shaggy
The cell surface receptor Notch is required during Drosophila embryogenesis for production of epidermal precursor cells. The secreted factor Wingless is required
for specifying different types of cells during differentiation of tissues from these epidermal precursor cells. The results reported here show that the full-length Notch
and a form of Notch truncated in the amino terminus associate with Wingless in S2 cells and in embryos. In S2 cells, Wingless and the two different forms of Notch
regulate expression of Frizzled 2, a receptor of Wg; hairy, a negative regulator of achaete expression; shaggy, a negative regulator of engrailed expression, and
patched, a negative regulator of wingless expression. Analyses of expression of the same genes in mutant N embryos indicate that the pattern of gene regulations
observed in vitro reflects regulations in vivo. These results suggest that the strong genetic interactions observed between Notch and wingless genes during
Drosophila development is at least partly due to regulation of expression of cuticle patterning genes by Wingless and the two forms of Notch (Wesley, 1999).
Genetic epistasis studies place shaggy between dishevelled (upstream) and armadillo (downstream) (Siegfried, 1994). Shaggy targets Armadillo, which modifies its cytoplasmic distribution in response to Wingless signals (Peifer, 1994b).
Ectopic expression of Wingless outside its normal ventral domain has been shown to reorganize the
dorsal-ventral axis of the leg in a non-autonomous manner. Cells that lack shaggy activity can influence the fate of neighboring cells to reorganize dorsal-ventral pattern in the leg, in the same manner as wingless-expressing cells. Therefore, clones of cells that lack shaggy activity exhibit all of the organizer activity previously attributed to wingless-expressing cells, but do so without expressing wingless. (Diaz-Benjumea, 1994).
In the leg disc, HH is secreted by posterior cells and acts at short range to induce dorsal anterior cells to secrete DPP and ventral anterior cells to secrete WG. Complementary patterns of dpp and wg expression are maintained by mutual repression. DPP signaling blocks wg transcription and WG signaling attenuates dpp transcription. This repression is essential for normal axial patterning because it ensures that the dorsalizing and ventralizing activities of DPP and WG are restricted to opposite sides of the leg primordium and meet only at the center of the primordium to distalize the appendage. A similar dorsoventral bias in the choice of dpp or wg expression is revealed by eliminating the activity of protein kinase A, an experimental intervention that mimics the reception of the HH signal. Constitutive activation of the WG signal transduction pathway by loss of Zeste white (Shaggy) kinase mimics the reception of WG signal, and is sufficient to bias dorsal cells to express wg rather than dpp (Jiang, 1996).
Shaggy acts as a repressor of engrailed autoregulation. Genetic epistasis experiments indicate that wg signaling operates by inactivating the zw3 repression of engrailed autoactivation (Siegfried, 1992).
In embryos mutant for armadillo, dishevelled and porcupine, the changes in engrailed expression are identical to those in wingless mutant embryos, suggesting that their gene products act in the wingless pathway (van den Heuvel (1993). dsh and porc act upstream of zw3, and arm acts downstream of zw3 (Siegfried, 1994).
Two genes expressed along the normal wing margin, vestigial and scalloped, are overexpressed at margin-like levels in shaggy-zeste white 3 clones. This phenotype does not depend upon the formation of ectopic bristle precursors and occurs in clones lacking both shaggy-zeste white 3 and the entire achaete-scute complex. As vestigial and scalloped are both involved in early patterning events prior to the stages of bristle specification, these results strongly suggest that shaggy-zeste white 3 is required for the normal specification or maintenance of regional identity in the developing wing blade. The margin-like transformation is only partial, since the expression of apterous (in pupal wings) and wingless and cut (at late third instar) was not reliably altered in shaggy-zeste white 3 clones. shaggy-zeste white 3 may act downstream of localized apterous and wingless expression to specify or maintain margin identity in the wing (Blair, 1994).
cut and achaete are targets of shaggy signaling in the wing margin region reflecting the activity of wg and probably mediating its function. The functional relationship between these genes and wg is the same as that which exists during the patterning of the larval epidermis (Couso, 1994).
Armadillo's role in signal transduction is normally negatively regulated by Shaggy/Zeste-white 3 kinase, which modulates Armadillo protein stability. Two sequences in the N-terminal domain of Armadillo are involved in its degradation. One is a consensus Shaggy/Zeste-white 3 phosphorylation site. The other is a sequence conserved between IkappaB and its fly homolog, cactus, surrounding the serines whose phosphorylation is thought to regulated ubiquitinization and control of protein stability. A mutant protein, Armadillo(S10), was generated with a 54 amino acid deletion in its N-terminal domain. Most of the wild-type Arm protein in an embryo is in adherens junctions, where it is highly phosphorylated; there is relatively little soluble Arm, which is less highly phosphorylated. In contrast, the least highly phosphorylated isoforms of ArmS10 predominate, resembling the pattern of accumulation of wild-type Arm in shaggy mutants. ArmS10 is constitutively active in Wingless signaling; its activity is independent of both Wingless signal and endogenous wild-type Armadillo. Armadillo(S10) is more stable than wild-type Armadillo, suggesting that it is less rapidly targeted for degradation. Armadillo(S10) is more stable and has escaped from negative regulation by Zeste white-3 kinase, and thus accumulates outside junctions even in the absence of Wingless signal. ArmS10 retains the Arm function in junctions even though it is constitutively active for Wg signaling. This suggests that the two Arm functions, the response to Wg signaling and acting as a structural protein in junctions, are independent. Even though overall levels of Arm phosphorylation are low in shaggy/zw3 mutants because the less phosphorylated isoform accumulates outside junctions, junctional Arm remains highly phosphorylated. It is concluded that kinases in addition to Zeste white-3 are implicated in Armadillo phosphorylation. Two models are discussed for the negative regulation of Armadillo in normal development. In one, the simple model, Shaggy/Zw3 negatively regulates Arm by direct phosphorylation within the N-terminus. Another model is suggested by the observation that other kinases besides Shaggy target Arm. An alternative direct target of Shaggy/Zw3 is the tumor suppressor APC (see Drosophila APC-like), which is readily phosphorylated by GSK. This phosphorylation regulates APC binding to beta-catenin, reducing beta-catenin stability. In this model, Shaggy is not required for phosphorylation of Arm in adherens junctions, suggesting that this phosphorylation is mediated by other kinases. The effect of Zw3 inactivation on Arm phosporylation may be solely due to its effects on the stability of soluble Arm (Pai, 1997).
The existence of homologous beta-catenin binding sites in Drosophila Apc raises a question whether Apc interacts with the Drosophila homolog of beta-catenin, the Armadillo protein. To test this possibility an in vitro binding assay was carried out using a bacterially expressed Apc fusion protein containing beta-catenin binding sites and Arm protein translated in vitro. Arm binds to the Apc fragment containing the beta-catenin binding sites, but not to a control composed of a beta galactosidase fusion protein, suggesting that binding between Arm and the Apc fragment is specific. Altogether these results indicate that the beta-catenin binding sites in Apc can substitute for human APC in the down-regulation of beta-catenin, and that the same region interacts directly with Arm (Hayashi, 1997).
shotgun (DE-cadherin] transcription level is regulated through the Wingless pathway. Drosophila genetic studies suggest that in the Wingless (Wg) signaling pathway, the segment polarity gene products, Dishevelled (Dsh), Zeste-white 3 (Zw-3), and Armadillo (Arm), work sequentially; wg and dsh negatively regulate Zw-3, which in turn down-regulates Arm. To biochemically analyze interactions between the Wg pathway and Shotgun, which binds to Arm, three proteins (Dsh, Zw-3, and Arm) were overexpressed in the Drosophila wing disc cell line (clone 8), which responds
to Wg signal. Dsh overexpression leads to accumulation of Arm primarily in the cytosol and elevation of Shotgun at cell junctions. Overexpression of wild-type and dominant-negative forms of Zw-3 decreases and increases Arm levels, respectively, indicating that modulation in Zw-3 activity negatively regulates Arm levels. Overexpression of an Arm mutant with an amino-terminal deletion elevates
Shotgun protein levels, suggesting that Dsh-induced Shotgun elevation is caused by the Arm accumulation induced by Dsh. Moreover, the Dsh-, dominant-negative Zw-3-, and truncated Arm-induced accumulation of Shotgun protein is accompanied by a marked increase in the steady-state levels of Shotgun mRNA, suggesting that transcription of shotgun is activated by Wg signaling. In addition, overexpression of shotgun elevates Arm levels by stabilizing Arm at cell-cell junctions (Yanagawa, 1997).
Armadillo's role in signal transduction is normally negatively regulated by Shaggy/Zeste-white 3 kinase, which modulates Armadillo protein stability. Two sequences in the N-terminal domain of Armadillo are involved in its degradation. One is a consensus Shaggy/Zeste-white 3 phosphorylation site. The other is a sequence conserved between IkappaB and its fly homolog, cactus, surrounding the serines whose phosphorylation is thought to regulated ubiquitinization and control of protein stability. A mutant protein, Armadillo(S10), was generated with a 54 amino acid deletion in its N-terminal domain. Most of the wild-type Arm protein in an embryo is in adherens junctions, where it is highly phosphorylated; there is relatively little soluble Arm, which is less highly phosphorylated. In contrast, the least highly phosphorylated isoforms of ArmS10 predominate, resembling the pattern of accumulation of wild-type Arm in shaggy mutants. ArmS10 is constitutively active in Wingless signaling; its activity is independent of both Wingless signal and endogenous wild-type Armadillo. Armadillo(S10) is more stable than wild-type Armadillo, suggesting that it is less rapidly targeted for degradation. Armadillo(S10) is more stable and has escaped from negative regulation by Zeste white-3 kinase, and thus accumulates outside junctions even in the absence of Wingless signal. ArmS10 retains the Arm function in junctions even though it is constitutively active for Wg signaling. This suggests that the two Arm functions, the response to Wg signaling and acting as a structural protein in junctions, are independent. Even though overall levels of Arm phosphorylation are low in shaggy/zw3 mutants because the less phosphorylated isoform accumulates outside junctions, junctional Arm remains highly phosphorylated. It is concluded that kinases in addition to Zeste white-3 are implicated in Armadillo phosphorylation. Two models are discussed for the negative regulation of Armadillo in normal development. In one, the simple model, Shaggy/Zw3 negatively regulates Arm by direct phosphorylation within the N-terminus. Another model is suggested by the observation that other kinases besides Shaggy target Arm. An alternative direct target of Shaggy/Zw3 is the tumor suppressor APC, which is readily phosphorylated by GSK. This phosphorylation regulates APC binding to beta-catenin, reducing beta-catenin stability. In this model, Shaggy is not required for phosphorylation of Arm in adherens junctions, suggesting that this phosphorylation is mediated by other kinases. The effect of Zw3 inactivation on Arm phosporylation may be solely due to its effects on the stability of soluble Arm (Pai, 1997).
Wnt signaling is a key pathway for tissue patterning during animal development. In Drosophila, the Wnt protein Wingless acts to stabilize
Armadillo inside cells where it binds to at least two DNA-binding factors that regulate specific target genes. One Armadillo-binding protein in
Drosophila is the zinc finger protein Teashirt. A 23 amino acid domain (between aa
692 and 715) in Arm as necessary for the interaction with Tsh. This domain lies in the
most conserved part of the C-terminal domain of Arm. Wingless signaling promotes the phosphorylation and the nuclear accumulation of
Teashirt. This process requires the binding of Teashirt to the C-terminal end of Armadillo. Evidence is presented that the serine/threonine
kinase Shaggy is associated with Teashirt in a complex (Gallet, 1999).
To investigate the effects of Wg signaling on Tsh phosphorylation, Western blots were performed on
proteins extracted from stage 9-11 embryos mutant for different components of the Wg pathway. Mutant embryos that constitutively transduce Wg and those lacking signal transmission were selected. In wild-type embryos, different hyperphosphorylated forms of Tsh are present. In constitutive Wg signaling mutant embryos, the most hyperphosphorylated forms
are predominant. Conversely, in Wg signaling loss-of-function mutants, the
upper band is fainter and the 116 kDa form is more apparent. By probing the
same blot with an anti-tubulin antibody and by densitometric analysis, the relative amount of Tsh in the different mutants can be correlated: there are equal amounts
of Tsh in wild-type and in embryos with gain of Wg signaling function, but there is less Tsh in mutants
lacking Wg signalling function. This is consistent with a decreased level of nuclear Tsh observed in the
absence of Wg function. Taken together, the results indicate that the Tsh phosphorylation and the increasing nuclear level of Tsh
is in part dependent on the Wg pathway. Nevertheless, even in mutants lacking signal transmission, Tsh
is still phosphorylated and localized in the nucleus, indicating that other
factors are acting on Tsh independently of Wg (Gallet, 1999).
