Striking circadian neuron diversity and cycling of Drosophila alternative splicing

Although alternative pre-mRNA splicing (AS) significantly diversifies the neuronal proteome, the extent of AS is still unknown due in part to the large number of diverse cell types in the brain. To address this complexity issue, this study used an annotation-free computational method to analyze and compare the AS profiles between small specific groups of Drosophila circadian neurons. The method, the Junction Usage Model (JUM), allows the comprehensive profiling of both known and novel AS events from specific RNA-seq libraries. The results show that many diverse and novel pre-mRNA isoforms are preferentially expressed in one class of clock neuron and also absent from the more standard Drosophila head RNA preparation. These AS events are enriched in potassium channels important for neuronal firing, and there are also cycling isoforms with no detectable underlying transcriptional oscillations. The results suggest massive AS regulation in the brain that is also likely important for circadian regulation (Wang, 2018).

Tissues of the nervous and germline systems, such as brain, testes and ovaries, have more complex transcriptomes than other cell types due to extensive alternative pre-mRNA splicing or AS. The nervous system especially exhibits vast numbers of AS isoforms, many of which are novel and are only beginning to be comprehensively identified. This increase in transcript isoform complexity likely contributes to the specification and functional diversity of cell types within the nervous system (Wang, 2018).

This study applied a novel computational algorithm called JUM to characterize the transcript isoform diversity generated by alternative splicing in three circadian neuronal subtypes (LNv, LNd and DN1), as well as a non-circadian dopaminergic neuron population (TH neurons) of the Drosophila central nervous system. JUM can comprehensively analyze, quantitate and compare tissue- or cell-type-specific AS patterns without requiring a priori annotations of known transcripts or transcriptomes. The analysis revealed a previously unappreciated diversity and complexity of alternatively spliced transcript isoform patterns in these four neuronal subtypes, suggesting that they contribute to neuronal identity, connectivity, activity and circadian functions. This is because many of these novel, previously undetected and unannotated isoforms were unique to a given neuronal population and occurred in transcripts from genes implicated in neuronal activity or circadian rhythms (Wang, 2018).

For example, the kinase Shaggy and the blue light photoreceptor Cryptochrome play central roles in circadian clock regulation and have novel AS patterns in discrete subsets of the circadian neurons. In addition, nine different transcripts involved in potassium transport undergo differential AS in circadian neurons compared to non-circadian neurons. These transcripts encode six different potassium channels. Many of these genes have a complex organization known to encode populations of functionally distinct proteins isoforms, which change the activation kinetics as well as calcium sensitivity of the channels. Neuronal firing is known to play a key role in the circadian circuit with recent studies illustrating that different subgroups of circadian neurons have characteristic time-of-day neuronal firing patterns. Although it is not yet fully understood which potassium channels play a critical role in each circadian neuron subgroup, several channel pre-mRNAs that undergo differential splicing in circadian neurons impact circadian behavior and sleep, such as slowpoke (slo), Shaker (Sh) and Hyperkinetic (Hk). It is therefore likely that AS adds diversity and distinct physiological properties to these protein isoforms, which then impacts neuron-specific firing patterns. From a more general perspective, AS augments transcriptional regulation in giving different circadian neurons individual identities and distinct functions (Wang, 2018).

Approximately 5% of the AS events identified in circadian neurons also undergo time-of-day dependent changes in alternative splicing (cycling splicing). It is important to note that all experiments carried out in this study were conducted under 12 hr of light and 12 hr of dark conditions, making it impossible to distinguish between light and clock control. Nonetheless, these data indicate that splicing adds a dramatic layer of gene regulation to diurnal changes in gene expression. Moreover, many of the cycling AS transcripts show constant overall mRNA levels, which suggests the existence of neuron-specific splicing factors that are expressed or activated only at specific times of the day. Indeed, this study has identified several candidate cycling neuron-enriched transcripts that encode RBPs that may help to drive cycling AS patterns (Wang, 2018).

A recent trend in biological research is to generate transcriptome profiles from single cells. For example, this strategy is part of the 'human cell atlas' project aimed at personalized genomic medicine or the 'brain initiative' project to generate profiles of all neurons in the mouse brain. One recent study was able to obtain about 20M sequence reads per isolated human iPS cell but only managed to analyze splicing patterns for the most highly expressed genes. The current study in contrast used ~100 isolated Drosophila neurons for each of the four neuron subtypes along with judicious use of both oligo-dT and random hexamer priming of the cDNA libraries. This strategy obtained about 10-30M sequence reads for each sample, including substantial information from the 5' ends of transcripts, and JUM was able to detect and classify a large number of previously unannotated pre-mRNA isoforms. Many of them are missing from the fly head RNA-seq data assayed and analyzed in parallel, indicating that these new isoforms are cell-type specific. Not surprisingly, the novel isoforms from the three circadian neuron groups fall into many gene ontology (GO) categories associated with specific circadian clock activity and function (Wang, 2018).

Taken together, the work presented in this study indicates that the number of alternative splicing events that take place in neuronal tissues is grossly underestimated, even though publically-funded genome projects, such as the NIH modENCODE projects deeply sequenced transcriptomes from a variety of Drosophila tissues and developmental stages. This is despite the appreciation of how much AS occurs in the nervous system, for example recent comprehensive analysis of splicing patterns through deep sequencing of ~50 mouse and human tissues revealed about 2500 neuronally-regulated alternative splicing events. It is therefore suggested that these events will need to be comprehensively evaluated by much deeper sequencing than is currently afforded by most contemporary single cell RNA-seq studies and by AS analysis software like JUM that is not constrained by a priori knowledge of known splicing events (Wang, 2018).

