Signal-induced phosphorylation of IkappaBalpha (Drosophila homolog: Cactus) targets this inhibitor of NF-kappaB for ubiquitination and subsequent degradation, thus allowing NF-kappaB to enter the nucleus to turn on its target genes. An IkappaB-ubiquitin (Ub) ligase complex has been identified that contains the F-box/WD40-repeat protein, beta-TrCP, a vertebrate homolog of Drosophila Slimb. beta-TrCP binds to IkappaBalpha only when the latter is specifically phosphorylated by an IkappaB kinase complex. Moreover, immunopurified beta-TrCP ubiquitinates phosphorylated IkappaBalpha at specific lysines in the presence of Ub-activating (E1) and -conjugating (Ubch5) enzymes. A beta-TrCP mutant lacking the F-box inhibits the signal-induced degradation of IkappaBalpha and subsequent activation of NF-kappaB-dependent transcription. Furthermore, Drosophila embryos deficient in slimb fail to activate twist and snail, two genes known to be regulated by the NF-kappaB homolog, Dorsal. These biochemical and genetic data strongly suggest that Slimb/beta-TrCP is the specificity determinant for the signal-induced ubiquitination of IkappaBalpha (Spencer, 1999).
The Hedgehog signal transduction pathway is involved in diverse patterning events in many organisms. In Drosophila, Hedgehog signaling regulates transcription of target genes by modifying the activity of the DNA-binding protein Cubitus interruptus (Ci). Hedgehog signaling inhibits proteolytic cleavage of full-length Ci (Ci-155) to Ci-75, a form that represses some target genes, and also converts the full-length form to a potent transcriptional activator. Reduction of Protein kinase A (PKA) activity also leads to accumulation of full-length Ci and to ectopic expression of Hedgehog target genes, prompting the hypothesis that PKA might normally promote cleavage to Ci-75 by directly phosphorylating Ci-155. A mutant form of Ci lacking five potential PKA phosphorylation sites (Ci5m) is not detectably cleaved to Ci-75 in Drosophila embryos. Moreover, changes in PKA activity dramatically alters levels of full-length wild-type Ci in embryos and imaginal discs, but does not significantly alter full-length Ci5m levels. These results are corroborated by showing that Ci5m is more active than wild-type Ci at inducing ectopic transcription of the Hh target gene wingless in embryos and that inhibition of PKA enhances induction of wingless by wild-type Ci but not by Ci5m. It is therefore proposed that PKA phosphorylation of Ci is required for the proteolysis of Ci-155 to Ci-75 in vivo. It is also showm that the activity of Ci5m remains Hedgehog responsive if expressed at low levels, providing further evidence that the full-length form of Ci undergoes a Hedgehog-dependent activation step (Price, 1999).
How does PKA phosphorylation of Ci-155 lead to its proteolysis? Loss of cos2 activity in wing disc clones induces high levels of Ci-155, suggesting that the integrity of the multiprotein cytoplasmic complex that contains Ci or the association of this complex with microtubules may be necessary in order for proteolysis to occur. It is possible that Ci phosphorylation also affects proteolysis by altering these interactions. A more direct role for PKA phosphorylation of Ci has been proposed based on the sequence and properties of the Slimb protein, which affects the conversion of Ci-155 to Ci-75. Slimb belongs to a family of F-box/ WD40-repeat proteins implicated in binding to and targeting phosphorylated molecules for ubiquitin-mediated degradation. It was recently shown that the vertebrate Slimb homolog, beta-TRCP, targets IkappaB and beta-catenin for ubiquitin-mediated degradation by binding specifically to a phosphorylated motif (DSGXXS, where both serines must be phosphorylated) present in both proteins. Whether Slimb participates in such a direct manner in Ci proteolysis is not clear. Slimb has not been shown to bind to Ci, and Ci proteolysis has not been shown to involve ubiquitination; Ci proteolysis is also unusual in being incomplete, leaving a stable 75 kDa product. Sequences around three Ci sites show some extended similarity to each other but are quite different from the IkappaB and beta-catenin consensus. It will be interesting to determine if Slimb, or another F-box protein, can bind directly to these regions of Ci when phosphorylated by PKA. Since Slimb recognition requires phosphorylation at multiple residues and the PKA site consensus in Ci contains additional serines, it is worth considering that the activity of another protein kinase in addition to PKA may also contribute to the regulation of Ci proteolysis (Price, 1999 and references).
In Drosophila, signaling by the protein Hedgehog (Hh) alters the activity of the transcription factor Cubitus interruptus (Ci) by inhibiting the proteolysis of full-length Ci (Ci-155) to its shortened Ci-75 form. Ci-75 is found largely in the nucleus and is thought to be a transcriptional repressor, whereas there is evidence to indicate that Ci-155 may be a transcriptional activator. However, Ci-155 is detected only in the cytoplasm, where it is associated with the protein kinase Fused (Fu), with Suppressor of Fused [Su(fu)], and with the microtubule-binding protein Costal-2. It is not clear how Ci-155 might become a nuclear activator. Mutations in Su(fu) cause an increase in the expression of Hh-target genes in a dose-dependent manner while simultaneously reducing Ci-155 concentration by some mechanism other than proteolysis to Ci-75. Conversely, eliminating Fu kinase activity reduces Hh-target gene expression while increasing Ci-155 concentration. It is proposed that Fu kinase activity is required for Hh to stimulate the maturation of Ci-155 into a short-lived nuclear transcriptional activator and that Su(fu) opposes this maturation step through a stoichiometric interaction with Ci-155 (Ohlmeyer, 1999).
Hh signaling thus elicits several changes that are required to convert Ci into an effective transcriptional activator. Hh spares Ci-155 from Protein kinase A- and Slimb-dependent protolysis to Ci-75, perhaps by modifying the phosphorylation status of Ci, and promotes dissociation of the Ci-155 complex from microtubules. It is proposed that in wing discs some of this 'primed' Ci-155 is not associated with Su(fu) and can activate dpp and ptc transcription but not anterior en expression. Most of the primed Ci-155 in wing discs and perhaps all of the primed Ci-155 in embryos is inactive while it is in complex with Su(fu) and signaling by Hh and Fused kinase are necessary for Ci-155 to become a transcriptional activator. This active form of Ci appears to be unstable and so is not detectable in the nuclei of cells responding to Hh. The lower levels of Ci-155 that are found in wing discs close to the source of Hh, as compared with levels in more distant regions of the Hh-signaling domain, may be explained by this model if more Hh is required to stimulate the Fu-kinase-dependent step in Ci activation than to protect Ci-155 from proteolytic degradation to Ci-75. This dosage dependence may account for the restricted range of engrailed induction relative to dpp and ptc in wing discs and the single-cell range of Hh signaling in embryonic ectoderm (Ohlmeyer, 1999).
