an active cell cycle, and becoming enlarged through re-replication. A final possibility is that there are partially redundant mechanisms for the regulation of Dup. As previously described, the SCF complex has been shown to be involved in the ubiquitination and subsequent degradation of Dup, and Gem will inhibit the remainder of the Dup that may be in the nucleus. There may be additional mechanisms which ubiquitinate or inhibit Dup, therefore avoiding re-replication. The smaller cells may have activated one of these mechanisms to aid the cell in proper cell cycle, ultimately avoiding cancer. The regulation of Dup is of vast importance, and there are several possibilities of alternate mechanisms to prevent the re-replication phenotype elicited by cells which have excess Dup in the nucleus (Kroeger, 2013).

To further implicate the necessity of Dup regulation in the proper cell cycle of plasmatocytes, a rescue experiment was performed by overexpressing the Dup inhibitor, Gem. By overexpressing this inhibitory protein, it was hypothesized that the nuclear localization of Gem would increase, the protein would bind Dup, and therefore decrease the re-replication that is observed in SCF complex knockdown. Performing immunohistochemistry experiments identified that there was an increase in nuclear Gem and a decrease in Dup. Additional experimental evidence supports this hypothesis as there is a decrease in size of plasmatocytes with genotype pxnGal4>UAS-Gem43>UAS-lin19 RNAiHM05197 compared with pxnGal4>UAS-lin19 RNAiHM05197. There is a drastic decrease in the number of giant cells, which are larger than 25.1 μm, in pxn>UAS-Gem43>UAS-lin19 RNAiHM05197 (8/100) plasmatocytes compared with SCF knockdown hemocytes (45/100). It was also noted that there was a significant decrease in the average size of plasmatocytes in hemolymph samples from Gem overexpression in the SCF knockdown background (p<0.001). These lines of evidence are all suggestive that knockdown of the SCF complex increased nuclear Dup leading to re-replication. By over-expressing its inhibitor, Gem, it is possible to partially rescue this enlarged cell phenotype generated by excess nuclear Dup. These data suggest the regulation of Dup is important in the proper cell cycle progression of plasmatocytes. Furthermore, these data support the hypothesis that the SCFSkp2 complex is responsible for the ubiquitination of Dup, allowing plasmatocytes to proliferate properly. Although this study provides substantial genetic evidence that the SCFSkp2 complex is necessary to ubiquitinate Dup allowing for proper hematopoietic cell cycle progression, future studies using biochemical techniques to show physical interactions are needed to support the model proposed here (Kroeger, 2013).

Furthermore, there are two ubiquitin ligase complexes known to be involved in the ultimate degradation of Dup: The SCFSkp2 complex, described in this manuscript, and the Cul4-DDB1-CDT2-PCNA (Cul4CDT2) complex. To vastly decrease the possibility that the Cul4CDT2 complex was responsible for the enlarged cell phenotype, both DDB1 and PCNA were knocked-down via RNAi and Cul4 mutants were also analyzed. Although DDB1 functional knockdown elicited a small number of enlarged cells, these cells had a different morphology than the SCFSkp2 knockdown. Additionally, none of the other analyses elicited any enlarged cells as observed when the SCFSkp2 complex was knocked-down. This further implicates the necessity of the SCFSkp2 complex in the proper plasmatocyte cell cycle (Kroeger, 2013).

To summarize, this manuscript identifies the SCF ubiquitin ligase complex as a novel regulator of plasmatocytes. Genetic evidence is presented that suggests that Dup is the main target for the SCFSkp2 complex. It is proposed that the SCFSkp2 complex plays an integral role in Drosophila hematopoiesis by ubiquitinating Dup, which is necessary for proper cell cycle progression. Knockdown of the SCF complex causes an accumulation of Dup in the nucleus, inducing the cell to undergo multiple rounds of replication without an intervening mitosis or cytokinesis. This causes some plasmatocytes to become vastly enlarged, with multiple centrioles and excessive DNA content. Taken together, these findings provide evidence that the SCF complex is necessary for proper cell cycle progression during plasmatocyte development in Drosophila. As the SCF complex is conserved from Drosophila to humans, these findings implicate the importance of the roles of ubiquitin ligase complexes in the cell cycle and their potential malfunctions in blood cell cancers (Kroeger, 2013).

Ter94 ATPase complex targets k11-linked ubiquitinated Ci to proteasomes for partial degradation

The Cubitus interruptus (Ci)/Gli family of transcription factors can be degraded either completely or partially from a full-length form [Ci155/GliFL] to a truncated repressor (Ci75/Gli(R)) by proteasomes to mediate Hedgehog (Hh) signaling. The mechanism by which proteasomes distinguish ubiquitinated Ci/Gli to carry out complete versus partial degradation is not known. This study shows that Ter94 ATPase and its mammalian counterpart, p97, are involved in processing Ci and Gli3 into Ci75 and Gli3R, respectively. Ter94 regulates the partial degradation of ubiquitinated Ci by Cul1-Slimb-based E3 ligase through its adaptors Ufd1-like and dNpl4. Cul1-Slimb-based E3 ligase, but not Cul3-Rdx-based E3 ligase, modifies Ci by efficient addition of K11-linked ubiquitin chains. Ter94Ufd1-like/dNpl4 complex interacts directly with Cul1-Slimb, and, intriguingly, it prefers K11-linked ubiquitinated Ci. Thus, Ter94 ATPase and K11-linked ubiquitination in Ci contribute to the selectivity by proteasomes for partial degradation (Zhang, 2013).

Hh signaling plays important roles in metazoan development, and its malfunction is implicated in numerous human congenital disorders and cancers. Secreted Hh proteins bind Patched (Ptc)-iHog coreceptors to relieve an inhibitory effect of Ptc on Smoothened (Smo), which leads to activation of the Ci/Gli family of zinc finger transcription factors. Biochemical and genetic studies in Drosophila have revealed several important steps in the regulation of Ci/Gli activity. In the absence of Hh, full-length Ci, Ci155, is sequentially phosphorylated by protein kinase A (PKA), glycogen synthase kinase 3 (GSK3), and casein kinase I (CKI) and then ubiquitinated by Cullin 1 (Cul1)-Supernumerary limbs (Slimb, known also as β-TrCP)-based E3 ligase. This results in partial degradation by proteasomes, leaving the N terminus of Ci intact (Ci75) as a transcriptional repressor. In the presence of Hh, unphosphorylated Ci155 accumulates and enters nucleus to activate Hh target genes. As a feedback control of the pathway, active Ci155 induces the expression of roadkill (rdx)/Hib to form Cul3-Rdx-based E3 ligase and promotes complete proteasomal degradation of Ci155 (Zhang, 2013).

Although it is well established that Ci is ubiquitinated by Cul1-Slimb and Cul3-Rdx-based E3 ligases under different conditions, it remains unknown how proteasomes distinguish ubiquitinated Ci for partial versus complete degradation. As ubiquitinated proteins are transferred to proteasomes by different pathways, it is hypothesized that some specific components are involved in the recruitment of ubiquitinated Ci for partial degradation. Transitional elements of the endoplasmic reticulum 94 kDa (Ter94) was identified as the Drosophila homolog of yeast CDC48, which is a member of the ATPase associated with various cellular activities (AAA) family. In mammals, the CDC48/Ter94 homolog p97 (also known as VCP) mainly functions in endoplasmic reticulum-associated degradation (ERAD). Proteomic analysis revealed that p97 might play a broad role in regulating the turnover of ubiquitin proteasome system (UPS) substrates. This study has shown that Ter94 is a component of the Ci processing machinery and is critical for Ci75 formation (Zhang, 2013).

The control of partial versus complete proteasomal degradation of Ci and Gli3 is a major regulatory step in Hh signal transduction. How proteasomes distinguish ubiquitinated Ci to carry out either partial or complete degradation is not known. Based on the current findings, the following model is proposed. In the absence of Hh, Ci155 is phosphorylated and ubiquitinated by Cul1-Slimb-based E3 ligase to generate Ci75. In this process, K11-linked ubiquitin chains are added onto Ci155. Ter94Ufd1-like/dNpl4 forms a complex with Cul1-Slimb-based E3 ligase through Ufd1-like and Roc1a, and another component dNpl4 is bound to the K11-linked ubiquitin chains on Ci155. Through ATP hydrolysis, Ter94Ufd1-like/dNpl4 facilitates the delivery of ubiquitinated Ci155 to the proteasomes for processing (Zhang, 2013).

Besides Ci and Gli3, the best example of partial degradation is the processing of human nuclear factor-κB (NF-κB) and its yeast homologs, SPT23 and MGA2. Previous studies have suggested that some common features of processing determinant domain (PDD) are involved in the partial proteasomal degradation of Ci and NF-κB. However, Ci also undergoes complete proteasomal degradation by Cul3-Rdx-based E3 ligase. Why such 'degradation stop signals' fail to work in such instances and how proteasomes make the decision for partial or complete degradation are unknown. Based on the current results and previous studies, it is proposed that Ter94/p97 complex may specifically target 'partial-degradation-proteins' to proteasomes through K11-linked ubiquitin chains. Further investigation is needed to provide direct evidence to support this hypothesis. As many similarities are shared between Ci and NF-κB precursors in partial degradation, it will be interesting to test whether p97 and K11-linked ubiquitination are also involved in the partial degradation and/or maturation of p100 in NF-κB signaling (Zhang, 2013).

This study found that K11-linked chains are added onto Ci by Cul1-Slimb-based E3 ligase in the absence of Hh pathway activity, whereas Cul3-Rdx-based E3 ligase mainly adds K48-linked chains on Ci when the pathway is active. This illustrates a phenomenon that the same protein can be modified with different types of ubiquitin chains by distinct E3 ligases. Although K11-linked chains added on APC substrates lead to complete degradation, the data demonstrate that K11-linked chains are involved in the partial degradation of Ci. These findings also raise the interesting possibility that the topology of ubiquitin chains may be recognized as a selective signal for proteasomal degradation. As mixed or heterologous ubiquitin chains may exist, further investigation is essential to determine whether mixed ubiquitin chains are formed by Cul1-Slimb-based E3 ligase on Ci (Zhang, 2013).

Squamous cell carcinoma related oncogene (SCCRO) family members regulate cell growth and proliferation through their cooperative and antagonistic effects on Cullin neddylation

SCCRO (squamous cell carcinoma related oncogene; a.k.a. DCUN1D1) is a highly conserved gene that functions as an E3 in neddylation. Although inactivation of SCCRO in yeast results in lethality, SCCRO-/- mice are viable. The exclusive presence of highly conserved paralogues in higher organisms led to an assessment of whether compensation by SCCRO's paralogues rescues lethality in SCCRO-/- mice. Using murine and Drosophila models, the in vivo activities of SCCRO and its paralogues were assessed in cullin neddylation. SCCRO family members were found to have overlapping and antagonistic activity that regulates neddylation and cell proliferation activities in vivo. In flies, both dSCCRO (CG7427) and dSCCRO3 (CG13322) promote neddylation and cell proliferation, whereas dSCCRO4 (CG6597) negatively regulates these processes. Analysis of somatic clones showed that the effects that these paralogues have on proliferation serve to promote cell competition, leading to apoptosis in clones, with a net decrease in neddylation activity. dSCCRO and, to a lesser extent, dSCCRO3 rescue the neddylation and proliferation defects promoted by expression of SCCRO4. dSCCRO and dSCCRO3 functioned cooperatively, with their coexpression resulting in an increase in both the neddylated cullin fraction and proliferation activity. In contrast, human SCCRO and SCCRO4 promotes and human SCCRO3 inhibits neddylation and proliferation when expressed in flies. These findings provide the first insights into the mechanisms through which SCCRO family members cooperatively regulate neddylation and cell proliferation (Fu, 2016).