Membrane bound GSK-3 activates Wnt signaling through Disheveled and Arrow

Wnt ligands and their downstream pathway components coordinate many developmental and cellular processes. In adults, they regulate tissue homeostasis through regulation of stem cells. Mechanistically, signal transduction through this pathway is complicated by pathway components having both positive and negative roles in signal propagation. This study examined the positive role of GSK-3/Zw3 in promoting signal transduction at the plasma membrane. Targeting GSK-3 to the plasma membrane activates signaling in Drosophila embryos. This activation requires the presence of the co-receptor Arrow-LRP5/6 and the pathway activating protein Disheveled. These results provide genetic evidence for evolutionarily conserved, separable roles for GSK-3 at the membrane and in the cytosol, and are consistent with a model where the complex cycles from cytosol to membrane in order to promote signaling at the membrane and to prevent it in the cytosol (Mannava, 2015).

Lithium promotes longevity through GSK3/NRF2-dependent hormesis

The quest to extend healthspan via pharmacological means is becoming increasingly urgent, both from a health and economic perspective. This study shows that lithium, a drug approved for human use, promotes longevity and healthspan. Lithium was shown to extend lifespan in female and male Drosophila, when administered throughout adulthood or only later in life. The life-extending mechanism involves the inhibition of glycogen synthase kinase-3 (GSK-3) and activation of the transcription factor nuclear factor erythroid 2-related factor (NRF-2). Combining genetic loss of the NRF-2 repressor Kelch-like ECH-associated protein 1 (Keap1) with lithium treatment revealed that high levels of NRF-2 activation confer stress resistance, while low levels additionally promote longevity. The discovery of GSK-3 as a therapeutic target for aging will likely lead to more effective treatments that can modulate mammalian aging and further improve health in later life (Castillo-Quan, 2016).

GSK-3 and CK2 kinases converge on Timeless to regulate the master clock

The molecular clock relies on a delayed negative feedback loop of transcriptional regulation to generate oscillating gene expression. Although the principal components of the clock are present in all circadian neurons, different neuronal clusters have varying effects on rhythmic behavior, suggesting that the clocks they house are differently regulated. Combining biochemical and genetic techniques in Drosophila, this study identified a phosphorylation program native to the master pacemaker neurons that regulates the timing of nuclear accumulation of the Period/Timeless repressor complex. GSK-3/SGG binds and phosphorylates Period-bound Timeless, triggering a CK2-mediated phosphorylation cascade. Mutations that block the hierarchical phosphorylation of Timeless in vitro also delay nuclear accumulation in both tissue culture and in vivo and predictably change rhythmic behavior. This two-kinase phosphorylation cascade is anatomically restricted to the eight master pacemaker neurons, distinguishing the regulatory mechanism of the molecular clock within these neurons from the other clocks that cooperate to govern behavioral rhythmicity (Top, 2016).

Shaggy functions downstream of dMyc and their concurrent downregulation confers additive rescue against tau toxicity in Drosophila

Neurodegenerative tauopathies such as Alzheimer's and Parkinson's diseases are characterized by hyperphosphorylation of tau protein and their subsequent aggregation in the forms of paired helical filaments and/or neurofibrillary tangles in specific areas of the brain. Despite several attempts, it remains a challenge to develop reliable biomarkers or effective drugs against tauopathies. It is increasingly evident now that due to the involvement of multiple cellular cascades affected by the pathogenic tau molecules, a single genetic modifier or a molecule is unlikely to be efficient enough to provide an inclusive rescue. Hence, multitargets based combinatorial approach(s) have been suggested to provide an efficient rescue against tauopathies. It has been reported that targeted downregulation of dmyc (a Drosophila homolog of human cmyc proto-oncogene) restricts tau etiology by limiting tau hyperphosphorylation and heterochromatin loss. Although, dmyc generates a significant rescue; however, it is not proficient enough to provide a complete alleviation against tauopathies. This study reports that tissue-specific concurrent downregulation of dmyc and gsk3β conveys a near-complete rescue against tau toxicity in Drosophila. It is noted that combinatorial downregulation of dmyc and gsk3β reduces tau hyperphosphorylation, restricts the formation of neurofibrillary tangles, and restores heterochromatin loss to the physiological level. Subsequent investigations revealed that dmyc regulates gsk3β via protein phosphatase 2A (dPP2A) in a dose-dependent manner to regulate tau pathogenesis. It is proposed that dmyc and gsk3β candidates can be utilized in a synergistic manner for the development of an efficient combinatorial therapeutic approach against the devastating human human tauopathies (Pragati, 2021).

Transcriptional Regulation

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).

Targets of Activity

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).