Wg signal acts by inhibiting the activity of Sgg, which would otherwise promote the degradation of Arm
inside the cell. Thus Arm accumulates inside the cell and can interact with its partners. Loss of Sgg
activity causes the stabilization of intracellular Arm everywhere in the segment promoting the
production of naked cuticle in the trunk. When Wg does not signal, Sgg is thought to promote phosphorylation of Arm on an
N-terminal motif, leading to Arm degradation via the ubiquitination pathway. In order to test the interaction between Tsh and Sgg, germ-line clones of sgg were induced. The distribution of Tsh was examined in such embryos. As expected, nuclear Tsh level is high, as in embryos
constitutively expressing the Wg pathway. In order to analyse the epistasis between tsh
and sgg, sgg cuticles were examined with or without tsh activity. Whereas sgg germ-line clones give
naked cuticle, absence of tsh gives larvae with
reduced naked cuticle and a lawn of denticles. Therefore Tsh acts downstream of Sgg.
Finally, mmunoprecipitations were performed to test whether Sgg and Tsh are in a complex. Using
affinity-purified anti-Tsh, Sgg co-immunoprecipitates with Tsh.
Together, these results show that Tsh is epistatic to Sgg; that the nuclear Tsh level is also
Sgg-dependent, and that in vivo Tsh is in a protein complex with Sgg (Gallet, 1999).
The protein-serine kinase Shaggy/Zeste-white3 (Sgg/Zw3) is the Drosophila homolog of mammalian glycogen synthase kinase-3
and has been genetically implicated in signal transduction pathways necessary for the establishment of patterning. Sgg/Zw3 is a
putative component of the Wingless (Wg) pathway, and analyses of epistasis suggest that Sgg/Zw3 function is repressed by Wg
signaling. The biochemical consequences of Wg signaling have been investigated with respect to the Sgg/Zw3 protein kinase
in two types of Drosophila cell lines and in embryos. Sgg/Zw3 activity is inhibited following
exposure of cells to Wg protein and by expression of downstream components of Wg signaling: Drosophila frizzled 2 and dishevelled. Wg-dependent inactivation
of Sgg/Zw3 is accompanied by serine phosphorylation. The level of Sgg/Zw3 activity regulates the stability of Armadillo protein and modulates the
level of phosphorylation of Drosophila Axin and Armadillo. Together, these results provide direct biochemical evidence in support of the genetic model of Wg signaling and
provide a model for dissecting the molecular interactions between the signaling proteins. (Ruel, 1999)
Using a yeast two-hybrid screen for proteins
that bind to Armadillo, the Drosophila beta-catenin homolog, a new Drosophila APC homolog, Adenomatous polypopsis coli tumor suppressor homolog 2 (Apc2), has been identified. Apc2 also binds to
Shaggy, the Drosophila GSK-3 homolog. Interference with Apc2 function produces embryonic phenotypes like those of shaggymutants.
Interestingly, Apc2 is concentrated in apicolateral adhesive zones of epithelial cells, along with Armadillo and E-cadherin, which are both
integral components of the adherens junctions in these zones. Various mutant conditions that cause dissociation of Apc2 from these zones also
obliterate the segmental modulation of free Armadillo levels that is normally induced by Wingless signaling. It is proposed that the
Armadillo-destabilizing protein complex, consisting of Apc2, Shaggy, and a third protein, Axin, is anchored in adhesive zones, and that
Wingless signaling may inhibit the activity of this complex by causing dissociation of Apc2 from these zones (Yu, 1999).
Wnt regulation of ß-catenin degradation is essential for development and carcinogenesis. ß-catenin degradation is initiated upon amino-terminal serine/threonine phosphorylation, which is believed to be performed by glycogen synthase kinase-3 (GSK-3) in complex with tumor suppressor proteins Axin and adenomatous polyposis coli (APC). Another Axin-associated kinase is described, whose phosphorylation of ß-catenin precedes and is required for subsequent GSK-3 phosphorylation of ß-catenin. This 'priming' kinase is casein kinase Ialpha (CKIalpha). CKIalpha phosphorylation of ß-catenin precedes and is obligatory for subsequent GSK-3 phosphorylation of ß-catenin. Depletion of CKIalpha inhibits ß-catenin phosphorylation and degradation and causes abnormal embryogenesis associated with excessive Wnt/ß-catenin signaling. This study uncovers distinct roles and steps of ß-catenin phosphorylation, and identifies CKIalpha as a component in Wnt/ß-catenin signaling (Liu, 2002).
The level of cytosolic ß-catenin determines the activation of Wnt responsive genes. Without Wnt stimulation, ß-catenin is constantly degraded by the proteosome. This degradation strictly depends upon ß-catenin phosphorylation, which occurs in a multiprotein complex composed of the following tumor suppressor proteins: adnomatous polyposis coli (APC), Axin, and glycogen synthase kinase-3 (GSK-3). It is believed that in this complex assembled by Axin, GSK-3 phosphorylates the ß-catenin amino-terminal region, thereby earmarking ß-catenin for ubiquitination-dependent proteolysis. Wnt signaling is suggested to inhibit ß-catenin phosphorylation, thus inducing the accumulation of cytosolic ß-catenin, which associates with the TCF/LEF (T cell factor/lymphocyte enhancer factor) family of transcription factors to activate Wnt/ß-catenin-responsive genes. Thus, ß-catenin phosphorylation controls ß-catenin protein level and Wnt signaling (Liu, 2002 and references therein).
Four serine (S)/threonine (T) residues (S33, S37, T41, and S45) at the amino-terminal region of ß-catenin are conserved from Drosophila to human and conform to the consensus GSK-3 phosphorylation site. Indeed, ß-catenin can be phosphorylated by GSK-3 in vitro, and these phospho-S/T residues are critical for ß-catenin recognition by the F box protein ß-Trcp (homolog of Drosophila Slimb), which is the specificity component of a ubiquitination apparatus. The significance of S33, S37, T41, and S45 phosphorylation in ß-catenin degradation is underscored by the observation that mutations at these S/T residues frequently occur in human colorectal cancer and several other malignancies, which are associated with and most likely caused by deregulated accumulation of ß-catenin (Liu, 2002 and references therein).
Whether CKIalpha regulates degradation of Armadillo (Arm), the Drosophila ortholog of ß-catenin, was investigated. Strikingly, RNAi depletion of Drosophila CKIalpha results in a dramatic increase of Arm protein in S2 cells. Furthermore, RNAi depletion of CKIalpha in Drosophila embryos generates a naked cuticle phenotype and a strong expansion of the expression domain of Wingless, which itself is an Arm target gene. This is reminiscent of the phenotype caused by loss-of-function mutations in Drosophila Axin or GSK-3 (zeste-white 3/shaggy) gene. Therefore, the Arm protein accumulation in S2 cells and the segment polarity phenotype in embryos resulting from CKIalpha RNAi together suggest that CKIalpha function is conserved and essential for ß-catenin degradation in both Drosophila and human (Liu, 2002).
Thus ß-catenin phosphorylation in vivo is sequentially carried out by two distinct kinases, CKIalpha and GSK-3. CKIalpha phosphorylation of S45 proceeds and is required for subsequent GSK-3 phosphorylation of T41, S37, and S33. These findings identify CKIalpha as an essential component that controls ß-catenin phosphorylation degradation. This understanding of ß-catenin phosphorylation at a single-residue resolution enables an examination of how ß-catenin mutations found in human cancers disrupt distinct steps in ß-catenin degradation. Thus, mutations surrounding S33 and S37 abolish ß-catenin recognition by ß-Trcp and the ubiquitination of ß-catenin; mutations at T41 prevent GSK-3 phosphorylation of S37 and S33 and thus ß-Trcp recognition; and mutations at S45 block the priming phosphorylation by CKIalpha and consequently all phosphorylation events by GSK-3. Each of these mutations causes ß-catenin to escape recognition by ß-Trcp and subsequent degradation (Liu, 2002).
CKIalpha was among the first protein kinase activities to be discovered, yet its function and regulation remain poorly understood. Like GSK-3, CKIalpha is expressed ubiquitously and appears to be constitutively active, consistent with its role in ß-catenin degradation. The finding that ß-catenin is a CKIalpha substrate in vivo therefore identifies CKIalpha as a central player in cell fate determination and growth control. This study shows that CKIalpha controls segment polarity during Drosophila embryogenesis. Interestingly, ß-catenin phosphorylation by CKIalpha and by GSK-3 are both stimulated by Axin. In fact, CKIalpha and GSK-3 bind to different regions of Axin such that they 'sandwich' ß-catenin in the Axin complex, thereby promoting effective ß-catenin phosphorylation. Since Wnt signaling inhibits only GSK-3 but not CKIalpha phosphorylation of ß-catenin, CKIalpha may represent a node at which other signaling pathways regulate ß-catenin protein level. Since depletion of CKIalpha causes ß-catenin accumulation in a manner similar to a lack of function of GSK-3, APC, or Axin, CKIalpha is a candidate tumor suppressor (Liu, 2002).
dpp expression has been examined in two groups of dorsal ectoderm cells at the posterior end of the embryo, in abdominal segment 8 and the telson. These dpp-expressing cells become tracheal cells in the posterior-most branches of the tracheal system (Dorsal Branch10, Spiracular Branch10, and the Posterior Spiracle). These branches are not identified by reagents typically used in analyses of tracheal development, suggesting that dpp expression confers a distinct identity upon posterior tracheal cells. dpp posterior ectoderm expression begins during germ band extension and continues throughout development. The sequences responsible for these aspects of dpp expression have been isolated in a reporter gene. An unconventional form of Wingless (Wg) signaling, Dpp signaling, and the transcriptional coactivator Nejire (CBP/p300) are required for the initiation and maintenance of dpp expression in the posterior-most branches of the tracheal system. These data suggest a model for the integration of Wg and Dpp signals that may be applicable to branching morphogenesis in other developmental systems (Takaesu, 2002).
dpp expression in posterior
tracheal branch anlagen appears to be initiated by prior episodes of wg
and dpp expression in the undifferentiated dorsal ectoderm.
The maintenance of dpp expression in posterior tracheal
branches appears to require continuous input from wg and
from a dpp feedback loop. The initiation and maintenance
of dpp expression in posterior tracheal branches also requires
continuous nej activity. Overall, the data are consistent
with the following combinatorial signaling model. The transcriptional activator Medea (Med, signaling for the Dpp
pathway) interacts with the transcriptional activator Arm
(signaling for the Wg pathway) via the transcriptional coactivator
Nej. This multimeric complex initiates and, with
continuous signaling, maintains dpp expression in posterior
tracheal branches with the help of Zw3. These data extend
previous studies of dpp expression and Dpp signaling in
several ways. nej has been reported to participate in
Dpp signaling. Expression from Dpp responsive
enhancers is reduced in nej zygotic mutant
embryos. While they show that nej3 enhances dpp wing
phenotypes, this study shows that Mad1 enhances nej3 embryonic phenotypes. The Dorsal Trunk Branch
forms normally in Mad12 zygotic mutant embryos, and the Dorsal Trunk Branch appears normal in
Med1 mutants. nej is involved in mediating combinatorial signaling by the Wg and Dpp pathways and the involvement
of nej in morphogenesis of Dorsal Branch, Spiracular Branch, and the Posterior Spiracle is demonstrated. A region of the histone acetyltransferase domain
of Nej binds to Mad. Further study is needed to reveal the mechanisms used by Nej to interact with Wg and Dpp signaling. Several questions remain
about the regulation of dpp expression by Wg, Dpp, and Nej.
Two questions arise about the mechanism of signal integration:
how is zw3 involved and how is Nej recruited to bridge
the two pathways? It is tempting to speculate that, in
response to a Wg or a Dpp signal, Zw3 (a serine-threonine
kinase) is involved in Nej recruitment. Numerous studies
have shown that p300/CBP transcriptional coactivation
functions are stimulated by its phosphorylation, but the
site of phosphorylation has never been mapped. Other questions concern the molecular nature of the enhancers that direct dpp expression in the
posterior tracheal branches. A 54-nucleotide region has been identified that contains two sets of
conserved, overlapping consensus binding sites for dTCF
and Mad/Med. Analyses of DNA-protein interactions predicted
by the data involving this candidate combinatorial
enhancer have begun (Takaesu, 2002).
In a function that seems to be distinct from its function in segment polarity, Shaggy can be considered a proneural gene. achaete and scute are expressed in a spatially restricted pattern. Both genes provide neural potential to cells. The domains of expression depend partly on extramacrochaetae, whose product is itself spatially restricted. extramacrochaetae acts as a negative post-translational regulator of achaete and scute. The protein kinase Shaggy also represses achaete and scute at many sites but may act via intermediate transcription factors. However shaggy and extramacrochaetae act synergistically; molecular studies suggest that they may be part of the same pathway. Shaggy is functionally homologous to the mammalian glycogen synthase kinase-3 and analogy with the known physiology of this enzyme, suggests that this function of Shaggy may result from the "constitutive" activity. At the site where a single neural precursor will develop, achaete and scute are initially expressed in a group of equivalent cells. shaggy is required downstream of Notch for transduction of the inhibitory signal. This second role of shaggy may be due to modulation of enzymatic activity during signaling (Simpson, 1993).
