supernumerary limbs


REGULATION

Protein Interactions

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 F-box protein Slimb controls the levels of clock proteins Period and Timeless

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).

NEMO/NLK phosphorylates PERIOD to initiate a time-delay phosphorylation circuit that sets circadian clock speed

The speed of circadian clocks in animals is tightly linked to complex phosphorylation programs that drive daily cycles in the levels of Period (Per) proteins. Using Drosophila, a time-delay circuit based on hierarchical phosphorylation was identified that controls the daily downswing in Per abundance. Phosphorylation by the Nemo/Nlk kinase at the 'per-short' phospho cluster domain on Per stimulates phosphorylation by Doubletime (Dbt/Ck1delta/epsilon) at several nearby sites. This multisite phosphorylation operates in a spatially oriented and graded manner to delay progressive phosphorylation by Dbt at other more distal sites on Per, including those required for recognition by the F box protein Slimb/β-TrCP and proteasomal degradation. Highly phosphorylated Per has a more open structure, suggesting that progressive increases in global phosphorylation contribute to the timing mechanism by slowly increasing Per susceptibility to degradation. These findings identify Nemo as a clock kinase and demonstrate that long-range interactions between functionally distinct phospho-clusters collaborate to set clock speed (Chiu, 2011).

This study shows that the per-short domain functions as a discrete hierarchical phospho-cluster that delays Dbt-mediated phosphorylation at the Slimb recognition site on Per, providing new insights into how clock protein phosphorylation contributes to circadian timing mechanisms. The cumulative effect of this delay circuit is to slow down the pace of the clock by ~8 hr. It is proposed that Dbt functions in a stepwise manner to phosphorylate clusters on Per that have distinct biochemical functions and effects on the rate of Per degradation, e.g., elements such as the per-short phospho-cluster that delays Per degradation and those such as the Slimb-binding site and global phosphorylation that enhance instability. Nmo plays a major role in the relative timing of Dbt activity at these different elements because it stimulates multisite phosphorylation at the per-short delay cluster by Dbt, which slows down the ability of Dbt to phosphorylate instability elements. Thus, a large portion of the phosphorylation events dictating when in a daily cycle Per is targeted for rapid degradation is not directly linked to binding ofSlimb per se. The current findings demonstrate that presumptive long-range interactions between distinct positively and negatively acting phospho-clusters collaborate to set clock speed and helps to explain why mutations in clock protein phosphorylation sites and/or the kinases that phosphorylate them can yield both fast and slow clocks (Chiu, 2011).

A proposed mechanism for the function of the per-short domain is supported by the congruence between in vitro biochemical studies based on purified recombinant Per protein from cultured S2 cells and in vivo changes in the pace of behavioral rhythms using transgenic models. This suggests that a primary biochemical effect of the per-short domain on clock speed in the fly is via modulating the rate of Dbt-mediated phosphorylation at the Slimb phospho-degron on Per. The physiological role of T583 phosphorylation is not clear, as mutating this site does not have detectable effects on the binding of Per to Slimb in S2 cells. In this regard, it is interesting to note that the original per-short domain was identified as encompassing aa 585-601 of Per (Baylies, 1992). Thus, it is likely that the 8 hr per-short delay circuit is governed by the dynamics underlying the phosphorylated status of three sites (i.e., S596, S589, and S585) (Chiu, 2011).

At present, it is not clear how phosphorylation in the per-short cluster slows down subsequent phosphorylation by Dbt at Ser47 and other sites. Inactivating the per-short cluster leads to increases in the rate of Dbt-mediated phosphorylation at not only the N terminus, but also the C terminus of Per, suggesting that it is a major control center for regulating the relative efficiency of Dbt phosphorylation at many sites on Per. It is suggested that the per-short phospho- cluster acts as a transient 'temporal trap' for Dbt. Once the sites in the per-short domain are phosphorylated by Dbt, this somehow allows it to continue its normal rate of phosphorylation at other phospho-clusters. Although speculative, progressive increases in phosphorylation at some of these other phospho-clusters might generate time-dependent local/overall conformational changes in Per, possibly via electrostatic repulsion, eventually leading to a more open Per structure that is more accessible to phosphorylation by Dbt at the Slimb-binding site and/or a more efficient substrate for degradation. Thus, the rapid degradation of Per during the early day is likely due to a combination of synchronous increases in the phospho-occupancy of Ser47 and overall phosphorylation of Per. Other factors such as protein phosphatases and the action of Timeless also play major roles in regulating the speed of the Per phosphorylation program (Chiu, 2011).

How might phosphorylation at S596 enhance phosphorylation at S589 and S585 by Dbt? Phosphorylation by the CK1 kinase family is generally enhanced by priming. However, phosphorylation at the per-short domain by Dbt does not follow the consensus priming-dependent recognition motif for the CK1 family of kinases (i.e., S/Tp-X-X-S/T, wherein S/Tp refers to the primed site, X is any amino acid, and the italicized residues the CK1 target site, as the S596 priming site is located C terminal to the Dbt sites. Thus, it is likely that phosphorylation of S596 by Nmo stimulates Dbt phosphorylation at the per-short region in a nonpriming-dependent manner (Chiu, 2011).

Ongoing studies are aimed at understanding the biochemical events underlying the ability of phosphorylation at S596 to enhance phosphorylation by Dbt in the per-short region. The discovery of a delay phospho-circuit also sheds light on why mutations in different phosphorylation sites on Per or Frq proteins, although affecting stability, can speed up or slow down the clock. The current findings also offer a logical explanation for why mutations that lower the kinase activity of CKI, which overall is expected to slow down the rate of PER degradation, can yield fast clocks. For example, although other mechanisms have been offered to explain the short-period phenotypes that are observed for the CKI3tau mutation in hamsters and a CKIdelta mutation associated with familial advanced sleep phase syndrome (FASPS) in humans, it is possible that phosphorylation at a per-short type delay cluster is preferentially compromised by the mutant kinase, which could appear as a substrate-specific gain-of-function mutation (Chiu, 2011).

Negatively acting phospho-clusters are likely to be a general feature of the timing mechanisms regulating the daily abundance cycles of clock proteins such as Pers in animals and Frq in Neurospora. However, other regulatory modules that operate in a phase-specific manner must participate to generate an ~24 hr oscillator. Most conspicuously, clock speed is intimately linked to the Per and Frq abundance cycles necessitating daily phases of de novo synthesis to replenish the pools of previously degraded proteins.Asrecently shown, the transcriptional negative feedback aspect of Per regulating Clk-Cyc-mediated transcription is also a component of the period-setting mechanism in Drosophila. Therefore, the ~24 hr Per abundance cycle is based on a combination of 'time constraints' that are generated using different regulatory modules. It is proposed that the per short- based timer mainly functions once Per has accumulated and begins participating in transcriptional repression, controlling Per abundance once it is disengaged from the dynamics of its cognate mRNA by setting in motion a series of sequential phosphorylation events that are calibrated to stimulate Per degradation in the nucleus at the appropriate time in a daily cycle, enabling the next round of circadian gene expression. In this context, it is interesting to note that a prior study analyzing the per-short domain suggested that it functions with a nearby 'perSD' domain to increase the transcriptional repressor function of Per. It is possible that the same phosphorylation events leading to Per degradation also function to increase its potency within the repressor complex (Chiu, 2011).

These studies also identify Nemo as a clock kinase. Nemo is the founding member of the evolutionarily conserved Nemo-like kinase (Nlk) family of proline-directed serine/threonine kinases closely related to mitogen-activated protein kinases (MAPK). It was originally characterized in Drosophila as required for planar cell polarity during eye development and is now known to function in many pathways. Nmo/Nlk is localized in the nucleus and is another factor in the circadian clock that also functions in the Wnt/Wg-signaling pathway, such as CKI3/Dbt, β-TrCP/Slimb, and GSK-3β/Sgg. It will be of interest to determine whether Nlk functions in the mammalian clock. Intriguingly, the phosphorylation sites on Per are largely clustered, and several of them have the same spatial arrangement as the per-short cluster, with a predicted pro-directed kinase site at the C-terminal end of the phospho-cluster. This suggests that Nmo and/or other pro-directed kinases serve as control points to activate spatially and perhaps functionally distinct phospho-clusters. Indeed, it has recently been shown that phosphorylation at Ser661 of Per by an as yet unidentified pro-directed kinase primes further phosphorylation by Sgg at Ser657 to regulate the timing of Per nuclear entry in key pacemaker neurons (Chiu, 2011).

In summary, a central aspect of circadian clocks is the presence of one or more clock proteins that provide a dual function by behaving as phospho-based timers and linking its timer role to gene expression by operating in a phase-specific manner to recruit repressor complexes that inhibit central clock transcription factors. These studies suggest that a major part of the timing mechanism underlying these phospho-clock proteins is based on spatially and functionally discrete phospho-clusters that interact to impose calibrated and sequentially ordered biochemical time constraints. In the case of Per, the per-short phospho-cluster functions as a central timing module by slowing down the ability of Dbt to phosphorylate instability elements regulating Per degradation and, hence, when Per repressor activity is terminated and the next round of circadian gene expression begins (Chiu, 2011).

Involvement of an SCFSlmb complex in timely elimination of E2F upon initiation of DNA replication 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).

Phosphorylation by double-time/CKIepsilon and CKIalpha targets cubitus interruptus for Slimb/beta-TRCP-mediated proteolytic processing

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).

Processing of the Drosophila hedgehog signaling effector Ci-155 to the repressor Ci-75 is mediated by direct binding to the SCF component Slimb

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).

Regulation of Ci-SCFSlimb binding, Ci proteolysis, and hedgehog pathway activity by Ci phosphorylation

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. The release of Ci-155 from repressive partners would therefore be expected 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).