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).
Transcription factor Ci mediates Hedgehog (Hh) signaling to determine the anterior/posterior (A/P) compartment of Drosophila wing disc. While Hh-inducible genes are expressed in A compartment cells abutting the A/P border, it is unclear how the boundaries of this region are established. Here, a Ci binding protein, Debra, has been identified that is expressed at relatively high levels in the band abutting the border of the Hh-responsive A compartment region. Debra mediates the polyubiquitination of full-length Ci, which then leads to its lysosomal degradation. Debra is localized in the multivesicular body, suggesting that the polyubiquitination of Ci directs its sorting into lysosome. Thus, Debra defines the border of the Hh-responsive region in the A compartment by inducing the lysosomal degradation of Ci (Dai, 2003).
Both the PKA phosphorylation sites and the processing site of the Ci protein are required for its Dbr-induced degradation. In addition, these sites are required for the proteasome-dependent processing of Ci-155, which also involves Slimb. Thus, Ci-155 levels are regulated by two separate degradative processes. That both processes share common regulatory elements suggests that it is likely that the events leading to the lysosomal degradation and proteasome processing of Ci-155 occur in parallel. Since Slimb contains an F box/WD40 repeat, and its vertebrate homolog is a component of the SCF ubiquitin ligase complex, Slimb is likely to act as an E3 ligase in transferring the ubiquitin moiety to Ci. In the absence of Dbr, Slimb induces the proteolytic processing of Ci-155 to Ci-75 via the proteasome, possibly by mediating limited Ci-155 ubiquitination that then serves as a proteolytic processing signal. When Dbr exists, Slimb cooperates to induce the full ubiquitination of Ci-155 that targets it for lysosomal degradation via MVBs. Dbr does not induce Ci-75 degradation. Slimb binds to both the N- and C-terminal regions of Ci-155. It may be that the binding of Slimb to Ci-75, which lacks the C-terminal region of Ci-155, is too weak to induce the ubiquitination of Ci-75, resulting in the maintenance of this form of Ci in the cell (Dai, 2003).
The Drosophila circadian clock is driven by daily fluctuations of the proteins Period and Timeless, which associate in a complex and negatively regulate the transcription of their own genes. Protein phosphorylation has a central role in this feedback loop, by controlling Per stability in both cytoplasmic and nuclear compartments as well as Per/Tim nuclear transfer. However, the pathways regulating degradation of phosphorylated Per and Tim are unknown. The product of the slimb (slmb) gene -- a member of the F-box/WD40 protein family of the ubiquitin ligase SCF complex that targets phosphorylated proteins for degradation -- is shown to be an essential component of the Drosophila circadian clock. slmb mutants are behaviorally arrhythmic, and can be rescued by targeted expression of Slmb in the clock neurons. In constant darkness, highly phosphorylated forms of the Per and Tim proteins are constitutively present in the mutants, indicating that the control of their cyclic degradation is impaired. Because levels of Per and Tim oscillate in slmb mutants maintained in light:dark conditions, light- and clock-controlled degradation of Per and Tim do not rely on the same mechanisms (Grima, 2002).
To test whether the SCF-mediated ubiquitin proteasome pathway is involved in the control of Per and Tim oscillations, circadian rhythms were examined of flies defective for genes encoding F-box proteins that are known to target phosphorylated substrates for degradation. The slimb (slmb) gene, which encodes an F-box/WD40 protein regulating transcription factors' levels in the wingless and hedgehog signaling pathways was examined. slmb8 mutants that normally die as early larvae were brought to adulthood by providing the slmb gene product throughout development under the control of a heat-shock promoter. The rescued HS-slmb slmb8 adult flies, hereafter referred to as slmbm mutants, were then tested for their locomotor activity rhythms in both light:dark (LD) and constant darkness (DD) conditions. slmbm mutants were completely arrhythmic in DD, whereas the heterozygous genotype displayed wild-type rhythms. The absence of anatomical defects of the PDF-expressing ventral lateral neurons (LNvs), which control the behavioral rhythms, strongly argues against a developmental origin of the mutants' rhythm defect. Furthermore, targeted slmb expression using well characterized LNvs-specific gal4 drivers restores near wild-type activity rhythms, whereas similarly targeted overexpression in a wild-type background lengthens the circadian period, indicating a cell-autonomous role of the slmb gene in circadian rhythmicity. In LD conditions, slmbm mutants did not display the light-off anticipation of activity that characterizes a functional clock, whereas it was observed in the flies expressing slmb under the LNvs-specific gal1118 driver. These experiments identify the F-box/WD40 protein Slmb as an essential component of the Drosophila brain clock (Grima, 2002).
To understand how Slmb might affect the circadian oscillator, slmbm mutants were analyzed for Per and Tim oscillations in the head. In wild-type flies maintained in LD cycles, Per and Tim proteins accumulate and are progressively phosphorylated during night time, with Tim disappearing at the end of the night whereas hyper-phosphorylated Per persists for a few hours in the morning. A similar temporal pattern persists in DD, and is required to sustain behavioral rhythmicity. In contrast, highly phosphorylated Per and Tim are present at all circadian times in slmbm mutants kept in DD, although low-amplitude oscillations of the hypo-phosphorylated forms indicate a weak residual activity of the molecular clock. In agreement with the persistence of weak protein cycling in slmbm heads, levels of per and tim transcripts displayed low-amplitude oscillations. Per immunoreactivity was examined in the LNvs that control behavioral rhythms. At circadian time (CT) 0 and CT 12, which correspond to the peak and trough of Per labelling in w flies at 20°C, slmbm mutants showed low levels of Per immunoreactivity, indicating that the oscillations of the proteins levels are also abolished in the clock cells. To determine whether Slmb acts at the protein level or through a transcriptional control, per was constitutively overexpressed through a transgene. High-molecular-mass Per proteins were observed to accumulate in head extracts of slmbm but not of wild-type flies carrying GMR-gal4 and UAS-per transgenes that drive strong Per expression in the eye. Altogether, these data indicate that Slmb is involved in the control of phosphorylated Per levels (Grima, 2002).
In LD conditions, Per and Tim degradation in the morning is driven by both the circadian cycle and by light. Light-induced Tim degradation involves ubiquitinylation of the protein, and is blocked by proteasome inhibitors. To test whether Slmb is involved in the light-induced degradation pathway of the clock proteins, Per and Tim levels were assayed in slmbm flies kept in LD conditions. In contrast to constant darkness, robust oscillations of Per and Tim amounts were observed in LD, with both proteins accumulating during the night and showing a strong day-time decrease. This shows that light-induced Per and Tim degradation does not occur through the same slmb-dependent mechanism as their circadian-cycle-controlled degradation in constant darkness. In addition, the absence of light-off anticipation in the slmbm activity profiles suggests that the mutants' altered temporal regulation of phosphorylated Per and Tim does not allow rhythmic outputs to be driven, although protein levels clearly cycle (Grima, 2002).