Within clusters mutant for shaggy, where several cells of a cluster follow the neural fate and differentiate bristles, it has been shown that cells display identical neuronal specificity: stimulation of the bristles evokes the same leg cleaning response; backfilling of single neurons reveals similar axonal projections in the central nervous system. This provides direct experimental evidence that the cells of a proneural cluster are developmentally equivalent (Simpson, 1990).
The secreted signaling molecule Hedgehog regulates gene expression in target cells in part by preventing proteolysis of the full-length Cubitus interruptus (Ci-155) transcriptional activator to the Ci-75 repressor form.
Ci-155 proteolysis depends on phosphorylation at three sites by Protein Kinase A (PKA). These phosphoserines prime further phosphorylation at adjacent Glycogen synthase kinase 3 (GSK3) and Casein kinase I (CK1) sites. Alteration of the GSK3 or CK1 sites prevents Ci-155 proteolysis and activates Ci in the absence of Hedgehog. Ci-155 proteolysis is also inhibited if cells lack activity of the Drosophila GSK3. Conversely, Ci-155 levels are reduced in Hedgehog-responding cells by overexpression of PKA and the Drosophila CK1, Double-time, a regulator of circadian rhythms. Thus Shaggy/GSK3 is implicated in the functioning of the Hedgehog pathway, in addition to its well known role in the Wingless pathway (Price, 2002).
Phosphorylation of Ci at three defined PKA sites primes further phosphorylation at adjacent GSK3 and CK1 sites. This PKA-primed phosphorylation could be catalyzed by purified mammalian GSK3ß and CK1delta enzymes or by activities in Drosophila embryo extracts. Changing the target serines of either GSK3 or CK1 consensus sites to alanines prevents proteolysis of Ci-155 to Ci-75 in flies. This result was demonstrated both by Western blots of embryo extracts and by assaying for the activity of Ci-75 as a transcriptional repressor in wing imaginal discs. It is argued that the resistance of these altered Ci molecules to proteolysis results from altered phosphorylation rather than a change in amino acid identity per se, because elimination of Sgg GSK3 activity produces a similar result and because the PKA sites required for priming further phosphorylation must themselves be intact in order for Ci-155 to be proteolyzed to Ci-75 (Price, 2002).
How extensively must Ci be phosphorylated in order to be proteolyzed? Whether each potential PKA, GSK3, or CK1 phosphoacceptor site is essential for Ci proteolysis has not been tested in flies. Evidence obtained in tissue culture cell studies suggests that Ci proteolysis is largely inhibited by alteration of single PKA sites, and at least one PKA site (site 1) is critical in flies. Alteration of two GSK3 sites, adjacent to PKA sites 2 and 3, prevents Ci-155 proteolysis. Hence, the view is favored that each of the phosphorylation sites in this region of Ci contributes significantly to Ci-155 proteolysis (Price, 2002).
Inhibition of the 26S proteosome in clone 8 tissue culture cells leads to the accumulation of highly phosphorylated full-length Ci forms of lowered gel mobility, especially if phosphatase activity is also inhibited. Ci-155 from untreated clone 8 cells can be separated into about six isoforms by isoelectric focusing. The location of phosphorylated residues was not mapped in either of these studies. However, it appears that Ci phosphorylated on a small number of sites, perhaps largely PKA sites, is stable enough to visualize, whereas subsequent, perhaps cooperative, phosphorylation, most likely on adjacent GSK3 and CK1 sites, leads to rapid Ci-155 proteolysis and is therefore evident only if proteolysis is artificially inhibited (Price, 2002).
The basic arrangement of PKA sites flanked by PKA-primed GSK3 and CK1 sites is conserved in Gli2 and Gli3 for each of the three PKA sites in Ci, with an additional fourth motif between PKA sites 2 and 3 of Ci. The identity of amino acids in each cluster extends beyond the consensus SGSK3RRXSPKAXXSCK1. For instance, PKA site 1 has an adjacent CK1 site followed by a second CK1-primed site (RRXSPKAXXSCK1XXSCK1), but there are no GSK3 sites. PKA sites 2 and 3 are flanked by GSK3 sites and CK1 sites (SGSK3RRXSPKAXXSCK1), but only site 3 includes a second GSK3 site (SGSK3XXXSGSK3RRXSPKA). Ignoring the possibility of additional interstitial phosphorylations in this region due to GSK3 priming of CK1 sites and vice versa, Ci contains a total of eight PKA-primed GSK3 or CK1 phosphorylation sites, whereas Gli2 and Gli3 contain eleven and nine, respectively, in this region of less than 80 amino acids. Gli1 has only two PKA sites in this region with three associated CK1 sites and only one GSK3 site. Commensurate with sequence conservation, both Gli2 and Gli3 appear to be proteolyzed when expressed in Drosophila, whereas Gli1 remains full-length. Processing of Gli proteins in flies appears to correspond, at least approximately, to their fate in their normal environment. These data are consistent with the proposal that a conserved mechanism of PKA-dependent proteolysis of Ci/Gli proteins depends on creating highly phosphorylated clusters of regularly spaced phosphoserine residues (Price, 2002).
How do multiple phosphorylations of Ci target it for degradation? Paired GSK3 phosphorylation sites are crucial for recognition of ß-catenin by Slimb/ß-TrCP, but they fall within a more specific consensus sequence DS(P)GXXS(P) that is conserved in IkappaB. None of the GSK3 or, of course, the CK1 or PKA sites in Ci conform to this consensus. It is possible that Slimb/ß-TrCP recognizes more epitopes than currently appreciated or that the presence of multiple weak binding sites collectively contributes to association with Slimb. The latter mechanism has been demonstrated for the recognition of yeast Sic1, which is phosphorylated within multiple suboptimal binding sequences, by the F box protein Cdc4. At least six such sites in Sic1 must be phosphorylated to exceed a physiological threshold for recognition (Price, 2002).
Ci phosphorylation might create a binding site for a Ci partner other than Slimb. The apparent requirement for extensive phosphorylation of Ci could easily be rationalized if the binding partner presented an extensive surface for electrostatic interaction, such as the armadillo repeat region of ß-catenin. The binding of this ß-catenin domain to repeated serine/threonine-rich motifs of APC (Adenomatous Polyposis Coli) protein is stimulated by phosphorylation of APC. Thus, structures analogous to the ß-catenin armadillo repeats might accommodate phosphorylation-dependent binding of repeated motifs in Ci. Binding of ß-catenin itself to phosphorylated Ci would, of course, provide a high-affinity Slimb binding partner within the Ci complex and could explain the observation that Ci degradation is proteosome dependent but does not involve detectable ubiquitination of Ci (Price, 2002).
Loss of Sgg activity in wing disc clones induces Ci-155 to levels at least as high as the A/P border, but slightly lower than in PKA mutant clones. It is inferred that elevated Ci-155 levels result from inhibition of proteolysis to Ci-75 because ci RNA levels were unchanged and because Ci-75 repressor activity was largely absent from posterior smo sgg mutant clones in wing discs expressing Ci ubiquitously. In these clones there appears to be a low level of repressor, raising the possibility of a second GSK3 contributing in a minor way to the phosphorylation and proteolysis of Ci (Price, 2002).
Anterior sgg mutant clones induce some ectopic expression of Hh target genes but do not reproduce the strong phenotypes of PKA mutant clones, as assessed by Hh target gene expression, disc morphology, and adult morphology. Two possible explanations for the differences between PKA and sgg mutant clone phenotypes are offered. One possibility is that Ci lacking PKA site phosphorylation (and hence GSK3 and CK1 site phosphoserines) is more active than Ci lacking only GSK3 site phosphorylation. No significant differences have been found in the activity of Ci lacking three PKA sites (Ci3m), as compared to Ci lacking both GSK3 sites (CiNm) for the transgenic lines tested in wing discs or embryos. However, it was found that Ci lacking five consensus PKA sites (Ci5m) is more active than Ci3m. The fourth and fifth PKA sites are not flanked by GSK3 or CK1 sites. The phosphorylation of these PKA sites might therefore reduce the activity of Ci in sgg mutant clones relative to PKA mutant clones (Price, 2002).
A second possibility is that sgg mutations may prevent Hh target gene expression despite generating a phosphoform of Ci that is adequately activated and protected from proteolysis. This hypothesis was investigated by examining the phenotype of clones lacking both PKA and Sgg activities. Since GSK3 phosphorylation of Ci depends on priming by PKA phosphorylation, the absence of Sgg activity should not alter the phosphorylation state of Ci from that in PKA mutant clones. Nevertheless, the levels of Ptc protein induced in sgg PKA smo mutant clones are much lower than for PKA smo mutant clones in the wing pouch cells of the disc, and pattern duplication of wings normally associated with PKA mutant clones is suppressed by the additional inactivation of sgg. Thus, it is possible that a Sgg substrate in addition to Ci can affect Hh target gene expression, at least in anterior presumptive wing cells. This might contribute to the failure of sgg mutant clones in the wing pouch to induce ectopic Ptc expression (Price, 2002).
It is important to note that the positive input of Sgg on Hh target gene expression inferred above is evident only in the artificial circumstances of eliminating PKA and Sgg activities by genetic mutation. PKA and Sgg are normally active in anterior cells away from the A/P border, as manifested by the proteolysis of Ci-155, and any regulation of their activities by Hh at the A/P border is unlikely to be as dramatic as mutational inactivation. Indeed, there was no consistent reduction in the expression of ptc or dpp reporters in sgg mutant clones at the A/P border. Hence, the relevance of Sgg substrates other than Ci during normal development remains to be established (Price, 2002).
Only one of the eight putative CK1 genes in Drosophila has been extensively investigated genetically. Weak alleles of this gene were named double-time because they alter circadian rhythms. Stronger alleles affect imaginal disc growth and patterning in a variety of ways, but relating these phenotypes to specific cellular processes or signaling pathways has been hampered by the limited growth and viability of cells homozygous for null and strong alleles. This property of dbt/dco also limits these investigations to showing that overexpression of Dbt can enhance the reductions of Ci-155 levels at the A/P border of wing discs due to PKA hyperactivity. This observation is consistent with the idea that increased PKA-primed phosphorylation of Ci by Dbt can promote Ci-155 proteolysis even in cells exposed to Hh, but it was not directly demonstrated that proteolysis is responsible for the reduced Ci-155 levels observed, nor does this result show that Dbt is normally involved in Ci phosphorylation. Dbt remains a good candidate for the CK1 homolog that phosphorylates Ci. It is a member of the CK1 delta/epsilon family, which has been implicated in Wnt signaling in Xenopus and in mammalian tissue culture cells (Price, 2002).
The identification of GSK3 and CK1 as components of the Hh signaling pathway extends previously noted similarities with the Wnt signaling pathway. In addition to these kinases, the F box protein Slimb is shared between the pathways, and both pathways include a component with similarity to the G protein-coupled receptors Smo on the Hh pathway and Frizzled, the Wg receptor. Finally, both pathways share the feature of constitutive phosphorylation-dependent degradation of a key effector that is reversed by ligand signaling. These shared components and other similarities invite speculations about 'crosstalk' and about conserved mechanisms (Price, 2002).
Even though reduced GSK3 activity can stabilize Ci-155 and ß-catenin in wing discs, in wild-type discs, Ci-155 levels are not elevated in cells where Wg signals and ß-catenin is not stabilized by Hh signaling. These observations are reminiscent of the independent transmission of insulin and Wnt signals in vertebrate cells. Insulin stimulation leads to inactivation of GSK3 by phosphorylation at a specific PKB site, but GSK3 in complex with Wnt pathway components is spared from phosphorylation. Wnt signaling does not inactivate GSK3 by the same phosphorylation, although some reduction in total cellular GSK3 activity can be measured and Wnt signaling does reduce the phosphorylation of specific Wnt pathway components by GSK3. The relevant substrates for GSK3 in the insulin pathway are primed by prior phosphorylation, as is the case for Ci; however, axin, APC, and ß-catenin GSK3 sites do not appear to depend on priming. Thus, a combination of sequestration of GSK3 subpopulations through binding interactions and the use of different substrate sites insulate the Wnt pathway from the insulin pathway and may similarly segregate Wnt and Hh signaling pathways despite the common use of GSK3 (Price, 2002).
The involvement of GSK3 and CK1 in Ci-155 proteolysis raises the exciting possibility that Hh might regulate the activity of one or both of these kinases. Regulation of GSK3 activity is a particularly appealing mechanism because the key regulatory event in Wnt signaling is generally thought to be the inhibition of GSK3 activity. The mechanism by which Wnt signaling regulates GSK3 is still not clear but appears to involve several ancillary proteins, other kinases, and a phosphatase. Thus, similar complexity might be anticipated in the Hh signaling pathway and perhaps the participation of yet more proteins previously known for their involvement in Wnt signaling (Price, 2002).