The phospho-occupancy of an atypical Slimb-binding site on Period that is phosphorylated by Doubletime controls the pace of the clock

A common feature of animal circadian clocks is the progressive phosphorylation of Period (Per) proteins, which is highly dependent on casein kinase Idelta/epsilon (CKIdelta/epsilon, termed Doubletime [Dbt] in Drosophila), and ultimately leads to the rapid degradation of hyperphosphorylated isoforms via a mechanism involving the F-box protein, beta-TrCP (Slimb in Drosophila). This study use the Drosophila model system shows that a key step in controlling the speed of the clock is phosphorylation of an N-terminal Ser (S47) by DBT, which collaborates with other nearby phosphorylated residues to generate a high-affinity atypical Slimb-binding site on Per. Dbt-dependent increases in the phospho-occupancy of S47 are temporally gated, dependent on the centrally located Dbt docking site on Per and partially counterbalanced by protein phosphatase activity. It is proposed that the gradual Dbt-mediated phosphorylation of a nonconsensus Slimb-binding site establishes a temporal threshold for when in a daily cycle the majority of Per proteins are tagged for rapid degradation. Surprisingly, most of the hyperphosphorylation is unrelated to direct effects on Per stability. This study also used mass spectrometry to map phosphorylation sites on Per, leading to the identification of a number of 'phospho-clusters' that explain several of the classic per mutants (Chiu, 2008).

To better understand the physiological role of phosphorylation in regulating PER stability, Drosophila was used as a model system. Using a range of strategies, including mutational analysis, mass spectrometry and phospho-specific antibodies S47 was identified as a key phospho-determinant regulating the efficiency of SLIMB binding to dPER. By evaluating the behavior of dPER mutants whereby amino acid 47 is constitutively 'nonphosphorylated' (S47A) or 'phosphorylated' (S47D), it was shown that the phospho-occupancy of S47 is a key biochemical throttle adjusting the pace of the clock. However, phosphorylation of S47 occurs within an atypical SLIMB-binding site. Additional DBT-dependent phosphorylated residues, which likely include one or more nearby Ser residues at amino acid 44/45 and possibly others within the first 100 amino acids, collaborate with pS47 to generate a high-affinity SLIMB-binding region on dPER. As such, the affinity of SLIMB for dPER is proportional to the degree of phospho-occupancy within an extended phosphorylation network centered on S47 that as a unit yields a graded response in the affinity of SLIMB-dPER interactions. Attaining a high proportion of dPER molecules that are phosphorylated at S47 and other key sites mediating SLIMB binding is progressive and occurs several hours after DBT stably interacts with dPER via the centrally located dPDBD, likely because DBT 'activity' is counterbalanced by TIM and protein phosphatases. It is proposed that the relatively slow assembly of a high-affinity SLIMB-binding site on dPER is at least partly 'designed' to extend the time that dPER acts as a transcriptional repressor, critical in generating transcriptional feedback loops with daily time frames. Finally, this mass spectrometry analysis identify 'hot spots' for phosphorylation, indicates that the majority of dPER phosphorylation is unrelated to direct effects on stability and sheds new insights into the underpinnings of several previously characterized mutants, including the classic perS allele (Chiu, 2008).

Early studies identified DpSGPhiX1+npS (pS, phosphorylated Ser; Phi, any hydrophobic amino acid; X, any amino acid) as the consensus motif for recognition by the β-TrCP/SLIMB F-box protein (Fuchs. 2004). Phosphorylation at both sites in this six amino acid consensus generally leads to a high-affinity β-TrCP/SLIMB-binding site. Indeed, the three negatively charged residues (Asp/Glu and two phospho-S/T residues) are important binding contacts underlying β-TrCP/substrate interactions (Wu, 2003). Furthermore, it is thought that the presence of an Asp/Glu at position 2 of the canonical binding domain can circumvent the need for a phospho-S/T at that position, as is the case for Wee1A (Watanabe, 2004). However, accumulating evidence indicates that β-TrCP-binding sites can deviate from this consensus. For example, recent work on the Ci/Gli family of transcription factors suggests a novel class of degenerate and weaker β-TrCP-binding sites that extend beyond the standard six-amino-acid binding motif, especially for those missing a Gly at the third position (Smelkinson, 2007). It was suggested that for these extended β-TrCP/SLIMB-binding motifs, significant contributions are made by the local presence of nonpolar residues, such as those found in the motifs for Ci, Gli, and Wee1A. Additional phosphorylation events at nearby regions are also thought to enhance the inherently weak binding affinities of extended β-TrCP-binding sites, enabling a more graded response compared with the standard sequence (Chiu, 2008).

The major SLIMB-dependent phospho-degron identified in this study [44p*Sp*SGpSSGYGG52; where p* = possible phosphorylation] seems to include signature elements found in both the standard and extended β-TrCP-binding motifs. A rather unique feature of the SLIMB-binding domain on dPER is that it includes two SSG repeats. S47 is phosphorylated, and based on mutational analysis and mass spectrometry, it is almost certain that either S44 and/or S45 are phosphorylated. A physiological role for S45 is further indicated by the perSLIH mutant (S45Y) that exhibits long periods, which based on the findings is likely due to reduced dPER-SLIMB interactions. Although changing S48 to Ala phenocopied the S47A mutation, the S48D mutation did not enhance binding to SLIMB, as was the case for S47D. Together with results showing that S48A did not modulate phosphorylation of S47, the data strongly suggest that S48 has a non-phosphorylation-dependent role as a crucial structural element. Mass spectrometry identified two phospho-residues in a dPER peptide from amino acids 40-48. Thus, there might only be two negatively charged residues in the major SLIMB-binding site on dPER. It is possible that the presence of a SSG tandem and a Tyr at position 50 can compensate for the lack of a third acidic residue normally found in β-TrCP-binding sites. It is also highly probable that other, yet to be identified, DBT-dependent phosphorylation sites besides those within the atypical SLIMB-binding site identified in this study contribute to enhancing SLIMB-dPER interactions (Chiu, 2008).

The presence of numerous suboptimal phospho-determinants is thought to generate a graded response in the binding efficiencies of F-box proteins to substrates. The general molecular framework is that progressive increases in the phospho-occupancy of multiple phosphorylated residues eventually reaches a threshold value that drives sufficient F-box protein/substrate interactions to yield desired outcomes. As such, regulating the kinetics of phosphorylation within the phospho-network mediating F-box recognition is a key determinant in the timing of substrate degradation. In the case of animal PER proteins, they undergo progressive increases in global phosphorylation that occur over an ~10-h time frame, whereby highly phosphorylated isoforms are associated with a rapid decline in levels (Chiu, 2008).

What accounts for the hours-long kinetics underlying the gradual increases in phosphorylation of S47 and likely many other DBT-dependent sites on dPER? Based on the in vitro ability of DBT to phosphorylate S47 despite phosphatase treatment of dPER, it is not believed that hierarchical phosphorylation based on prior priming is a major component in regulating the timing of when S47 is phosphorylated in vivo. Rather, the findings strongly suggest that the gradual build-up in the phospho-occupancy of S47 and other sites is at least partly based on a dynamic balance between DBT-mediated phosphorylation and the opposing activities of TIM and protein phosphatases. In agreement, blocking phosphatase activity strongly enhanced the abundance of phosphorylated S47. Recent evidence suggests that the ability of TIM to stabilize dPER might be by acting as a bridge that facilitates the targeting of protein phosphatase activity toward dPER (Fang, 2007). Indeed, it is likely that the strong protective function of TIM on dPER partially overrides the destabilizing effects of the S47D mutant as it attains peak levels comparable with those of wild-type dPER. Following this line of reasoning, it is suggested that a major reason for the advanced dper RNA and protein cycles in the S47D mutant is that as TIM levels decline in the late night the 'released' dPER(S47D) protein is no longer protected (or less so) and undergoes accelerated nuclear clearance, leading to an earlier disengagement from transcriptional repression, which advances the subsequent dper RNA and protein cycles. Likewise, while this manuscript was under review a recent report showed that the CKIepsilon tau mutation, which shortens rhythms in mice, has an 'asymmetrical' effect on PER protein stability, preferentially accelerating nuclear clearance and hence advancing the molecular oscillations underpinning the clockworks (Meng, 2008). Thus, although differential phosphorylation plays a major role in setting the intrinsic stabilites of PER proteins, additional variables, such as phase-specific protein-protein interactions, are critical in the 'readout' from these phospho-signals (Chiu, 2008).

Many of the DBT-dependent phosphorylation sites that were identified using mass spectrometry do not lie within optimal CKI sites, suggesting that inefficient phosphorylation by DBT might also contribute to the overall rate of progressive increases in dPER phosphorylation. It is also possible that the strong binding of DBT to the centrally located dPDBD, while increasing the local concentration of DBT, could function as a slow 'time-release capsule' whereby the disengagement of DBT is first required prior to phosphorylation of dPER residues at more distantly located sites (Chiu, 2008).

Although the phosphorylation requirements and in vivo significance of regions on mPER1 and mPER2 that interact with β-TrCP are not known, it is likely to also be based on noncanonical β-TrCP-binding sites. In addition, hyperphosphorylation of mammalian PER proteins requires a centrally located CKI-binding site. Therefore, mammalian PER proteins, especially mPER1 and mPER2, are likely to be targeted by β-TrCP to the 26S proteasome in a manner similar to that described here for dPER. This type of mechanism might also apply to other clock proteins such as Frequency (Frq) in Neurospora that undergoes daily changes in phosphorylation and stability that are remarkably similar to those observed for PER proteins. In addition, the phosphorylated state of FRQ is regulated by casein kinases, protein phosphatases, and the rapid degradation of highly phosphorylated isoforms is mediated by the F-box protein FWD1, a homolog of β-TrCP (Chiu, 2008).