Clock-dependent Per and Tim degradation occurs at the end of the circadian cycle, and relieves the transcriptional repression that the proteins exert on their own genes. Per degradation has also been proposed to take place during the rising phase of the protein levels in the early night, and to be responsible for the shift (of 5 h) between per messenger RNA and Per protein peaks. In order to determine whether Slmb levels vary during a circadian cycle and may therefore affect Per and Tim only during a limited time window, anti-Slmb antibodies were raised and the Slmb protein was followed in head extracts at different circadian times. A strongly reacting protein, as well as a faintly reacting one slightly above, were detected at a relative molecular mass of 45,000 (Mr 45K) in wild-type flies, and did not show any oscillations of their levels over a 24-h time course. Similarly, slmb mRNA did not show any cycling. Slmb therefore appears not to be circadianly regulated, and could therefore act on different steps of the cycle (Grima, 2002).
Both early- and late-night Per degradation steps appear to depend upon Per phosphorylation, which requires the casein kinase I encoded by the double-time (dbt) gene. To find out how Slmb could affect Per and Tim phosphorylation, Tests were performed to see whether Dbt, and Shaggy (Sgg), that has been shown to phosphorylate Tim, are affected in slmbm mutants. No alterations of the level or the mobility of these kinases were detected in slmbm head extracts. Next, whether Slmb could associate with the Per protein was examined, by searching for Per-Slmb interactions in co-immunoprecipitation experiments on head extracts. The Slmb protein was found to be co-precipitated by anti-Per antibodies, and anti-Slmb can precipitate Per in wild-type flies collected at CT 0. Similar results were obtained with pooled extracts. In addition, Slmb co-precipitates with Dbt . Because Per, but not Dbt, is profoundly affected in slmbm mutants, these results support Per rather than Dbt as a Slmb target for ubiquitinylation, and suggest that the three proteins constitute a complex. Slmb was co-immunoprecipitated by anti-Per antibodies in tim0 flies, indicating that Per-Slmb complexes can form in the absence of Tim. Although twice as much extract was used for tim0 flies to compensate for the low Per levels in this genotype, the amount of immunoprecipitated Slmb suggests that the absence of Tim may favor Per-Slmb complexes. These results fit well with Slmb being involved in the control of unbound Per, either during its cytoplasmic accumulation at the beginning of the protein cycle or during its nuclear degradation at the end. To test whether the formation of Per-Slmb complexes is circadianly controlled, co-immunoprecipitations were performed at the beginning of the night when Per is mostly hypo-phosphorylated, or at the end of the night when Per is highly phosphorylated. All time points showed comparable levels of Per-Slmb complexes, and several forms of Per were immunoprecipitated by the anti-Slmb antibodies (compare CT 1 and CT 13). This indicates that differently phosphorylated Per molecules can be committed to Per-Slmb complexes (Grima, 2002).
Possible explanations for the accumulation of highly phosphorylated Per in slmbm mutants would be that partially phosphorylated Per is the relevant Slmb substrate for degradation, or that Slmb targets some Per kinase that is bound to Per. The presence of highly phosphorylated Per in slmbm indicates that Slmb is required for the control of phosphorylated Per accumulation in the early night. Moreover, Slmb overexpression in the LNvs results in a lengthening of the circadian period. In agreement with the behavioral data, Slmb overexpression slows down the oscillations of Per immunoreactivity in these cells, which showed a ~6 hour delay compared to wild-type controls after two days. These data can be explained by high levels of cytoplasmic Slmb inducing too much degradation of cytoplasmic Per, thus further delaying the night accumulation of the protein, whereas high levels of nuclear Slmb would rather precipitate the fall of the Per protein and shorten the circadian period. It is therefore thought that Slmb is, at least, involved in the control of cytoplasmic Per accumulation in the early night (Grima, 2002).
The presence of low-mobility Tim proteins at all circadian times in slmbm mutants indicates that the accumulation of phosphorylated Tim is also Slmb-dependent. Remarkably, the Tim kinase Sgg controls the Slmb-dependent proteolysis of Cubitus interruptus and degradation of Armadillo. The results suggest that phosphorylated Tim could be a Slmb target or that Tim is phosphorylated by a Slmb-dependent kinase. Because Tim is hypo-phosphorylated in per0 flies, it is also possible that the accumulation of hyper-phosphorylated Per in slmbm influences Tim phosphorylation (Grima, 2002).
Although protein degradation is commonly believed to have a major role in the control of the oscillations of clock proteins, the present work is the first to implicate a characterized component of the ubiquitin proteasome pathway. Because cycling of phosphorylated Per proteins also occurs in the mammalian clock, it would be interesting to determine whether the Slmb mammalian homolog ß-Trcp is involved in the control of phosphorylated Per levels. F-box proteins have been shown to be important at the G1/S transition of the cell cycle, by targeting phosphorylated cyclins and inhibitors of cyclin kinases for degradation by the proteasome. This study therefore suggests that the cell-cycle and the circadian-clock machineries share mechanisms to control the oscillations of phosphorylated proteins (Grima, 2002).
Protein phosphorylation has a key role in modulating the stabilities of circadian clock proteins in a manner specific to the time of day. A conserved feature of animal clocks is that Period (Per) proteins undergo daily rhythms in phosphorylation and levels, events that are crucial for normal clock progression. Casein kinase Iepsilon (CKIepsilon) has a prominent role in regulating the phosphorylation and abundance of Per proteins in animals. This was first shown in Drosophila with the characterization of Doubletime (Dbt), a homolog of vertebrate casein kinase Iepsilon. However, it has not been clear how Dbt regulates the levels of Per. Using a cell culture system, this study shows that Dbt promotes the progressive phosphorylation of Per, leading to the rapid degradation of hyperphosphorylated isoforms by the ubiquitin–proteasome pathway. Slimb, an F-box/WD40-repeat protein functioning in the ubiquitin–proteasome pathway interacts preferentially with phosphorylated Per and stimulates its degradation. Overexpression of slimb or expression in clock cells of a dominant-negative version of slimb disrupts normal rhythmic activity in flies. These findings suggest that hyperphosphorylated Per is targeted to the proteasome by interactions with Slimb (Ko, 2002).
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).