The Drosophila protein Shaggy (Sgg, also known as Zeste-white3,
Zw3) and its vertebrate ortholog glycogen synthase kinase 3
(GSK3) are inhibitory components of the Wingless (Wg) and
Wnt pathways. Sgg is also a negative regulator
in the Hedgehog (Hh) pathway. In Drosophila, Hh acts both by
blocking the proteolytic processing of full-length Cubitus interruptus,
Ci (Ci155), to generate a truncated repressor form(Ci75),
and by stimulating the activity of accumulated Ci155.
Loss of sgg gene function results in a cell-autonomous accumulation
of high levels of Ci155 and the ectopic expression of Hh-responsive
genes including decapentaplegic and wg. Simultaneous
removal of sgg and Suppressor of fused, Su(fu), results
in wing duplications similar to those caused by ectopic Hh
signaling. Ci is phosphorylated by GSK3 after a primed phosphorylation
by protein kinase A (PKA), and mutating GSK3
phosphorylation sites in Ci blocks its processing and prevents the
production of the repressor form. It is proposed that Sgg/GSK3 acts
in conjunction with PKA to cause hyperphosphorylation of Ci,
which targets it for proteolytic processing, and that Hh opposes
Ci proteolysis by promoting its dephosphorylation (Jia, 2002).
During Drosophila limb development, posterior (P)-compartment cells express and secrete Hh that induces adjacent anterior (A)-compartment cells to express target
genes including dpp, wg (leg only) and patched (ptc) by regulating the transcription factor Ci. In A-compartment cells distant
from the AP compartment boundary, Ci is processed to generate a
truncated repressor form (Ci75) that represses a subset of Hh-responsive genes including dpp. In A-compartment cells adjacent
to the AP compartment border, Hh signaling blocks Ci processing
to generate Ci75, and causes the accumulation of full-length Ci
(Ci155). In addition, high levels of Hh stimulate a distinct
transcriptional activation activity of Ci155, which is required for the expression of Hh-responsive genes such as ptc (Jia, 2002 and references therein).
In both wing and leg discs, loss of sgg function in the A
compartment either by using sgg mutations or by overexpressing
a dominant negative form of GSK3 (DN-GSK3) causes the
accumulation of high levels of Ci155 in a cell-autonomous fashion
without affecting ci-lacZ expression. In wing discs, anterior sgg- cells or DN-GSK3-expressing cells located outside the wing pouch region
ectopically express dpp, which is repressed by Ci75.
However, anterior sgg-
cells do not ectopically activate ptc, which is
activated by Ci155. In leg discs, anterodorsal sgg-
cells distant from the AP boundary ectopically express wg and low levels
of dpp, a phenotype similar to that associated with sgg
PKA double-mutant cells in which both Wg and Hh signaling
pathways are ectopically activated. As in the case of wing discs,
sgg- cells seem to transduce low levels of Hh signaling activity, because wg is not fully activated, and little, if any, ptc is expressed. One hypothesis that accounts for these
observations is that loss of sgg function affects Ci processing to
generate Ci75 but does not stimulate the activity of Ci155 (Jia, 2002).
The activity of Ci155 is regulated by several mechanisms including
attenuation by Su(fu). Whereas loss of Su(fu) function does
not cause any significant phenotypes, it dramatically enhances sgg- phenotypes. For example, anterior sgg;Su(fu) double-mutant clones organize wing duplication, whereas sgg
single-mutant clones form only small outgrowths. In
wing discs, anterior sgg;Su(fu) double-mutant cells activate low
levels of ptc, which is not ectopically expressed in sgg
single-mutant cells. In leg discs, anterior sgg;Su(fu) double-mutant
cells express ptc and high levels of wg. Hence, Ci155 accumulated in sgg- cells is largely inactive and its activity is
stimulated by removal of Su(fu) function (Jia, 2002).
Ectopic activation of the Wg pathway by overexpression of a
constitutively active form of Armadillo (DeltaArm) does not cause the
accumulation of high levels of Ci155 or activate any Hh-responsive
genes. Hence, the constitutive Hh
signaling activity in sgg-
cells is not secondary to aberrant activation
of the Wg pathway in these cells. Hh induces stabilization
of Smoothened (Smo), a seven-transmembrane protein that
transduces the Hh signal, but anterior sgg-
cells do not stabilize Smo. In addition, sgg;smo double-mutant cells, like sgg single-mutant cells, accumulate high levels of Ci155,
suggesting that Sgg acts downstream of Smo to regulate Ci processing (Jia, 2002).
The proteolytic processing of Ci requires the activities of
several intracellular Hh signaling components, including PKA and
the kinesin-related protein Costal2 (Cos2). Overexpressing
either Cos2 or a constitutively active form of PKA (mC*) blocks the
accumulation of Ci155 induced by Hh. In contrast,
wing discs overexpressing mC* or Cos2 accumulate high levels of
Ci155 after treatment with 50 mMLiCl, a specific inhibitor of GSK3
kinase activity. These observations suggest that Sgg acts downstream of, or in parallel with, PKA and Cos2 to regulate Ci processing (Jia, 2002).
PKA promotes Ci processing by directly phosphorylating it at
multiple sites in its carboxy-terminal region. Whether Sgg/GSK3 also regulates Ci processing by direct phosphorylation was also investigated. The canonical GSK3-phosphorylation site
consists of two Ser/Thr residues separated by three amino acids:
(Ser/Thr 0)-X-X-X-(Ser/Thr +4). Phosphorylation of Ser/Thr at
the +4 position by a priming kinase allows GSK3 to phosphorylate
Ser/Thr at the 0 position. Examination of Ci sequence reveals
three GSK3 consensus sites (Ser 852, Ser 884 and Ser 888) adjacent
to two previously identified PKA phosphorylation sites (Ser 856 and
Ser 892). To determine whether Ci can be a direct
substrate of Sgg/GSK3, an in vitro kinase assay was carried out. Three
glutathione S-transferase (GST)-Ci fusion proteins containing Ci
fragments from amino acids 441-1,065 were generated: GST-Ci
contains wild-type sequence; GST-Ci-3P has three PKA sites
mutated (S838A, S856A and S892A); and GST-Cim3 has three
GSK3 consensus sites mutated (S852A, S884A and S888A).
GST-Ci is specifically phosphorylated by PKA but not by GSK3
without prior PKA treatment; however, it
becomes a good substrate for GSK3 after primed phosphorylation
by PKA. In contrast, neither GST-Ci-3P nor GST-Cim3 can be phosphorylated by GSK3 even after PKA treatment. These results suggest that primed phosphorylation by PKA at Ser 856 and Ser 892 allows GSK3 to phosphorylate Ci at Ser 852, Ser 884 and Ser 888 (Jia, 2002).
To determine whether Sgg/GSK3 phosphorylates Ci in vivo, Ci phosphorylation was examined in cl-8 cells treated with or without
LiCl, which specifically blocks GSK3 kinase activity. Treating cl-8 cells with
both a proteasome inhibitor, MG132, and a phosphatase inhibitor,
okadaic acid (OA), results in the accumulation of hyperphosphorylated
forms of Ci155, which exhibit much slower electrophoretic
mobility on SDS polyacrylamide gel than unphosphorylated or
hypophosphorylated forms. Treating cells with
MG132 and OA in the presence of 50 mMLiCl results in hypophosphorylation
of Ci because it eliminates the slowest-migrating forms
of Ci155. These observations suggest that inhibition
of Sgg/GSK3 kinase activity affects Ci phosphorylation in intact
cells. The residual phosphorylation of Ci155 in the presence of LiCl
is probably due to phosphorylation by PKA because LiCl does not
inhibit PKA kinase activity (Jia, 2002).
If Ci processing is regulated by GSK3 phosphorylation, one
would predict that mutating GSK3-phosphorylation sites in Ci
should block its processing to generate the Ci75 repressor. To test
this, UAS transgenes were generated containing hemagglutinin
(HA)-tagged wild-type (UAS-HA-Ci) or mutant Ci (UAS-HA-Cim3);
these were expressed in wing discs using the Gal4/UAS
system. Whereas HA-Ci is partially processed into Ci75, HA-Cim3
does not give rise to detectable Ci75. The
effect on the production of Ci75 of mutating GSK3-phosphorylation
sites was also examined using an in vivo function assay. Either wild-type or mutant Ci was ectopically expressed in the P-compartment of wing discs
carrying smo- clones, and hh-lacZ expression, which is
inhibited by Ci75, was examined. P-compartment smo-
cells expressing HA-Ci block hh-lacZ expression, indicating that wild-type
Ci is processed to generate Ci75 in the absence of Hh signaling. In
contrast, P-compartment smo- cells expressing HA-Cim3 do not
repress hh-lacZ expression, indicating that HA-Cim3 does
not produce Ci75 in vivo. Hence, mutating GSK3-phosphorylation
sites in Ci affects its proteolytic processing to generate the repressor
form (Jia, 2002).
To assess the importance of individual GSK3-phosphorylation
sites for Ci processing, two additional mutant forms of
Ci (HA-Cim1 and HA-Cim2) were examined using the in vivo function
assay. P-compartment smo- cells expressing HA-Cim2 partially
block hh-lacZ expression, suggesting that lack of phosphorylation
at Ser 884 and Ser 888 attenuates Ci processing. P-compartment
smo- cells expressing HA-Cim1 also partially inhibit
hh-lacZ expression, however, to a lesser extent than those expressing
HA-Cim2, suggesting that mutating Ser 852 greatly impedes, although does not completely abolish, Ci processing. Hence, efficient processing of Ci seems to require phosphorylation at all three GSK3 sites (Jia, 2002).
Taken together, these data suggest that Sgg/GSK3 acts in conjunction
with PKA to promote hyperphosphorylation of Ci, which is
essential for efficient Ci processing to generate its repressor form.
The requirement for multiple phosphorylation seems to be a general
mechanism to regulate proteolysis of regulatory proteins. The
involvement of multiple phosphorylation may provide a way for Hh
to differentially regulate Ci by controlling its level of phosphorylation.
For example, low levels of Hh may cause partial dephosphorylation
of Ci by opposing Sgg, resulting in an inhibition of Ci
processing to generate Ci75 but leaving Ci155 inactive. In contrast,
high levels of Hh may cause complete dephosphorylation of Ci by
opposing PKA, which not only blocks Ci processing but also
stimulates the activity of accumulated Ci155 (Jia, 2002).
GSK3 is involved in multiple signaling pathways, raising the
question of how its activity is selectively regulated by individual
pathways. An emerging theme is that GSK3 is present, together with
its substrates, in distinct complexes that are regulated by different
upstream stimuli. Future study will determine whether Sgg/GSK3
forms a complex with Cos2 or Ci and whether Hh regulates Sgg/
GSK3 within the complex. In vertebrates, three Gli proteins (Gli1,
Gli2 and Gli3) are implicated in transducing Hh signals. Interestingly,
all three Gli proteins contain multiple GSK3-phosphorylation
consensus sites adjacent to PKA sites, raising the possibility that GSK3 may regulate Gli proteins in vertebrate Hh pathways. Hh and Wnt signaling pathways act in synergy in certain developmental contexts. The finding that GSK3
is involved in both Hh and Wnt pathways raises the possibility that these two pathways might converge at GSK3 in certain developmental processes (Jia, 2002).
Hedgehog (Hh) proteins control animal development by regulating the Gli/Ci family of transcription factors. In Drosophila, Hh counteracts phosphorylation by PKA, GSK3, and CKI to prevent Cubitus interruptus (Ci) processing through unknown mechanisms. These kinases physically interact with the kinesin-like protein Costal2 (Cos2) to control Ci processing and Hh inhibits such interaction. Cos2 is required for Ci phosphorylation in vivo, and Cos2-immunocomplexes (Cos2IPs) phosphorylate Ci and contain PKA, GSK3, and CKI. By using a Kinesin-Cos2 chimeric protein that carries Cos2-interacting proteins to the microtubule plus end, it was demonstrated that these kinases bind Cos2 in intact cells. PKA, GSK3, and CKI directly bind the N- and C-terminal regions of Cos2, both of which are essential for Ci processing. Finally, it was shown that Hh signaling inhibits Cos2-kinase complex formation. It is proposed that Cos2 recruits multiple kinases to efficiently phosphorylate Ci and that Hh inhibits Ci phosphorylation by specifically interfering with kinase recruitment (Zhang, 2005).