An interesting feature of the distribution in phosphorylation sites on dPER that were identified using mass spectrometry is that they seem to concentrate in clusters, suggesting the presence of'phospho-modules' with different functions. Most of these clusters appear anchored by proline-directed phosphorylation sites, which are phosphorylated by endogenous kinases expressed in S2 cells. Of note, one such cluster is located in the dPER 'short domain' (T585-T610). Mutations in this region result in animals with short periods. In fact, the mutated residues of two classic per mutants that have short periods, perS (S589N, 19-h period) and perT (G593D, 16-h period), are right in the heart of this cluster. S589 is phosphorylated in a DBT-dependent manner; and G593, when mutated, may affect phosphorylation at nearby S589 and/or S596. Although the perS mutants was isolated more than 35 years ago, the current results provide the first biochemical understanding for the short period phenotype, suggesting that phosphorylation events in the 'short domain,' some of which are DBT-dependent, may collaboratively function to slow down the clock. It is now becoming apparent that phosphorylation at different sites on PER proteins can result in differential effects on the pace of the clockworks, whereby some lead to faster clocks while others slow it down. The presence of phosphorylated residues with opposing outcomes on the speed of the clock can explain why mutations in CKIepsilon/delta/DBT can yield a variety of period-altering phenotypes from short to long, despite the fact that overall enzymatic activity is generally reduced (Chiu, 2008).

A rather unanticipated finding is that the majority of dPER phosphorylation is unrelated to direct effects on stability. This is supported by the lack of detectable SLIMB binding to a dPER fragment only missing the first 100 amino acids despite extensive phosphorylation as inferred from being the region underlying the majority of phosphorylation-dependent electrophoretic mobility shifts and confirmed by mass spectrometric analysis. Other lines of evidence also imply that a significant amount of multiphosphorylation is not linked to direct effects on PER stability. For example, abolishing phosphorylation at many centrally located sites on mPER3 does not attenuate CKI-mediated in vitro interactions with β-TrCP. Also, a trans-dominant version of CKII reduced global hyperphosphorylation of dPER without major effects on its levels (Chiu, 2008).

Thus, there are likely to be at least two functionally distinct DBT-dependent phosphorylation programs regulating different aspects of PER metabolism and activity: one that controls β-TrCP/SLIMB binding, and another that integrates with other kinases, such as CKII, to modulate nuclear entry/accumulation and/or ability to function as a transcriptional repressor. Indeed, mass spectrometric analysis of dPER identified numerous phosphorylation sites in a putative nuclear localization site and within the CCID mediating dPER inhibition of CLK-mediated transcription. Variants of dPER missing the major DBT docking site are hypophosphorylated and weak repressors. However, the relationship between hyperphosphorylation and repressor potency is not clear, since the DBT docking site on dPER also functions as a molecular scaffold for DBT and perhaps CKII-mediated inhibition of CLK-dependent transcription. Nonetheless, it is clear that the DBT docking site is a critical nexus for coordinating multiple phosphorylation programs. A challenge is to examine the functions of the newly identified phosphorylation sites and dissect the mechanisms by which they regulate dPER metabolism and activity (Chiu, 2008).

The SCF/Slimb ubiquitin ligase limits centrosome amplification through degradation of SAK/PLK4

Centrioles are essential for the formation of microtubule-derived structures, including cilia and centrosomes. Abnormalities in centrosome number and structure occur in many cancers and are associated with genomic instability. In most dividing animal cells, centriole formation is coordinated with DNA replication and is highly regulated such that only one daughter centriole forms close to each mother centriole. Centriole formation is triggered and dependent on a conserved kinase, SAK/PLK4. Downregulation and overexpression of SAK/PLK4 is associated with cancer in humans, mice, and flies. This study shows, in Drosophila cultured cells, that centrosome amplification is normally inhibited by degradation of SAK/PLK4, mediated by the SCF/Slimb ubiquitin ligase. This complex physically interacts with SAK/PLK4, and in its absence, SAK/PLK4 accumulates, leading to the striking formation of multiple daughter centrioles surrounding each mother. This interaction is mediated via a conserved Slimb binding motif in SAK/PLK4, mutations of which lead to centrosome amplification. This regulation is likely to be conserved, because knockout of the ortholog of Slimb, beta-Trcp1 in mice, also leads to centrosome amplification. Because the SCF/beta-Trcp complex plays an important role in cell-cycle progression, these results lead to new understanding of the control of centrosome number and how it may go awry in human disease (Cunha-Ferreira, 2009).

Substrates of the SCF/Slimb complex show a conserved degron that is recognized by Slimb. SAK/PLK4 protein has a modified Slimb recognition site in Drosophila (DSGIIT; position 293) and humans (DSGHAT; position 285). In previous studies, human SAK/PLK4 was shown to be ubiquitinated and removal of a region encompassing amino acids 272–311 led to stabilization of the human SAK/PLK4, although no analysis was performed on centrosome number. The conserved Slimb recognition site was mutated to DAGIIA in Drosophila SAK/PLK4, which is called SAK-ND (nondegradable). This mutation abolished SAK's ability to interact with Slimb and led to a decrease in SAK's ubiquitinated species. Moreover, SAK-ND is more stable in comparison to its WT counterpart. Thus, this mutation allowed testing of the biological significance of SAK/PLK4 degradation by the SCF/Slimb complex. Upon expression of the mutated SAK/PLK4 construct, a very similar phenotype was observed to the one registered after Slimb RNAi, i.e, rosette-like structures. These structures contained centriole percursors or elongating centrioles. Those results suggest a common centriole-amplification phenotype observed after Slimb RNAi and after expression of the SAK-ND mutant. The observation of at least five centrioles clustered at spindle poles in mitosis suggests that some of the supranumerary centrioles formed in one cycle after impairment of SAK/PLK4 degradation by the SCF/Slimb complex become proper microtubule-organizing centers. The formation of supranumerary centrosomes was quantified at the light microscope level. Upon transient low-level expression of the SAK-ND construct fused to GFP, a statistical significant increase in centrosome number was systematically observed in transfected cells compared to cells expressing low levels of the WT SAK fused to GFP. Indeed, expression of low levels of SAK-ND led to a 2-fold increase in centrosome amplification and after expression of a dominant-negative form of Slimb, suggesting that high levels of SAK/PLK4 underlie those phenotypes (Cunha-Ferreira, 2009).

Together, these results show that degradation of SAK/PLK4 by the SCF/Slimb complex is critical to restrict its function, preventing centrosome amplification. Although other mechanisms for regulation of SAK/PLK4 activity may exist, proteolytic degradation is likely to be conserved across species and be relevant in the animal. The Slimb-binding phosphodegron in SAK/PLK4 is conserved in vertebrates. Moreover, knockout of the ortholog of Slimb in mice, β-Trcp1, and both SkpA and Slimb Drosophila mutants show an increase in centrosome number, as do flies that overexpress SAK/PLK4 (Cunha-Ferreira, 2009).

It has been suggested that centriole overduplication is normally prevented by a 'licensing event,' the disengagement of centrioles that occurs at the exit of mitosis, required for the next duplication cycle. Requirement for this event ensures that centrioles duplicate only once in every cell cycle. However, it was recently shown that centriole amplification can also occur through the simultaneous formation of many daughters from a single mother, as it is after overexpression of SAK/PLK4 and SAS-6. These data suggest that proteolysis mediated by SCF/Slimb plays an important role in limiting the amount of SAK/PLK4, but not SAS-6, available to trigger multiple daughter formation. When SAK/PLK4 is not degraded, multiple daughter centrioles may be generated, which led to centrosome amplification. The regulation of centriole number thus emerges as a multistep mechanism, where proteolysis controls the activity of key players, SAK/PLK4 and SAS-6 (Cunha-Ferreira, 2009).

The mammalian F-box counterpart of Slimb, β-Trcp, plays a role in the DNA damage response by halting the cycle in response to genotoxic inputs. Misregulation of β-Trcp has been observed in cancer cells in which centrosome number is often increased after stress. It is thus possible that Slimb/β-Trcp coordinates checkpoints that monitor the status of DNA replication with centrosome number. The misregulation of this F-box protein would therefore result in changes in levels of SAK/PLK4, which are associated with mitotic abnormalities and oncogenesis. Given that other substrates of the Slimb/β-Trcp F-box protein require to be phosphorylated and the current results with the SAK-ND mutant, it is likely that SAK/PLK4 also requires such a modification at the serine and threonine residues in its DSGIIT degron to mark it for degradation. Future research on the identity and regulation of the kinase that phosphorylates SAK/PLK4 and on the localization of Slimb and SAK/PLK4 phosphorylated at the DSGIIT degron should indicate how different signaling events are coordinated in the cell to prevent centrosome amplification. These results open new avenues for understanding the mechanisms underlying centrosome misregulation that are of direct relevance to human disease (Cunha-Ferreira, 2009).