Hedgehog (Hh) proteins govern animal development by regulating the Gli/Ci family of transcription factors. In Drosophila, Hh signaling blocks proteolytic processing of full-length Ci to generate a truncated repressor form. Ci processing requires sequential phosphorylation by PKA, GSK3, and a casein kinase I (CKI) family member(s). This study shows that Double-time (DBT)/CKIε and CKIα act in conjunction to promote Ci processing. CKI phosphorylates Ci at three clusters of serine residues primed by PKA and GSK3 phosphorylation of other residues. CKI phosphorylation of Ci confers binding to the F-box protein Slimb/β-TRCP, the substrate recognition component of the SCFSlimb/β-TRCP ubiquitin ligase required for Ci processing. CKI phosphorylation sites act cooperatively to promote Ci processing in vivo. Substitution of Ci phosphorylation clusters with a canonical Slimb/β-TRCP recognition motif found in β-catenin renders Slimb/β-TRCP binding and Ci processing independent of CKI. It is proposed that phosphorylation of Ci by CKI creates multiple Slimb/β-TRCP binding sites that act cooperatively to recruit SCFSlimb/β-TRCP (Jia, 2005).
Regulation of Ci/Gli processing is a key regulatory step in the Hh signal transduction pathway; however, the underlying mechanism is still not fully understood. This study provides evidence that two CKI isoforms, DBT/CKIε and CKIα, act additively to promote Ci processing. It was found that CKI phosphorylates multiple Ser residues arranged in three clusters in the C-terminal half of Ci, and that CKI can phosphorylate sites primed by PKA or GSK3 phosphorylation. In addition, DBT/CKIε and CKIα are required for Ci phosphorylation in vivo. CKI sites in different phosphorylation clusters act cooperatively to promote Ci processing in vivo. More importantly, Slimb/β-TRCP was shown to directly bind CKI-phosphorylated Ci through its WD40 repeats. Finally, substitution of multiple CKI sites with a Slimb/β-TRCP binding motif found in β-catenin renders Ci processing independent of CKI. Based on these and other observations, it is proposed that PKA- and GSK3-primed CKI phosphorylation of Ci creates docking sites for Slimb/β-TRCP that recruit SCFSlimb/β-TRCP to regulate Ci processing (Jia, 2005).
This study employed dominant-negative kinase, genetic mutations, and heritable RNAi knockdown to investigate the role of two CKI isoforms in Ci processing in vivo. Overexpression of a dominant-negative DBT/CKIε (DN-DBT) caused cell-autonomous accumulation of Ci155 and ectopic dpp expression, suggesting that interference with DBT/CKIε activity impairs Ci processing. As a further support, it was found that A compartment dbt/dco mutant cells accumulate high levels of Ci155. The phenotypes associated with dbt/dco mutations differ depending on the alleles used. The hypomorphic allele, dco3, does not seem to affect Ci processing, although it does affect cell growth and proliferation. By contrast, more severe alleles, including dcoP103 and dcole88, affect Ci processing. The lack of Hh-related phenotypes associated with the weak allele of dbt/dco is likely due to compensation by other CKI isoforms. This may explain why RNAi knockdown of DBT/CKIε does not affect Hh signaling in cultured cells, since RNAi knockdown usually does not completely eliminate the function of the targeted genes, and hence often resembles hypomorphic genetic mutations. Alternatively, other CKI isoforms might be expressed in cultured cells at higher levels than in imaginal discs, so that they can compensate for the complete loss of DBT/CKIε in cultured cells (Jia, 2005).
To investigate the role of CKIα in Ci processing, the heritable RNAi approach was used, and two CKIα RNAi constructs were generated: CRS and CRL. CRL knocks down CKIα more effectively than CRS, likely due to its larger targeting sequence; however, it also knocks down DBT/CKIε. In contrast, CRS appears to be more specific for CKIα. Expressing CRL in wing discs induces high levels of Ci155 accumulation and ectopic dpp expression. In contrast, expressing CRS resulted only in a modest increase in Ci155 without inducing ectopic dpp expression. However, expressing CRS in DBT/CKIε hypomorphic (dco3/dcole88) wing discs completely blocked Ci processing, as evident by the accumulation of high levels of Ci155 and ectopic dpp expression in these discs. These data suggest that CKIα and CKIε play partially redundant roles in Ci processing, and that they act additively to provide optimal CKI kinase activity required for efficient Ci phosphorylation and processing. Consistent with this notion, CKIα and CKIε bind equally well to Cos2. This is in contrast to what has been proposed for the Wnt pathway, where CKIε and CKIα appear to play opposing roles and act on distinct protein substrates. Since CKI sites are conserved in Gli proteins, it awaits to be determined whether CKIε or CKIα or both are involved in Gli regulation (Jia, 2005).
Using an in vitro kinase assay, two types of CKI phosphorylation events were uncovered: one primed by PKA and the other by GSK3 phosphorylation. CKI phosphorylation sites are arranged in three clusters. Whereas cluster 1 contains only PKA-primed CKI sites, both cluster 2 and 3 contain PKA- and GSK3-primed CKI sites. Using an in vivo functional assay, it was demonstrated that both PKA- and GSK3-primed CKI sites are involved in Ci processing. For example, the two types of CKI sites in cluster 2 appear to have overlapping function; mutations in either one only partially blocked Ci processing, whereas mutations in both completely blocked Ci processing (Jia, 2005).
CKI sites in different phosphorylation clusters appear to act cooperatively to promote Ci processing. Strikingly, mutating the two CKI sites in cluster 1 (CiSA12) completely abolishes Ci processing. Similarly, mutating all the CKI sites in cluster 2 also abolishes Ci processing. A dosage-sensitive interaction was observed between two phosphorylation clusters. For example, partial loss of function of both cluster 2 and cluster 3 nearly abolish Ci processing. Based on these and other observations, it is proposed that each phosphorylation cluster acts as a functional module, and Ci processing requires cooperative action among the three modules (Jia, 2005).
Ci lacks the canonical Slimb/β-TRCP binding motif (DSGXXS) found in other SCFSlimb/β-TRCP substrates such as β-catenin and Iκ-B, inviting speculation that Ci phosphorylation could recruit a protein(s) other than Slimb/β-TRCP and that the involvement of SCFSlimb/β-TRCP in Ci processing could be indirect. This study assessed whether hyperphosphorylation of Ci directly recruits Slimb/β-TRCP. It was found that a GST-Ci fusion protein binds Slimb/β-TRCP efficiently after it is phosphorylated by CKI, following primed phosphorylation by the other kinases. In addition, binding of GST-Ci to Slimb is compromised when a subset of CKI sites was mutated to Ala. These observations support the hypothesis that phosphorylation of Ci at CKI sites confers Slimb/β-TRCP binding. The in vivo relevance of Slimb/β-TRCP binding was demonstrated by the finding that a single canonical Slimb/β-TRCP binding site can substitute for the three phosphorylation clusters to promote Ci processing. Strikingly, Ci variants bearing the DSGXXS motif can undergo processing even when CKI activity is blocked. These observations suggest that the major function of CKI in Ci processing is to recruit SCFSlimb/β-TRCP by phosphorylating Ci at multiple Ser residues that function as docking sites for Slimb/β-TRCP (Jia, 2005).