Clustering and negative feedback by endocytosis in planar cell polarity signaling is modulated by ubiquitinylation of Prickle

The core components of the planar cell polarity (PCP) signaling system, including both transmembrane and peripheral membrane associated proteins, form asymmetric complexes that bridge apical intercellular junctions. While these can assemble in either orientation, coordinated cell polarization requires the enrichment of complexes of a given orientation at specific junctions. This might occur by both positive and negative feedback between oppositely oriented complexes, and requires the peripheral membrane associated PCP components. However, the molecular mechanisms underlying feedback are not understood. This study found that the E3 ubiquitin ligase complex Cullin1(Cul1)/SkpA/Supernumerary limbs(Slimb) regulates the stability of one of the peripheral membrane components, Prickle (Pk). Excess Pk disrupts PCP feedback and prevents asymmetry. Pk was found to participate in negative feedback by mediating internalization of PCP complexes containing the transmembrane components Van Gogh (Vang) and Flamingo (Fmi), and that internalization is activated by oppositely oriented complexes within clusters. Pk also participates in positive feedback through an unknown mechanism promoting clustering. these results therefore identify a molecular mechanism underlying generation of asymmetry in PCP signaling (Cho, 2015).

This study has shown that Cul1 complex-mediated ubiquitinylation of Pk is required for correct function of the core PCP signaling module, thereby ensuring proper alignment of hairs on the Drosophila wing. Ubiquitinylation by the Cul1 complex targets Pk for proteasome-dependent degradation, and in its absence, excess Pk accumulates, resulting in disruption of core PCP function. In several previous reports, ubiquitinylation has been recognized to regulate PCP signaling. In a mouse model, Smurf E3 ligases were shown to regulate PCP signaling by modulating Pk levels. However, mutation of Drosophila smurf failed to show PCP defects. In Drosophila, Cul3 E3 ligase-BTB protein-mediated regulation of Dsh ubiquitinylation modulates PCP signaling, as does the de-ubiqutinylating enzyme Faf, possibly acting on or upstream of Fmi, or more recently proposed to act on Pk. Loss of either activity shows subtle effects on final PCP outcomes in Drosophila. In no case is there a demonstrated mechanism for how these events impact the characteristic asymmetric subcellular localization of PCP proteins that underlies cell polarization (Cho, 2015).

Slimb was found to be the F-box protein that mediates Pk and Cul1 complex association in vivo. It appears likely that the motif that mediates interaction between Pk and Slimb resides in the C-terminal half of the protein, as do the Vang interaction domain and the farnesylation (CaaX) motif. Of note, the amount of Slimb protein in the cell was also dependent on Pk. In previous cell culture studies, F-box proteins themselves were targeted for ubiquitinylation by their own Cul complexes when not bound by other substrates, and this appears to be the case here, as Slimb levels are increased in cul1 knock-down clones. Furthermore, this result supports the idea that Pk is the major target of the Cul1 complex during pupal wing development (Cho, 2015).

If the Cul1-SkpA-Slimb complex targets Pk for degradation, why do Slimb and Pk accumulate together on the proximal side of wildtype cells? Pk is known to bind to Vang, and to localize with it in the proximal complex. Slimb adapts the Cul1 complex to Pk and is seen to colocalize with Vang on the proximal side, as well as with overexpressed Pk. However, this suggests that the Pk in this location is resistant to Cul1 complex-dependent degradation. Pk levels have long been known to be limited by a Vang-dependent activity. Recently, it has been shown that farnesylation of Pk is required for Pk to interact with Vang and promote its degradation, and that levels of Pk also depend on SkpA, leading to the suggestion that farnesylation-dependent Pk-Vang interaction results in SkpA-dependent Pk degradation. This study provides evidence suggesting that the Cul1-SkpA-Slimb E3 complex directly targets Pk for destruction, but in contrast, the finding that Pk with deleted CaaX domain accumulates to elevated levels in cul1 knock-down cells indicates that Cul1/SkpA/Slimb-dependent degradation is independent of farnesylation. Furthermore, the finding that Pk promotes internalization of Fmi-Vang-Pk during mutual exclusion of oppositely oriented core PCP complexes leads to a model, that is consistent with the shared observation that Pk associated with stable intercellular complexes ([Dsh-Fz-Fmi]-[Fmi-Vang-Pk]) is protected from degradation (Cho, 2015).

In theory, generation of cell polarity requires the combination of a local self-enhancement of a cell polarity factor and a long range inhibition of the same factor. In isolated cells, likely the evolutionarily more ancient mechanism, intracellular local self-enhancement can arise through cooperativity among P proteins. Intracellular long range inhibition is most easily accomplished by limiting amounts of a component of the P complex, such that aggregation of P complexes in one location decreases the probability of aggregation elsewhere by depletion of that component (Cho, 2015).

Cell polarization within a multicellular system introduces additional possible intercellular mechanisms for both the local self-enhancement and the long range inhibition (see Cell polarity establishment and the involvement of Pk-mediated endocytosis). If two polarity complexes, P and Q, exist, and can interact at junctions between adjacent cell boundaries, then both the local and long range effects can be mediated through these intercellular interactions. If P complexes recruit Q complexes to opposing sides of junctions, and if mutual antagonism between P and Q occurs, then long range inhibition can occur by P recruiting Q to the neighbor, where P is then excluded. Similarly, exclusion of P decreases Q in that region of the original cell, enabling the accretion of more P (in effect, cooperativity) (Cho, 2015).

The peripheral membrane associated core PCP proteins Pk, Dsh and Dgo appear to mediate these polarization events, but how they do so is not known. They are not required for assembly of asymmetric [Fz-Fmi]-[Fmi-Vang] complexes, but were known to share the ability to induce clustering, and are all required for the feedback amplification that results in the asymmetric subcellular localization of PCP signaling complexes. While their action somehow promotes the assortment of proximal and distal core proteins to opposite sides of the cell, how they carry out this function, and in particular whether this is through intracellular or intercellular mechanisms, is unclear (Cho, 2015).

To understand how excess Pk resulting from mutation of the Cul1 E3 complex disrupts PCP, Pk's role in establishment of core asymmetry was studied further. pk mutation causes symmetric distribution of other core proteins without substantially diminishing or enhancing their junctional recruitment. On the other hand, Pk overexpression causes both accumulation of higher levels of all proximal and distal core proteins and induces their clustering at apical membrane domains, generating discrete puncta. A Pk induced clustering of similarly oriented core complexes could explain both the aggregated punctate appearance and the increased levels of accumulated proteins if one assumes a steady state relationship between free asymmetric complexes and unassembled components as asymmetric complexes are sequestered into puncta (Cho, 2015).

Pk over-expression study shows that Fz is not required for making Fmi clusters, but Vang is. This suggests an intracellular mechanism in which Pk interacts with Vang at the apical membrane to induce clustering. However, since Vang over-expression does not cause accumulation of other core proteins, a specific function for Pk beyond stabilization of Vang must be considered to explain the accumulation of other core proteins. Furthermore, the depletion of Fmi from the membrane achieved by the very high levels of Pk upon simultaneous Pk overexpression and Cul1 depletion argues for a function for Pk beyond clustering (Cho, 2015).

Pk might stimulate amplification simply by promoting clustering, with long range inhibition mediated by other mechanisms, or perhaps by limiting amounts of Pk. However, the data suggest an alternative interpretation, as Pk-dependent mutual exclusion of oppositely oriented complexes is observed, forcing local accumulation of distal proteins induced the Pk-dependent removal of proximal proteins within the same cell. Exclusion is associated with Pk mediated internalization of Pk-Vang-Fmi complexes, suggesting that this exclusion involves endocytosis. The requirement for Vang in this internalization is consistent with a previous study showing that Vang contributes to Fmi internalization. It is therefore proposed that Pk is involved in an intercellular long range inhibition to promote feedback amplification (Cho, 2015).

Like clustering, the Pk-induced routing of Fmi into intracellular vesicles was dependent on Vang, and Pk, Vang and Fmi colocalize in vesicles both apically and more basally, indicating that Fmi-Vang complex trafficking is regulated by associated Pk. However, unlike clustering, it is also dependent on Fz. This suggests a model for feedback inhibition in which oppositely oriented asymmetric complexes interact within clusters, leading to endocytosis and removal of Pk-Vang-Fmi. Competitive interaction between the proximal protein Pk and the distal protein Dgo for Dsh binding is known to occur, suggesting that these interactions might result in either of two alternative outcomes, one of which would be disruption of the proximal complex, and the other disruption of the distal complex. It is proposed that if the distal complex 'wins,' thus remaining stable, the proximal Pk-Vang-Fmi complex becomes internalized in a Pk-dependent step. Once there is a predominance of complex in a given orientation, Vang will be enriched on one side of the intercellular boundary with relatively little Fz present. Since Pk and Slimb associate with Vang, they too will be enriched, but the absence of competitive interactions from the Fz complex allows them to remain within clusters, accounting for the accumulation of Pk and Slimb on the proximal side of wildtype wing cells. According to this model, Pk and Slimb are observed primarily at sites where they are inactive and therefore not internalized (Cho, 2015).

Modest levels of Pk overexpression both enhance accumulation of PCP protein complexes at the membrane and disrupt the normal orientation of polarization. This may be explained by enhanced feedback amplification that overwhelms the ability to interpret directional inputs. In contrast, the depletion of Fmi from the membrane observed with the very high levels of Pk induced by simultaneous Pk overexpression and Cul1 depletion suggests that sufficient Pk can induce internalization even without the competitive interactions from the Fz complex that normally stimulate internalization (Cho, 2015).

The mechanism for Pk-dependent clustering is not known. As previously proposed, clustering may result from a scaffolding effect; the possibility of decreased endocytosis accounting for clustering was previously discounted. Whatever the mechanism, clustering by Pk must occur independent of Fz. Furthermore, Pk must enable the multimeric aggregation of complexes containing [Vang-Fmi]-[Fmi] or [Vang-Fmi]-[Fmi-Fz]. Induction of multimeric clustering would also provide a context for the dose-dependent competition that determines internalization of either the proximal or distal complex. Additional work will be required to determine how Pk facilitates clustering (Cho, 2015).

Since Cul1 depletion increases the amount of Pk, and excess Pk produces clustering and amplification, how Cul1 might produce the observed phenotype is now considered. The simplest possibility is that in the Cul1 mutant, excess Pk produces excess clustering and amplification that overwhelms the directionality in the system. However, because Pk is associated with Slimb and yet stable in the polarized state, and because Pk degradation is dependent on Vang, the possibility is also entertained that Cul1-dependent degradation is somehow functionally coupled to Pk-mediated internalization. Additional studies will be required to distinguish these possibilities (Cho, 2015).

In summary, a model is proposed in which Pk-dependent internalization of proximal complexes provides an intercellular long range inhibition that contributes to amplification of core protein asymmetric localization. At the same time, Pk provides a local cooperative effect by inducing clustering and accumulation of proximal complexes. The mechanism for clustering are not known, but a simple model is that Pk mediates closely related internalization events (Cho, 2015).