The Protein Phosphatase 2A regulatory subunit Twins stabilizes Plk4 to induce centriole amplification

Centriole duplication is a tightly regulated process that must occur only once per cell cycle; otherwise, supernumerary centrioles can induce aneuploidy and tumorigenesis. Plk4 (Polo-like kinase 4) activity initiates centriole duplication and is regulated by ubiquitin-mediated proteolysis. Throughout interphase, Plk4 autophosphorylation triggers its degradation, thus preventing centriole amplification. However, Plk4 activity is required during mitosis for proper centriole duplication, but the mechanism stabilizing mitotic Plk4 is unknown. This paper shows that PP2A [Protein Phosphatase 2A(Twins)] counteracts Plk4 autophosphorylation, thus stabilizing Plk4 and promoting centriole duplication. Like Plk4, the protein level of PP2A's regulatory subunit, Twins (Tws), peaks during mitosis and is required for centriole duplication. However, untimely Tws expression stabilizes Plk4 inappropriately, inducing centriole amplification. Paradoxically, expression of tumor-promoting simian virus 40 small tumor antigen (ST), a reported PP2A inhibitor, promotes centrosome amplification by an unknown mechanism. ST actually mimics Tws function in stabilizing Plk4 and inducing centriole amplification (Brownlee, 2011).

Plk4 protein is maintained at near-undetectable levels for the majority of the cell cycle by ubiquitin-mediated proteolysis. The ubiquitin ligase SCFSlimb is responsible for Plk4 degradation and recognizes an extensively phosphorylated degron situated immediately downstream of the kinase domain (KD; ~50 amino acids containing the Slimb-binding domain)KD. Slimb is appropriately positioned on centrioles throughout the cell cycle to promote rapid Plk4 destruction, but centrioles are not required for its activity. In any case, Plk4 degradation is critical in blocking all pathways of centriole amplification. Unlike other Polo kinase members, Plk4 is a homodimer capable of autophosphorylating its downstream regulatory element (DRE), a serine-rich region surrounding its SBD, in trans to promote Slimb binding. Autoregulation is a conserved feature of Plk4. Moreover, a RNAi screen of the fly kinome suggests that no other kinase is required for Plk4 degradation. The continuous and efficient degradation of Plk4 indicates that Plk4 is immediately active when expressed and that control of Plk4’s protein level is key to regulating its activity (Brownlee, 2011).

However, surprisingly little is known about the converse event: how Plk4 is activated. The results reveal the existence of a previously unknown facet of the regulation of centriole duplication, a process which transiently stabilizes and activates Plk4 specifically during mitosis. Serine/threonine phosphatases were investigated as possible effectors to counteract Plk4 autophosphorylation. PP2A is an excellent candidate to fulfill this role as it has important functions in mitosis and localizes to mitotic centrioles in cultured fly cells and centrosomes in dividing Caenorhabditis elegans embryos. A previous study found that the number of γ-tubulin foci in mitotic S2 cells was diminished after PP2A RNAi, but whether this resulted from a bona fide loss of centrioles or instead reflects a requirement for PP2A for centrosome maturation was not determined. Subsequently, a role for PP2A in centrosome maturation was identified in a genome-wide RNAi screen. The current results indicate that PP2A and the regulatory subunit Tws are required for centriole duplication by dephosphorylating and stabilizing Plk4. Without PP2ATws, Plk4 cannot be stabilized, and centrioles fail to duplicate. PP2A is also required for centriole assembly in C. elegans embryos but functions downstream in the centriole assembly process (Kitagawa, 2011; Song, 2011). Although the catalytic and structural PP2A subunits are abundant, regulatory subunits are needed for intracellular targeting and recognition of a myriad of substrates. Tws overexpression is sufficient to stabilize Plk4 in a dose-dependent manner, causing centriole amplification and multipolar spindle formation. Like Plk4, Tws protein levels are low during interphase but rise and peak during mitosis. Accordingly, the results suggest that PP2ATws stabilizes mitotic Plk4 by counteracting Plk4 autophosphorylation, enabling cells to switch Plk4 activity (and thus centriole duplication) on and off. This mechanism is inherently highly sensitive to the presence of Tws, a rate-limiting component. Moreover, this is likely a conserved mechanism because overexpression of human Tws also stabilizes fly Plk4 in S2 cells. Clearly, an important goal for future studies is to establish whether the regulation of Tws levels and cell cycle control are linked. In addition, the results suggest that up-regulation of Tws could be a means to amplify centrioles in multiciliated cells and that increased Tws activity could be a condition found in cancerous cells (Brownlee, 2011).

Centrosome amplification is a hallmark of cancer and is also observed upon expression of DNA tumor virus proteins, which include SV40 ST, human papillomavirus E7, human T cell leukemia virus type-1 Tax, hepatitis B virus oncoprotein X, and human adenovirus E1A. However, mechanisms for centrosome amplification by viral oncoproteins are not known. SV40 ST has been found to directly bind the highly conserved Drosophila catalytic and structural PP2A subunits and to induce centrosome overduplication in cultured fly cells (Kotadia, 2008). Notably, ST is a well-established PP2A inhibitor and is known to bind structural PP2A subunits, forcing endogenous PP2A regulatory subunits to be displaced and inhibiting PP2A activity. However, the current results demonstrate that ST expression does not inhibit all PP2A activities but, instead, stimulates PP2A stabilization of Plk4. This represents the first evidence that ST mimics the function of a PP2A regulatory subunit in cells. It will be important to determine whether ST targets additional PP2A substrates during tumorigenesis and whether other tumorigenic viruses (e.g., human papillomavirus and hepatitis B) known to promote centrosome amplification exploit this same mechanism. Intriguingly, human papillomavirus E7 oncoprotein binds PP2A catalytic and structural subunits and prevents PP2A from dephosphorylating Akt. Although a previous study has suggested that PP2A may function as a tumor suppressor, these findings indicate that unregulated PP2A activity leads to centriole amplification and chromosomal instability and should therefore be considered as a potential oncogenic factor (Brownlee, 2011).

Cullin-3 controls Timeless oscillations in the Drosophila circadian clock

Eukaryotic circadian clocks rely on transcriptional feedback loops. In Drosophila, the Period and Timeless proteins accumulate during the night, inhibit the activity of the Clock (Clk)/Cycle (Cyc) transcriptional complex, and are degraded in the early morning. The control of Per and Tim oscillations largely depends on post-translational mechanisms. They involve both light-dependent and light-independent pathways that rely on the phosphorylation, ubiquitination, and proteasomal degradation of the clock proteins. Slmb, which is part of a CULLIN-1-based E3 ubiquitin ligase complex, is required for the circadian degradation of phosphorylated Per. This study shows that Cullin-3 (Cul-3) is required for the circadian control of Per and Tim oscillations. Expression of either Cul-3 RNAi or dominant negative forms of Cul-3 in the clock neurons alters locomotor behavior and dampens Per and Tim oscillations in light-dark cycles. In constant conditions, Cul-3 deregulation induces behavioral arrhythmicity and rapidly abolishes Tim cycling, with slower effects on Per. Cul-3 affects Tim accumulation more strongly in the absence of Per and forms protein complexes with hypo-phosphorylated Tim. In contrast, Slmb affects Tim more strongly in the presence of Per and preferentially associates with phosphorylated Tim. Cul-3 and Slmb show additive effects on Tim and Per, suggesting different roles for the two ubiquitination complexes on Per and Tim cycling. This work thus shows that Cul-3 is a new component of the Drosophila clock, which plays an important role in the control of Tim oscillations (Grima, 2012).

Hipk proteins dually regulate Wnt/Wingless signal transduction

The Wnt/Wingless (Wg) pathway is an evolutionarily conserved signaling system that is used reiteratively, both spatially and temporally, to control the development of multicellular animals. The stability of cytoplasmic β-catenin/Armadillo, the transcriptional effector of the pathway, is controlled by sequential N-terminal phosphorylation and ubiquitination that targets it for proteasome-mediated degradation. Orthologous members of the Homeodomain-interacting protein kinase family from Drosophila to vertebrates have been implicated in the regulation of Wnt/Wingless signaling. In Drosophila, as a consequence of Hipk activity, cells accumulate stabilized Armadillo that directs the expression of Wg-specific target genes. Hipk promotes the stabilization of Armadillo by inhibiting its ubiquitination (and hence subsequent degradation) by the SCF(Slimb) E3 ubiquitin ligase complex. Vertebrate Hipk2 impedes β-catenin ubiquitination to promote its stability and the Wnt signal in a mechanism that is functionally conserved. Moreover, this study describes that Hipk proteins have a role independent of their effect on β-catenin/Armadillo stability to enhance Wnt/Wingless signaling (Verheyen, 2012).

In summary, Hipk proteins play multiple important roles during developmental signaling events, and have the capacity to simultaneously impact the outcomes of both Wg and Hh signal transduction. The finding that Hipk proteins act by blocking Slimb/β-TrCP-mediated ubiquitination of substrates suggests that additional targets may also be affected through the action of these kinases. Additionally a nuclear role for Hipk was observed that is independent of its role in stabilizing the Wg pathway effector Arm. The data suggest that Hipk can enhance the transcriptional activation by the Arm/TCF complex. This role thought to be independent of its role in blocking substrate ubiquitination (Verheyen, 2012).

Multiple roles have been described for Hipk family members in regulation of Wnt/Wg pathway activity. Such findings are reminiscent of the work performed to decipher the roles of several other kinases in the Wnt pathway. Both GSK3 and CK1 have distinct roles at different steps in the transduction of Wnt signaling and act to either promote or inhibit pathway activity and as a result gene expression. The finding that Hipk-stabilized Arm is phosphorylated by GSK3 and CK1 reveals a specific point of action in the targeting and destruction of Arm/β- catenin. Future work should reveal whether the stabilized N-terminally phosphorylated Arm is associated with the destruction complex, in effect blocked from being transferred to the proteasome as a result of Hipk acting on Slimb. The regulation of β-catenin stability and activity has important implications for normal growth and patterning, tissue homeostasis and the development of cancer (Verheyen, 2012).