The recently solved crystal structure of the β-TRCP/β-catenin phospho-peptide complex reveals that the two phospho-Ser and the aspartate residues in the DSGXXS motif make critical contacts with several basic residues from the WD40 repeats of β-TRCP that form a single substrate binding pocket. Although none of the three phosphorylation clusters in Ci contains a DSGXXS motif, they all contain related sequences. For example, cluster 1 contains DSQNSTAS, cluster 2 contains SSQSS and SSQVSS, and cluster 3 contains SSQMS. It is proposed that these phospho-Ser motifs represent low-affinity or suboptimal sites for Slimb/β-TRCP recognition, and optimal binding of Slimb/β-TRCP to Ci is achieved by cooperative binding among multiple low-affinity sites. The high local concentration of binding sites greatly increases the probability of interaction so that Ci is unable to diffuse away from Slimb/β-TRCP before rebinding occurs. Hence, Ci becomes kinetically trapped in close proximity to Slimb/β-TRCP once the binding is engaged. Alternatively, phosphorylation of Ci could recruit a cofactor that binds cooperatively with Slimb/β-TRCP to hyperphosphorylated Ci. Both models can explain the observed high cooperativity among multiple phosphorylation clusters in Ci processing (Jia, 2005).
The ability to bind a single high-affinity site or multiple low-affinity sites appears to be a general feature for the SCF family of ubiquitin ligases. Another well-characterized SCF complex, SCFCDC4, can bind certain substrates such as Cyclin E through a single high-affinity site and other substrates such as Sic1 through multiple low-affinity sites. In the case of Sic1, phosphorylation at multiple sites appears to set a threshold for kinase activity that converts a smooth temporal gradient of kinase activity into a switch-like response for degradation of Sic1 and onset of S phase. In the case of Ci/Gli regulation, first, the requirement for hyperphosphorylation may render Ci processing highly dependent on the activity of individual kinases and hence highly sensitive to Hh, since low levels of Hh suffice to block Ci processing although such levels of Hh may only cause a small reduction in Ci phosphorylation levels. Second, cooperativity among multiple phosphorylation sites may convert a smooth spatial Hh activity gradient into a sharp response for Ci processing, since a small drop in the level of Ci phosphorylation could result in a dramatic reduction in Ci processing and hence the level of Ci75. Third, employing multiple phosphorylation events may allow the levels of Ci phosphorylation to be fine-tuned by different thresholds of Hh signaling activity, leading to differential regulation of Ci processing and activity, as the activity of Ci155 appears to be regulated by phosphorylation independent of its processing. Finally, employing multiple kinases to regulate Ci/Gli may provide opportunities for crosstalk between the Hh and other signaling pathways in certain developmental contexts (Jia, 2005).
Signaling by extracellular Hedgehog (Hh) molecules is crucial for the correct allocation of cell fates and patterns of cell proliferation in humans and other organisms. Responses to Hh are universally mediated by regulating the activity and the proteolysis of the Gli family of transcriptional activators such that they induce target genes only in the presence of Hh. In the absence of Hh, the sole Drosophila Gli homolog, Cubitus interruptus (Ci), undergoes partial proteolysis to Ci-75, which represses key Hh target genes. This processing requires phosphorylation of full-length Ci (Ci-155) by protein kinase A (PKA), casein kinase 1 (CK1), and glycogen synthase kinase 3 (GSK3), as well as the activity of Slimb. Slimb is homologous to vertebrate ß-TRCP1, which binds as part of an SCF (Skp1/Cullin1/F-box) complex to a defined phosphopeptide motif to target proteins for ubiquitination and subsequent proteolysis. Phosphorylation of Ci at the specific PKA, GSK-3, and CK1 sites required in vivo for partial proteolysis stimulates binding to Slimb in vitro. Furthermore, a consensus Slimb/ß-TRCP1 binding site from another protein can substitute for phosphorylated residues of Ci-155 to direct conversion to Ci-75 in vivo. From this, it is concluded that Slimb binds directly to phosphorylated Ci-155 to initiate processing to Ci-75. The phosphorylated motifs in Ci that are recognized by Slimb have been explored and some evidence is provided that silencing of Ci-155 by phosphorylation may involve more than binding to Slimb (Smelkinson, 2006).
The mechanism and consequences of Hh signaling have been studied extensively in the developing Drosophila wing imaginal disc, where Hh, secreted from posterior compartment cells, induces a strip of nearby, responsive anterior cells (AP border cells) to express a small set of target genes, including decapentaplegic (dpp), that subsequently pattern the developing wing. In anterior cells far from Hh, Ci-155 is processed slowly to Ci-75, which crucially represses potential Hh target genes, including hh itself and dpp, to ensure that they are not ectopically expressed. Even low-level Hh signaling at the AP border blocks Ci-75 production, thereby also increasing the concentration of Ci-155. Hh further activates Ci-155 in a dose-dependent manner by facilitating its nuclear accumulation and potentially also by modifying its binding partners in the nucleus. Because formation of Ci-75 requires both Ci-155 phosphorylation and the activity of Slimb, it was proposed that Slimb might promote partial proteolysis of Ci-155 by directly binding to phosphorylated Ci-155 and catalyzing its ubiquitination. However, despite some support for this hypothesis, Ci-155 contains no obvious consensus binding site for Slimb/β-TRCP1: there are only two well-studied examples where proteasomal degradation of a ubiquitinated protein is incomplete (NF-KappaB precursors, p100 and p105), and ubiquitinated Ci-155 has not been detected when Ci-155 is stabilized by inhibiting the proteasome (Smelkinson, 2006).
To determine whether Slimb can bind to Ci in a manner dependent on phosphorylation, a purified GST fusion protein was used that includes the key phosphorylation sites of Ci. This GST-Ci protein undergoes a significant mobility shift in SDS polyacrylamide gels when phosphorylated by PKA and CK1 together and an even greater shift if GSK3 is also included. GST-Ci binds more avidly than GST alone to 35S-labeled full-length Slimb produced by in vitro translation in a reticulocyte lysate and to HA-tagged full-length Slimb from crude extracts of transiently transfected Drosophila Kc cells. This binding is reproducibly increased by using PKA together with CK1, and to a much greater extent by using all three protein kinases to phosphorylate GST-Ci prior to the binding assay. The synergistic contribution of GSK3 was clearest in the HA-Slimb binding assay, and so this assay was used to investigate further the characteristics of Ci binding to Slimb (Smelkinson, 2006).
