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 SCF^Slimb 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 p27^Kip1 (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 (Ci^FL) 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 Ci^FL, 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 Ci^FL stability, it was found that there is mechanistic difference in controlling Ci^FL stability between anterior and posterior cells separated by the MF. Whereas the Cul1-based SCF^Slimb complex controls Ci^FL stability in anterior cells, a Cul3-dependent protein degradation mechanism controls Ci^FL 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, Ci^FL proteolytic processing requires the activity of the Nedd8-modified, Cul1-based SCF^Slimb complex. This Ci^FL proteolytic processing is inhibited by Smo signaling and promoted by PKA phosphorylation on Ci^FL. The mechanism by which Ci^FL is proteolyzed from Ci^FL to Ci75 is not clear. It is proposed that Nedd8 modifies and activates SCF^Slimb for Ci ubiquitination and then proteolysis, as evidenced by Cul1 modification by Nedd8 and Ci^FL accumulation in Nedd8, Cul1, and slimb mutants. Consistently, proteolysis of Ci^FL 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 Ci^FL 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 Ci^FL 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 Ci^FL 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 SCF^Slimb-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 Ci^FL 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 Ci^FL 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. Ci^FL 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 Ci^FL (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 Ci^FL 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 SCF^ßTrCP 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).

GENE STRUCTURE

cDNA clone length - 3250

Bases in 5' UTR - 414

Exons - 5

Bases in 3' UTR - 511

PROTEIN STRUCTURE

Amino Acids - 774

Structural Domains

See SMART (Simple Modular Architecture Research Tool) for information on Cullins.

date revised: 31 January 2002

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