RNAi screening for kinases and phosphatases identifies Shaggy as a FoxO activator

FoxO transcription factors are key regulators of growth, metabolism, life span, and stress resistance. FoxOs integrate signals from different pathways and guide the cellular response to varying energy and stress conditions. FoxOs are modulated by several signaling pathways, e.g., the insulin-TOR signaling pathway and the stress induced JNK signaling pathway. This study reports a genome wide RNAi screen of kinases and phosphatases aiming to find regulators of dFoxO activity in Drosophila S2 cells. By using a combination of transcriptional activity and localization assays several enzymes were identified that modulate dFoxO transcriptional activity, intracellular localization and/or protein stability. Importantly, several currently known dFoxO regulators were found in the screening, confirming the validity of the approach. In addition, several interesting new regulators were identified, including protein kinase C and glycogen synthase kinase 3beta, two proteins with important roles in insulin signaling. Furthermore, several mammalian orthologs of the proteins identified in Drosophila also regulate FOXO activity in mammalian cells. These results contribute to a comprehensive understanding of FoxO regulatory processes (Mattila, 2008).

By using a combination of transcriptional reporter and localization assays, twenty one dFoxO regulators were discovered. Some positive hits from the screen had an effect in dFoxO activity, localization, and protein stability, whereas other hits affected only transcriptional activity, suggesting that more mechanisms beyond subcellular localization and degradation are used by cells to regulate dFoxO activity. In addition to the 18 proteins that affected dFoxO transcriptional activity, the screening produced three more hits. Two of them seem to affect only dFoxO localization (dgkd and ptp69d), and one, neurospecific receptor kinase (nrk), affected exclusively dFoxO protein stability. It is possible that these proteins regulate dFoxO transcription on specific promoters in conjunction with other activators and that such factors are missing in Drosophila S2 cells. This would explain their lack of effect on the dInR promoter. Alternatively, they could affect dFoxO stability resulting in a net effect of dFoxO protein accumulation in the nucleus (Mattila, 2008).

Initially, the screening strategy was designed to identify both positive and negative regulators of dFoxO activity; however, no dFoxO repressors were found. Putative dFoxO repressors were present in the primary hit list of 31 targets, but those were later excluded in the secondary screen. This surprising observation suggests that the screen may be biased against dFoxO repressors. dFoxO is a well known inhibitor of protein biosynthesis in vivo, so under conditions of increased dFoxO activity, a reduction of general translation is expected that could affect GFP and luciferase translation too. Therefore, it is hypothesized that in the case of enhanced dFoxO activity it is possible that the concomitant inhibition of protein biosynthesis overruled a slight increase in reporter accumulation. This would explain the lack of dFoxO repressors among the targets of the screen. Moreover, the design of the screening based on S2 cells excludes the identification of regulatory mechanisms specific for other cell types, and instances where dFoxO is acting as a cofactor thereby regulating transcription indirectly (Mattila, 2008).

The results demonstrate that Drosophila PKC53E isoform is a dFoxO activator. Similar results were obtained in mammalian cells pointing out that the interaction is conserved. PKC isoforms have very important roles in insulin signaling, and each of the isoforms has been shown to be activated by insulin stimulation or conditions important for effective insulin stimulation. Importantly, PKC isoforms can both activate or inhibit insulin signaling: Atypical PKC isoforms are required for insulin-stimulated glucose transport in muscle and adipocytes. In contrast, certain conventional and novel PKC isoforms are known to antagonize insulin signaling in vertebrates. This interaction is implicated in the pathogenesis of free fatty acid mediated insulin resistance. Drosophila possesses six PKC isoforms whose role in this context has not yet been addressed. PKC53E homolog is closest to human conventional PKCα. Interestingly, it has been shown that PKCα inhibits insulin signaling through binding and phosphorylation of IRS1. Thus, PKCα would serve as a constitutively active inhibitory regulator of the insulin cascade through its association with IRS1. On stimulation with insulin, PKCα would dissociate from IRS1, thus releasing this protein from its down-regulated state. This would open the 'gate' for transmission of the insulin signal. It has been found that dFoxO/FOXO1 increases insulin sensitivity by up-regulating insulin receptor transcription. The observation that Drosophila PKCα activates dFoxO adds an additional twist in the complex regulatory network that dFoxO has on insulin signaling. Interestingly, in the experimental system used in this study AKT dependent dFoxO bandshift and AKT Ser-505 phosphorylation was not affected by PKC53E, indicating that PKC53E regulation of dFoxO is independent of AKT signaling (Mattila, 2008).

Another well known enzyme implicated in the control of metabolism identified as a regulator of dFoxO transcriptional activity is the Drosophila ortholog of Glycogen synthase kinase 3β (GSK-3β, Shaggy). GSK-3β is a regulator of glucose metabolism through the phosphorylation and inhibition of glycogen synthase, the rate limiting enzyme of glycogen deposition. GSK-3β is inhibited by AKT, so it was not surprising to see that GSK-3β activates dFoxO. GSK-3β protein level and activity is elevated in type II diabetic skeletal muscle cells reflecting the impairment of whole body glucose uptake characteristic to this disease. In addition, selective inhibition of GSK-3β by lithium chloride represses the expression of g6pase and pepck in rat hepatoma cells, both known targets of FoxO. Taken together, these observations suggest that some of the metabolic effects of GSK-3β are achieved by directly modulating dFoxO activity (Mattila, 2008).