Three PKA sites ('P1-3'), the neighboring three PKA-primed CK1 sites ('C1-3') and the two adjacent PKA-primed GSK3 sites ('G2,3') are required for Ci-155 to be converted to Ci-75 in Drosophila embryos and wing discs. When the serine residues at these PKA, CK1, or GSK3 sites were replaced with alanines, significant stimulation of binding of GST-Ci to HA-Slimb was no longer seen by any combination of PKA, CK1, and GSK3. Thus, strong binding of HA-Slimb to a Ci fragment in vitro requires the same protein kinases and the same phosphorylation sites that are required in vivo to convert Ci-155 to Ci-75 (Smelkinson, 2006).
Whether a defined, minimal Slimb binding site from another protein could direct processing of Ci-155 to Ci-75 was tested. β-catenin is a prototypical substrate for the β-TRCP1 SCF complex, in which a dually phosphorylated motif (DpSGIHpS, where pS stands for phosphoserine) is the critical recognition element for binding. This motif is conserved in Drosophila β-catenin (Armadillo), and Armadillo proteolysis depends on both this sequence and Slimb activity. The motif is also expected to serve as a direct binding site for Slimb, the Drosophila homolog of β-TRCP1. Tests revealed that this consensus Slimb/β-TRCP1 binding site, engineered into Ci, is functional and directs Slimb binding in vitro with an apparent avidity similar to that seen for fully phosphorylated wild-type Ci (Smelkinson, 2006).
Binding assays were used to search for the direct Slimb recognition elements in Ci because no established Slimb/β-TRCP consensus binding sites are apparent in the sequence of Ci or Gli proteins. Each of the three PKA sites in Ci is required for detectable processing to Ci-75 in Kc tissue-culture cells; therefore, whether each site contributes to Slimb binding in vitro was tested. Replacement of all three PKA-primed CK1 sites (and the two predicted CK1-primed CK1 sites) with acidic residues abolished any stimulation of HA-Slimb binding by phosphorylation of GST-Ci (Smelkinson, 2006).
It cannot be readily determined whether the PKA sites and PKA-primed CK1 sites are directly recognized by Slimb. However, the evident contribution of each PKA site to Slimb binding implies that each must nucleate at least one direct Slimb binding site. Surrounding the three PKA sites there are two types of motifs that are related to previously recognized or postulated Slimb/β-TRCP binding motifs (DSGXXS, DSGXXXS, TSGXXS, EEGXXS, DDGXXD, and DSGXXL. (1) The six-amino-acid motif (D/pS)(pS/pT)(Q/Y)XX(pS/pT) might be created in three places if phosphorylation occurs, as is suspected, at some nonconsensus sites. These motifs most closely resemble the β-TRCP1 binding site postulated for p100 (DpSAYGpS), but the presence of glutamine or tyrosine at the third position in place of glycine in the ideal consensus would be expected to reduce binding by more than an alanine substitution. (2) Many five-amino-acid motifs are created in which two acidic residues (DpS, pSpS, or pSpT) are separated from a phosphorylated residue (pS or pT) by only two amino acid residues. Four of these eight motifs include a glutamine at the third position. However, this residue does not appear to be instrumental in Slimb binding because substitution with alanine has little effect. On the basis of the crystal structure of β-TRCP1, it is hard to predict the affinity of the designated five-amino-acid motifs for Slimb, but it is likely to be lower than for any of the motifs cited above. Regardless of which precise motifs contribute most significantly to Slimb binding, it is clear from the mutational analysis that Ci must be highly phosphorylated over a region spanning almost sixty amino acid residues to generate several suboptimal binding sites that must collaborate to provide physiologically significant affinity for Slimb. This follows a precedent established for degradation of the yeast cell-cycle regulator Sic1 by binding of the SCFCDC4 complex to multiple low-affinity phosphodegrons. The requirement for extensive Ci phosphorylation could account for the essential role of the scaffolding protein Cos2 in facilitating phosphorylation and for the relatively slow conversion of Ci-155 to Ci-75 seen in vivo (Smelkinson, 2006).
In summary, processing of Ci-155 to Ci-75 is initiated by direct binding of Slimb to Ci-155 molecules that have been extensively phosphorylated by PKA, GSK3, and CK1. This presumably leads to ubiquitination of Ci-155 and its partial proteolysis by the proteasome, generating a transcriptional repressor that plays a key developmental role in cells that are not exposed to Hh. Whether phosphorylation also prevents Ci-155 from activating transcription through an additional mechanism remains to be explored, as does the mechanism by which proteolysis of Ci-155 is limited to preserve its N-terminal domains as Ci-75 (Smelkinson, 2006).
Hedgehog (Hh) proteins signal by inhibiting the proteolytic processing of Ci/Gli family transcription factors and by increasing Ci/Gli-specific activity. When Hh is absent, phosphorylation of Ci/Gli triggers binding to SCF ubiquitin ligase complexes and consequent proteolysis. This study shows that multiple successively phosphorylated CK1 sites on Ci create an atypical extended binding site for the SCF substrate recognition component Slimb. GSK3 enhances binding primarily through a nearby region of Ci, which might contact an SCF component other than Slimb. Studies of Ci variants with altered CK1 and GSK3 sites suggest that the large number of phosphorylation sites that direct SCFSlimb binding confers a sensitive and graded proteolytic response to Hh, which collaborates with changes in Ci-specific activity to elicit a morphogenetic response. When Ci proteolysis is compromised, its specific activity is limited principally by Su(fu), and not by Cos2 cytoplasmic tethering or PKA phosphorylation (Smelkinson, 2007).
The central task of Hh signal transduction in Drosophila is to regulate the activity of the transcription factor Ci. This is accomplished by regulating the levels of Ci-155 activator and Ci-75 repressor and the specific activity of Ci-155. To examine the regulation of Ci-155-specific activity in isolation, a Ci variant (Ci-S849A) was developed that escapes PKA-dependent proteolysis but has a minimally altered pattern of PKA-initiated phosphorylation. At 29°C, Ci-S849A was expressed in wing discs (via C765-GAL4) at roughly the same level as endogenous Ci, but at 20°C, the levels of Ci-S849A were distinctly lower. At 29°C, Ci-S849A induced the Hh target gene reporter ptc-lacZ in A cells that are not stimulated by Hh. Since ptc induction requires Ci-155 activator and is not accomplished simply by loss of Ci-75 repressor, it is concluded that elevated levels of Ci-155 can suffice to confer some activator function. At 20°C, Ci-S849A induced ptc-lacZ in A cells only when Su(fu) was removed. By contrast, Ci-S849A activity was not detectably increased by loss of either PKA activity or Cos2 activity in P smo mutant clones, where Hh signaling is blocked. Thus, Su(fu) is the principal component that limits Ci-155-specific activity when Ci-155 is protected from PKA-dependent proteolysis, whereas PKA and Cos2 normally limit Ci-155 activity simply by promoting its proteolysisby promoting its proteolysis.