To facilitate detection of protein-protein interaction between Cos2 and its binding partners in vivo, a Kinesin/Cos2 chimeric protein (Kinco) was generated in which the microtubule binding domain of Cos2 is replaced by the motor domain of Drosophila KHC. Kinco moves to the microtubule plus end and accumulates near the basal surface of imaginal disc epithelial cells. Strikingly, Kinco carries all the known Cos2 binding proteins to the same subcellular compartment, leading to colocalization. PKAc, GSK3, and two CKI isoforms, CKIα and CKIϵ, all colocalize with Kinco at the microtubule plus end, demonstrating that these kinases associate with Cos2 in intact cells. Hence, Kinco provides a powerful tool to determine if a protein interacts with Cos2 in vivo. In addition, Kinco colocalizes with Cos2-interacting proteins in cultured Drosophila cells such as S2 and cl8 cells. It is conceivable that one can use such a cell-based colocalization assay to identify additional proteins that form a complex with Cos2. Furthermore, it is also possible to extend this approach to other protein complexes by generating appropriate Kinesin chimeric 'bait' proteins (Zhang, 2005).
By using immunoprecipitation and GST pull-down assays, the kinase interaction domains were mapped to the microtubule binding (MB) and C-terminal (CT) of Cos2. GST fusion proteins containing either of these domains bind purified recombinant PKAc, GSK3, and CKI, suggesting that these kinases directly bind Cos2. However, the possibility cannot be rule out
that these kinases may have additional contacts with other components in the Cos2 complex. Indeed, it was found that CKI can bind Ci in yeast (Zhang, 2005).
Several lines of evidence suggest that Cos2/kinase interaction plays an important role in regulating Ci phosphorylation and processing: (1) Ci phosphorylation is compromised in cos2 mutants; (2) the kinase-interacting domains in Cos2 are essential for Ci processing; (3) overexpressing multiple kinases can bypass the requirement of Cos2 for Ci processing (Zhang, 2005).
PKAc, GSK3, and CKI appear to bind competitively to Cos2; however, since Cos2 can dimerize and each Cos2 protein contains two kinase binding domains, a Cos2 dimer could in principle bind all three kinases simultaneously. It is possible that these kinases might not form a tight complex with Cos2 in a stoichiometric manner, which could explain why purification of endogenous Cos2 complexes failed to identify any of these kinases. However, by using in vitro kinase assay and Western blot analysis, the association of PKAc, GSK3, and CKI with endogenous Cos2 was detected. It is likely that interactions between Cos2 and kinases are transient; however, such interactions could increase local concentrations of these kinases; this greatly facilitates Ci hyperphosphorylation (Zhang, 2005).
It has been demonstrated that Hh induces Ci dephosphorylation in cl8 cells; however, it is not clear whether Hh blocks Ci phosphorylation by all three kinases or a subset of them. By using an antibody that specifically recognizes a phosphorylated PKA site in Ci, it was found that Hh partially inhibits PKA phosphorylation of Ci in wing discs. Consistent with this, Hh only partially blocks Cos2/PKA interaction. In contrast, Hh appears to have a more profound influence on the interaction between Cos2 and CKI or GSK3. Furthermore, CKI and GSK3 kinase activities associated with endogenous Cos2 diminishes upon Hh stimulation and Cos2IPs phosphorylates Ci to a lesser extent after Hh treatment. These observations suggest that Ci phosphorylation by CKI and GSK3 is likely to be inhibited by Hh in vivo (Zhang, 2005).
Several mechanisms may contribute to the regulation of Cos2-Ci-kinase complex formation by Hh. (1) The finding that PKAc, GSK3, and CKI bind Cos2 domains that also interact with Smo raises a possibility that Smo/Cos2 interaction may exclude kinases from binding to Cos2. Indeed, a membrane-tethered form of SmoCT (Myr-SmoCT) interferes with Cos2-Ci-kinase complex formation. (2) Smo/Cos2 interaction at the cell surface may induce conformational change in Cos2, which could mask its kinase interacting domains. (3) Cos2 is phosphorylated in response to Hh. Phosphorylation of Cos2 could regulate its interaction with one or more kinases. (4) There is evidence that Hh induces dissociation of Ci from Cos2, which may further decrease the accessibility of Ci to the kinases. This may explain why Hh induces more significant dissociation of PKAc from Ci than from Cos2. (5) Hh induces degradation of Cos2 in P compartment cells as well as in cells immediately adjacent to the A/P boundary; this may lead to a chronic destruction of Cos2-Ci-kinase complexes. However, it appears that only high levels of Hh induce Cos2 degradation in vivo. Low levels of Hh may prevent Ci phosphorylation with different mechanisms such as those described above (Zhang, 2005).
The following model is proposed for the regulation of Ci phosphorylation by Cos2 and Hh. In the absence of Hh, Cos2 scaffolds multiple kinases and Ci into proximity, thus increasing the accessibility of Ci to these kinases and facilitating extensive phosphorylation of Ci. Upon Hh stimulation, Cos2 complexes are recruited to the cell surface via Smo, leading to disassembly of Cos2-Ci-kinase complexes. As a consequence, Ci phosphorylation is compromised and Ci processing is blocked. This model has several interesting parallels to that proposed for the Wnt pathway. In quiescent cells, both pathways employ large protein complexes to bring kinases and their substrates in close proximity, resulting in phosphorylation and proteolysis of the transcription factor (Ci) or effector (β-catenin). Upon ligand stimulation, both pathways recruit the cytoplasmic signaling complex to the cell surface and cause dissociation of the complex, leading to dephosphorylation and stabilization of the transcription factor/effector. Interestingly, both pathways use common kinases, including GSK3 and CKI. However, these kinases together with their substrates form distinct signaling complexes assembled by pathway-specific scaffolding proteins (Cos2 and Axin in the Hh and Wnt pathways, respectively). Pathway activation is achieved by a specific interaction between the receptor system and the scaffolding protein (Smo/Cos2 interaction in the Hh pathway and LPR5/6/Axin interaction in the Wnt pathway). Thus, each pathway only controls the pool of kinases in the same complex with the pathway effector, leading to pathway-specific regulation of substrate phosphorylation. The combinatorial mechanism by which pathway-specific scaffolds bring common kinases into proximity with their substrates thus appears to be a general one and may apply to other signaling pathways that utilize a common set of kinases (Zhang, 2005).
Multisite hyperphosphorylation of tau has been implicated in the pathogenesis of neurodegenerative diseases including Alzheimer's disease (AD). However, the phosphorylation events critical for tau toxicity and mechanisms regulating these events are largely unknown. Drosophila PAR-1 kinase is shown to initiate tau toxicity by triggering a temporally ordered phosphorylation process. PAR-1 directly phosphorylates tau at S262 and S356. This phosphorylation event is a prerequisite for the action of downstream kinases, including glycogen synthase kinase 3 (GSK-3) and cyclin-dependent kinase-5 (Cdk5), to phosphorylate several other sites and generate disease-associated phospho-epitopes. The initiator role of PAR-1 is further underscored by the fact that mutating PAR-1 phosphorylation sites causes a much greater reduction of overall tau phosphorylation and toxicity than mutating S202, one of the downstream sites whose phosphorylation depends on prior PAR-1 action. These findings begin to differentiate the effects of various phosphorylation events on tau toxicity and provide potential therapeutic targets (Nishimura, 2004).
Drosophila has established itself as a model system for studying human neurodegenerative disorders. Fly models of tauopathy have been created by expressing wild-type or FTDP-linked mutant forms of h-tau. Using such models and based largely on overexpression experiments, it has been shown that Shaggy (GSK-3) can promote neurofibrillary tangle (NFT) pathology in photoreceptor neurons (Jackson, 2002). Whether GSK-3 and NFT are necessary for tau-mediated neurodegeneration, however, remains uncertain. Other studies have shown that tau-mediated neurodegeneration could occur without NFT and that GSK-3ß-induced tau hyperphosphorylation in mice could correlate inversely with neuropathology (Nishimura, 2004 and references therein).
Critical testing for a functional role of phosphorylation in tau-mediated neuropathology will require identifying the physiological tau kinase and assessing the consequence of removing this kinase activity on the disease process. Through loss-of-function and overexpression genetic studies and biochemical analysis, it has been shown that PAR-1 is a physiological tau kinase that plays a central role in regulating tau phosphorylation and toxicity in Drosophila. PAR-1 is a Ser/Thr kinase originally identified in C. elegans for its role in regulating cell polarity and asymmetric cell division. PAR-1 homologs have been found in eukaryotes ranging from yeast to mammals and exert essential cellular and developmental functions. MARK kinase, the mammalian homolog of PAR-1, regulates MT dynamics, epithelial cell polarity, and neuronal differentiation. Drosophila PAR-1 plays important roles in MT organization, oocyte differentiation, anterior-posterior axis formation, and Wingless signaling. While analyzing the neuronal function of PAR-1, it was found that Drosophila PAR-1 is a physiological kinase for fly Tau and h-tau. Overexpression of PAR-1 leads to elevated tau phosphorylation and enhanced toxicity, whereas removing PAR-1 function or mutating PAR-1 phosphorylation sites in tau abolishes tau toxicity. Furthermore, an initiator role for PAR-1 has been uncovered in a multisite phosphorylation process that generates pathogenic forms of tau. In this process, phosphorylation by PAR-1 precedes and is obligatory for downstream phosphorylation events, including those carried out by GSK-3 and Cdk5, to generate toxic tau. Consistent with PAR-1 playing an initiator role in the process, mutating PAR-1 phosphorylation sites causes a much more dramatic reduction of overall tau phosphorylation and toxicity than mutating one of the downstream Cdk5/GSK-3 phosphorylation sites. These findings have important implications for understanding the biogenesis of pathogenic tau in neurons and for developing mechanism-based therapeutic strategies (Nishimura, 2004).
Recent transgenic animal studies have implicated two kinases, GSK-3 and Cdk5, in the phosphorylation of tau in vivo. Analyses of tau phosphorylation status in transgenic mice overexpressing GSK-3 or Cdk5 have detected increased phosphorylation at certain sites previously identified as their in vitro phosphorylation sites. For example, S202 and PHF-1 sites (S396 and S404) have been shown to be prominent Cdk5 and GSK-3 phosphorylation sites, respectively, and the two kinases may have overlapping specificity at these sites. Tests were performed to see whether these sites in h-tauM were also phosphorylated by the corresponding fly kinases. The activity of Cdk5 is regulated by its binding with neuron-specific activators. Overexpression of Drosophila P35 activator has been shown to elevate endogenous Cdk5 activity. In P35 and h-tauM coexpression flies, the level of phosphorylation at S202 recognized by CP13 antibody is elevated. In addition, phosphorylation at AT270 sites was also significantly increased. Phosphorylation at AT100, AT180, and PHF-1 sites was relatively unchanged. Thus, phosphorylation at S202 and T181 responds to changes in Cdk5 levels. The eye morphology of P35 and h-tauM coexpressing flies appearssimilar to that of flies expressing h-tauM alone, suggesting that elevated Cdk5 activity does not significantly enhance tau toxicity. Shaggy and h-tauM coexpression flies were analyzed next. Coexpression of Shaggy and h-tau results in enhanced eye degeneration phenotypes. In the coexpression flies, significantly increased tau phosphorylation was observed at PHF-1, CP13, AT180, and AT100 sites. It is concluded that these phospho-epitopes contain GSK-3 phosphorylation sites and that elevated phosphorylation at these sites enhances tau toxicity (Nishimura, 2004).
The fact that many of the above-tested phosphorylation sites for GSK-3 and Cdk5 kinases are affected in S2A suggests that phosphorylation by the two kinases is regulated by prior PAR-1 action. To test this idea further, the phosphorylation status of GSK-3 and Cdk5 phosphorylation sites was analyzed in PAR-1 and h-tauM coexpression flies. In addition to 12E8 sites, significant increase of phosphorylation was observed at CP13 and PHF-1 sites in these flies. In contrast, phosphorylation at other sites such as AT100 sites was little changed, suggesting that PAR-1 is not a rate-limiting factor for these phosphorylation events. Since in vitro kinase assays showed that PAR-1 is incapable of directly phosphorylating the CP13 and PHF-1 sites, the elevated phosphorylation at these sites in PAR-1 coexpressing flies are likely mediated by downstream kinases such as Cdk5 and GSK-3 (Nishimura, 2004).
Whether coexpression of PAR-1, GSK-3, or Cdk5 has any modulating effect on S2A toxicity was further tested in vivo. PAR-1 and S2A coexpression flies showed a mild rough eye phenotype similar to PAR-1 overexpression alone, indicating that PAR-1 overexpression does not confer additional toxicity to S2A. Co-overexpression of GSK-3 or Cdk-5 also did not change S2A toxicity. These results further support the notion that phosphorylation by PAR-1 at S262 and S356 is a prerequisite for the subsequent phosphorylation by downstream kinases such as GSK-3 and Cdk5 to generate toxic tau species (Nishimura, 2004).