It is noted that a similar intercellular long range inhibition was initially discussed long ago, except that [Vang-Pk] was proposed to disrupt [Fz-Dsh]. This interpretation was based largely on inference. The current study provides evidence that [Fz-Dsh] disrupts [Vang-Pk] (by promoting internalization). On theoretical grounds, either one would be sufficient to cause polarization, but the possibility cannot be excluded that both may occur. Indeed, vesicles containing Fz, Dsh and Fmi have been shown to be transcytosed in a microtubule-dependent fashion with a directional bias, and these vesicles appear to derive from apical junctions, where they may arise by exclusion (Cho, 2015).

Although knock-down of smurf in flies reveals no function in PCP; the mechanism described in this study is similar to that inferred for Smurf in mouse PCP. Mice mutant for both Smurf1 and Smurf2 show PCP defects and lose asymmetric localization of core PCP proteins. Furthermore, biochemical evidence was provided that Smurfs, in the presence of the Dsh homolog Dvl2 (and Par6) mediated ubiquitinylation of mouse Pk1. From this, a model was proposed that proximal complexes containing Pk1, and presumably Vang and Celsr (Fmi), are disrupted upon proximity to distal complexes containing Fzd and Dvl2. This model is similar to the model of mutual exclusion, except that the mode of disruption was not directly addressed. While this study proposes disruption by internalization, perhaps coupled to degradation, the mouse stud was only able to address degradation. Furthermore, it is not known if, in mouse, Pk1 mediates clustering, perhaps by a related mechanism, as as is described in flies (Cho, 2015).

The de-ubiquitinase USPX9 was recently identified as a regulator of Pk in the context of Pk's role in epilepsy in human, mouse, zebrafish and flies. Similarly, the orthologous Drosophila de-ubiquitinase Faf modulates the pksple dependent seizure phenotype in flies. These observations suggest that while the ubiquitinylating and de-ubiquitinylating activities of Smurf and USPX9 control the ubiquitinylation state of vertebrate Pk's, Cul1 and Faf may serve the analogous function to regulate ubiquitinylation of Drosophila Pk (Cho, 2015).


DEVELOPMENTAL BIOLOGY

The Cul1-based SCFSlimb complex controls CiFL processing in the anterior cells of the eye disc. In contrast, Cul3-mediated Ci degradation mechanism is specific to the posterior cells. These specific activities in controlling Ci protein stability are not caused by differential gene expression of Cul1 or Cul3 in the eye disc. Ubiquitous mRNA expression patterns of both Cul1 and Cul3, and ubiquitous Cul1 protein expression are found all along the eye disc, suggesting that control of specificity is mediated by mechanisms other than regulation of Cul1 and Cul3 expression (Ou, 2002).

Regulation of Hh signal transduction as Drosophila eye differentiation progresses

Differentiation of the Drosophila retina occurs as a morphogenetic furrow sweeps anteriorly across the eye imaginal disc, driven by Hedgehog secretion from photoreceptor precursors differentiating behind the furrow. A BTB protein, Roadkill, is expressed posterior to the furrow and targets the Hedgehog signal transduction component Cubitus interruptus for degradation by Cullin-3 and the proteosome. Clonal analysis and conditional mutant studies establish that roadkill transcription is activated by the EGF receptor and Ras pathway in most differentiating retinal cells, and by both EGF receptor/Ras and by Hedgehog signaling in cells that remain unspecified. These findings outline a circuit by which Hedgehog signal transduction is modified as Hedgehog signaling initiates retinal differentiation. A model is presented for regulation of the Cullin-3 and Cullin-1 pathways that modifies Hedgehog signaling as the morphogenetic furrow moves and the responses of retinal cells change (Baker, 2009).

As the morphogenetic furrow crosses the eye disc, Ci155 accumulates most highly just anterior to the morphogenetic furrow, even though Hh is secreted posterior to the morphogenetic furrow. The sharp reduction in Ci155 as the furrow passes is associated with a switch from Cul1-dependent processing to Cul3-dependent degradation (Ou, 2002). The posterior eye expresses rdx, encoding a BTB protein that couples Ci 155 to the Cul3 pathway (Kent, 2006; Zhang, 2006). This study identified the signals that induce rdx and that process Ci155 in the posterior eye (Baker, 2009).

The induction of rdx transcription couples Ci155 processing to Cul3 (Kent, 2006; Zhang, 2006). rdx transcription is regulated by both Hh signaling and Ras signaling, and there were distinctions between cell types. The smo mosaic and hhts2 experiments show that Hh signaling is continuously required for rdx transcription in unspecified cells with basal nuclei. In the absence of smo, EGFR-dependent rdx transcription occurs in differentiating photoreceptor cells only, not in unspecified cells. The egfr mosaics show that EGFR is essential for rdx transcription in all cells except the R8 photoreceptor class. Thus, EGFR-dependent differentiation was sufficient to induce rdx in photoreceptors even without Hh signaling, but Hh was not sufficient to induce rdx anywhere without EGFR signaling, except for the R8 cells. Undifferentiated cells might require both the Ras and Hh signaling pathways to induce rdx because the level of Ras signaling is lower in unspecified cells than in differentiating cells of the ommatidia. Alternatively, there may be a combinatorial requirement for both pathways in unspecified cells (Baker, 2009).

There has been some discussion of whether proteolysis of Ci155 by Cul-3 is regulated directly by Hh, as is Cul-1 dependent Ci processing. The current studies provide no support for this idea. In all the genotypes examined, Ci proteolysis correlates with the expression of rdx, and the simplest explanation is that the only effect of Hh on the Cul-3 pathway is through rdx transcription, directly in unspecified cells, and indirectly via EGFR-mediated differentiation in most specified cells (Baker, 2009).

Two mechanisms, acting in different cells, appear to reduce Hh responses through Ci155 after the furrow passes. One also occurs in wing development, where rdx is transcribed only by cells experiencing high Hh signaling levels close to the source of Hh. In wing development, rdx and the Cul3-pathway modulate the amount of Ci155 available for Cul1-dependent processing, lowering the maximum level of Ci155 activity at high Hh levels. Rdx could lower Ci155 levels in unspecified eye cells posterior to the furrow by this mechanism, in which an equilibrium between Hh-dependent induction of rdx, and rdx- and Cul3-dependent degradation of Ci155, leads to a lower level of Ci155 protein than anterior to the furrow. By contrast, in the specified, differentiating eye cells, rdx transcription becomes independent of Hh signaling, and Ci155 is degraded more completely (Baker, 2009).

If there is Hh signaling posterior to the furrow, as these studies find maintains rdx transcription in unspecified retinal cells, why are genes such as atonal that are induced by Hh signaling ahead of the furrow not also expressed posterior to the furrow? There are at least three possible explanations. First, rdx may dampen Ci155 accumulation in unspecified cells such that the threshold necessary for ato expression is not achieved posterior to the furrow. This is unlikely to be the sole explanation, since mutating rdx or cul3 permits Ci155 accumulation but does not lead to ectopic R8 specification, but it could contribute in conjunction with other mechanisms. Secondly, other genes may interfere posterior to the furrow. This could include egfr induction of Bar gene expression, since Bar genes antagonize ato expression. There seem to be multiple respects in which EGFR-dependent differentiation renders cells unable to continue anterior responses to Hh, and it is also envisaged that egfr might play a role in further mechanisms that modulate the response to Dpp signaling posterior to the furrow, should such mechanisms exist. Finally, recent evidence suggests that induction of ato by Hh is not so simple as the induction of a target gene above a threshold in a morphogen gradient, but depends indirectly on Hh repressing Eyeless and activating Sine Oculis, so that these transcription factors are coexpressed and turn on ato only in a domain ahead of the furrow. In this case, persistent Hh signaling would not be expected to activate ato expression once Ey had been repressed (Baker, 2009).

Recently, Hh has been discovered to induce compensatory proliferation in response to eye disc cell death, a further example of post-furrow Hh function. The current results now suggest the model that loss of EGFR-dependent rdx expression elevates Ci155 locally to permit Hh responses when photoreceptor cells that secrete EGFR ligands are lost. Consistent with this idea, loss of rdx or cul3 also result in proliferation of eye disc cells (Baker, 2009).

The regulation of rdx expression and thus degradation of Ci by Cullin-3 may not be sufficient to explain Ci regulation posterior to the furrow. In order for Ci155 to be stable, as observed in cul3 mutant clones and egfr mutant clones, Ci155 must escape processing to Ci75 by Cul-1. Ahead of the furrow, and in most other tissues, rdx is not expressed, Ci is not coupled to Cul3, and Ci155 is stabilized wherever Hh inhibits Smo and the Cul1 pathway. The observation that Ci155 is stable in cul3 clones, or in the genotypes where rdx is not expressed, shows that Ci155 escapes processing by the Cul1 pathway in the posterior eye as well, but this is not due to Hh. Ci155 accumulates in smo egfr mutant clones that do not express rdx and cannot respond to Hh (Baker, 2009).

One model would be that once rdx is induced, Ci155 is sequestered and not available to be processed by Cul1. This model cannot explain why Ci155 accumulates in egfr clones that lack rdx expression, where Ci155 should be available for Cul1. Therefore Ci155 must escape Cul1-mediated processing in the posterior eye by a distinct mechanism. This could be explained by the induction of a component distinct from Rdx that inhibits the processing of Ci155 by Cul1, or sequesters Ci155. It is equally possible that a component essential for processing of Ci155 by Cul1 is repressed posterior to the morphogenetic furrow (Baker, 2009).

Previous studies show that Ci155 never accumulates in smo tkv clones that are unable to respond to either Hh or Dpp signaling. Clones of cells unable to respond to Dpp, but able to respond to Hh and Ras, show only a subtle change in Ci155 labeling. These previously published observations suggest that Ci155 remains a target of Cul1 in the absence of both Dpp and Hh signaling, perhaps through failure to transcribe or repress transcription of a gene that modulates Ci155 proteolysis by Cul1 posterior to the furrow (Baker, 2009).

It is now possible to account for why smo clones affect Ci155 levels differently from cul3 clones, a previously puzzling observation. In cul3 clones, or egfr clones that do not express rdx, the Cul3 pathway cannot degrade Ci155 and the Cul1 pathway is inactivated posterior to the furrow exactly as in wild type discs, so Ci155 accumulates. In smo clones, Ci155 transiently accumulates in those cells in which processing by Cul1 has been lost but rdx not yet induced. In such cells, Ci155 is not coupled to any cullin, and is stable. Eventually, differentiation spreads into the posterior of smo clones, leading to rdx expression, and Cul3-dependent Ci degradation. If differentiation and rdx expression are prevented, as in smo egfr clones, then Ci155 remains stable. Because there is a delay in expressing rdx in smo clones compared to wildtype, Ci155 is not subject to Cul3-mediated processing as soon as in wild type, and there is a period when Ci155 has been uncoupled from Cul1-processing but not yet coupled to the Cul3 pathway. It is during this period that Ci155 accumulates in smo mutant cells (Baker, 2009).

These findings help explain how a wave of differentiation moves across the eye disc uni-directionally. Hh, secreted from differentiating photoreceptor cells, must be present at highest concentrations posterior to the furrow. Indeed, ahead of the furrow Ci155 is stabilized in a decreasing posterior-to-anterior gradient, consistent with a gradient of Hh protein coming from a source posterior to the furrow. Yet, the cell-autonomous responses to Hh signaling that are seen ahead of the furrow, such as cell cycle arrest and atonal expression, do not occur posterior to the furrow, where Ci is rendered unstable by Rdx and Cul3, induced both directly by Hh itself, and indirectly by the photoreceptor differentiation that is largely induced by EGFR posterior to the furrow (Baker, 2009).