An interesting dFoxO regulator is Polo-like kinase. Polo-like kinases (Plks) are known regulators of cell cycle progression. In addition, Plks have a role in the protection against cellular stress through the transcription factor HSF1. Recently it was proposed that an intricate tradeoff between lifespan and cancer results from opposing effects of enzymes regulating FoxO and p53 activity. Plks are known to inhibit p53 transcriptional activity, so the results raise the possibility that Plks mediate the common but opposing regulators of p53 and FoxO. Interestingly, FoxOs are necessary in the completion of the cell cycle, which is partly mediated by cell cycle dependent activation of Plk expression by FOXO3a. The results show that Drosophila dFoxO is regulated by Polo, suggesting the existence of a positive feedback mechanism that has a role in achieving periodic M-phase gene expression and proper cell cycle exit (Mattila, 2008).

dFoxO localization was affected by eight modulators; however, band shifts demonstrated that none of these proteins phosphorylated dFoxO in the three conserved Ser/Thr amino acids known to regulate nuclear/cytoplasmic status through AKT. This observation raises the possibility that some of the newly identified dFoxO regulators could affect dFoxO nuclear/cytoplasmic localization by phosphorylating dFoxO in additional residues that do not alter its electrophoretic mobility, or that dFoxO regulation by these proteins is indirect. Further studies will be needed to clarify this point (Mattila, 2008).

In summary, this study has identified 21 dFoxO modulators. The results underscore the complexity underlying dFoxO regulation and establish dFoxO as a transcription factor controlled exquisitely by an intricate network of kinases and phosphatases achieving a perfect balance of activity. This balance ensures the correct execution of key cellular processes in metabolism, response to stress, and life span (Mattila, 2008).

Neuronal activity and Wnt signaling act through Gsk3-β to regulate axonal integrity in mature Drosophila olfactory sensory neurons

The roles played by signaling pathways and neural activity during the development of circuits have been studied in several different contexts. However, the mechanisms involved in maintaining neuronal integrity once circuits are established are less well understood, despite their potential relevance to neurodegeneration. This study demonstrates that maintenance of adult Drosophila olfactory sensory neurons requires cell-autonomous neuronal activity. When activity is silenced, development occurs normally, but neurons degenerate in adulthood. These detrimental effects can be compensated by downregulating Glycogen synthase kinase-3β (Gsk-3β). Conversely, ectopic expression of activated Gsk-3β or downregulation of Wnt effectors also affect neuron stability, demonstrating a role for Wnt signaling in neuroprotection. This is supported by the observation that activated adult neurons are capable of increased Wingless release, and its targeted expression can protect neurons against degeneration. The role of Wnt signaling in this process is non-transcriptional, and may act on cellular mechanisms that regulate axonal or synaptic stability. Together, evidence is provided that Gsk-3β is a key sensor involved in neural circuit integrity, maintaining axon stability through neural activity and the Wnt pathway (Chiang, 2009).

During development, electrical activity and cell signaling pathways have been shown to collaborate to eliminate neurons or synaptic connections. In the mature vertebrate nervous system, many neurons are eliminated, although the majority survive through the life of the animal. Little is known about the mechanisms underlying the maintenance of mature neurons in a circuit, or, indeed, whether there is any active requirement at all for maintenance. Given the fundamental role of electrical activity in neuronal function, it was asked whether activity is the sensor for the integrity of the nerve cell. In order to test this rigorously, a preparation was required from which both spontaneous and evoked activity could be removed and the consequences on neuronal maintenance assessed. The OSNs are unique in that they allow the manipulation of spontaneous and odor-induced activity in the mature animal. Experiments in this system implicate a role for activity in neuronal maintenance. Further, this study showed that Gsk-3β, a molecule that is a sensor for nutrients and Wnt signals, acts 'downstream' of neuronal activity in this process. The results thus integrate a key property of neurons, electrical activity, with Gsk-3β signaling. Although Gsk-3β can receive inputs from a variety of pathways, the expression of Wg in the adult antennal lobe and the observation that degeneration could be rescued by expression of Dsh and Wg, suggested that this is a major signaling pathway acting on neuronal survival in the mature animal. It was also established that Wnt signaling acts through a non-transcriptional pathway involving Gsk-3β. The action of Wnt signaling during the formation and plasticity of neuronal circuits is mediated through the 'divergent canonical pathway', as demonstrated by several studies in both vertebrates and Drosophila (Chiang, 2009).

Spontaneous activity is essential for both the development and maintenance of OSN projections in the mouse olfactory bulb. In Drosophila, the formation of the peripheral olfactory map is independent of ORs or activity, and is possibly hardwired. However, as in mammals, OSN maintenance requires neural activity. The availability of Or83b-null mutants and the conditional TARGET system was exploited to demonstrate that OSN terminals within the antennal lobe glomeruli develop normally in the absence of activity, but show local degeneration in older animals in which the most distal ends exhibit signs of beading, blebbing and, eventually, fragmentation, which are hallmarks of axon degeneration (Chiang, 2009).

In vertebrates, electrically silent visual system neurons and OSNs retract when placed in an environment of active neurons because of synaptic competition for target sites. When all neurons in a given field are silenced to the same extent, elimination does not occur, suggesting that differences in activity, rather than the absolute activity state, determine the stability of connections. Contrary to expectation from these findings, neurons innervating a single glomerulus retracted their contacts and showed degeneration when silenced. Further, clones that drive the light chain of tetanus toxin (TNT-G) in small subsets of OSNs also degenerate, indicating that activity influences neuron survival by exerting autonomous effects at the level of individual cells (Chiang, 2009).