Regulation of Autophosphorylation Controls PLK4 Self-Destruction and Centriole Number

Polo-like kinase 4 (PLK4) is a major player in centriole biogenesis: in its absence centrioles fail to form, while in excess leads to centriole amplification. The SCF-Slimb/betaTrCP-E3 ubiquitin ligase controls PLK4 levels through recognition of a conserved phosphodegron. SCF-Slimb/betaTrCP substrate binding and targeting for degradation is normally regulated by phosphorylation cascades, controlling complex processes, such as circadian clocks and morphogenesis. This study shows that PLK4 is a suicide kinase, autophosphorylating in residues that are critical for SCF-Slimb/betaTrCP binding. A multisite trans-autophosphorylation mechanism, likely to ensure that both a threshold of PLK4 concentration is attained, is demonstrated, and a sequence of events is observed before PLK4 can autodestruct. It was shown that PLK4 trans-autophosphorylates other PLK4 molecules on both Ser293 and Thr297 within the degron and that these residues contribute differently for PLK4 degradation, the first being critical and the second maximizing auto-destruction. Second, PLK4 trans-autophosphorylates a phospho-cluster outside the degron, which regulates Thr297 phosphorylation, PLK4 degradation, and centriole number. Finally, the importance was shown of PLK4-Slimb/betaTrCP regulation as it operates in both soma and germline. As betaTrCP, PLK4, and centriole number are deregulated in several cancers, this work provides novel links between centriole number control and tumorigenesis (Cunha-Ferreira, 2013).

Polo-like Kinase 4 autodestructs by generating its Slimb-binding phosphodegron

Polo-like kinase 4 (Plk4) is a conserved master regulator of centriole assembly. Previous study has found that Drosophila Plk4 protein levels are actively suppressed during interphase. Degradation of interphase Plk4 prevents centriole overduplication and is mediated by the ubiquitin-ligase complex SCFSlimb/betaTrCP. Since Plk4 stability depends on its activity, the consequences were examined of inactivating Plk4 or perturbing its phosphorylation state within its Slimb-recognition motif (SRM). Plk3 was shown to be directly responsible for extensively autophosphorylating and for generating its Slimb-binding phosphodegron (the residues that direct the starting place of degradation). Phosphorylatable residues within this regulatory region were systematically mutated to determine their impact on Plk4 protein levels and centriole duplication when expressed in S2 cells. Notably, autophosphorylation of a single residue (Ser293) within the SRM is critical for Slimb binding and ubiquitination. These data also demonstrate that autophosphorylation of numerous residues flanking S293 collectively contribute to establishing a high-affinity binding site for SCFSlimb. Taken together, these findings suggest that Plk4 directly generates its own phosphodegron and can do so without the assistance of an additional kinase(s) (Klebba 2013).

The SCFSlimb E3 ligase complex regulates asymmetric division to inhibit neuroblast overgrowth

Drosophila larval brain neuroblasts divide asymmetrically to balance between self-renewal and differentiation. This study demonstrates that the SCFSlimb E3 ubiquitin ligase complex, which is composed of Cul1, SkpA, Roc1a and the F-box protein Supernumerary limbs (Slimb), inhibits ectopic neuroblast formation and regulates asymmetric division of neuroblasts. Hyperactivation of Akt leads to similar neuroblast overgrowth and defects in asymmetric division. Slimb associates with Akt in a protein complex, and SCFSlimb acts through SAK and Akt to inhibit neuroblast overgrowth. Moreover, Beta-transducin repeat containing, the human ortholog of Slimb, is frequently deleted in highly aggressive gliomas, suggesting a conserved tumor suppressor-like function (Li, 2014).

It has been shown that Akt associates with Slimb and can be ubiquitinated by Slimb. It was determined which domain of Akt is important for its association with Slimb. The central region of Akt, which contains its protein kinase domain (T2), but not the N-terminus (T1, containing a pleckstrin homology domain) or the C-terminus (T3, containing an AGC kinase domain), interacted with Slimb in S2 cells by co-immunoprecipitation. Taken together, these biochemical and genetic data suggest that the SCFSlimb complex inhibits ectopic NB formation in part through an association with Akt (Li, 2014).

Beta-transducin repeat containing (BTRC/β-TrCP; human homologue of Slimb) showed a significant loss in 72.5% of glioma patients. Its copy number was an independent predictor of prognosis in a multivariate analysis. BTRC copy loss was observed in patients with glioblastoma (82%) and oligodendroglioma (68%). It was also observed in patients with mesenchymal and proliferative (85 and 88%, respectively), which are frequently associated with activated AKT signaling, a central oncogenic pathway regulating glioblastoma (GBM) growth and survival. The BTRC functional module was derived by mining mRNA expression databases and mapped to 544 mRNA transcripts. This module was able to stratify patients into two subgroups and was significantly associated with survival. A multivariate Cox Regression model confirmed that the BTRC functional subgroup was independently associated with survival. Furthermore, BTRC copy changes have a significant inverse correlation with the gene expression of AKT2 and PIK3CD (Li, 2014).

β-TRCP, the mammalian Slimb homolog, is speculated to play a greater role as an oncogene than as a tumor suppressor. This study has demonstrated that the SCFSlimb complex plays an important role in NB self-renewal and asymmetric division. It acts at least in part through the oncogenic protein Akt, a key player that regulates NB self-renewal and asymmetric division. The activation of Akt in NBs appears to be independent of Phosphatase and tensin homologue (PTEN), a negative regulator of PI3K/Akt signaling, because no NB overgrowth were observed in two loss-of-function alleles, PTENC494 and PTEN1. A recent report suggested a role for Target of rapamycin (TOR) signaling in restraining tumorigenesis in larval brains. PI3K/TOR also interacts genetically with Pins in the suppression of tumor growth in larval brains, suggesting the complex role of PI3K/Akt and the interconnected TOR pathway in NB homeostasis (Li, 2014).

Crumbs promotes expanded recognition and degradation by the SCFSlimb/beta-TrCP ubiquitin ligase

In epithelial tissues, growth control depends on the maintenance of proper architecture through apicobasal polarity and cell-cell contacts. The Hippo signaling pathway has been proposed to sense tissue architecture and cell density via an intimate coupling with the polarity and cell contact machineries. The apical polarity protein Crumbs (Crb) controls the activity of Yorkie (Yki)/Yes-activated protein, the progrowth target of the Hippo pathway core kinase cassette, both in flies and mammals. The apically localized Four-point-one, Ezrin, Radixin, Moesin domain protein Expanded (Ex) regulates Yki by promoting activation of the kinase cascade and by directly tethering Yki to the plasma membrane. Crb interacts with Ex and promotes its apical localization, thereby linking cell polarity with Hippo signaling. This study shows that, as well as repressing Yki by recruiting Ex to the apical membrane, Crb promotes phosphorylation-dependent ubiquitin-mediated degradation of Ex. Skp/Cullin/F-boxSlimb/beta-transducin repeats-containing protein (SCFSlimb/beta-TrCP) was identifed as the E3 ubiquitin ligase complex responsible for Ex degradation. Thus, Crb is part of a homeostatic mechanism that promotes Ex inhibition of Yki, but also limits Ex activity by inducing its degradation, allowing precise tuning of Yki function (Ribeiro, 2014).

Recent work in flies and mammals has implicated the apical polarity determinant Crb as a transmembrane receptor for Hpo signaling. Accordingly, clonal loss of crb function leads to Yki derepression and increased growth. However, the observation that Crb is required for Ex membrane localization apparently conflicts with the finding that Crbintra overexpression reduces Ex levels and, like crb loss of function, leads to increased Yki activity. This study reconciles these findings by showing that Crb is not only required for Ex tethering at the apical membrane but also for promoting its degradation via the SCFSlimb/β-TrCP E3 ubiquitin ligase. Indeed, immediately downstream of its FERM domain, Ex contains a sequence that conforms to the D/S/TSGφXS consensus sequence for canonical Slmb targets, which is conserved in Ex orthologs from arthropod species but absent from related FERM domain proteins such as Moe and Mer. In addition, loss of Slmb increases Ex levels in vivo, whereas Slmb depletion prevents Crbintra-induced Ex degradation in cell culture. Thus, in crb mutants, Ex no longer reaches the apical membrane and is protected from degradation in the cytoplasm, where it accumulates but is presumably unable to repress Yki. When Crbintra is overexpressed, Ex turnover at the membrane (or in an endocytic compartment if Ex degradation occurs after Crb internalization) is accelerated, leading to its depletion and consequent Yki activation. Therefore, in both cases, the outcome is Yki derepression, albeit for different reasons (Ribeiro, 2014).

How is the Slmb:Ex association regulated by Crb? Previous observations point to the involvement of a phosphorylation-dependent degradation mechanism, because Crb induces Ex phosphorylation. Accordingly, mutation of the phosphodegron (Ser453) or the putative priming site (Ser462) leads to loss of Slmb:Ex association and Ex stabilization. The most obvious candidate Ex kinase in this context is aPKC, which is known to interact with the Crb polarity complex and to phosphorylate Crb at its FBM. However, aPKC is thought to be recruited to the Crb complex via Par-6, which interacts with Crb through the PBM at the C terminus of the Crb intracellular domain. This is inconsistent with the fact that the PBM is dispensable for Crb to promote both Ex phosphorylation and degradation. Moreover, a dominant-negative version of aPKC is unable to rescue the overgrowth phenotype of Crbintra overexpression in the wing. The Hpo downstream kinase Wts is another attractive candidate, because mammalian LATS1/2 promotes YAP phosphorylation on Ser381, providing priming for phosphorylation by CK1δ/ε and triggering degradation by SCFβ-TrCP. However, neither Ser462 nor Ser453 conform to the Wts/LATS consensus. The identity of the kinases therefore remains open (Ribeiro, 2014).