There are other examples where Hh-secreting tissues are not the targets of Hh signaling. For, example, in Drosophila wing development, anterior compartments respond to Hh secreted by posterior compartments, but posterior compartment cells do not respond because ci transcription is repressed by the posterior-specific protein Engrailed. In vertebrate development, notochord cells express Shh but the responses seen in the nearby spinal cord are not seen in notochord. Such segregation of Hh-producing cells from fields competent to respond to Hh makes sense, if the purpose of Hh signaling in development is to pattern new body regions. Hh signaling is also deregulated in many tumors. Whether any of these tumors activate Hh signaling by affecting GLI protein stability, or other normal down-regulatory mechanisms, remains to be seen. In any case, mechanisms that render cells unresponsive to Hh by coupling Ci155 to the proteosome might prove useful in the treatment of cancers that depend on Hh signaling (Baker, 2009).


EFFECTS OF MUTATION

The genetic evidence suggests that Nedd8 is directly required for CiFL proteolytic processing, consistent with the hypothesis that neddylation affects CiFL proteolysis through regulating SCFSlimb activity. Cullin proteins are the identified targets for Nedd8 modification. In the Drosophila genome, six Cullin proteins are identified, each corresponding to a mammalian homolog. Among them, Cul1 is involved in the formation of SCF complexes that function as E3 ligase. The null Cul1 allele, Cul1EX was generated. In Cul1EX homozygous larvae in the first instar stage, the Cul1 signal detected by anti-Cul1 antibodies is almost completely absent. The residual Cul1 protein in Cul1EX larvae is probably maternally contributed (Ou, 2002).

Whether Nedd8 conjugates to Cul1 in Drosophila was investigated further. Cell extracts of eye-antennal discs and brain lobes isolated from wild-type third instar larvae were immunoprecipitated by anti-Cul1 antibodies and immunoblotted by anti-Nedd8 antibodies. The Nedd8-positive signal suggests that Cul1 is modified by Nedd8 in Drosophila. Consistently, the Nedd8-modified Cul1 signal is absent in cell extracts prepared from the first instar Nedd8 mutant larvae (Ou, 2002).

In addition, it was found that unmodified Cul1 accumulates in the Nedd8 hypomorphic mutants Nedd8EP(2)2063/AN015. This effect of Cul1 accumulation is also observed in Nedd8 null mutant clones in third-instar eye discs. These data suggested that Nedd8 modification of Cul1 might modulate Cul1 stability (Ou, 2002).

To test whether Nedd8 activity in controlling protein stability is mediated through Cul1, whether depleting Cul1 would exhibit the same phenotypes as depleting Nedd8 was examined. Cul1EX clones were generated in developing wing discs, and the protein levels of CiFL and Arm were examined. Accumulations of CiFL and Arm were found in Cul1EX mutant cells, identical to the phenotypes observed in Nedd8 mutant clones. In the eye disc, CycE accumulates in Cul1 mutant cells. These results suggest that Nedd8 and Cul1 function together for controlling the protein stability of Ci and Arm in the wing disc and CycE in the eye disc (Ou, 2002).

Whether the Cul1-based SCF complex is involved in controlling CiFL stability in the eye disc was examined. When located anterior to the MF, Cul1EX cells accumulate high levels of CiFL, a phenotype similar to Nedd8 mutant cells. However, Cul1EX cells that located posterior to the MF only expressed a basal level of CiFL, suggesting that the Cul1-based SCF complex may not control Ci stability in the posterior cells (Ou, 2002).

In addition to the Nedd8-Cul1 core component, the SCF complex also includes a substrate-specific F-box protein. To investigate whether SCF activity in CiFL processing is limited to the anterior cells of the eye disc, the mutant phenotype of slimb was examined. slimb is required for CiFL proteolytic processing in tissues such as wing and leg discs. When slimb1 mutant clones were generated in eye discs, high levels of CiFL accumulation were detected exclusively in clones located anterior to the MF. No accumulation of CiFL could be detected in posterior slimb1 clones. Suppression of CiFL accumulation in the posterior cells was not due to possible residual activity present in hypomorphic slimb1, because identical results of CiFL accumulation were observed in the strong hypomorphic allele slimb2 and the null allele slimbP (Ou, 2002).

In summary, the results strongly suggested that in vivo, the Nedd8-modified, Cul1-based SCFSlimb complex controls CiFL proteolysis in anterior cells. Following the sweep of the MF, CiFL stability in the posterior cells is controlled by an SCFSlimb-independent mechanism (Ou, 2002).

A ubiquitin-proteasome pathway represses the Drosophila immune deficiency signaling cascade

The inducible production of antimicrobial peptides is a major immune response in Drosophila. The genes encoding these peptides are activated by NF-κB transcription factors that are controlled by two independent signaling cascades: the Toll pathway that regulates the NF-κB homologs, Dorsal and DIF; and the IMD pathway that regulates the compound NF-κB-like protein, Relish. Although numerous components of each pathway that are required to induce antimicrobial gene expression have been identified, less is known about the mechanisms that either repress antimicrobial genes in the absence of infection or that downregulate these genes after infection. In a screen for factors that negatively regulate the IMD pathway, two partial loss-of-function mutations were isolated in the SkpA gene that constitutively induce the antibacterial peptide gene, Diptericin, a target of the IMD pathway. These mutations do not affect the systemic expression of the antifungal peptide gene, Drosomycin, a target of the Toll pathway. SkpA encodes a homolog of the yeast and human Skp1 proteins. Skp1 proteins function as subunits of SCF-E3 ubiquitin ligases that target substrates to the 26S proteasome, and mutations affecting either the Drosophila SCF components, Slimb and Cullin1, or the proteasome also induce Diptericin expression. In cultured cells, inhibition of SkpA and Slimb via RNAi increases levels of both the full-length Relish protein and the processed Rel-homology domain. It is concluded that in contrast to other NF-κB activation pathways, the Drosophila IMD pathway is repressed by the ubiquitin-proteasome system. A possible target of this proteolytic activity is the Relish transcription factor, suggesting a mechanism for NF-κB downregulation in Drosophila (Khush, 2002).

In wild-type flies Diptericin is tightly controlled by the IMD pathway. Therefore, to identify genes that normally function to repress the IMD pathway, 2,000 yellow, white (y,w) F1 male progeny from male flies mutagenized with ethyl methanesulfonate were screened for constitutive expression of a Green Fluorescent Protein (GFP) reporter gene under the control of the Diptericin promoter. Two males, J6 and G49, expressed Diptericin-GFP, and this gene was constitutively expressed in larvae and adults in homozygous lines derived from these males. Although flies carrying the J6 and G49 mutations are viable and fertile at 25°C, G49 is pupal lethal at 29°C, indicating temperature-sensitive phenotypes associated with this mutation (Khush, 2002).

Using recombination mapping, the J6 and G49 mutations were shown to be tightly linked to the y locus on the proximal tip of the X chromosome. To further localize the two mutations, deletions were used to determine that J6 falls in the area defined by the overlap of Df(1)74k24.1, Df(1)svr, and Df(1)su(s)83, placing it in cytological region 1B10 near the Dredd gene. Two lethal P-element insertions in the Bloomington stock center collection, l(1)G0389 and l(1)G0109, which map near this region, were shown to not complement the constitutive Diptericin expression in the J6 and G49 lines. By sequencing DNA flanking the P elements in the two insertion lines, both elements were ascertained to lie within 200 bp of each other in the 5' untranslated region of the SkpA gene. To confirm that J6 and G49 are mutations in SkpA, a wild-type SkpA transgene on the second chromosome was shown to suppress the constitutive Diptericin expression phenotype in G49 flies. The J6 and G49 lines were shown to each contain a point mutation in the SkpA gene that generates a single amino acid change in the SkpA protein: J6, renamed SkpAJ6, converts threonine 98 to an isoleucine, and G49, renamed SkpAG49, replaces glutamic acid 101 with a lysine. These alleles are hypomorphic mutations of SkpA since the P-element insertions are pupal lethal at 25°C. SkpAG49 is pupal lethal at 29°C, and homozygous SkpAG49 adults transferred to 29°C express Diptericin at similar levels as flies heterozygous for SkpAG49 and either the P-element insertions or deletions that remove SkpA. At 29°C, therefore, SkpAG49 behaves like a null mutation, which probably reflects the significant change from the negatively charged glutamic acid to the positively charged lysine in this allele (Khush, 2002).

The SkpA gene encodes a protein that is highly similar to Skp1 proteins in humans and yeast. Skp1 proteins are components of SCF ubiquitin ligases that target substrates to the proteasome, and crystal structures of human Skp1 complexed with the F-box protein Skp2 and the cullin protin Cul1 have been solved. SkpAJ6 and SkpAG49 both affect a conserved region of SkpA that corresponds to helix 5 of Skp1; helix 5 forms part of the core interface between Skp1, the F-box region of Skp2, and the amino-terminal domain of Cul1, with some amino acids in this helix making direct contact with residues in Skp2 and Cul1. This suggests that the SkpAJ6 and SkpAG49 mutations disrupt interactions between SkpA and the F-box protein and cullin components of an SCF complex. Protein interaction studies indicate that SkpA functions with the F-box protein Slimb and the Cullin-like protein Cullin1 (Cul1) in a Drosophila SCF complex. In support of this model, slimb1 and dcul1l(2)02074 mutant larvae, as well as larvae carrying the DTS5 mutation, a dominant-negative mutation that affects the β6 subunit of the 26S proteasome, were shown to express Diptericin at levels comparable to those in the SkpA mutants. To further test the DTS5 phenotype, the UAS-Gal4 system was used to overexpress a UAS-DTS5 transgene in larval fat bodies: DTS5 overexpression induces Diptericin to levels that are comparable to those generated by bacterial infection with Erwinia carotovora carotovora 15 (Ecc15). Flies heterozygous for mutations at both the SkpA and slimb loci were generated: these flies constitutively express Diptericin, indicating a synergistic interaction between SkpA and slimb. The constitutive Diptericin expression in the slimb1, dcul1l(2)02074, and DTS5 mutants and the interaction between SkpA and slimb together suggest that an SCFSkpA/Cul1/Slimb ubiquitin ligase represses Diptericin expression by targeting a regulatory factor for degradation by the 26S proteasome (Khush, 2002).

To determine if the constitutive Diptericin expression in the SCF complex mutants is mediated through the IMD pathway, Diptericin levels were examined in larvae homozygous for mutations in either SkpA, or slimb and various genes of the IMD pathway: SkpAJ6;imd1 and SkpAG49;dtak11 double mutants display constitutive Diptericin expression, although Diptericin levels are slightly reduced in the SkpAG49;dtak11 larvae. Mutations in DmIkkγ, DmIkkβ, and Relish, however, completely block Diptericin expression in the SkpAJ6 background, and a Dredd mutation completely blocks Diptericin expression in the slimb1 background. The constitutive Diptericin expression observed in SkpA and slimb mutants, therefore, does not require IMD and dTak1, but it is dependent on the DmIKK complex, Dredd, and Relish. These results imply that, in wild-type flies, the SCFSkpA/Cul1/Slimb negatively regulates the IMD pathway by targeting one of these factors, or an additional unidentified component of the IMD pathway, for degradation by the proteasome. In contrast to fat body cells, the IMD pathway is the primary regulator of all antimicrobial genes, including Drosomycin, in surface epithelial tissues. A Drosomycin-GFP transgene is constitutively expressed in tracheal cells but not in fat body cells of slimb1 mutant larvae; this expression pattern further demonstrates that the IMD pathway, but not the Toll pathway, is constitutively activated when the SCFSkpA/Cul1/Slimb complex is compromised (Khush, 2002).