Since the S2A mutation disrupts tau phosphorylation at multiple downstream sites, it does not allow distinguishing the contribution of individual phosphorylation sites to tau toxicity. This issue was addressed by making point mutations in the downstream phosphorylation sites. Focus was placed on the S202 site because it is phosphorylated by Cdk5 and GSK-3 in vivo and because AT8 antibody, which is sensitive to phosphorylation at this site, was considered an Alzheimer-diagnostic antibody. Transgenic flies were generated that express h-tauM containing an Ala substitution at S202 (S202A). Western blot analysis demonstrated that, as predicted, S202A protein was no longer recognized by CP13 or AT8 antibodies. Significantly, phosphorylation at 12E8, AT100, PHF-1, AT180, and AT270 sites was unaffected by S202A mutation. This suggests that unlike S262 and S356 sites, the phosphorylation state of S202 does not influence that of other sites. Examination of external eye morphology by SEM and photoreceptor staining of eye sections has shown that, unlike S2A, S202A is as toxic as h-tauM. This suggests that phosphorylation by GSK-3 and Cdk5 at S202 site plays a rather limited role in conferring tau toxicity. This result supports the notion that PAR-1 plays an initiator role in the pathogenic phosphorylation process and further suggests that phosphorylation at downstream sites other than S202 or a combination of those downstream phosphorylation events makes a major contribution to tau toxicity (Nishimura, 2004).
Thus PAR-1, the fly homolog of mammalian MARK kinase, plays a central role in conferring tau toxicity in vivo. This study reveals PAR-1 function in triggering a temporally ordered phosphorylation process that is responsible for generating toxic forms of tau. This multisite phosphorylation process involves downstream kinases such as Cdk5 and GSK-3, whose action depends on prior phosphorylation of h-tau by PAR-1. A nonphosphorylatable mutation at S202, one of the downstream GSK-3/Cdk5 target sites whose phosphorylation depends on prior PAR-1 action, has a much smaller impact on overall tau phosphorylation and toxicity than mutations at PAR-1 phosphorylating sites. This strongly supports the initiator role of PAR-1 in generating toxic species of tau and further implies that the toxic form of tau may be phosphorylated at a subset or all of the other downstream sites (Nishimura, 2004).
It was previously shown that PAR-1 regulates the Wingless/Wnt pathway in Drosophila and Xenopus by phosphorylating the core component Dishevelled. It is thus interesting that GSK-3, another core component of Wingless pathway, acts downstream of PAR-1 to phosphorylate h-tau. These results are consistent with the notion that the Wingless pathway may be involved in regulating tau phosphorylation. It has been proposed that the pathway components are utilized differently in tau phosphorylation than in canonical Wnt signaling. The data indicate that PAR-1 and GSK-3 directly phosphorylate tau in an ordered fashion, with PAR-1 action preceding that of GSK-3. One parsimonious explanation for the requirement of prior phosphorylation by PAR-1 is that PAR-1 phosphorylation reduces the affinity of tau for MT and releases it from the MT network, therefore allowing easy access by other kinases. If that is the case, the mechanism may operate in a region-specific manner since certain phosphorylation sites do not depend on prior PAR-1 action. The data are also consistent with the idea that PAR-1 phosphorylation at 12E8 sites provides docking sites for intermediary kinase(s) and/or adaptor molecule(s), which facilitate subsequent phosphorylation by GSK-3 and Cdk5. It appears that the phosphorylation at certain downstream sites is achieved through a complex process. For example, phosphorylation at AT100 sites depends on prior PAR-1 action, but PAR-1 co-overexpression does not increase phosphorylation at these sites. Instead, co-overexpression of GSK-3 can lead to increased phosphorylation at AT100 sites. Previous in vitro studies have shown that the generation of AT100 epitope requires a PHF-like conformation of tau and the sequential phosphorylation by GSK-3 and PKA. It remains to be determined whether GSK-3 and PKA act downstream of PAR-1 to phosphorylate AT100 sites in flies (Nishimura, 2004).
Protein Phosphatase 2A (PP2A) has a heterotrimeric-subunit structure, consisting of a core dimer of ~36 kDa catalytic and ~65 kDa scaffold subunits complexed to a third variable regulatory subunit. Several studies have implicated PP2A in Wg/Wnt signaling. However, reports on the precise nature of the PP2A role in Wg/Wnt pathway in different organisms are conflicting. twins (tws), which codes for the B/PR55 regulatory subunit of PP2A in Drosophila, is shown to be a positive regulator of Wg/Wnt signaling. In tws- wing discs both short- and long-range targets of Wingless morphogen are downregulated. Analyses of tws- mitotic clones suggest that requirement of Tws in Wingless pathway is cell-autonomous. Epistatic genetic studies indicate that Tws functions downstream of Dishevelled and upstream of Sgg and Armadillo. These results suggest that Tws is required for the stabilization of Armadillo/ß-catenin in response to Wg/Wnt signaling. Interestingly, overexpression of, otherwise normal, Tws protein induces dominant-negative phenotypes. The conflicting reports on the role of PP2A in Wg/Wnt signaling could be due to the dominant-negative effect caused by the overexpression of one of the subunits (Bajpai, 2004).
Results of these studies show that Twins is involved in
modifying Wg signaling. Partial to complete downregulation of
short- (Ct and Sca) and long-range (Dll and Vg) targets of Wg pathway is observed in tws- background. The downregulation of Wg signaling in wing discs is reflected in adult phenotypes, such as serrated wing margin in mitotic clones of tws. Loss-of-Wg
phenotypes (induced by the overexpression of DN-TCF/pan or Sgg or
Cadintra) are enhanced in tws heterozygous mutant
background. In addition, mutation in tws suppresses the phenotypes
induced by Dsh, a positive component of Wg signaling. Finally, some of the
phenotypes induced by the overexpression of Tws are characteristic of
gain-of-Wg phenotypes. These results suggest that Tws functions as a positive regulator of Wg signaling (Bajpai, 2004).
Overexpression of otherwise normal Tws protein
induces dominant-negative phenotypes. The dominant-negative phenotype is
unlikely to be neomorphic or antimorphic, since UAS-Tws rescues tws
alleles (at the levels of both Wingless-dependent and independent
developmental events) and also induces gain-of-Wg phenotypes. The
dominant-negative phenotype is probably due to imbalance in the relative
amounts of the three subunits in the heterotrimeric complex, proper formation of which is obligatory for PP2A function. Thus, the conflicting reports on the role of PP2A in Wnt signaling could be due to the dominant negative effect caused by the overexpression of one of the subunits (Bajpai, 2004).
In tws mutant background, cytoplasmic Arm levels are
downregulated. Even overexpressed Arm is degraded in
tws- background. Furthermore, loss of tws had no
effect on the degradation-resistant form of Arm, which suggests that Tws
functions upstream of Arm to mediate Wg signaling. These
results could not be confirmed directly by Western blotting, since only a very small fraction (such as DV cells) of wing disc shows changes in Arm levels in response to Wg signaling. Nevertheless, results presented in this report suggest that stabilization of the cytoplasmic form of Arm by Wg signaling is dependent on Tws function (Bajpai, 2004).
A dominant-negative form of Sgg/GSK-3ß is able to rescue
tws- phenotype at the level of Dll expression. However,
overexpression of Dsh failed to rescue Dll expression in
tws- discs, suggesting that Tws functions downstream of
Dsh and upstream of Sgg to stabilize cytoplasmic Arm in response to Wg
signaling. Preliminary results presented here suggest that function of Tws in Wg pathway is inactivation of Sgg. Normally, overexpressed APC sequesters Arm only in those cells in which Sgg activity is downregulated. In other cells, APC participates in Arm-degradation
machinery. In tws- wing discs, overexpressed APC fails to sequester Arm in DV cells, suggesting that loss of tws results in upregulation of Sgg activity. However, it has been reported that PR/B56epsilon functions upstream of Dsh to regulate Wnt signaling in Xenopus embryos. The PR/B56epsilon homolog in Drosophila is widerborst (with 80% identity at the protein level), which is involved in the determination of planar cell polarity. widerborst is also known to be functional upstream of Dsh, but not in the canonical Wg/Wnt pathway. Although Tws homologs in other organisms have not been well characterized, the current studies are consistent with a role for PP2A in dephosphorylation of Axin (Bajpai, 2004).
The next question regards the substrate of PP2A function in the Wg pathway. In mammalian cells, Axin is dephosphorylated in response to Wnt signaling. Furthermore, dephosphorylated Axin binds ß-catenin less efficiently than the phosphorylated form. Thus, dephosphorylation of Axin would free ß-catenin from the degradation machinery. Thus,
Tws may function by inhibiting the activity of Axin, which acts a scaffold
protein to bring Sgg and Arm to close proximity. Further biochemical work is in progress to determine phosphorylated status of Arm in
tws- background and to determine if Tws directly binds to Sgg or Axin or both (Bajpai, 2004).
The biochemical machinery that underlies circadian rhythms is conserved among animal species and drives self-sustained molecular oscillations and functions, even within individual asynchronous tissue-culture cells. Yet the rhythm-generating neural centres of higher eukaryotes are usually composed of interconnected cellular networks, which contribute to robustness and synchrony as well as other complex features of rhythmic behaviour. In mammals, little is known about how individual brain oscillators are organized to orchestrate a complex behavioural pattern. Drosophila is arguably more advanced from this point of view: a group of adult brain clock neurons expresses the neuropeptide PDF and controls morning activity (small LNv cells; M-cells), whereas another group of clock neurons controls evening activity (CRY+, PDF- cells; E-cells). Transgenic mosaic animals were generated with different circadian periods in morning and evening cells. This study shows by behavioural and molecular assays, that the six canonical groups of clock neurons are organized into two separate neuronal circuits. One has no apparent effect on locomotor rhythmicity in darkness, but within the second circuit the molecular and behavioural timing of the evening cells is determined by morning-cell properties. This is due to a daily resetting signal from the morning to the evening cells, which run at their genetically programmed pace between consecutive signals. This neural circuit and oscillator-coupling mechanism ensures a proper relationship between the timing of morning and evening locomotor activity (Stoleru, 2005).
Overexpression of the Timeless (Tim) kinase Shaggy (Sgg; Drosophila GSK3) shortens the period by 3-4 h. Sgg expression was driven in all clock cells by crossing tim-GAL4 with flies carrying an EP element inserted at the Sgg locus (EP1576, referred to as UAS-Sgg). The locomotor activity rhythm of tim-GAL4/UAS-Sgg (timSgg) flies in constant darkness (DD) confirmed previous results, in that the period was about 3 h shorter than that of control flies (Stoleru, 2005).
Sgg was expressed exclusively in LNv cells by constructing a Pdf-GAL4/UAS-Sgg genotype. The Pdf-GAL4 driver is well characterized and drives gene expression only in two clock-cell groups: the PDF+ small LNv (s-LNv) cells (that is, M-cells) and the PDF+ large LNv (l-LNv) cells. The driver is inactive in the CRY+PDF- evening cells. Pdf-GAL4/UAS-Sgg (PdfSgg) flies also manifested a short period. The period shortening was less than that of timSgg flies, probably because of weaker expression from Pdf-GAL4 driver in LNv cells. Sgg expression from an even weaker driver, cry13-GAL4, did not affect behavioural period (Stoleru, 2005).
A close inspection of the behavioural actograms revealed that the period of evening activity is significantly shorter in PdfSgg flies (with a daily advance of about 2 h). This indicates that the pace of E-cells was accelerated, although the period manipulation was restricted to M-cells. An advanced evening peak, without an increase in E-cell Sgg expression, indicates that the faster M oscillator might be setting the E-cell pace. It is therefore proposed that the PDF+ cells influence molecular circadian events within E-cells (Stoleru, 2005).
To investigate this possibility, the molecular period (cycle duration) of each clock-cell group was estimated in these different genotypes: UAS-Sgg (control), timSgg and PdfSgg. Fly brains were analysed by in situ hybridization for tim RNA expression pattern after 4 days in DD, so that a barely detectable daily advance by 2-3 h would result in an aggregate advance of 8-12 h on DD4 (fourth day of DD). Indeed, Sgg overexpression in all clock neurons (timSgg) markedly shifted the interval of high tim mRNA expression on DD4 by about 12 h, from between CT10 and CT18 to before CT6. (CT is the circadian time within a constant-darkness experiment; CT0 is the hour of the last lights-on event.) All neurons expressing clock genes showed a similar temporal pattern, consistent with the expected Sgg-induced period shortening in all clock cells, and with a deterministic relationship between the molecular period and the locomotor activity period (Stoleru, 2005).