Regulation of tissue growth during development and adult life depends on the maintenance of tissue architecture, which, in turn, relies on cell-cell and cell-matrix interactions. Due to its intimate coupling to polarity and cytoskeletal regulators, the Hpo signaling pathway is thought to sense epithelial integrity and couple tissue architecture to growth control. In particular, YAP has been shown to sense contact inhibition in cell culture, such that its progrowth activity is silenced as cultured epithelial cells reach confluence. This is thought to depend on the assembly of tight junctions, leading to repression of YAP by the mammalian Crb complex. Recent work in flies and zebrafish has suggested that Crb can form homodimers in trans. This suggests that Crb mediates local cell-cell communication in epithelial tissues. Indeed, clonal loss of Crb leads to Crb depletion in the junctions of wild-type cells abutting the crb mutant tissue. This loss of Crb at the clone boundary is mirrored by loss of Ex, which is dependent on Crb for its apical localization. Thus, loss of Crb caused by loss of polarity or cell death can be transmitted to neighboring cells. This has been proposed as a means of cell- cell communication to induce regenerative proliferation through Yki activation. Indeed, genetic induction of epithelial wounds in imaginal discs has been shown to up-regulate Yki activity in cells neighboring the wound. In addition, Yki/ YAP is activated and promotes tissue regeneration upon injury in the vertebrate and fly intestine, as well as in the mouse liver (Ribeiro, 2014).

These findings suggest that Crb (and perhaps other polarity proteins) functions as a sensor of cell density and tissue integrity during development. In this model, disruption of Crb function would lead to Yki/YAP derepression, which, upon tissue injury, would allow regenerative growth to ensue. Another interesting question is whether liganded Crb behaves differently to unliganded Crb with respect to regulation of Ex stability. For example, it is possible that unliganded Crb promotes Ex turnover faster than its liganded counterpart, which might provide a sensitive means of responding to the status of neighboring cells. Further work will be needed to resolve this issue. The present work indicates that Crb fulfills a dual function in Hpo signaling, both recruiting Ex apically to repress Yki activity and promoting Ex turnover through phosphorylation and Slmb-dependent degradation. This mechanism could ensure constant turnover of Ex at the apical membrane, allowing Yki activity to rapidly respond to changing environmental conditions. This dynamic equilibrium could be particularly important to promote fast tissue regeneration upon injury (Ribeiro, 2014).

The F-box protein Slmb restricts the activity of aPKC to polarize epithelial cells

The Par-3/Par-6/aPKC complex is the primary determinant of apical polarity in epithelia across animal species, but how the activity of this complex is restricted to allow polarization of the basolateral domain is less well understood. In Drosophila, several multiprotein modules antagonize the Par complex through a variety of means. This study identified a new mechanism involving regulated protein degradation. Strong mutations in supernumerary limbs (slmb), which encodes the substrate adaptor of an SCF-class E3 ubiquitin ligase, cause dramatic loss of polarity in imaginal discs accompanied by tumorous proliferation defects. Slmb function is required to restrain apical aPKC activity in a manner that is independent of endolysosomal trafficking and parallel to the Scribble module of junctional scaffolding proteins. The involvement of the Slmb E3 ligase in epithelial polarity, specifically limiting Par complex activity to distinguish the basolateral domain, points to parallels with polarization of the C. elegans zygote (Skwarek, 2014).

This study extends the mechanisms involved in epithelial polarity to include a new function: targeted protein degradation. Targeted degradation can create spatial asymmetries in protein distributions, and there is precedent for roles of E3 ubiquitin ligases, including SCFSlmb, in polarizing different aspects of cells. The involvement of Slmb in Drosophila apicobasal polarity has gone unnoticed due to the previous use of hypomorphic alleles. The strong alleles described in this study display potent expansion of the apical pole of imaginal epithelia, demonstrating that Slmb is a new polarity regulator that functions to restrict the apical domain (Skwarek, 2014).

Loss of Slmb phenocopies the polarity defects associated with mutations in two classes of 'apical antagonists': the Scrib module of core polarity regulators, and endocytic regulators that control trafficking through the early endosome. Despite the similar polarity defects, slmb mutations do not alter endolysosomal cargo traffic, nor do they display protein recruitment defects characteristic of Scrib module mutants; furthermore, no genetic interactions are seen with either pathway. Nevertheless, the downstream consequences of polarity misregulation - including tumor-like transformation and the upregulation of specific target genes - are again shared between slmb and the other apical antagonists, and, moreover, slmb and Scrib module mutant cells share a distinctive trafficking defect associated with elevated aPKC activity. It is therefore suggestrf that Slmb acts in parallel to the Scrib module to antagonize the Par complex and other apical regulators (Skwarek, 2014).

The role for Slmb defined in this study points to the existence of an apical polarity-regulating protein substrate, the levels of which must be controlled. A number of validated Slmb substrates have been ruled out as the relevant target. Bioinformatic scans of Drosophila proteins for Slmb degron sequences suggest other candidates, including Expanded (Ex), but overexpression of Ex is not sufficient to induce polarity defects resembling those of slmb. Although a contribution from the elevation of multiple substrates cannot be ruled out, slmb-like polarity phenotypes can be induced by the elevated activity of individual proteins, including Crb or aPKC. Despite evidence that aPKC undergoes ubiquitin-mediated degradation in embryos, neither aPKC nor Crb levels appear to be controlled by Slmb-mediated degradation in imaginal discs. Nevertheless, the data together suggest that the substrate of Slmb in polarity regulation will function as a positive regulator of aPKC-driven outcomes (Skwarek, 2014).

The demonstration that Slmb limits aPKC activity to distinguish the epithelial basolateral domain reveals intriguing parallels to polarization of the worm zygote. In this context, Par-2 is the primary antagonist that restricts aPKC/Par activity, while Lgl homologs function in a parallel, redundant role. Par-2 contains a RING finger domain that is characteristic of single-subunit E3 ligases, but Par-2 homologs have not been identified outside of nematodes, Par-2 does not affect aPKC/Par levels, and a degraded substrate in polarity regulation has yet to be identified. The discovery of a Drosophila E3 ligase with a similar function to Par-2 raises the possibility of a conserved molecular logic to polarity in these two paradigmatic systems; determination of the relevant substrate will shed further light on this question (Skwarek, 2014).

Slmb antagonises the aPKC/Par-6 complex to control oocyte and epithelial polarity

The Drosophila anterior-posterior axis is specified when the posterior follicle cells signal to polarise the oocyte, leading to the anterior/lateral localisation of the Par-6/aPKC complex and the posterior recruitment of Par-1, which induces a microtubule reorganisation that localises bicoid and oskar mRNAs. This study shows that oocyte polarity requires Slmb, the substrate specificity subunit of the SCF E3 ubiquitin ligase that targets proteins for degradation. The Par-6/aPKC complex is ectopically localised to the posterior of slmb mutant oocytes, and Par-1 and oskar mRNA are mislocalised. Slmb appears to play a related role in epithelial follicle cells, as large slmb mutant clones disrupt epithelial organisation, whereas small clones show an expansion of the apical domain, with increased accumulation of apical polarity factors at the apical cortex. The levels of aPKC and Par-6 are significantly increased in slmb mutants, whereas Baz is slightly reduced. Thus, Slmb may induce the polarisation of the anterior-posterior axis of the oocyte by targeting the Par-6/aPKC complex for degradation at the oocyte posterior. Consistent with this, overexpression of the aPKC antagonist Lgl strongly rescues the polarity defects of slmb mutant germline clones. The role of Slmb in oocyte polarity raises an intriguing parallel with C. elegans axis formation, in which PAR-2 excludes the anterior PAR complex from the posterior cortex to induce polarity, but its function can be substituted by overexpressing Lgl (Morais-de-Sa, 2014).

Very little is known about how the posterior follicle cells signal to polarise the AP axis of the oocyte, except that signalling is disrupted when the germline is mutant for components of the exon junction complex, such as Mago nashi. The current results reveal that Slmb also plays an essential role in this pathway, where it acts to establish the complementary cortical domains of Baz/Par-6/aPKC and Par-1. Although Slmb might act in a variety of ways to establish this asymmetry, the observation that it regulates the levels of the Par-6/aPKC complex suggests a simple model in which Slmb directly or indirectly targets a component of the complex for degradation at the posterior of the oocyte. Since aPKC phosphorylates Par-1 to exclude the latter from the cortex, the degradation of aPKC would allow the posterior recruitment of Par-1, which would then maintain polarity by phosphorylating and antagonising Baz. Indeed, this might explain the observation that Par-6 is excluded from the posterior cortex before Baz. The polarisation of the oocyte therefore appears to occur in two phases. During the initiation phase, Slmb removes the Par-6/aPKC complex from the posterior cortex to allow the recruitment of Par-1. Par-1 then maintains and reinforces this asymmetry by phosphorylating Baz to exclude it from the posterior cortex, thereby removing the cortical anchor for the Par-6/aPKC complex (Morais-de-Sa, 2014).

Slmb is usually recruited to its targets by binding to phosphorylated residues that lie 9-14 amino acids downstream from the ubiquitylated lysine. Although both aPKC and Par-6 contain several sequences that could serve as atypical Slmb binding sites, neither contains a classic Slmb-dependent degron sequence. It is therefore unclear whether the SCFSlmb complex directly ubiquitylates either protein to target it for degradation or whether it targets another, unknown component of the complex that is required for the stability of Par-6 and aPKC. Nevertheless, this model leads to the prediction that the polarising signal from the follicle cells will induce the activation of a kinase that phosphorylates a Slmb substrate at the posterior of the oocyte, thereby triggering the local degradation of the Par-6/aPKC complex (Morais-de-Sa, 2014).