Although the genetic results do not allow differentiation between the DmIKK complex, Dredd, Relish, or other unidentified downstream components of the IMD pathway as targets of the ubiquitin-proteasome pathway, the mammalian Relish homolog, P105, is regulated by an SCF complex that contains the Slimb homolog β-TrCP/E3RSIκB. Consequently, RNA-mediated interference (RNAi), an effective technique for specifically inhibiting targeted proteins, was used in cultured Drosophila S2 cells to test for interactions between the SCFSkpA/Cul1/Slimb complex and Relish. Initially, SkpA and Slimb activity were blocked in S2 cells via RNAi; then, transient expression of a full-length Relish protein, modified by an N-terminal FLAG tag, was induced in the same S2 cells and the effects of the SkpA and slimb RNAi treatments on FLAG-Relish protein stability was monitored using Western blots and anti-FLAG antibodies (Khush, 2002).

Reducing Slimb activity, in the absence of LPS stimulation, visibly increases steady-state levels of both full-length Relish and the active N-terminal Rel-homology domain; levels of both polypeptides are further increased by inhibiting Slimb and SkpA simultaneously. This effect is specific since RNAi of the SkpA homologs, SkpB and SkpD, does not increase Relish levels. Dredd RNAi does increase Relish levels at day 1, but this is probably because Dredd inhibition blocks Relish processing. Previous studies show that Relish processing in S2 cells is induced by lipopolysaccharide (LPS) and requires Dredd activity. As expected, therefore, RNAi of Dredd blocks LPS-induced Relish processing. Simultaneous RNAi of SkpA and Slimb in the presence of LPS, however, results in higher steady-state levels of the Rel-homology domain up to 4 days after Relish induction. Higher levels of the Rel-homology domain after SkpA and Slimb RNAi could be caused by increased processing of full-length Relish. However, because full-length Relish levels also mount, the explanation is favored that Rel-homology domain turnover is reduced. Although the Slimb and SkpA RNAi treatments appear to inhibit Relish turnover, Relish levels do eventually diminish. This suggests that RNAi efficiency decreases with time, possibly due to degradation of the transfected double-stranded RNA. These RNAi experiments indicate that the constitutive antimicrobial gene expression in SkpA and slimb mutant flies is caused by higher Relish levels, and they suggest that the SCFSkpA/Cul1/Slimb complex represses the IMD pathway by promoting the degradation of both full-length and processed Relish proteins (Khush, 2002).

If the constitutive antimicrobial gene expression in flies carrying mutations that affect the SCFSkpA/Cul1/Slimb complex or proteasome is due to higher Relish levels, this would imply some level of steady-state Relish activation. Low levels of the Rel-homology domain have been reported in nuclear extracts from unstimulated S2 cells, and these low levels indicate that Relish is constitutively processed. Increasing Relish levels in larvae and adults via the Gal4-UAS system is sufficient to induce low levels of Diptericin expression. These results indicate that Relish is constitutively processed and activated to some level, supporting the hypothesis that Relish activity, in the absence of infection, is countered by ubiquitination and degradation (Khush, 2002).

CSN maintains the germline cellular microenvironment and controls the level of stem cell genes via distinct CRLs in testes of Drosophila melanogaster

Stem cells and their daughters are often associated with and depend on cues from their cellular microenvironment. In Drosophila testes, each Germline Stem Cell (GSC) contacts apical hub cells and is enclosed by cytoplasmic extensions from two Cyst Stem Cells (CySCs). Each GSC daughter becomes enclosed by cytoplasmic extensions from two CySC daughters, called cyst cells. CySC fate depends on an Unpaired (Upd) signal from the hub cells, which activates the Janus Kinase and Signal Transducer and Activator of Transcription (Jak/STAT) pathway in the stem cells. Germline enclosure depends on Epidermal Growth Factor (EGF) signals from the germline to the somatic support cells. Expression of RNA-hairpins against subunits of the COnstitutively Photomorphogenic-9- (COP9-) signalosome (CSN; see CSN5) in somatic support cells disrupted germline enclosure. Furthermore, CSN-depleted somatic support cells in the CySC position next to the hub had reduced levels of the Jak/STAT effectors Zinc finger homeotic-1 (Zfh-1) and Chronologically inappropriate morphogenesis (Chinmo). Knockdown of CSN in the somatic support cells does not disrupt EGF and Upd signal transduction as downstream signal transducers, phosphorylated STAT (pSTAT) and phosphorylated Mitogen Activated Protein Kinase (pMAPK), were still localized to the somatic support cell nuclei. The CSN modifies fully formed Cullin RING ubiquitin ligase (CRL) complexes to regulate selective proteolysis. Reducing cullin2 (cul2) from the somatic support cells disrupted germline enclosure, while reducing cullin1 (cul1) from the somatic support cells led to a low level of Chinmo. It is proposed that different CRLs enable the responses of somatic support cells to Upd and EGF (Qian, 2014).


EVOLUTIONARY HOMOLOGS

Structural studies of the cullin componenet of the SCF complex

SCF complexes are the largest family of E3 ubiquitin-protein ligases and mediate the ubiquitination of diverse regulatory and signalling proteins. The crystal structure of the Cul1-Rbx1-Skp1-F boxSkp2 SCF complex is presented; Cul1 is shown to be an elongated protein that consists of a long stalk and a globular domain. The globular domain binds the RING finger protein Rbx1 through an intermolecular beta-sheet, forming a two-subunit catalytic core that recruits the ubiquitin-conjugating enzyme. The long stalk, which consists of three repeats of a novel five-helix motif, binds the Skp1-F boxSkp2 protein substrate-recognition complex at its tip. Cul1 serves as a rigid scaffold that organizes the Skp1-F boxSkp2 and Rbx1 subunits, holding them over 100 Å apart. The structure suggests that Cul1 may contribute to catalysis through the positioning of the substrate and the ubiquitin-conjugating enzyme, and this model is supported by Cul1 mutations designed to eliminate the rigidity of the scaffold (N. Zheng, 2002).

Function of the substrate recognizing F-box protein component of ubiquitin E3 ligases

SCFCdc4 (Skp1, Cdc53/cullin, F-box protein) defines a family of modular ubiquitin ligases (E3s) that regulate diverse processes including cell cycle, immune response, and development. Mass spectrometric analysis of proteins copurifying with Cdc53 identified the RING-H2 finger protein Hrt1 as a subunit of SCF. Hrt1 shows striking similarity to the Apc11 subunit of anaphase-promoting complex. Conditional inactivation of hrt1(ts) results in stabilization of the SCFCdc4 substrates Sic1 and Cln2 and cell cycle arrest at G1/S. Hrt1 assembles into recombinant SCF complexes and individually binds Cdc4, Cdc53 and Cdc34, but not Skp1. Hrt1 stimulates the E3 activity of recombinant SCF potently and enables the reconstitution of Cln2 ubiquitination by recombinant SCFGrr1. Surprisingly, SCF and the Cdc53/Hrt1 subcomplex activate autoubiquitination of Cdc34 E2 enzyme by a mechanism that does not appear to require a reactive thiol. The highly conserved human HRT1 complements the lethality of hrt1Delta, and human HRT2 binds CUL-1. It is concluded that Cdc53/Hrt1 comprise a highly conserved module that serves as the functional core of a broad variety of heteromeric ubiquitin ligases (Seol, 1999).

The SCF complex containing Skp1, Cul1, and the F-box protein FWD1 (the mouse homolog of Drosophila Slimb and Xenopus beta-TrCP) functions as the ubiquitin ligase for IkappaBalpha. FWD1 associates with Skp1 through the F-box domain and also recognizes the conserved DSGXXS motif of IkappaBalpha. The structural requirements for the interactions of FWD1 with IkappaBalpha and with Skp1 have now been investigated further. The D31A mutation (but not the G33A mutation) in the DSGXXS motif of IkappaBalpha abolishes the binding of IkappaBalpha to FWD1 and its subsequent ubiquitination without affecting the phosphorylation of IkappaBalpha. The IkappaBalpha mutant D31E still exhibits binding to FWD1 and undergoes ubiquitination. These results suggest that, in addition to site-specific phosphorylation at Ser(32) and Ser(36), an acidic amino acid at position 31 is required for FWD1-mediated ubiquitination of IkappaBalpha. Deletion analysis of Skp1 reveals that residues 61-143 of this protein are required for binding to FWD1. In contrast, the highly conserved residues Pro(149), Ile(160), and Leu(164) in the F-box domain of FWD1 are dispensable for binding to Skp1. Together, these data delineate the structural requirements for the interactions among IkappaBalpha, FWD1, and Skp1 that underlie substrate recognition by the SCF ubiquitin ligase complex (Hattori, 1999).

Ubiquitin-conjugation targets numerous cellular regulators for proteasome-mediated degradation. Thus, the identification of ubiquitin ligases and their physiological substrates is crucially important, especially for those cases in which aberrant levels of regulatory proteins (e.g., beta-catenin, p27) result from a deregulated ubiquitination pathway. In yeast, the proteolysis of several G1 regulators is controlled by ubiquitin ligases (or SCFs) formed by three subunits: Skp1, Cul A (Cdc53), and one of many F-box proteins. Specific F-box proteins (Fbps) recruit different substrates to the SCF. Although many Fbps have been identified in mammals, their specific substrates and the existence of multiple SCFs have not yet been reported. One human Fbp, beta-Trcp (beta-Transducin repeat containing protein), does indeed form a novel SCF with human Skp1 and Cul1. Consistent with recent reports indicating that Xenopus and Drosophila beta-Trcp homologs act as negative regulators of the Wnt/beta-catenin signaling pathway, human beta-Trcp interacts with beta-catenin in vivo. Furthermore, beta-catenin is specifically stabilized in vivo by the expression of a dominant negative beta-Trcp. These results indicate that the Cul1/Skp1/beta-Trcp complex forms a ubiquitin ligase that mediates the degradation of beta-catenin (Latres, 1999).

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 identified 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).

Characterization of the SKP1 component of SCF ubiquitin complexes

The SCF ubiquitin ligase complex of budding yeast triggers DNA replication by catalyzing ubiquitination of the S phase cyclin-dependent kinase inhibitor SIC1. SCF is composed of three proteins -- ySKP1, CDC53 (Cullin), and the F-box protein CDC4 -- that are conserved from yeast to humans. As part of an effort to identify components and substrates of a putative human SCF complex, hSKP1 was isolated in a two-hybrid screen with hCUL1, the closest human homologue of CDC53. hCUL1 associates with hSKP1 in vivo and directly interacts with both hSKP1 and the human F-box protein SKP2 in vitro, forming an SCF-like particle. Moreover, hCUL1 complements the growth defect of yeast cdc53(ts) mutants, associates with ubiquitination-promoting activity in human cell extracts, and can assemble into functional, chimeric ubiquitin ligase complexes with yeast SCF components. Taken together, these data suggest that hCUL1 functions as part of an SCF ubiquitin ligase complex in human cells. Further application of biochemical assays similar to those described here can now be used to identify regulators/components of hCUL1-based SCF complexes, to determine whether the hCUL2-hCUL5 proteins also are components of ubiquitin ligase complexes in human cells, and to screen for chemical compounds that modulate the activities of the hSKP1 and hCUL1 proteins (Lyapina, 1998).