The prior assertion that PKA limits Ci-155-specific activity by direct phosphorylation was based on the use of an inappropriate reagent (Ci-U), which was mistakenly thought to be inert to PKA-dependent proteolysis. A similar role for Cos2 was previously inferred principally from the observation that Ci-155 accumulated more rapidly in the nuclei of anterior Leptomycin B-treated wing disc cells when those cells lack . It appears that the inferred cytoplasmic retention of Ci-155 by Cos2 contributes very little quantitatively to limiting the activity of stabilized Ci-155. That conclusion is supported by the observation that loss of Cos2 function did not enhance the weak induction of En seen in pka mutant clones of otherwise WT wing discs. The remaining, long-standing observations suggesting roles for PKA and Cos2 in limiting Ci-155-specific activity are the different degrees of Hh target gene induction in pka (strongest), cos2 (intermediate), and slimb (weakest) mutant wing disc clones. It is suggested that this might result from different degrees of disruption of PKA-dependent proteolysis in these clones. This suggestion is consistent with the proposed role of Cos2 in facilitating Ci-155 phosphorylation and with the observation that ptc-lacZ can be induced in slimb mutant clones when PKA activity is halved (Smelkinson, 2007).
Both GST-Ci association with SCFSlimb and Ci-155 proteolysis depend on two phosphorylated regions of Ci. The first region provides an essential Slimb binding site that can be created by five successive CK1 phosphorylations primed initially by PKA site 1. Of the four phosphorylated residues within the motif (844SpTpYYGSpMQSp852) that interacts directly with Slimb, at least one (S849) is essential for binding, and two others (S844 and S852) enhance binding (T845 is essential, but priming and binding functions have not been separated). The second critical phosphorylated region of Ci includes two GSK3 sites (S884 and S888) that are primed by PKA site 3. This region enhances, but is not sufficient for, binding to SCFSlimb (Smelkinson, 2007).
The requirement for multiple successive phosphorylations by PKA, CK1, and GSK3 to create a high-affinity SCFSlimb binding domain on Ci-155 has two important consequences. First, it demands a special mechanism for facilitating Ci phosphorylation that is met by Cos2. Second, it provides a mechanism through which a small change in Ci-155 phosphorylation, induced for example by limited dissociation of protein kinases from Cos2, can be translated into a substantial inhibition of Ci-155 proteolysis. The sharp increase in Ci-155 levels at the anterior limit of Hh signaling territory shows that a low dose of Hh does indeed severely curtail PKA-dependent Ci-155 proteolysis. Hh could inhibit proteolytic processing of Ci variants driven by either PKA-primed GSK3 sites (Ci-SL) or PKA-primed CK1 sites (Ci-G2,3E and Ci-Y846G), but complete inhibition was observed only for the latter pair. This suggests that the sensitive response of Ci proteolysis to Hh depends principally on CK1 (Smelkinson, 2007).
How does inhibition of Ci-155 proteolysis affect Ci-155 activity? Previously, the properties of Ci-U and slimb mutant clones were taken as evidence that inhibition of proteolysis does not suffice for Ci activation. Since Ci-U is subject to PKA-dependent proteolysis and PKA does not affect the specific activity of Ci-155, instead the properties of Ci-S849A and pka mutant clones were relied upon to conclude that complete inhibition of PKA-dependent proteolysis does suffice to induce the Hh target gene ptc. This, in turn, suggests that the high Ci-155 levels anterior to the stripe of elevated ptc expression at the AP border of wing discs result from substantial, but incomplete, inhibition of Ci-155 proteolysis (Smelkinson, 2007).
It has generally been assumed that inhibition of Ci-155 proteolysis is uniformly strong throughout the AP border and that the activation of ptc and en in nested domains is due solely to changes in Ci-155-specific activity elicited by increasing levels of Hh. However, several factors suggest that there may also be a significant gradient of residual PKA-dependent proteolysis at the AP border that contributes to morphogen action (Smelkinson, 2007).
First, the precise degree of substantially inhibited Ci-155 proteolysis can determine whether Hh target genes are induced or not. This is evident from differences in ptc-lacZ induction among proteolytically impaired Ci variants and between pka and slimb mutant clones (Smelkinson, 2007).
Second, Su(fu) is the principal regulator of Ci-155 activity when Ci-155 levels are elevated, yet Hh instructs an almost unchanged morphogenetic response in the absence of Su(fu). It is likely that a proteolytic gradient is critical under these conditions, although it is also possible that Cos2 assumes a more significant role in regulating Ci-155-specific activity when Su(fu) is absent (Smelkinson, 2007).
Third, it was found that the loss of any one of four phosphoserines that contribute to Ci-Slimb binding (S844, S852, S884, and S888) diminishes, but does not abolish, Slimb binding. For S888A (G3A) this results in elevated Ci-155 levels and an increased activity, but residual proteolysis is clearly evident from the generation of sufficient Ci-75 to repress hh-lacZ. Thus, dispersion of direct Slimb binding determinants among several phosphorylatable residues provides a mechanism for Hh to elicit graded inhibition of Ci-155 proteolysis. It is speculated that in response to high levels of Hh, most Ci-155 molecules will not bind to SCFSlimb at all because they lack at least two of the six key phosphorylated residues, whereas a large proportion of Ci-155 molecules may bind SCFSlimb with intermediate affinity in response to low or intermediate Hh levels because they lack only one critical phosphoserine (Smelkinson, 2007).
Fourth, regulation of Ci-155-specific activity depends on Ci-155 levels. Thus, Ci-155 is only activated by loss of Su(fu) when Ci-155 levels are elevated by Hh or appropriate mutations, presumably because other stoichiometric binding partners such as Cos2 act redundantly with Su(fu) when their Ci-155 binding capacity is not saturated. We would therefore expect the release of Ci-155 from repressive partners to be progressively facilitated as the relative levels of Ci-155 increase. This would allow increasing Hh levels to enhance Ci-155 activity through synergistic effects on Ci-155 levels and Ci-155-specific activity (Smelkinson, 2007).