The demonstration that Slmb is required for the exclusion of the Par-6/aPKC complex from the posterior of the Drosophila oocyte raises interesting parallels with AP axis formation in C. elegans. Although Drosophila does not have an equivalent of the main symmetry-breaking step in the worm, in which a contraction of the actomyosin cortex removes the anterior PAR proteins from the posterior, the function of Slmb is analogous to that of PAR-2 in the alternative polarity induction pathway. Both proteins act to remove the Par-6/aPKC complex from the posterior cortex to allow the posterior recruitment of Par-1, which then reinforces polarity by excluding Baz/PAR-3 by phosphorylation. Furthermore, the polarity phenotypes of both slmb and par-2 mutants can be rescued by the overexpression of Lgl. Slmb and PAR-2 act by different mechanisms, since the former is a subunit of the SCF ubiquitin ligase complex and promotes the degradation of the Par-6/aPKC complex, whereas the latter functions by recruiting PAR-1. Nevertheless, it is intriguing that PAR-2 contains a RING finger domain that is typically found in ubiquitin ligases, suggesting that it might have lost this activity during evolution (Morais-de-Sa, 2014).

The Drosophila Bcl-2 family protein Debcl is targeted to the proteasome by the beta-TrCP homologue Slimb

The ubiquitin-proteasome system is one of the main proteolytic pathways. It inhibits apoptosis by degrading pro-apoptotic regulators, such as caspases or the tumor suppressor p53. However, it also stimulates cell death by degrading pro-survival regulators, including IAPs. In Drosophila, the control of apoptosis by Bcl-2 family members is poorly documented. Using a genetic modifier screen designed to identify regulators of mammalian bax-induced apoptosis in Drosophila, this study identified the ubiquitin activating enzyme Uba1 as a suppressor of bax-induced cell death. Uba1 was demonstrated to regulate apoptosis induced by Debcl, the only counterpart of Bax in Drosophila. Furthermore, these apoptotic processes were shown to involve the same multimeric E3 ligase-an SCF complex consisting of three common subunits and a substrate-recognition variable subunit identified in these processes as the Slimb F-box protein. Thus, Drosophila Slimb, the homologue of beta-TrCP targets Bax and Debcl to the proteasome. These new results shed light on a new aspect of the regulation of apoptosis in fruitfly that identifies the first regulation of a Drosophila member of the Bcl-2 family (Colin, 2014).

This paper reports the regulation of bax- and debcl-induced apoptosis by the ubiquitin-proteasome pathway. The stimulation of this pathway by overexpressing Uba1, which encodes the ubiquitin activating enzyme, leads to an almost complete loss of bax-induced cell death. This regulation seems conserved through evolution, as debcl-induced apoptosis is also regulated in this way. However, since Bax seems to necessitate Debcl in order to kill Drosophila eye cells, one could wonder whether suppression of Bax-induced cell death depends on the direct effect of Uba1 on Debcl. Nevertheless, since both Bax and Debcl proteins are degraded when Uba1 is overexpressed, this seems unlikely unless Debcl stabilizes Bax (Colin, 2014).

Since Buffy inhibits autophagy in response to starvation, it is hypothesized that Debcl induces an autophagic cell death. Autophagy was monitored by using a UAS-Atg8-GFP transgene. It was found that actually no autophagy could be detected upon Debcl expression. Uba1 has been shown in the literature to be required for autophagy and reduction of cell size in the intestine. This study shows that Debcl-induced cell death in the wing disc is not only suppressed by Uba1 but also by proteasome mutants. These data suggest that the UPS pathway is the main proteolytic pathway involved in the suppression of Bax and Debcl-induced apoptosis by Uba1. However, given that slmb has been shown to regulate Wg and Dpp pathways, it cannot be excluded that these pathways are partially involved in phenotypic suppression (Colin, 2014).

Studies of the proteasome-dependent regulation of members of the Bcl-2 family in mammals have only rarely led to the identification of the specific E3 ligases. This study identified an SCFSlmb complex as the E3 ubiquitin ligase that regulates the Debcl pathway and may target it to proteasomal degradation. It would be interesting to determine whether the mammalian homologue of Slmb, β-TrCP, targets Bax to the proteasome in mammalian cells (Colin, 2014).

This study has show Debcl is a target of the UPS, thus finding a new regulation of apoptosis that differs from the control of Dronc, Drice and RHG protein levels by Diap1. Indeed, Diap1 is a key enzyme that decides of cell fate by degrading either pro-apoptotic regulators or itself, leading to either cell survival or apoptosis. Since Diap1 levels are downregulated by the F-box protein Morgue in presence of Rpr or Grim, it could be hypothesized that the Morgue/Diap1 pathway is involved in bax- and debcl-induced apoptosis regulation. This does not seem to be the case because a hypomorphic allele of morgue did not increase bax- and debcl-induced apoptosis. Furthermore, overexpression of diap1 does not inhibit bax- and debcl-induced apoptosis. Thus, the proteasome-dependent regulation that this study has identified is independent of Diap1 and differs from the Morgue/Diap1 regulation of cell death. The existence of different UPS-modulated cell death pathways is also supported by the reported absence of genetic interaction between RHG pathway components and Debcl (Colin, 2014).

The results indicate an anti-apoptotic role of Uba1 in the wing tissue. However, two other studies revealed a pro-apoptotic role of Uba1 in the eye; strong Uba1 loss-of-function alleles lead to apoptosis and compensatory proliferation in the developing eye. As previously shown in other systems, these processes seem to involve the RHG/Diap1/Dronc pathway. Hypomorphic alleles of Uba1 have shown opposite effects as they suppressed hid- or grim-induced apoptosis in the eye. These results are consistent with previous data indicating that Uba1 overexpression, using the Uba1 EP2375 allele, increases RHG-induced apoptosis. In principle, this apparent contradiction may result from either the cell death signal or the studied tissue. The use of a GMR-Gal4 driver shows that Uba1 overexpression also inhibits debcl-induced apoptosis in the eye tissue, which suggests that the Uba1 effect is specific of debcl-induced apoptosis. In contrast, rpr-induced cell death is enhanced by Uba1 overexpression in the eye, whereas it is suppressed by Uba1 in the wing. These results suggest that Debcl could be involved in rpr-induced cell death in the wing but not in the eye. RHG proteins are known to mediate their pro-apoptotic function by stimulating Diap1 degradation by the UPS while Debcl is a direct target of ubiquitination. Therefore, RHG-induced degradation of Diap1 through its ubiquitination by Morgue could explain the pro-apoptotic role of Uba1 in the eye whereas the anti-apoptotic Uba1 function mediated by Slmb in Debcl-induced cell death would rely on Debcl degradation. By showing that different pro-apoptotic pathways are regulated by the UPS in Drosophila, this work suggests that the tissue-dependent effect of the pleiotropic enzyme Uba1 must result from a change in the balance between UPS pro- and anti-apoptotic effects. It is proposed that this change relies on the availability in E3 enzymes, the incoming signals and relative amounts of pro- and anti-apoptotic regulators of cell fate (Colin, 2014).

Protection of Armadillo/beta-Catenin by Armless, a novel positive regulator of Wingless signaling

The Wingless (Wg/Wnt) signaling pathway is essential for metazoan development, where it is central to tissue growth and cellular differentiation. Deregulated Wg pathway activation underlies severe developmental abnormalities, as well as carcinogenesis. Armadillo/β-Catenin plays a key role in the Wg transduction cascade; its cytoplasmic and nuclear levels directly determine the output activity of Wg signaling and are thus tightly controlled. In all current models, once Arm is targeted for degradation by the Arm/β-Catenin destruction complex, its fate is viewed as set. This study identified a novel Wg/Wnt pathway component, Armless (Als; CG5469) that is required for Wg target gene expression in a cell-autonomous manner. Genetic and biochemical analyses showed that Als functions downstream of the destruction complex, at the level of the SCF/Slimb/βTRCP E3 Ub ligase. In the absence of Als, Arm levels are severely reduced. Biochemical and in vivo studies showed that Als interacts directly with Ter94, an AAA ATPase known to associate with E3 ligases and to drive protein turnover. It is suggested that Als antagonizes Ter94's positive effect on E3 ligase function, and it is proposed that Als promotes Wg signaling by rescuing Arm from proteolytic degradation, spotlighting an unexpected step where the Wg pathway signal is modulated (Reim, 2014).

The wingless (wg) gene was found nearly forty years ago with the characterization of a Drosophila mutant without wings. The gene encodes a secreted glycoprotein, the founding member of the Wnt family of signaling proteins. In the decades following its discovery, Wg/Wnt signaling has been shown to be essential during embryogenesis. Indeed, it is important throughout an organism's life, controlling also the homeostasis of different organs, for example, regeneration of epithelial cells in the intestine - the aberrant behavior of these cells in cancer is caused by constitutive Wg/Wnt signaling, which is consequently a key focus of medical and translational research (Reim, 2014).

The relay of the Wg signal is controlled at different levels. However, the pivotal step is the regulation of the levels of Armadillo (Arm)/β-Catenin, the key transducer of the Wg/Wnt pathway. A multiprotein complex consisting of the scaffold proteins Axin and APC and the kinases Shaggy/GSK3β and Casein kinase I (CKI) recruits and phosphorylates Arm/β-Catenin. This marks Arm/β-Catenin for ubiquitination by the SCF/Slimb/βTRCP E3 ubiquitin ligase and subsequent degradation by the ubiquitin-proteasome system (UPS). When Wg/Wnt binds its receptors at the cell membrane, degradation of Arm/β-Catenin is prevented, presumably by protein interactions that lead to the dissociation of the E3 ubiquitin ligase from the Arm/β-Catenin destruction complex. As a consequence, Arm/β-Catenin translocates into the nucleus, where it adopts its role as a transcriptional effector of Wg/Wnt signaling. Although this step is crucial, and is a potential point of regulation, little is known about the players involved in the processing of Arm/β-Catenin and its ultimate degradation (Reim, 2014).