ROC1 component of ubiquitin E3 ligases

Activation of the transcription factor NF-kappaB in response to proinflammatory stimuli requires the phosphorylation-triggered and ubiquitin-dependent degradation of the NF-kappaB inhibitor, IkappaB alpha. The phosphorylation-dependent ubiquitination of IkappaB alpha has been reconstituted in vitro with purified components. ROC1, a novel SCF-associated protein, is recruited by cullin 1 to form a quatemary SCFHOS-ROC1 holenzyme (with Skp1 and the beta-TRCP homolog HOS). SCFHOS-ROC1 binds IKK beta-phosphorylated IkappaB alpha and catalyzes its ubiquitination in the presence of ubiquitin, E1, and Cdc34. ROC1 plays a unique role in the ubiquitination reaction by heterodimerizing with cullin 1 to catalyze ubiquitin polymerization (Tan, 1999).

ROC1 is a common component of a large family of ubiquitin E3 ligases that regulate cell cycle progression and signal transduction pathways. Evidence is presented suggesting that a conserved RING-H2 structure within ROC1 is critical for its ubiquitin ligation function. Mercury-containing sulfhydryl modification agents (rho-hydroxymercuribenzoate and mercuric chloride) irreversibly inhibit the ROC1-CUL1 ubiquitin ligase activity without disrupting the complex. Consistent with this, these reagents also eliminate the ability of the Skp1-CUL1-HOS-ROC1 E3 ligase complex to support the ubiquitination of IkappaBalpha. Site-directed mutagenesis analysis identifies RING-H2 finger residues Cys(42), Cys(45), Cys(75), His(77), His(80), Cys(83), Cys(94), and Asp(97) as being essential for the ROC1-dependent ubiquitin ligase activity. Furthermore, C42S/C45S and H80A mutations reduce the ability of ROC1 to interact with CUL1 in transfected cells and diminish the capacity of ROC1-CUL1 to form a stable complex with Cdc34 in vitro. However, C75S, H77A, C94S, and D97A substitutions have no detectable effect on ROC1 binding activities. Thus, the ROC1 RING-H2 finger may possess multiple biochemical properties that include stabilizing an interaction with CUL1 and recruiting Cdc34. A possible role of the RING finger in facilitating the Ub transfer reaction is discussed (Chen, 2000).

A purified ubiquitination system is described capable of rapidly catalyzing the covalent linkage of polyubiquitin chains onto phosphorylated IkappaBalpha. The initial ubiquitin transfer and subsequent polymerization steps of this reaction require the coordinated action of Cdc34 and the SCF(HOS/beta-TRCP)-ROC1 E3 ligase complex, comprised of four subunits (Skp1, cullin 1 [CUL1], HOS/beta-TRCP, and ROC1). Deletion analysis reveals that the N terminus of CUL1 is both necessary and sufficient for binding Skp1 but is devoid of ROC1-binding activity and, hence, is inactive in catalyzing ubiquitin ligation. Consistent with this, introduction of the N-terminal CUL1 polypeptide into cells blocks the tumor necrosis factor alpha-induced and SCF-mediated degradation of IkappaB by forming catalytically inactive complexes lacking ROC1. In contrast, the C terminus of CUL1 alone interacts with ROC1 through a region containing the cullin consensus domain, to form a complex fully active in supporting ubiquitin polymerization. These results suggest the mode of action of SCF-ROC1, where CUL1 serves as a dual-function molecule that recruits an F-box protein for substrate targeting through Skp1 at its N terminus, while the C terminus of CUL1 binds ROC1 to assemble a core ubiquitin ligase (Wu, 2000a).

Effects of Cul1 mutation

The sequential timing of cell-cycle transitions is primarily governed by the availability and activity of key cell-cycle proteins. Recent studies in yeast have identified a class of ubiquitin ligases (E3 enzymes) called SCF complexes, which regulate the abundance of proteins that promote and inhibit cell-cycle progression at the G1-S phase transition. SCF complexes consist of three invariable components -- Skp1, Cul-1 (Cdc53 in yeast) and Rbx1 -- and a variable F-box protein that recruits a specific cellular protein to the ubquitin pathway for degradation. To study the role of Cul-1 in mammalian development and cell-cycle regulation, mice deficient for Cul1 were generated and null embryos and heterozygous cell lines were analyzed. Cul1 is shown to be required for early mouse development and Cul1 mutants fail to regulate the abundance of the G1 cyclin, cyclin E (encoded by Ccne), during embryogenesis (Dealy, 1999).

The stability of many proteins is controlled by the ubiquitin proteolytic system, which recognizes specific substrates through the action of E3 ubiquitin ligases. The SCFs are a class of ubiquitin ligase that target a number of cell cycle regulators and other proteins for degradation in both yeast and mammalian cells. Each SCF complex is composed of the core protein subunits Skp1, Rbx1 and Cul1 (known as Cdc53 in yeast), and substrate-specific adaptor subunits called F-box proteins. To understand the physiological role of SCF complexes in mammalian cells, mice carrying a deletion in the Cul1 gene were generated. Cul1(-/-) embryos arrest around embryonic day 6.5 (E6.5) before the onset of gastrulation. In all cells of the mutant embryos, cyclin E protein, but not mRNA, is highly elevated. Outgrowths of Cul1(-/-) blastocysts have limited proliferative capacity in vitro and accumulate cyclin E in all cells. Within Cul1(-/-) blastocyst cultures, trophoblast giant cells continue to endocycle despite the elevated cyclin E levels. These results suggest that cyclin E abundance is controlled by SCF activity, possibly through SCF-dependent degradation of cyclin E (Wang, 1999).

Transcriptional regulation of Cul1

The c-Myc oncoprotein plays an important role in the growth and proliferation of normal and neoplastic cells. To execute these actions, c-Myc is thought to regulate functionally diverse sets of genes that directly govern cellular mass and progression through critical cell cycle transitions. Evidence is provided that c-Myc promotes ubiquitin-dependent proteolysis by directly activating expression of the Cul1 gene, encoding a critical component of the ubiquitin ligase SCF(SKP2). The cell cycle inhibitor p27(kip1) is a known target of the SCF(SKP2) complex, and Myc-induced Cul1 expression matches well with the kinetics of declining p27(kip1) protein. Enforced Cul1 expression or antisense neutralization of p27(kip1) is capable of overcoming the slow-growth phenotype of c-Myc null primary mouse embryonic fibroblasts (MEFs). In reconstitution assays, the addition of in vitro translated Cul1 protein alone is able to restore p27(kip1) ubiquitination and degradation in lysates derived from c-myc(-/-) MEFs or density-arrested human fibroblasts. These functional and biochemical data provide a direct link between c-Myc transcriptional regulation and ubiquitin-mediated proteolysis and together support the view that c-Myc promotes G(1) exit in part via Cul1-dependent ubiquitination and degradation of the CDK inhibitor, p27 (O'Hagan, 2000).

Function of the Cul1 based SCF complex in cell cycle regulation

Deregulation of cell proliferation is a hallmark of cancer. In many transformed cells, the cyclin A/CDK2 complex that contains S-phase kinase associated proteins 1 and 2 (SKP1 and SKP2) is highly induced. To determine the roles of this complex in the cell cycle regulation and transformation, the composition of this complex was analyzed. This complex contains an additional protein, human CUL-1, a member of the cullin/CDC53 family. The identification of CUL-1 as a member of the complex raises the possibility that the p19(SKP1)/p45(SKP2)/CUL-1 complex may function as the yeast SKP1-CDC53-F-box (SCF) protein complex that acts as a ubiquitin E3 ligase to regulate the G1/S transition. In mammalian cells, cyclin D, p21(CIP1/WAF1), and p27(KIP1) are short-lived proteins that are controlled by ubiquitin-dependent proteolysis. To determine the potential in vivo targets of the p19(SKP1)/p45(SKP2)/CUL-1 complex, the specific antisense oligodeoxynucleotides were used against either SKP1, SKP2, or CUL-1 RNA to inhibit their expression. Treatment of cells with these oligonucleotides causes the selective accumulation of p21 and cyclin D proteins. The protein level of p27 is not affected. These data suggest that the human p19(SKP1)/p45(SKP2)/CUL-1 complex is likely to function as an E3 ligase to selectively target cyclin D and p21 for the ubiquitin-dependent protein degradation. Aberrant expression of human p19(SKP1)/p45(SKP2)/CUL-1 complex thus may contribute to tumorigenesis by regulating the protein levels of G1 cell cycle regulators (Yu, 1998).

Centrosomes organize the mitotic spindle to ensure accurate segregation of the chromosomes in mitosis. The mechanism that ensures accurate duplication and separation of the centrosomes underlies the fidelity of chromosome segregation, but remains unknown. In Saccharomyces cerevisiae, entry into S phase and separation of spindle pole bodies each require CDC4 and CDC34, which encode components of an SCF (Skp1-cullin-F-box) ubiquitin ligase, but a direct (SCF) connection to the spindle pole body is unknown. Using immunofluorescence microscopy, it has been shown that in mammalian cells the Skp1 protein and the cullin Cul1 are localized to interphase and mitotic centrosomes and to the cytoplasm and nucleus. Deconvolution and immunoelectron microscopy suggest that Skp1 forms an extended pericentriolar structure that may function to organize the centrosome. Purified centrosomes also contain Skp1, and Cul1 modified by the ubiquitin-like molecule NEDD8, suggesting a role for NEDD8 in targeting. Using an in vitro assay for centriole separation in Xenopus extracts, antibodies to Skp1 or Cul1 block separation. Proteasome inhibitors block both centriole separation in vitro and centrosome duplication in Xenopus embryos. Candidate centrosomal F-box proteins have been identified, suggesting that distinct SCF complexes may direct proteolysis of factors mediating multiple steps in the centrosome cycle (Freed, 1999).

The periodic expression of cell cycle proteins is important for the regulation of cell cycle progression. The amount of CDK inhibitor, p27(kip1), one such protein, seems to be regulated by the ubiquitin-proteasome system. The ubiquitin ligase (E3) toward p27(kip1) is thought to be SCF(skp2). The activity of SCF(skp2) is increased by the addition of Roc1 protein to the complex. Furthermore, the ubiquitination of p27(kip1) seems to be dependent on the phosphorylation of T187 of p27(kip1) because the mutant T187A is not ubiquitinated at all in an in vitro ubiquitination system. Cullin-1, a component of SCF, is modified by ubiquitin-like protein Nedd8. The modification site of cullin-1 was shown to be K696; the K696R mutant is not modified. When the effect of the Nedd8 modification on the SCF(skp2) activity toward p27(kip1) was investigated, the activity was markedly decreased by using the Nedd8-unmodified mutant cullin-1 (K696R), indicating that the modification may play an important role on the SCF(skp2) activity toward p27(kip1) (Morimoto, 2001).

The Cdc25 dual-specificity phosphatases control progression through the eukaryotic cell division cycle by activating cyclin-dependent kinases. Cdc25A regulates entry into S-phase by dephosphorylating Cdk2, it cooperates with activated oncogenes in inducing transformation and is overexpressed in several human tumors. DNA damage or DNA replication blocks induce phosphorylation of Cdc25A and its subsequent degradation via the ubiquitin-proteasome pathway. The regulation of Cdc25 A in the cell cycle has been investigated. Cdc25A degradation during mitotic exit and in early G(1) is found to be mediated by the anaphase-promoting complex or cyclosome (APC/C)(Cdh1) ligase; a KEN-box motif in the N-terminus of the protein is required for its targeted degradation. Interestingly, the KEN-box mutated protein remains unstable in interphase and upon ionizing radiation exposure. Moreover, SCF (Skp1/Cullin/F-box) inactivation using an interfering Cul1 mutant accumulates and stabilizes Cdc25A. The presence of Cul1 and Skp1 in Cdc25A immunocomplexes suggests a direct involvement of SCF in Cdc25A degradation during interphase. A dual mechanism of regulated degradation is proposed that allows for fine tuning of Cdc25A abundance in response to cell environment (Donzelli, 2002).