However, the PdfSgg tim RNA profiles were strikingly different and unexpected. Whereas the s-LNv cells showed a roughly 8 h advance in DD4, expected from a period shortening of 2 h per day, the l-LNv cells showed no appreciable change from those in control flies; that is, their molecular program is apparently unaffected by Sgg overexpression within these cells. Also surprising were the DN1 and DN3 profiles, which showed a roughly 8 h advance, as were the LNd cells, which were advanced by about 6 h relative to those in control flies. Since PdfSgg flies do not overexpress Sgg in these three cell groups, their molecular programs behave in a non-cell-autonomous manner. Because the E-cells are included within these groups and because the s-LNv cells (the M-cells) are the only cells with a cell-autonomous program that match the behavioural period of the flies, the M-cells apparently determine the clock pace of these other neuronal groups, including the E-cells (Stoleru, 2005).
The l-LNv cells and DN2 cells emerged as the only clock-gene-expressing neurons that evaded control of the M-cells and maintained a wild-type-like phase of tim RNA cycling in PdfSgg flies. Because DN2 cells are genotypically wild type in these flies, it is inferred that they oscillate with cell-autonomous properties and are the best candidates for determining the non-cell-autonomous wild-type-like characteristics of the l-LNv cells. As a consequence there are at least two parallel clock-cell circuits in the Drosophila brain in constant darkness: the M-E circuit controls locomotor activity rhythms and is driven by the M-cells (s-LNv cells), whereas the DN2-l-LNv circuit has as yet unknown functions and is driven by the DN2 cells (Stoleru, 2005).
To verify and extend these concepts, a genotype was generated in which the E-cells should run faster than M-cells. By adding the previously described Pdf-GAL80 repressor construct to the tim-GAL4;UAS-Sgg background, Sgg was expected to be overexpressed in all clock neurons with the exception of PDF-expressing cells. As these include the M-cells (s-LNv cells), they should run more slowly (24 h) than the E-cells (about 21 h). A 'faster takes all' rule predicts that the short-period E-cells will dominate over the normal 24 h M-cells in this genotype and generate a behavioural rhythm of about 21 h. Alternatively, dominant M-cells will give rise to a behavioural period of 24 h despite the faster endogenous oscillator in the E-cells (Stoleru, 2005).
Consistent with a dominant M-cell model was the observation that timSgg/PdfGAL80 flies had an almost wild-type period in DD. The molecular analysis is also consistent, since the s-LNv cells manifested a wild-type-like program: tim mRNA peaked between CT12 and CT20 on DD4. Despite Sgg overexpression, the LNd cells, DN1 cells and DN3 cells had a similar and wild-type-like pattern of tim expression. As described above, this indicates that all three cell groups behave non-autonomously and are probably driven by the s-LNv cells. This result is supported by the anatomical pattern of s-LNv neuronal processes, which project towards the brain regions populated by LNd, DN1 and DN3 cells. DN2 cells were again the only Sgg-overexpressing cells in which the phase of tim RNA oscillation corresponded to the predicted accelerated pace. The l-LNv cells, despite lacking Sgg overexpression (because of the PdfGAL80 transgene), also showed a comparable advance of tim expression. These timSgg/PdfGAL80 results confirm that the s-LNv cells determine the phase of LNd, DN1 and DN3 cells and that an independent cellular network includes the DN2 and l-LNv cells. Because the behavioural period was wild-type-like and paralleled the molecular clock within the s-LNv cells, the results confirm that these M-cells assign the circadian period in the absence of light cues (Stoleru, 2005).
To confirm the lack of a contribution of DN2/l-LNv to the E–M network function and to locomotor rhythms, the timSgg/cryGAL80 genotype was also examined. It is similar to the timSgg/PdfGAL80 genotype described above, except that Sgg overexpression is repressed in a wider group of cells. These include most if not all of the E-cells and l-LNv cells as well as the M-cells. Since DN2 cells are the only clock cells in which cry promoter-driven expression was not detected, it is expected that the faster clock in timSgg/cryGAL80 would be limited to CRY- cells, including the apparently cell-autonomous DN2 cells (Stoleru, 2005).
Indeed, tim hybridization in situ confirmed that the period of DN2 rhythm was shortened by about 2-3 h per day. The l-LNv neurons were shifted to about the same extent, which is consistent with the notion that they behave non-cell-autonomously and follow the pace of the DN2 clock program. All other clock cells maintained a pattern similar to that of control flies. Because timSgg/cryGAL80 flies had a normal behavioural period, these results confirm that l-LNv and DN2 cells do not contribute detectably to locomotor activity rhythms. This conclusion is in agreement with previous results showing that wild-type flies have persistent DD behavioural rhythms, despite protein oscillation idiosyncrasies of the l-LNv and DN2 cells (Stoleru, 2005).
How does the M-cell (s-LNv) clock determine the period of E-cells (LNd cells/DN cells)? Although previous work indicated possible oscillator coupling and a direct effect of LNv on the transcriptional oscillations of other clock cells, it was difficult to envision how the M-cells could override the intrinsic molecular timing of the E-cells. A second possibility is therefore considered, namely that the E-cells maintain an unaltered intrinsic clock program but receive a daily resetting signal from the M-cells. This model predicts that the timing of the evening activity within every cycle (between two consecutive mornings) reflects the status of the endogenous clock of E-cells, whereas the overall period exhibited by the evening peaks reflects the pace of the M-cell resetting signal (Stoleru, 2005).
To examine this possibility, the different transgenic strains were assayed for their average evening activity phase within a cycle, by using the leading morning peak as a reference and then measuring the average time until the subsequent evening peak. The overall DD period correlated with the genotype of M-cells as expected, but the length of the subjective day (M-E interval) correlated only with the genotype of the E-cells. In control flies with a period of about 24 h, the subjective day was roughly 12 h, similar to the duration of subjective day of PdfSgg. The latter strain features a wild-type-like E-oscillator but a fast, Sgg-expressing M-oscillator and a period of about 22 h. In contrast, timSgg flies express Sgg in both E-cells and M-cells, and both the average length of subjective day and the period (M-M) are reduced. The results indicate that the E-cells run an autonomous clock program whose starting (or ending) points are determined by daily resetting signals from the M-cells (Stoleru, 2005).
A DD unidirectional M ---> E resetting mechanism also predicts that a slower (24 h) M-cell clock and a faster E-cell clock will have a normal morning peak phase but an advanced evening peak phase. To test this prediction, the behavioural outputs of timSgg/PdfGAL80 and timSgg/cryGAL80 flies, which differ only in the genotypes of their E-cells, were compared. Both strains have periods of about 24 h, but the former should give rise to a fast E-cell molecular program, whereas the latter should have an E-clock of 24 h as a result of suppression of Sgg expression (Stoleru, 2005).
Indeed, the evening phase of timSgg/cryGAL80 is similar to that of control flies, and it always occurs about 2.5 h later than that of timSgg/PdfGAL80. The evening phase of timSgg/PdfGAL80 is more similar to that of timSgg, although the latter genotype has a much shorter period than the former. The length of subjective day of timSgg/PdfGAL80 flies further confirms that the evening phase within each cycle is a reflection of the endogenous E-cell rhythm, whereas the period of the cycle (M-M) correlates with the intrinsic M-cell clock (Stoleru, 2005).
These comparisons indicate that the circadian network is modulated by intercellular communication signals, which achieve and maintain circadian coherence -- the proper relationship between morning and evening activity. The dominant M-clock determines the period of the entire system by providing a daily reset signal to the E-clock in darkness and is therefore a true cellular Zeitgeber. Because the M-cells can delay as well as advance E-cells, the resetting signal may be required for E-cell oscillations. The usual candidate for this signal is the M-cell-specific neuropeptide PDF. It contributes to the normal synchrony and/or rhythmicity in constant darkness, with a striking similarity to the mammalian neuropeptide VIP. Moreover, injecting PDF into the cockroach brain causes circadian phase delays. Other principles and/or molecules may also be relevant to the M-E subnetwork, because E-cells can drive clockless M-cells to manifest cyclical behavioural outputs under 12 h light/12 h dark (LD) conditions (Stoleru, 2005).
The l-LNv and DN2 cells are the two neuronal groups that escape the M-cell reset signal in DD. They constitute a second circadian subnetwork with no apparent effect on locomotor activity rhythms and no known function. The DN2 cells are among the few clock-gene-expressing brain cells in larvae and are also the only clock cells that do not change their morphology after eclosion. Larval DN2 cells are apparently devoid of CRY and manifest anti-phase oscillations of Tim and PER. It is therefore likely that both the DN2 cells and the l-LNv cells impart circadian regulation to unknown physiological functions relevant to both larvae and adults. More generally, it is expected that the organizational principles of the two subnetworks described in this study will also be relevant to mammalian neuronal networks with important behavioural functions, for example the relationship between different oscillators in the SCN (Stoleru, 2005).
Work in Drosophila has defined two populations of circadian brain neurons, morning cells (M-cells) and evening cells (E-cells), both of which keep circadian time and regulate morning and evening activity, respectively. It has long been speculated that a multiple oscillator circadian network in animals underlies the behavioral and physiological pattern variability caused by seasonal fluctuations of photoperiod. This study manipulated separately the circadian photoentrainment pathway within E- and M-cells and shows that E-cells process light information and function as master clocks in the presence of light. M-cells in contrast need darkness to cycle autonomously and dominate the network. The results indicate that the network switches control between these two centers as a function of photoperiod. Together with the different entraining properties of the two clock centers, the results suggest that the functional organization of the network underlies the behavioral adjustment to variations in daylength and season (Stoleru, 2007).
Two populations of circadian brain neurons, morning cells (M-cells) and evening cells (E-cells), have been connected to morning and evening locomotor activity, respectively (Grima, 2004; Stoleru, 2004). Interactions between the two oscillator populations were studied by selectively overexpressing sgg to speed up the clock in only one cell population or the other (Stoleru, 2005). This study has found that sgg overexpression gives rise to LL rhythmicity, which led to a search for the cellular substrates of entrainment. The rhythmicity is predominantly due to sgg overexpression in E-cells, which suggested that this subset of the clock network is particularly important in the light and that Sgg affects the biochemical pathway through which light impacts clock molecules and adjusts phase to the correct time of day. Indeed, strong evidence is presented that Sgg modulates Cry function, which affects in turn the core clock proteins Per and Tim. The separate manipulation of the Sgg/Cry pathway within E- and M-cells also reveals that the E-clocks drive the behavioral rhythm in light, with prominent Per oscillations of nuclear localization. This light dependence of E-cells contrasts with M-cells, which need darkness to cycle autonomously and dominate the activity output pathway. This distinction suggests a simple dual-oscillator model for how the clock adjusts to photoperiod changes, and support for this seasonal model was obtained by examining E- and M-cell cooperation under different photoperiods (Stoleru, 2007).
The free-running pacemaker and entrainment are two important and increasingly understood aspects of circadian rhythms. In contrast, little information exists about seasonal adjustment, namely, how a constant ~24-hr timekeeper accommodates dramatically different photoperiods. This study shows that the previously defined dual oscillator system in Drosophila, M-cells and E-cells, creates different rhythmic patterns by alternating master clock roles. This understanding emerged from restricting Sgg overexpression to E-cells, which allowed the E-oscillator to function and render flies rhythmic in LL. Sgg probably modulates Cry activity and, when overexpressed, provides sufficient Per and Tim to allow E-oscillator function under constant illumination conditions. The E-clocks therefore manifest free-running properties and function as the master pacemakers in LL, analogous to a previous finding that the M-oscillator is the master in DD (Stoleru, 2005). Nonetheless, these constant conditions, and even the perfect standard LD cycles commonly used in the laboratory, are poor approximations of the changing LD environments found in nature. Circadian oscillators and their entrainment mechanisms have adapted to the dramatic seasonal changes in photoperiod. The previous strategy of using oscillators with different speeds, combined with different photoperiods, has led to a model of alternating control between the M-oscillator and E-oscillator (Stoleru, 2007).
Sgg appears to attenuate, rather than inactivate, Cry activity in E-cells. This is because the LL period of timSgg/PdfGAL80 (~23.5 hr) is longer than the intrinsic period of Sgg-expressing E-clocks in DD (~21 hr) (Stoleru, 2005). A longer period in light is compatible with attenuated light perception under high light intensity conditions (1600 lx, which renders wild-type flies completely arrhythmic) and the application of Aschoff's rule to insects [Aschoff, 1979; One of the earliest observations in the study of circadian rhythms was that continuous light (LL) lengthens circadian period in most nocturnal animal species. 'Aschoff's Rule' posits that there is a positive log-linear relationship between the LL intensity and period]. As there is also a prominent effect on Cry stability, Sgg may be the regulator previously predicted to bind to the Cry C terminus (Busza, 2004; Dissel, 2004). Although Cry is favored as the major circadian substrate of Sgg, there may be others, e.g., the serotonin receptor. Biochemical support for GSK3 involvement in mammalian rhythms has recently been obtained (Yin, 2006). Since GSK3 is a proposed therapeutic target of lithium, the relationship between Sgg and Cry reported in this study recalls the intriguing relationship between mood disorders, light sensitivity, and circadian rhythms (Stoleru, 2007).