Enhanced neuronal activity has been shown to trigger the activation of a wide range of genes including transcription factors, cell adhesion molecules, membrane excitability proteins, translational regulators such as dFmr (Fmr1) and signaling molecules such as Wnt. The observation that Wg is expressed in the adult brain led to a test of the role of Wg/Wnt signaling in stability. It was found that downregulation of Wg pathway members compromises OSN stability, resulting in phenotypes similar that resulting from a lack of neuronal activity. Several studies argue for a link between neuronal activity and Wnt/Wg signaling during formation of LTP and in activity-dependent dendritic morphogenesis. At the Drosophila neuromuscular junction, activity-dependent Wg secretion results in structural outgrowth mediated by Gsk-3β in the motoneurons and nuclear localization of the cleaved C terminus of DFz2 in the postsynaptic muscle cells (Ataman, 2008; Chiang, 2009 and references therein).

Evidence is provided for a requirement of Wg/Wnt signaling for stabilization of adult OSNs, although a transcriptional output of the pathway is not required. Non-transcriptional roles for Wnt signaling have been demonstrated previously, well-studied examples being in Wnt7a-induced growth cone and axon remodeling of the mossy fibers and in Drosophila neuromuscular junction synaptogenesis and plasticity (Ataman, 2008). The output of signaling in these systems is mediated by Gsk-3β, which regulates microtubule cytoskeletons. Gsk-3β phosphorylates the Microtubule-associated protein 1B (mammalian homolog of Futsch) and Tau, thereby influencing microtubule stability. Neuronal activity regulates Gsk-3β enzymatic activity through a series of phosphorylation and dephosphorylation events, whereby activity-regulated PP1 phosphatase and PI3K-Akt kinase regulate phosphorylation of Gsk-3β serine 9 (Chiang, 2009).

Activation of Gsk-3β in rat hippocampus inhibits LTP with associated synaptic impairments reminiscent of Alzheimer's disease. A genetic link between late-onset Alzheimer's disease and the Wnt pathway co-receptor LRP6 has been demonstrated in human subjects and there is a possibility that Alzheimer's disease-associated synaptic impairments are due to aberrant GSK-3β kinase activity. The phenotypes observed in OSNs with activated Gsk-3β or a chronic blockage of activity are tantalizingly similar to those described during neurodegeneration in Alzheimer's disease models(Chiang, 2009).

A model is proposed whereby neuronal activity acts together with Gsk-3β to maintain circuit stability in the adult olfactory system. Activity appears to lead to Wg release, which acts in an autocrine manner to impinge upon Gsk-3β activity. It is also possible that autonomous activity could signal to Gsk-3β through other pathways, one of them being the energy status of the neuron. In adult OSNs, Wnt signaling appears to be non-transcriptional and Gsk-3β possibly acts cell-autonomously to regulate the dynamics of the microtubule cytoskeleton. The results support the emerging view of a role for the Wnt pathway in neuroprotection, and the approach provides a system in which to examine the structural and molecular mechanisms that operate during altered physiological states in a genetically tractable organism (Chiang, 2009).

The kinase Sgg modulates temporal development of macrochaetes in Drosophila by phosphorylation of Scute and Pannier

Evolution of novel structures is often made possible by changes in the timing or spatial expression of genes regulating development. Macrochaetes, large sensory bristles arranged into species-specific stereotypical patterns, are an evolutionary novelty of cyclorraphous flies (see The development and evolution of bristle patterns in Diptera) and are associated with changes in both the temporal and spatial expression of the proneural genes achaete (ac) and scute (sc). Changes in spatial expression are associated with the evolution of cis-regulatory sequences, but it is not known how temporal regulation is achieved. One factor required for ac-sc expression, the expression of which coincides temporally with that of ac-sc in the notum, is Wingless (Wg). Wingless downregulates the activity of the serine/threonine kinase Shaggy (Sgg; also known as GSK-3). This study demonstrates that Scute is phosphorylated by Sgg on a serine residue and that mutation of this residue results in a form of Sc with heightened proneural activity that can rescue the loss of bristles characteristic of wg mutants. It is suggested that the phosphorylated form of Sc has reduced transcriptional activity such that sc is unable to autoregulate, an essential function for the segregation of bristle precursors. Sgg also phosphorylates Pannier, a transcriptional activator of ac-sc, the activity of which is similarly dampened when in the phosphorylated state. Furthermore, it was shown that Wg signalling does not act directly via a cis-regulatory element of the ac-sc genes. It is suggested that temporal control of ac-sc activity in cyclorraphous flies is likely to be regulated by permissive factors and might therefore not be encoded at the level of ac-sc gene sequences (Yang, 2012).

achaete-scute products become detectable in wing discs only at mid third larval instar. The known upstream regulators, Pnr and the Iro-C genes, are selector genes that pattern the medial and lateral halves of the notum, respectively. Therefore their activity is not restricted to ac-sc activation and bristle patterning and they are expressed for a considerable period before ac-sc gene products are detected. Furthermore, although activation of ac-sc in proneural clusters by Pnr and Iro-C dramatically increases transcription at these sites, the ac-sc genes are also expressed at low levels over the entire disc epithelium, presumably through activity of the basal promoters. Indeed maintenance of proneural genes in an active state of basal transcription is a general feature of neuroepithelia. So what prevents accumulation of Ac-Sc at earlier stages in disc development (Yang, 2012)?