The anaphase-promoting complex or cyclosome (APC) is an unusually complicated ubiquitin ligase, composed of 13 core subunits and either of two loosely associated regulatory subunits, Cdc20 and Cdh1. The architecture of the APC was analyzed using a recently constructed budding yeast strain that is viable in the absence of normally essential APC subunits. The largest subunit, Apc1, serves as a scaffold that associates independently with two separable subcomplexes, one that contains Apc2 (Cullin), Apc11 (RING), and Doc1/Apc10, and another that contains the three TPR subunits (Cdc27, Cdc16, and Cdc23). The three TPR subunits display a sequential binding dependency, with Cdc27 the most peripheral, Cdc23 the most internal, and Cdc16 between. Apc4, Apc5, Cdc23, and Apc1 associate interdependently, such that loss of any one subunit greatly reduces binding between the remaining three. Intriguingly, the cullin and TPR subunits both contribute to the binding of Cdh1 to the APC. Enzymatic assays performed with APC purified from strains lacking each of the essential subunits revealed that only cdc27Δ complexes retain detectable activity in the presence of Cdh1. This residual activity depends on the C-box domain of Cdh1, but not on the C-terminal IR domain, suggesting that the C-box mediates a productive interaction with an APC subunit other than Cdc27. The IR domain of Cdc20 is dispensable for viability, suggesting that Cdc20 can activate the APC through another domain. This study has provided an updated model for the subunit architecture of the APC (Thornton, 2006).

Cullins and checkpoints

Genomic integrity is maintained by checkpoints that guard against undesired replication after DNA damage. CDT1, a licensing factor of the pre-replication complex (preRC), is rapidly proteolysed after UV- or gamma-irradiation. The preRC assembles on replication origins at the end of mitosis and during G1 to license DNA for replication in S phase. Once the origin recognition complex (ORC) binds to origins, CDC6 and CDT1 associate with ORC and promote loading of the MCM2-7 proteins onto chromatin, generating the preRC. Radiation-mediated CDT1 proteolysis is independent of ATM and CHK2 and can occur in G1-phase cells. Loss of the COP9-signalosome (CSN) or CUL4-ROC1 complexes completely suppresses CDT1 proteolysis. CDT1 is specifically polyubiquitinated by CUL4 complexes and the interaction between CDT1 and CUL4 is regulated in part by gamma-irradiation. This study reveals an evolutionarily conserved and uncharacterized G1 checkpoint that induces CDT1 proteolysis by the CUL4-ROC1 ubiquitin E3 ligase and CSN complexes in response to DNA damage (Higa, 2003).

Cullins assemble a potentially large number of ubiquitin ligases by binding to the RING protein ROC1 to catalyse polyubiquitination, as well as binding to various specificity factors to recruit substrates. The Cul4A gene is amplified in human breast and liver cancers, and loss-of-function of Cul4 results in the accumulation of the replication licensing factor CDT1 in Caenorhabditis elegans embryos and ultraviolet (UV)-irradiated human cells. Human UV-damaged DNA-binding protein DDB1 associates stoichiometrically with CUL4A in vivo, and binds to an amino-terminal region in CUL4A in a manner analogous to SKP1, SOCS and BTB binding to CUL1, CUL2 and CUL3, respectively. With SKP1-CUL1, the DDB1-CUL4A association is negatively regulated by the cullin-associated and neddylation-dissociated protein, CAND1. Recombinant DDB1 and CDT1 bind directly to each other in vitro, and ectopically express DDB1 bridges CDT1 to CUL4A in vivo. Silencing DDB1 prevents UV-induced rapid CDT1 degradation in vivo and CUL4A-mediated CDT1 ubiquitination in vitro. It is suggested that DDB1 targets CDT1 for ubiquitination by a CUL4A-dependent ubiquitin ligase, CDL4A(DDB1), in response to UV irradiation (Hu, 2004).

SCF complex targets IkappaB

Regulation of NF-kappaB occurs through phosphorylation-dependent ubiquitination of IkappaBalpha, which is degraded by the 26S proteasome. Recent studies have shown that ubiquitination of IkappaBalpha is carried out by a ubiquitin-ligase enzyme complex called SCF[beta(TrCP)] . Nedd8 modification of the Cul-1 component of SCF[beta(TrCP)] is important for function of SCF[beta(TrCP)] in ubiquitination of IkappaBalpha. In cells, Nedd8-conjugated Cul-1 was complexed with two substrates of SCF[beta(TrCP)] , phosphorylated IkappaBalpha and beta-catenin, indicating that Nedd8-Cul-1 conjugates are part of SCF[beta(TrCP)] in vivo. Although only a minute fraction of total cellular Cul-1 is modified by Nedd8, the Cul-1 associated with ectopically expressed betaTrCP was highly enriched for the Nedd8-conjugated form. Moreover, optimal ubiquitination of IkappaBalpha requires Nedd8 and the Nedd8-conjugating enzyme, Ubc12. The site of Nedd8 ligation to Cul-1 is essential, since SCF[beta(TrCP)] containing a K720R mutant of Cul-1 only weakly supports IkappaBalpha ubiquitination compared to SCF[beta(TrCP)] containing WT Cul-1, suggesting that the Nedd8 ligation of Cul-1 affects the ubiquitination activity of SCF[beta(TrCP)] . These observations provide a functional link between the highly related ubiquitin and Nedd8 pathways of protein modification and show how they operate together to selectively target the signal-dependent degradation of IkappaBalpha (Read, 2000).

SCF complex targets p130

p130 is a tumor suppressor of the pocket protein family whose expression is posttranscriptionally regulated and largely G0 restricted. The mechanism of down-regulation of p130 expression in proliferating cells was investigated. The decline of p130 expression as G0 cells reenter the cell cycle is due to a decrease in protein stability. The enhancement of p130 turnover in late G1 and S phase compared with G0 and early G1 phase is dependent on Cdk4/6-specific phosphorylation of p130 on Serine 672, and is independent of Cdk2 activity. The activity of the ubiquitin ligase complex Skp1-Cul1/Cdc53-F-box protein Skp2 [SCF(Skp2)] and the proteasome are necessary for p130 degradation. In vitro, recombinant Skp2 is able to bind hyperphosphorylated but not dephosphorylated p130. Furthermore, in vitro polyubiquitination of p130 by SCF(Skp2) is specifically dependent on phosphorylation of p130 on Serine 672. Thus, like the Cdk inhibitor p27(Kip1), p130 turnover is regulated by Cdk-dependent G1 phosphorylation leading to ubiquitin-dependent proteolysis (Tedesco, 2002).

BTB/POZ domain proteins are substrate adaptors for Cullin 3 ubiquitin ligases

Cullins (CULs) are subunits of a prominent class of RING ubiquitin ligases. Whereas the subunits and substrates of CUL1-associated SCF complexes and CUL2 ubiquitin ligases are well established, they are largely unknown for other cullin family members. S. pombe CUL3 (Pcu3p) forms a complex with the RING protein Pip1p and all three BTB/POZ domain proteins encoded in the fission yeast genome. The integrity of the BTB/POZ domain, which shows similarity to the cullin binding proteins SKP1 and elongin C, is required for this interaction. Whereas Btb1p and Btb2p are stable proteins, Btb3p is ubiquitylated and degraded in a Pcu3p-dependent manner. Btb3p degradation requires its binding to a conserved N-terminal region of Pcu3p that precisely maps to the equivalent SKP1/F box adaptor binding domain of CUL1. It is proposed that the BTB/POZ domain defines a recognition motif for the assembly of substrate-specific RING/cullin 3/BTB ubiquitin ligase complexes (Geyer, 2003).

These results identified BTB/POZ proteins as components of Pcu3p/Pip1p ubiquitin ligase complexes. Four pieces of evidence suggest that BTB/POZ domain proteins are functionally equivalent to the SKP1/F box adaptor dimers determining the substrate specificity of CUL1-associate SCF complexes: (1) all three BTB/POZ proteins present in the fission yeast genome interact with Pcu3p/Pip1p complexes; (2) BTB/POZ domains are structurally related to SKP1; (3) N-terminal residues invariably conserved in all CUL3 homologs, including Pcu3p, cluster in the same region of CUL1 that mediates its interaction with SKP1/F box adaptor dimers. Both the Btb3p/Pcu3p interaction and Pcu3p-dependent Btb3p degradation depend on the integrity of this conserved N-terminal region. (4) Btb3p is ubiquitylated in vitro in a Pcu3p-dependent manner, a finding reminiscent of CUL1-dependent ubiquitylation and degradation of F box proteins. Taken together, these findings strongly suggest that the BTB/POZ domain proteins ubiquitously present in eukaryotes define a family of substrate-specific adaptors for CUL3. Since fission yeast encodes three different BTB/POZ domain proteins, all of which interact with Pcu3p and Pip1p, it may form a minimum of three distinct RING/cullin 3/BTB complexes (Geyer, 2003).

Cullins and meiosis

The faithful segregation of chromosomes during meiosis is vital for sexual reproduction. Currently, little is known about the molecular mechanisms regulating the initiation and completion of meiotic anaphase. Inactivation of CUL-2, a member of the cullin family of ubiquitin ligases, delays or abolishes meiotic anaphase II with no effect on anaphase I, indicating differential regulation during the two meiotic stages. In cul-2 mutants, the cohesin REC-8 is removed from chromosomes normally during meiosis II and sister chromatids separate, suggesting that the failure to complete anaphase results from a defect in chromosome movement rather than from a failure to sever chromosome attachments. CUL-2 is required for the degradation of cyclin B1 in meiosis and inactivation of cyclin B1 partially rescued the meiotic delay in cul-2 mutants. In cul-2 mutants, the failure to degrade cyclin B1 precedes the metaphase II arrest. CUL-2 is also required for at least two aspects of embryonic polarity. The extended meiosis II in cul-2 mutants induces polarity reversals that include reversed orientation of polarity proteins, P granules, pronuclei migration and asymmetric cell division. Independent of its role in meiotic progression, CUL-2 is required to limit the initiation/spread of the polarity protein PAR-2 in regions distant from microtubule organizing centers. Inactivation of the leucine-rich repeat protein ZYG-11 (Drosophila homolog CG12084) produces meiotic and polarity reversal defects similar to those observed in cul-2 mutants, suggesting that the two proteins function in the same pathways (Liu, 2004).