The archetypal β-TRCP/Slimb substrates, β-catenin and IKB, contain a single, dually phosphorylated, high-affinity binding site (DSpGxxSp) that triggers rapid substrate proteolysis. The primary direct Slimb binding motif that has been defined (SpTpYYGSpMQSp) in Ci differs notably by the presence of Tyr instead of Gly at the third position, by the inclusion of a fourth electronegative residue at its C terminus, and by binding with lower affinity, permitting additional influences on Ci-SCFSlimb association. The fourth electronegative residue (pS852) likely interacts with at least one of two positively charged Slimb surface residues (R333 and R353) based on their potential proximity and reduced GST-Ci binding to the R333A/R353A Slimb variant (Smelkinson, 2007).
Most known β-TRCP substrates include phosphorylated or acidic residues that are two to four residues C-terminal to the standard six amino acid binding motif (DSpGXXSp), but their contribution to binding has not generally been assessed. Even β-catenin includes such phosphorylated residues that are known to have an essential priming role but have not been tested rigorously for direct interactions with β-TRCP. The variant β-TRCP binding motif (EEGFGSpSSp) of mammalian Wee1A presents a notable exception, in which a β-TRCP Arg residue equivalent to R353 of Slimb interacts with the phosphoserines at position 6 and 8 of this motif. This suggests that positive surface residues of β-TRCP/Slimb may commonly stabilize association with extended binding motifs. It is speculated that extended β-TRCP/Slimb binding motifs are likely to be especially important and prevalent in substrates lacking Gly at the third position because it was found that R333 or R353 (or both) of Slimb promotes binding to Ci-WT, but not to Ci-SL, and pS852 contributes significantly to Slimb binding in Ci-WT, but not in Ci-Y846G (Smelkinson, 2007).
Vertebrate Gli homologs of Ci also have a residue other than Gly at the third position (generally Ala) and a potentially phosphorylated Ser at the C terminus of a putative extended β-TRCP binding motif of 9 to 11 residues (SSAYx(x)SRRSS). Both a second Wee1A binding motif (DSAFQEPDS) and a β-TRCP binding motif of the p100 precursor of NFKB p52 (DSAYGSQSVE) also lack a Gly residue at the third position and include residues beyond the six amino acid core motif that might, by analogy to Ci, potentiate binding (Smelkinson, 2007).
Studies of Ci provide a clear precedent for the use of an extended β-TRCP/Slimb binding motif to translate regulated substrate phosphorylation into regulated proteolysis. However, it was also found that Slimb binding cannot be predicted by focusing on only the interactions of charged residues. Thus, fully phosphorylated Ci includes two sequences with a distribution of charged residues similar to that of the primary SCFSlimb binding site (837DSpQNSpTpASpTp and 858SpSpQVSpSpIPTp compared with 844SpTpYYGSpMQSp), but those sites neither suffice for Slimb binding (in Ci-S849A) nor enhance binding significantly in vitro (as revealed by D837A, T842A, S858A, S859A, and Ds2 variants). This probably reflects significant binding contributions of nonpolar residues in positions 3-5 of an extended Slimb/β-TRCP binding motif, as suggested by the presence of Tyr or Phe at position 4 of the functional motifs of Ci, Gli, p100, and Wee1A (Smelkinson, 2007).
Several F-box proteins that use WD40 repeats to bind substrate (FBW proteins) also include a dimerization domain that directs assembly of higher-order SCF complexes. Some substrates of these SCF complexes (for example, Cyclin E for Fbw7 and Wee1A for β-TRCP) contain more than one phosphorylated region capable of interacting with the same WD40 binding surface of the FBW protein. This raises the question of whether the cooperative function of two or more such regions depends on SCF dimerization and simultaneous interaction with two FBW subunits of a dimeric complex (Smelkinson, 2007).
This study found that Ci also contains two phosphorylated regions that contribute to SCF association, and Slimb molecules can bind to each other within higher-order functional SCF complexes. It was also found that Slimb self-association enhanced binding to GST-Ci relative to GST-Ci-SL, which contains a single DSGxxS motif; however, it was not required for both phosphorylated regions of GST-Ci to stimulate binding. Hence, simultaneous binding to separate Slimb monomers within a larger complex can be excluded as a requisite mechanism for cooperativity between the two phosphorylated regions of Ci. This is consistent with recent structural studies that predict a wider separation of WD40 binding surfaces within an SCF dimer than can be spanned readily by the two critical phosphorylated regions of a single Ci molecule (Smelkinson, 2007).
Does the region preceding PKA site 3 of Ci bind directly to the FBW component (Slimb) of an SCF complex as for Cyclin E and Wee1A? That model was proposed for Gli-2/3 proteins, which include a recognizable, potentially extended, variant Slimb-binding motif (DSYDPISTDAS). The analogously positioned sequence in Drosophila (SFYDPISPGCS) retains the YDPIS sequence and the two GSK3 sites at position 7 and 11 (underlined) but lacks a Ser at position 2 (italics), which is required in Gli-2/3 for normal β-TRCP association and proteolysis. Also, Ala substitution of the first Ser in the Drosophila motif (together with three other Ser residues) had only a minor effect on Slimb binding in vitro and Ci-155 proteolysis in vivo. Thus, this region of Ci does not have a clearly recognizable and demonstrably functional, conventional β-TRCP/Slimb binding site. It is, nevertheless, conceivable that the conserved elements of the putative β-TRCP binding motif of Gli-2/3 might provide a very weak direct interaction with the WD40 domain of Slimb that is sufficient to enhance SCFSlimb association (Smelkinson, 2007).
However, this study also found that the GSK3 enhancement of Ci-Slimb binding conferred by the GSK3 sites preceding PKA site 3 was lost if Slimb lacked an F-box domain and consequent direct association with SkpA and its SCF complex partners. This result is interpreted with some caution because Slimb-ΔF also bound less well than wild-type Slimb to a canonical β-catenin substrate and to GST-Ci that was phosphorylated only at its primary Slimb binding site. Nevertheless, the result suggests that the region of Ci immediately preceding PKA site 3 might augment SCF association by binding directly to an SCF component other than Slimb (Smelkinson, 2007).
Whether GSK3 stimulates Ci binding to SCFSlimb via a direct interaction with Slimb, an unprecedented interaction with another SCF component, or a conformational effect on the primary Slimb binding site of Ci, the Ci-G3A transgene reveals that the stimulation conferred by GSK3 phosphorylation is critical for efficient Ci-155 proteolysis and for Hh pathway silencing. Since Slimb self-association enhanced GST-Ci, but not GST-Ci-SL, binding in vitro, it is suspected that this may also be important for Ci-155 proteolysis in vivo. It is not known if SCFSlimb dimerization (or oligomerization) is regulated, but the different modes of association of SCFSlimb with Ci and β-catenin certainly provide several opportunities for SCF regulatory mechanisms or mutations to affect the Hh pathway without altering the Wnt/β-catenin pathway (Smelkinson, 2007).
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