This study has shown that Sc is phosphorylated by Sgg, an enzyme that is expressed constitutively. Furthermore a mutated form of Sc that is resistant to phosphorylation has significantly greater bristle-forming activity than the wild-type protein. This suggests reduced transcriptional activity of phospho-Sc. One possibility is that the turnover of phospho-Sc is rapid, owing to phosphorylation-dependent ubiquitination and degradation. It has been reported that mutations in the GSK-3β consensus motif in β-catenin abolishes ubiquitination and leads to protein stability. GSK-3β also induces ubiquitination and degradation of Drosophila myc protein through the proteasome pathway and mutation of residues in the phosphorylation domain affects stability of this protein. Indeed it has been shown that mutation of the phosphorylation site SPTS to APAA stabilizes the Sc protein. This suggests that before expression of wg at the mid third larval instar, the stability and transcriptional activity of any Sc present, whether derived from transcription mediated by the basal promoter or enhanced by Pnr and the Iro-C proteins, would be reduced through phosphorylation by Sgg (Yang, 2012).

Development of neural precursors requires high levels of Sc, which are needed for the process of lateral inhibition and singling out of precursors as well as for autoregulation. During this process in Drosophila, Sc binds its own promoter, through a specific regulatory sequence, the sensory organ precursor enhancer (SOPE), to further activate transcription in presumptive precursors (Culi, 1998). Therefore, any factors that diminish the activity of Sc itself have the potential to prevent sufficient accumulation to allow selection of precursors and maintenance of precursor cell fate. Expression of wg at mid third larval instar would lead to inactivation of Sgg. The consequent accumulation of a more active nonphosphorylated form of Sc might allow levels of Sc to accumulate sufficiently for precursor cell development. Achaete does not appear to be a target for Sgg. However, this protein has been shown to be dispensable for bristle development (Yang, 2012).

Pnr is also a target for phosphorylation by Sgg and, like Sc, a mutated phosphorylation-resistant form of Pnr is hyperactive. So phosphorylation of Pnr might also result in ubiquitination and increased degradation, a situation that would be modified by Wg signalling at mid third larval instar. The effects of phosphorylation on Pnr and Sc appear to be quantitative, rather than all or nothing. Pannier has other targets before Wg signalling and activation of ac-sc (the iro genes and wg itself) and if the sole function of Wg were to be the inactivation of Sgg then one would expect loss of sgg function to have no bristle phenotype. So de-phosphorylation might just give an extra little boost to the system. Interestingly it has been shown that the Drosophila transcription factor Mad is also a target of Sgg and that phosphorylation-resistant Mad proteins are hyperactive (Eivers, 2009). Mad is activated by Dpp/TGFβ signalling, which in turn regulates expression of both pnr and the Iro-C genes in the thorax. Thus, it appears that inactivation of Sgg by the Wg signal can stimulate the levels of Sc via multiple routes: by increasing the levels of expression of pnr and the Iro-C genes as well as the activity of Pnr and Sc themselves. Thus, expression of wg at mid third larval instar might result in levels of Sc sufficient for macrochaete development. It is not known how the second phase of ac-sc expression for microchaetes is regulated (Yang, 2012).

Wingless is unlikely to be the only factor regulating temporal ac-sc expression. Indeed, although loss of sgg function can affect bristles over the entire notum, the effects of wg appear to be restricted to the medial notum. Other factors must be involved on the lateral notum. One possibility is NFkappa-B/Rel, a factor that is required for functioning of the the sensory organ precursor enhancer (SOPE) and singling out of precursors, and that also indirectly affects the stability of sc transcripts (Culi, 1998; Ayyar, 2007). Another event that coincides with the accumulation of ac-sc products at mid third larval instar is a small peak of 20H-ecdysteroid (not associated with a moult). Indeed ecdysone has been implicated in temporal regulation of expression of the proneural gene atonal and the development of atonal-dependent sense organs (Yang, 2012).

Wingless signalling has important functions in the thorax, likely to be ancient, that are linked to the development and patterning of flight muscles. So wg was probably already expressed on the notum of the ancestor of the Cyclorrapha, before the evolution of macrochaetes. The rapid development of the notum and short pupal period in many Nematocera leaves little requirement for any temporal control of expression. By contrast, the prolonged period of growth and patterning during the larval and pupal life of Drosophila allows time for two discrete phases of proneural gene expression. Wingless might then have been co-opted for the regulation of ac-sc and the evolution of macrochaetes in the lineage leading to the Cyclorrapha. The current results suggest that the Wg signal does not involve transcriptional regulation of target genes but instead is mediated simply through inactivation of Sgg. The phosphorylation sites are strongly conserved in the sc genes of C. vicina and C. capitata, two other species of Schizophora, suggesting a conserved mechanism of regulation by Wg and Sgg. By contrast, the same sites are not conserved in the other genes of the Drosophila AS-C, or in the ac-sc homologues of A. gambiae, although other potential Sgg phosphorylation sites can be detected in these proteins. Phosphorylation of Sc by Sgg could have been recently acquired in the Cyclorrapha. The ac and sc genes themselves have arisen from duplication events thought to have taken place during evolution of the Cyclorrapha (see Negre, 2009). Phosphorylation of Pnr by Sgg might also have been acquired in the lineage leading to the Schizophora, as one of the sites is conserved in the pnr protein of C. vicina, but not that of Megaselia abdita or A. gambiae (Yang, 2012).