The mechanisms that ensure coupling between meiotic cell cycle progression and subsequent developmental events, including specification of embryonic axes, are poorly understood. Here, zyg-11 and the cullin cul-2 promote the metaphase-to-anaphase transition and M phase exit at meiosis II in C. elegans. ZYG-11 acts with a CUL-2-based E3 ligase that is essential at meiosis II and that functions redundantly with the anaphase-promoting complex/cyclosome at meiosis I. The data also indicate that delayed M phase exit in zyg-11(RNAi) embryos is due to accumulation of the B type cyclin CYB-3. PAR proteins and P granules become polarized in an inverted manner during the meiosis II delay resulting from zyg-11 or cul-2 inactivation, and zyg-11 and cul-2 can regulate polarity establishment independent of a role in cell cycle progression. Furthermore, microtubules appear dispensable for ectopic polarity during the meiosis II delay in zyg-11(RNAi) embryos, as well as for AP polarity during the first mitotic cell cycle in wild-type embryos. These findings suggest a model in which a CUL-2-based E3 ligase promotes cell cycle progression and prevents polarity establishment during meiosis II, and in which the centrosome acts as a cue to polarize the embryo along the AP axis after exit from the meiotic cell cycle (Sonneville, 2004).

Function of COP9 signalsome: neddulation and the Cul1 subunit of SCF

Members of the cullin and RING finger ROC protein families form heterodimeric complexes to constitute a potentially large number of distinct E3 ubiquitin ligases. The highly conserved C-terminal sequence in CUL1 is dually required, both for nuclear localization and for modification by NEDD8. Disruption of ROC1 binding impairs nuclear accumulation of CUL1 and decreases NEDD8 modification in vivo but has no effect on NEDD8 modification of CUL1 in vitro, suggesting that ROC1 promotes CUL1 nuclear accumulation to facilitate its NEDD8 modification. Disruption of NEDD8 binding has no effect on ROC1 binding, nor does it affect nuclear localization of CUL1, suggesting that nuclear localization and NEDD8 modification of CUL1 are two separable steps, with nuclear import preceding and required for NEDD8 modification. Disrupting NEDD8 modification diminishes the IkappaBalpha ubiquitin ligase activity of CUL1. These results identify a pathway for regulation of CUL1 activity-ROC1 and the CUL1 C-terminal sequence collaboratively mediate nuclear accumulation and NEDD8 modification, facilitating assembly of active CUL1 ubiquitin ligase. This pathway may be commonly utilized for the assembly of other cullin ligases (Furukawa, 2000).

The SCF-ROC1 ubiquitin-protein isopeptide ligase (E3) ubiquitin ligase complex targets the ubiquitination and subsequent degradation of protein substrates required for the regulation of cell cycle progression and signal transduction pathways. ROC1-CUL1 is a core subassembly within the SCF-ROC1 complex, capable of supporting the polymerization of ubiquitin. The CUL1 subunit of the bacterially expressed, unmodified ROC1-CUL1 complex is conjugated with Nedd8 at Lys-720 by HeLa cell extracts or by a purified Nedd8 conjugation system (consisting of APP-BP1/Uba3, Ubc12, and Nedd8). This covalent linkage of Nedd8 to CUL1 is both necessary and sufficient to markedly enhance the ability of the ROC1-CUL1 complex to promote ubiquitin polymerization. A mutation of Lys-720 to arginine in CUL1 eliminates the Nedd8 modification, abolishes the activation of the ROC1-CUL1 ubiquitin ligase complex, and significantly reduces the ability of SCF(HOS/beta)(-TRCP)-ROC1 to support the ubiquitination of phosphorylated IkappaBalpha. Thus, although regulation of the SCF-ROC1 action has been shown to preside at the level of recognition of a phosphorylated substrate, this study demonstrates that Nedd8 is a novel regulator of the efficiency of polyubiquitin chain synthesis and, hence, promotes rapid turnover of protein substrates (Wu, 2000b).

COP9 signalosome (CSN) cleaves the ubiquitin-like protein Nedd8 from the Cul1 subunit of SCF ubiquitin ligases. The Jab1/MPN domain metalloenzyme (JAMM) motif in the Jab1/Csn5 subunit (see Drosophila Csn5) was found to underlie CSN's Nedd8 isopeptidase activity. JAMM is found in proteins from archaea, bacteria, and eukaryotes, including the Rpn11 subunit of the 26S proteasome. Metal chelators and point mutations within JAMM abolished CSN-dependent cleavage of Nedd8 from Cul1, yet have little effect on CSN complex assembly. Optimal SCF activity in yeast and both viability and proper photoreceptor cell (R cell) development in Drosophila melanogaster required an intact Csn5 JAMM domain. It is proposed that JAMM isopeptidases play important roles in a variety of physiological pathways (Cope, 2002).

Cullin proteins assemble a large number of RING E3 ubiquitin ligases and regulate various physiological processes. Covalent modification of cullins by the ubiquitin-like protein NEDD8 activates cullin ligases through an as yet undefined mechanism. p120CAND1 selectively binds to unneddylated CUL1 and is dissociated by CUL1 neddylation. CAND1 forms a ternary complex with CUL1 and ROC1. CAND1 dissociates SKP1 from CUL1 and inhibited SCF ligase activity in vitro. Suppression of CAND1 in vivo increases the level of the CUL1-SKP1 complex. It is suggested that by restricting SKP1-CUL1 interaction, CAND1 regulates the assembly of productive SCF ubiquitin ligases, allowing a common CUL1-ROC core to be utilized by a large number of SKP1-F box-substrate subcomplexes (Liu, 2002).

With approximately 50 genes encoding F box proteins in mammals, the incorporation of individual F box proteins into the active CUL1 ligase complex has to be tightly controlled. CUL1 is expressed at a much lower concentration than SKP1 and F box proteins, indicating that it is a rate-limiting factor in the assembly of a large number of SCF ligases. While there is a clear necessity for tight control over the dynamic and destructive ubiquitin ligase complexes, all the previously characterized subunits of the SCF-ROC ligases (CUL1, SKP1, F box, and ROC) are involved in either catalytic activity or substrate recruitment. NEDD8 modification of CUL1 was the only factor known to play a role in regulating cullin ligases, but its mechanism remains elusive. CAND1 functions as a negative regulator of SCF activity and is regulated by neddylation on CUL1. By regulating the assembly of SCF ubiquitin ligases, CAND1 allows a large number of SKP1-F box-substrate subcomplexes to be utilized by the same CUL1-ROC core. CAND1 may also interact with and similarly regulate other cullins (Liu, 2002).

The SCF ubiquitin E3 ligase regulates ubiquitin-dependent proteolysis of many regulatory proteins such as p27(Kip1), IkappaB, and beta-catenin. A CUL1 binding protein, p120(CAND1), has been isolated. The majority of CUL1 is in a complex with CAND1 and ROC1 independent of SKP1 and F box protein SKP2. Both in vivo and in vitro, CAND1 prevents the binding of SKP1 and SKP2 to CUL1 while dissociation of CAND1 from CUL1 promotes the reverse reaction. Neddylation of CUL1 or the presence of SKP1 and ATP causes CAND1 dissociation. These data suggest that CAND1 regulates the formation of the SCF complex, and its dissociation from CUL1 is coupled with the incorporation of F box proteins into the SCF complex, causing their destabilization (J. Zheng, 2002).

Cullin family proteins organize ubiquitin ligase (E3) complexes to target numerous cellular proteins for proteasomal degradation. Neddylation, the process that conjugates the ubiquitin-like polypeptide Nedd8 to the conserved lysines of cullins, is essential for in vivo cullin-organized E3 activities. Deneddylation, which removes the Nedd8 moiety, requires the isopeptidase activity of the COP9 signalosome (CSN). This study shows that in cells deficient for CSN activity, cullin1 (Cul1) and cullin3 (Cul3) proteins are unstable, and that to preserve their normal cellular levels, CSN isopeptidase activity is required. It is further shown that neddylated Cul1 and Cul3 are unstable - as suggested by the evidence that Nedd8 promotes the instability of both cullins - and that the unneddylatable forms of cullins are stable. The protein stability of Nedd8 is also subject to CSN regulation and this regulation depends on its cullin-conjugating ability, suggesting that Nedd8-conjugated cullins are degraded en bloc. It is proposed that while Nedd8 promotes cullin activation through neddylation, neddylation also renders cullins unstable. Thus, CSN deneddylation recycles the unstable, neddylated cullins into stable, unneddylated ones, and promotes cullin-organized E3 activity in vivo (Wu, 2005).

Structure of the Cand1-Cul1-Roc1 complex

The SCF ubiquitin ligase complex regulates diverse cellular functions by ubiquitinating numerous protein substrates. Cand1 (Cullin-associated and neddylation-dissociated protein: Drosophila homolog: CG5366), a 120 kDa HEAT repeat protein, forms a tight complex with the Cul1-Roc1 SCF catalytic core, inhibiting the assembly of the multisubunit E3 complex. The crystal structure of the Cand1-Cul1-Roc1 complex shows that Cand1 adopts a highly sinuous superhelical structure, clamping around the elongated SCF scaffold protein Cul1. At one end, a Cand1 ß hairpin protrusion partially occupies the adaptor binding site on Cul1, inhibiting its interactions with the Skp1 adaptor and the substrate-recruiting F box protein subunits. At the other end, two Cand1 HEAT repeats pack against a conserved Cul1 surface cleft and bury a Cul1 lysine residue, whose modification by the ubiquitin-like protein, Nedd8, is able to block Cand1-Cul1 association. Together with biochemical evidence, these structural results elucidate the mechanisms by which Cand1 and Nedd8 regulate the assembly-disassembly cycles of SCF and other cullin-dependent E3 complexes (Goldenberg, 2004).

Yeast COP9/Signalosome suppresses Cullin activity through recruitment of the deubiquitylating enzyme Ubp12p

The COP9/signalosome (CSN) is known to remove the stimulatory NEDD8 modification from cullins. The activity of the fission yeast cullins Pcu1p and Pcu3p is dramatically stimulated when retrieved from csn mutants but inhibited by purified CSN. This inhibition is independent of cullin deneddylation but mediated by the CSN-associated deubiquitylating enzyme Ubp12p, which forms a complex with Pcu3p in a CSN-dependent manner. In ubp12 mutants, as in csn mutants, Pcu3p activity is stimulated. CSN is required for efficient targeting of Ubp12p to the nucleus, where both cullins reside. Finally, the CSN/Ubp12p pathway maintains the stability of the Pcu1p-associated substrate-specific adaptor protein Pop1p. It is proposed that CSN/Ubp12p-mediated deubiquitylation creates an environment for the safe de novo assembly of cullin complexes by counteracting the autocatalytic destruction of adaptor proteins (Zhou, 2003).

The COP9 signalosome complex and COP1 in plants

The COP9 signalosome (CSN) is an evolutionarily conserved multiprotein complex that mediates the repression of photomorphogenesis in the dark in Arabidopsis through the degradation of transcription factors such as HY5 and HYH. CSN-mediated HY5 and HYH degradation also requires the activity of the putative E3 ubiquitin ligase (E3) component COP1 and the E2-conjugating enzyme variant COP10. CSN is also required for auxin responses mediated by the SCF-type E3 SCF(TIR1). To determine whether Arabidopsis CSN is required for E3-mediated processes in a more general manner, plants were generated with reduced E3 function by suppressing AtRBX1, an essential core subunit of SCF-type E3s. AtRBX1 transgenic plants share multiple phenotypes with CSN reduced-function plants, such as morphological defects and reduced responses to auxin, jasmonic acid, and cold stress, suggesting that CSN is required for multiple AtRBX1-mediated processes. Furthermore, mutants with defects in AXR1, a protein that had been described only as a regulator of SCF(TIR1) function, also is required for other E3-mediated processes and for the COP1/COP10/CSN-mediated repression of photomorphogenesis in the dark. It is concluded that CSN and AXR1 are of general importance for different pathways that are controlled by E3-mediated protein degradation (Schweichheimer, 2002).


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