The cryb genotype markedly affects DD period in some of the rhythmic genotypes described in this study. Although Cry is probably unnecessary for M-cell rhythmicity, this could reflect some redundancy or assay insensitivity. Moreover, the DD period of cryb is slightly shorter than that of wild-type (23.7 versus 24.4), suggesting that 'dark Cry' makes some contribution to pacemaker function in M-cells as well as E-cells. For these reasons, it is suggested that Drosophila Cry is closer to the central pacemaker than previously believed, and therefore closer to the level of importance of its mammalian paralogs in influencing free-running pacemaker activity. Unlike mammalian Cry, however, Drosophila Cry still appears to function predominantly at a posttranslational level. Indeed, the effects of cryb on Sgg overexpression in DD suggest that the proposed effect of Sgg on Tim stability is really an effect of Sgg on Cry followed by an altered Cry-Tim interaction. It is noted that there is a recent proposal (Collins, 2006) that Drosophila Cry, like mammalian Cry, also functions as a transcription factor in peripheral clocks (Stoleru, 2007).
The importance of E-cells in LL rhythmicity is underscored by the staining results of timSgg/PdfGAL80 brains. Only some E-cells and DN2s manifest robust cycling. It has been suspected that E-cells are important in light because they can rescue the output of arrhythmic M-cells in LD, but not in DD (Stoleru, 2004). Indeed, all of these observations make it attractive to view E-cells as autonomous pacemakers. There is, however, evidence that M-cells may not be completely dispensable. Moreover, a synchronizing or stabilization function is compatible with previous observations under different conditions (Stoleru, 2007).
In the timSgg/PdfGAL80 genotype, only Per nuclear localization changes were detectable near the end of LL cycle. The nature of the assay makes it hard to conclude that there were no differences in total Per staining intensity, i.e., no oscillations in Per levels, so the unique nature of the Per nuclear localization cycling is a tentative conclusion. The same caveat applies to the absence of Tim oscillations and nuclear staining, i.e., negative results cannot exclude low-amplitude oscillations; it is noted, however, that Tim cytoplasmic sequestration has been previously observed in cryb flies after several days in LL. Furthermore, the circadian nuclear accumulation of Tim has been shown to respond differently than that of Per to changes in photoperiod. Nonetheless, Tim could be shuttling with a predominant steady-state cytoplasmic localization, nuclear Tim could be rapidly degraded to create a low nuclear pool, or both (Stoleru, 2007).
The importance of E-cells in entrainment is strongly supported by the potent effect of restricted Cry rescue of cryb: E-cell rescue is much more impressive than M-cell rescue. Moreover, the differences between the two rescued phase response curves (PRCs) are striking; E-cell rescue is virtually complete, whereas the M-cell rescue is notably deficient in the delay zone. In addition, flies with Sgg overexpression in E-cells show altered PRCs, whereas flies with Sgg overexpression in M-cells respond normally to light. The results are strikingly different in darkness, as M-cell-restricted expression causes the typical short period determined by Sgg overexpression, whereas E-cell overexpression has no systemic effect (Stoleru, 2007).
The PRC delay zone is the region impacted most strongly by E-cell Sgg overexpression, indicating that the lights-off early night region is most important to E-cell function and light entrainment. Exposure to light in this interval should mimic long days (summer), which, it is speculated, will delay phase by many hours so that “evening” output of the following day will coincide with the objective evening of the environment. Even the short nights of summer are probably enough time for E-clocks to accumulate sufficient Tim and Per, shuttle them into the nucleus, and reconstitute the rhythmic substrate observed in the Sgg-overexpressing brains in LL. In contrast, M-cells need darkness to cycle robustly. They will become the master clocks and drive the system whenever lights fail to turn on more than 12 hr past lights-off, i.e., during the long nights of winter that mimic the beginning of a DD cycle. Since the intrinsic pacemaker program of M-cells in darkness relies on the changing nature of clock proteins during the night, it is hypothesized that the activity phases under long nights (winter) are locked to lights-off. This suggestion is supported by preliminary data and previous observations showing that per transcription remains locked to lights-off under different entrainment regimes. M-cells are also capable of fully entraining the system in the PRC interval that determines a phase advance (late night). This is consistent with their predicted role in generating an advanced evening output, coincident with the early evenings typical of winter. Otherwise put, long summer days should underlie light primacy as well as long and prominent evening delay zones; both suggest E-cell dominance. Night primacy and M-cells should dominate under winter conditions. This concept endows E- and M-cells with the properties originally envisioned by the Pittendrigh and Daan (1976) dual-oscillator model of entrainment (Stoleru, 2007).
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).
Entrainment of the Drosophila circadian clock to light involves the light-induced degradation of the clock protein timeless (Tim). This entrainment mechanism is inhibited by serotonin, acting through the Drosophila serotonin receptor 1B (5-HT1B). 5-HT1B is expressed in clock neurons, and alterations of its levels affect molecular and behavioral responses of the clock to light. Effects of 5-HT1B are synergistic with a mutation in the circadian photoreceptor cryptochrome (Cry) and are mediated by Shaggy (Sgg), Drosophila glycogen synthase kinase 3beta (GSK3beta), which phosphorylates Tim. Levels of serotonin are decreased in flies maintained in extended constant darkness, suggesting that modulation of the clock by serotonin may vary under different environmental conditions. These data identify a molecular connection between serotonin signaling and the central clock component Tim and suggest a homeostatic mechanism for the regulation of circadian photosensitivity in Drosophila (Yuan,2005).
Serotonin regulates the entrainment of circadian behavioral rhythms in Drosophila by affecting the molecular response to light. By modulating the expression of the 5-HT1B receptor in clock neurons, a role of this receptor subtype has been established in the regulation of Drosophila circadian photosensitivity. The data also demonstrate that the molecular connection between 5-HT1B signaling and the clock is GSK3β, which directly phosphorylates the central clock component Tim. It is proposed that serotonin signaling is a part of the homeostatic regulation that prevents dramatic fluctuations in the phase of the circadian clock. In addition, given the altered levels of serotonin in extended DD, it may confer selectivity on the response of the clock to light under different environmental conditions (Yuan, 2005).
The expression pattern of 5-HT1B, as determined by both UAS-Gal4 experiments and by immunostaining, provides some clues to its functions in Drosophila. Besides LNvs and SE5HT-IR neurons, major compartments of the fly brain that express the 5-HT1B receptor include the optic lobes, PI neurons, and mushroom bodies. Interestingly, expression in each of these locations is consistent with functions proposed for serotonin signaling in other organisms. In the housefly, the neuropil of the optic lobes undergoes daily structural changes regulated possibly by serotonin and PDF. PI neurons are neurosecretory cells that may also participate in the ocellar phototransduction pathway. The mushroom body is important for olfactory learning and memory in Drosophila. Therefore, in addition to its postsynaptic function in the LNvs, 5-HT1B may be involved in other aspects of physiology and behavior (Yuan, 2005).
The effect of 5-HT1B on Tim was especially pronounced in the small LNvs. One of the differences between the large and small LNvs is in the timing of nuclear entry, which is delayed in the small subgroup. If delayed nuclear entry accounts for the increased resistance of Tim to light in the small LNvs, it would suggest that 5-HT1B signaling largely affects cytoplasmic Tim (Yuan, 2005).
In addition to its effect on the light response, 5-HT1B overexpression influences free-running behavioral rhythms of cryb flies. It is speculated that this is due to the loss of synchrony among LNs. The mutual coupling of oscillators within an organism is important for the generation and synchronization of circadian rhythms, and serotonin is implicated in this process in some insects. Decreased synchrony may also result from the reduced photosensitivity produced by 5-HT1B overexpression. Interestingly, a significant number of glass, cryb double mutants, which lack CRY as well as all visual photoreceptors, are arrhythmic in DD (Yuan, 2005).
5-HT1B not only affects circadian photosensitivity when over- or under-expressed, it also appears to be the major receptor subtype required for the inhibitory effects of serotonin on entrainment. Notably, when 5-HT1B was knocked down with the RNAi transgene driven by tim-Gal4, the effect on photosensitivity was not as pronounced as with the 5-HT1B-Gal4 driver. This might be due to some background differences in flies carrying the tim-Gal4 transgene, or to nonspecific effects produced by expressing the RNAi construct in irrelevant cells. Also, the possibility that cells other than clock neurons participate in the regulation of light sensitivity via 5-HT1B cannot be excluded. However, clock cells clearly have a major role in this effect, in particular since the circadian response to serotonin is eliminated in the tim-Gal4/RNAi flies (Yuan, 2005).
Effects of serotonin on circadian photosensitivity have been demonstrated in other systems, but the underlying mechanisms were not identified. These studies in Drosophila address this issue by demonstrating an effect of 5-HT1B signaling on the posttranslational modification of Tim via Sgg. In 5-HT1B-overexpressing flies, Tim phosphorylation is reduced, and its stability is increased. In contrast, Sgg phosphorylation is increased (i.e., its activity is decreased) in response to elevated levels of 5-HT1B as well as in response to serotonin treatment. Consistent with this effect of 5-HT1B on Sgg, increased Sgg activity abolishes effects of 5-HT1B overexpression on circadian photosensitivity, while 5-HT1B attenuates the period shortening produced by excess Sgg activity. These reciprocal effects in genetic experiments strongly support the regulation of Sgg activity by 5-HT1B. Expression data indicate that Sgg is expressed predominantly in the cytoplasm. The regulation of cytoplasmic Sgg by 5-HT1B is predicted to affect the phosphorylation status of Tim mainly in the cytoplasm; Sgg-phosphorylated Tim is transported to the nucleus more effectively and is also a better substrate for light-induced degradation (Yuan, 2005).
5-HT1B alone does not significantly affect circadian period, suggesting that its effects on Sgg are limited. In this context, it is noted that, while sgg hypomorphs have a period of ~26 hr, flies hemizygous for the locus have wild-type periods. It is inferred that small (up to 50%) changes in Sgg activity do not alter circadian period but can affect circadian photosensitivity. A role for Sgg in circadian photosensitivity was previously suggested by Martinek (2001) who found that forms of Tim phosphorylated by Sgg were selectively degraded in response to light. In fact, phosphorylated Tim is known to be more sensitive to light. While Sgg appears to be the primary kinase that increases photic sensitivity of Tim, the actual process of light-induced Tim degradation involves the activity of a tyrosine kinase (Yuan, 2005).
These results provide a new mechanism for circadian regulation by a G protein-coupled signaling pathway. A role for GSK3β in the mammalian circadian system was recently reported (Iwahana, 2004). In addition, the mammalian 5-HT1A receptor affects phosphorylation of GSK3β in the mouse brain. It is possible that inhibition of GSK3β activity is a conserved mechanism in the regulation of circadian entrainment in mammals and insects (Yuan, 2005).
Slow dark adaptation has been described in Drosophila, whereby circadian sensitivity to light increases more than 10-fold over 3 days in DD. Increased light responsiveness during dark adaptation occurs in rodents, but the mechanism underlying these effects has not been addressed. Elevated responsiveness to light after prolonged exposure to darkness could be due either to a gain in sensitivity in the sensory system or to an increase in sensory output, which may be caused by a reduction in an inhibitory mechanism. In this study, lower serotonin levels were observed in flies maintained in DD. Given that serotonin signaling modulates circadian light sensitivity, it may be the reduction in this inhibitory mechanism that at least partially accounts for the enhanced light response in prolonged DD (Yuan, 2005).
It is proposed that serotonin signaling, which is itself upregulated by light, is a part of a homeostatic mechanism that regulates circadian light sensitivity. A recent study using human subjects also suggested that serotonin levels in the brain reflect the duration of prior light exposure. This change in serotonin levels with light may be relevant to the etiology and treatment of seasonal affective disorder (SAD), a mood disorder related to the reduced hours of sunlight in winter, particularly at northern latitudes. SAD patients respond to antidepression drug treatments, as well as to light therapy, both of which may produce an increase in serotonin. The interplay of serotonin, light, and the circadian system suggests a close relationship between circadian regulation and mental fitness (Yuan, 2005).
Serotonin modulates the entrainment of the circadian system. In contrast, the current results, and studies done in mammalian systems also, suggest circadian effects on serotonin signaling. (1) Based upon the differences seen in LD versus DD in the fly brain, the level of serotonin is affected by the environmental light cycle. (2) Receptor levels are modulated by circadian components, since 5-HT1B levels are altered in fly circadian mutants. In addition, serotonin release and receptor activity are regulated in a circadian fashion in mammals. Mutual regulation of the circadian and serotonin systems may be necessary to maintain the normal physiological functions of both systems (Yuan, 2005).
Caspases are well known for their role in the execution of apoptotic programs, in which they cleave specific target proteins, leading to the elimination of cells, and for their role in cytokine maturation. In this study, a novel substrate was identified, that, through cleavage by caspases, can regulate Drosophila neural precursor development. Shaggy (Sgg)46 protein, an isoform e