In a genome-wide RNA interference (RNAi) screen Armless (Als) was isolated as a regulator of proximodistal growth of Drosophila limbs, and has been shown in subsequent analyses to exert its function in the Wg pathway. Detailed genetic studies demonstrate that Als acts downstream of the destruction complex, at the level of the SCF/Slimb/βTRCP E3 Ub ligase. Cells depleted for Als exhibit strongly reduced Arm protein levels. Importantly, the activity of a constitutively active form of Arm, which cannot be phosphorylated and hence escapes ubiquitination and proteasomal degradation, is insensitive to depletion of Als. Using immunopurification and mass spectrometry analysis this study found that Ter94 interacts with Als. Ter94 is an AAA ATPase associated with protein turnover and proteasomal degradation. In sum, these data suggest that Als acts downstream of the Arm/β-Catenin destruction complex to positively regulate Arm protein levels, possibly by rescuing Arm from ubiquitination via Slimb. The human ortholog of Als, UBXN6, can substitute for Als in Drosophila, and Wnt target gene expression was impaired upon knock-down of UBXN6 in HEK-293 cells. It is thus infered that Als and UBXN6 represent regulators of a conserved mechanism that ensures appropriate levels of Armadillo/β-Catenin by antagonizing its entry into the UPS (Reim, 2014).

A prevalent mechanism for controlling information flow in signaling pathways is the alteration of the protein levels of key components. In the Wg/Wnt pathway, the Arm/β-Catenin destruction complex targets Arm/β-Catenin for ubiquitination by the SCF/Slimb/βTRCP E3 Ub ligase, resulting in proteasomal degradation and low cytoplasmic levels of Arm/β-Catenin in the Wnt pathway off state. If the pathway is turned on, Slimb-mediated ubiquitination is prevented, thus rescuing Arm from its proteasomal fate and causing a concomitant increase in Arm protein levels. This study describes Als as a new component of this control system; Als was found to be required to prevent the degradation of Arm/β-Catenin (Reim, 2014).

This study has identified als in a genome-wide in vivo RNAi screen in Drosophila. Because no EMS- or P-element-induced null allele was isolated, and because another gene overlaps with als, particularly thorough evidence validating als gene function was obtained. (1) The als phenotypes could be reproduced by nine different UAS-RNAi transgenes encoding independent RNA target sites. Together with an extended off target analysis, unintentional RNAi was ruled out as a cause for the als phenotypes. (2) RNAi-mediated inhibition of als expression was ascertained by monitoring als mRNA expression via real-time PCR and antisense mRNA in situ hybridization. (3) Expression of Als with different RNAi-insensitive rescue transgenes, as well as with its human ortholog UBXN6, rescued als phenotypes (Reim, 2014).

These analyses show that als encodes an essential positive Wg signaling component. This conclusion is based on the following evidence. als depletion caused wings with notched wing margins and loss of sensory bristles, which is characteristic of impaired Wg signaling. The distal wing region is most sensitive to als levels, as is the case for other positive components of Wg signaling. In agreement with this, increased als expression was found in the central wing pouch, at least in earlier L3 larval stages. Stimulation of the Wg pathway in wing imaginal discs or Kc-167 cells caused higher als expression, suggesting that als can be positively controlled by Wg signaling. However, Als levels must be precisely controlled since already mild overexpression of UAS-als elicits a dominant-negative effect on Wg signaling. The function of als for Wg signaling is not restricted to the wing: also in other tissues, such as the thorax, eyes, legs, and the embryo, als phenotypes are identical to those seen when Wg signaling is disturbed. Also in human HEK-293 cells UBXN6/UBXD1, the ortholog of Als, was found to be required for Wnt signaling, and human UBXN6 largely rescues the als phenotypes in Drosophila, which suggests their functional conservation. Depletion of als also enhanced Wg-sensitized phenotypes, further supporting the notion that its product is a Wg pathway component. Moreover, the expression of positively regulated Wg target genes is reduced or abolished upon loss of als function, while Wg-repressed target gene expression is ectopically activated. Importantly, while interfering with als function suppressed Wg signaling, it did not affect other pathways, such as Notch and Hh, Jak/Stat, or EGFR signaling. However, it cannot be ruled out that als is not required in another pathway in a different biological context. In humans, UBXN6 is reported to play a role in diverse scenarios: for example, it was shown to play a role in Caveolin turnover in human osteosarcoma U2OS cells. This might indicate a broader role of UBXN6 in mammalians (Reim, 2014).

The data show that Als regulates Armadillo protein levels. Based on epistasis experiments, Als acts downstream of Shaggy/GSK3β and upstream of the SCF/Slimb/βTRCP E3 Ub ligase, which is known to ubiquitinate Arm, a prerequisite for proteasomal degradation. Consistent with this, the degradation-resistant form of Arm could completely bypass the requirement for als, in contrast to the wild-type form of Arm. This suggests that proteasomal degradation acts downstream of als; however, this cannot be taken as an unambiguous proof. Importantly, depletion of ubiquitin and overexpression of CSN6, a negative regulator of SCF/Slimb/βTRCP E3 Ub ligase, could ameliorate the als phenotype (as well as phenotypes based on the overexpression of Axin or Shaggy, which overactivate the destruction complex, thus resulting in enhanced Arm degradation). In contrast, altering these factors did not ameliorate the Lgs phenotype, which is caused by interfering more downstream in the Wg pathway. These findings suggest that als works upstream of proteasomal degradation. A further informative experiment was monitoring Wg pathway components with respect to protein levels: Arr, Fz, Axin, APC, Sgg, and Arm. The only change in the absence of Als function was Arm: its levels were strongly reduced upon als depletion. The effects on Arm levels could be due either to a direct effect on Arm or to an indirect effect on a negative component. Importantly, the rate-limiting factor Axin as well as other key negative components of the Arm/β-Catenin destruction complex were unaltered uponals depletion (Reim, 2014).

Some further mechanistic insight was obtained with the finding that Ter94 interacts in vitro and in vivo with Als. Interestingly, Als-Ter94 was found to localize at the cell cortex, as was similarly observed for the Arm/β-Catenin destruction complex. The studies are consistent with earlier work that showed that the human ortholog of Ter94, p97, interacts with UBXN6. Ter94/p97/Cdc48 is a conserved and highly abundant AAA ATPase that was found to associate with SCF/Slimb/βTRCP E3 Ub ligases or proteasomal shuttle factors to mediate UPS-mediated protein degradation. Specifications of the diverse activities of Ter94/p97 and the fate of its substrates are mainly exerted by UBX domain protein co-factors, which eventually either promote or hinder p97's function in protein turnover; an example of the latter involves the dissociation of the SCF/Slimb/βTRCP E3 Ub ligase complex, eventually leading to its inactivation. Interestingly, it was recently reported that inactivation of the E3 ligase complex upon Wnt signaling is achieved by its dissociation from the destruction complex. Based on the current experiments and what is known about Ter94/p97, a possible mechanism is suggested that Als antagonizes Ter94's positive effect on E3 ligase function, thereby rescuing Arm levels. No increased protein levels were observed of Slimb, Axin, Shaggy, or APC in this analyses; thus, the results favor a model in which Als antagonizes Ter94 to hinder the transfer of Arm to the proteasome by interfering with the SCF/Slimb/βTRCP E3 Ub ligase function or its assembly. Importantly, no interaction was found between Arm and Als. This is consistent with the finding that UBX domain family members lacking an UBA domain, such as UBXN6/Als, do not directly interact with substrate proteins, but are necessary for the activity or fate of the Ter94/p97 (Reim, 2014).

Interestingly, another study found that ter94 depletion affected the partial proteolysis of Ci. However, that study observed neither any typical consequence of disturbed Hh signaling per se (i.e., no alteration of Hh target gene expression in genes such as ptc) nor any phenotypical consequence upon overexpression of a dominant negative form of Ter94 (i.e., aberrant wing patterning and growth typical for Hh signaling). This is consistent with the current data that neither Ci target expression nor Hh signaling was affected upon als or ter94 depletion (Reim, 2014).

p97/Ter94 is known as a highly pleiotropic AAA ATPase associated with many cellular functions. Further, p97/Ter94 acts in multifaceted and large protein–protein complexes, and it is its regulatory co-factors, including UBX domain proteins, that render p97/Ter94 specific for a certain task in a particular cellular context. For example, p47/Shp1 is a co-factor of p97/Ter94 that blocks other co-factors from Ter94 binding. Interestingly, in Kc-167 cell mass spectroscopy experiments, this study found p47 in Ter94/Als protein complexes, but only in the absence of Wg stimulation. On the other hand, als transcript and Als protein levels were elevated upon Wg signaling. These findings suggest a dynamic regulation of the Ter94 complex upon signaling inputs. The identification and functional analysis of all key components of the Als-Ter94 complex will be needed to obtain a refined insight into Als-Ter94's molecular mechanism (Reim, 2014).

Critically, this work spotlights an underappreciated facet in the control of the output of the entire canonical Wg/Wnt pathway - how Arm/β-Catenin is handed over to the proteasome— and the potential for regulating this step; this works also indicates that this step, in contrast to the conventional wisdom, is tunable. The identification and characterization of the UBX protein Als as a positive regulator of Wg/Wnt signaling contributes to this layer of pathway control (Reim, 2014).

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).


supernumerary limbs: Biological Overview | Evolutionary Homologs | Developmental Biology | Effects of Mutation | References

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