Uniform proneural gene expression, together with Notch-mediated lateral inhibition, is sufficient to generate a pattern of evenly spaced, but randomly positioned, bristles such as that seen in Nematocera and for the microchaetes of the Cyclorrapha. For this process, the SOPE, a very ancient regulatory element that predates the Diptera (Ayyar, 2010), is the only cis-regulatory element of ac-sc that would be required. Factors that act through the SOPE could be co-opted to modulate the temporal activity of ac-sc. This includes factors regulating activity of Sc, which itself binds the SOPE (Culi, 1998). Control at this level could be superimposed on the ancestral state without the need to acquire new regulatory sequences for the binding of novel transcriptional repressors and activators. By contrast, the spatially restricted expression underlying the macrochaete pattern is linked to changes at the AS-C complex and the acquisition of novel cis-regulatory elements that possibly arose in association with gene duplication events. This illustrates the power of evolution to make use of factors acting both in cis and in trans to effect morphological change (Yang, 2012).

Mad is required for wingless signaling in wing development and segment patterning in Drosophila

A key question in developmental biology is how growth factor signals are integrated to generate pattern. This study investigated the integration of the Drosophila BMP and Wingless/GSK3 signaling pathways via phosphorylations of the transcription factor Mad. Wingless was found to regulate the phosphorylation of Mad by GSK3 in vivo. In epistatic experiments, the effects of Wingless on wing disc molecular markers (senseless, distalless and vestigial) were suppressed by depletion of Mad with RNAi. Wingless overexpression phenotypes, such as formation of ectopic wing margins, were induced by Mad GSK3 phosphorylation-resistant mutant protein. Unexpectedly, Mad phosphorylation by GSK3 and MAPK was found to occur in segmental patterns. Mad depletion or overexpression produced Wingless-like embryonic segmentation phenotypes. In Xenopus embryos, segmental border formation was disrupted by Smad8 depletion. The results show that Mad is required for Wingless signaling and for the integration of gradients of positional information (Eivers, 2009; full text of article).

Transgenic flies expressing forms of Mad resistant to GSK3 phosphorylation displayed high BMP and Wg signaling phenotypes. Mad contains 74 serines/threonines, yet phosphorylation-resistant mutations of a single MAPK or of two GSK3 sites generated hyperactive transcription factors. Previous work in Drosophila had identified that phosphorylation by the Nemo/NLK kinase in the MH1 domain of Mad inhibits its activity, and that a neomorphic human Smad4 mutation can produce Wg-like phenotypes when overexpressed in the wing. During Drosophila early embryogenesis, Mad linker phosphorylations tracked the priming activity of MAPK/EGFR, particularly in the ventral side, suggesting that Mad may be regulated independently of dorsal Dpp signals. Drosophila EGFR activates MAPK in a broad ventral region which corresponds to the neurogenic ectoderm. Although this study focused on the role of Wg/GSK3 on Mad regulation, the priming phosphorylation for GSK3 is provided by EGFR signaling and is critical for Mad to be polyubquitinated and degraded in the centrosome (Eivers, 2009).

Mad MGM (Mad GSK mutant; see Phosphorylation-Resistant Mad Proteins are Hyperactive), which mimics Mad receiving a maximal Wg signal, phenocopied known Wg overexpression phenotypes. In the wing, MGM caused the formation of ectopic rows of Senseless-expressing cells, sensory bristles, and entire ectopic wing margins. In larval cuticles, MGM caused the reduction of ventral denticle belts, which were replaced by naked cuticle regions, an indicator of Wg signaling. The mad gene product was demonstrated to be involved in Wg signaling in multiple in vivo assays. In the wing disc, Wg overexpression strongly increased senseless, distalless, optomotor blind, and vestigial transcripts, and co-expression of Mad RNAi inhibited this effect, without affecting Wg expression levels. The induction of ectopic neurogenic ectoderm tissue positive for SoxNeuro by RNAi was epistatic to the inhibition of SoxNeuro expression caused by Wg overexpression. Taken together, these results suggest that Mad is a required component for several Wg signaling events in Drosophila (Eivers, 2009).

Segmentation phenotypes were observed when Mad RNAi was expressed maternally using the pUASp vector. Segment fusions were generated in which larval naked cuticle was replaced by large denticles of the same type (row 5) as those seen in Wg nulls. In gain-of-function experiments, overexpression of GSK3-resistant Mad caused denticle belts to be replaced by naked cuticle, mimicking Wg signaling. Thus, depletion or overexpression of Mad generated Wg-like phenotypes, indicating that Mad functions in the Wg signaling pathway during segmental patterning (Eivers, 2009).

The MAPK pathway, which during Drosophila embryonic segmentation is regulated by EGFR activity, would decrease the duration of the Mad signal by promoting Mad polyubiquitination and degradation. The EGFR-activating genes rhomboid and spitz are activated in the anterior of each segment and Wg in the posterior border of the anterior compartment. Wg/Wnt signals would increase the duration of the signal by inhibiting GSK3 phosphorylations, generating a double gradient of GSK3 and MAPK activities that would regulate Mad stability and signaling within each segment. This may occur in a Dpp-independent fashion, but it is also possible that BMP signals might be active during larval segmentation, since the expression of the BMP receptor thickveins has a segmental pattern of expression. In addition, Dpp is expressed in the ectoderm during segmentation stages, and its promoter contains segmentation modulation elements (Eivers, 2009).

Finding a role for Mad in segmentation was remarkable, because this process has been extensively studied in Drosophila genetic screens and Mad had not been previously implicated as part of the segmentation machinery. This new role for Mad can be explained by the fact that Mad appears to also function independently of Dpp and that the Mad10 and Mad12 null alleles are nulls only for the BMP pathway. This persistence of a Mad linker regulation by phosphorylation could explain results in the literature showing that Mad10 mutant clones can result in Wg-like effects in the Drosophila wing. Overexpression of Mad12 synthetic mRNAs mutated in the GSK3 phosphorylation sites have strong posteriorizing activity in Xenopus embryos. This indicates that Mad mutants previously thought to be nulls retain BMP-independent functions (Eivers, 2009).

There were previous indications of a role for Dpp during segmentation in the Drosophila literature. It has been noted that in hypomorphic mutations of the BMP antagonist short gastrulation (sog, the homolog of Chordin), four copies of Dpp caused loss of some denticles and an increase in naked cuticle. In addition, it has been reported that in Dpp null mutants the posterior spiracles are replaced by an ectopic denticle belt. As noted in this study, Dpp nulls can present denticle belt fusions, a phenotype that has been observed previously in embryos injected with noggin mRNA. Dpp null phenotypes and those of Mad10 and Mad12 mutants (which lose C- terminal, but not linker phosphorylations) are not identical to those of Mad RNAi. It is suggested that this is because Mad also has Dpp-independent functions. Dissecting which effects of Mad are Dpp-dependent and which ones are independent will be an interesting area of future investigation. This study presents evidence for both taking place (Eivers, 2009).

Future work will have to address the level at which Mad regulation by MAPK and GSK3 interacts with other intracellular components of the Wg transduction pathway that result in similar phenotypes. The phenotypes observed for Mad loss-of function and mad phosphorylation-resistant linker mutants overexpression were very similar to those found for the armadillo/β-catenin, pangolin/lef1, legless/bcl9 and pygopus genes. The present study does not resolve the issue of whether the stabilized forms of Mad interact directly at the protein-protein binding level, thus modifying the core Wg pathway, or at level of DNA enhancers. Wnt responsive enhancers frequently contain Smad binding sites near TCF/Pangolin binding sites. In the vertebrates, direct binding between Lef1/Tcf and Smads 1 to 4 at the level of enhancer binding sites has been known for some time. What this study now shows is that Mad is also directly regulated at the level of its phosphorylation at GSK3 sites by Wg signaling. The possibility that Wg-stabilized Mad may bind to Armadillo/β-catenin, Pangolin/lef1, Legless/bcl9 and Pygopus independently of nearby Mad binding sites cannot be excluded at present. Mechanistic studies will have to explain the remarkable similarities between the stabilized Mad phenotypes and those of canonical Wg phenotypes in wing discs and bristles, segments and in neurogenic ectoderm in Drosophila, which suggest a widespread requirement for Mad in Wg signaling. Another aspect that will need to be addressed is why in Xenopus Wnt signaling through Smad1 has a complete requirement for β-Catenin, and in Drosophila the MGM can induce senseless only in regions in which β-catenin is also stabilized (Eivers, 2009).

Many developmental mechanisms have been conserved during evolution, but segmentation is one in which commonalities between Drosophila and the vertebrates have not been found. Segmentation in vertebrates relies on the cyclic oscillation of Notch pathway transcripts in the posterior paraxial mesoderm. In theory, Smad1/5/8 could provide an attractive regulator of the segmentation clock, because BMP signals have a duration of 1-2 hours in cultured cells, which can be extended by inhibiting GSK3. Wnt pathway genes cycle rhythmically in vertebrates, offering an interesting possibility for regulating Smad5/8 activity. Notch is required for segmentation in spiders, but not in Drosophila (Damen, 2007). Recently, it has been found that in the cockroach, an insect in which the segments are formed sequentially in a posterior growth zone (and not simultaneously as in Drosophila), stripes of Delta and Hairy mRNA (two genes of the Notch pathway) cycle rhythmically as in the vertebrates (Damen, 2007). This study has now found that Smad5/8 is required for the formation of segmental boundaries in Xenopus somites and that Mad is required for Drosophila segment patterning. However, the results do not establish whether similar molecular steps are affected in both organisms. The conservation of this unexpected conserved role for Mad/Smad is important from an Evo-Devo perspective because it suggests that the last common ancestor shared between Drosophila and vertebrates, Urbilateria, might have been segmented (Eivers, 2009).

These studies on Drosophila Mad have uncovered an unexpected role for Mad in the Wg signaling pathway. Mad/Smads are transcription factors that have low binding affinity for DNA and require other DNA binding proteins as co-factors in order to recognize the promoters and enhancers of hundreds of target genes. Future work will have to address how Mad or its partner Medea/Smad4 interact with proteins such as Armadillo/β-Catenin and Pangolin/Lef1 on Wnt-responsive promoters in Drosophila. The present study shows that Mad is required for Wg to signal, through its GSK3 phosphorylation sites, in a number of different in vivo assays. These include wing margin formation, sensory bristle induction in the wing, induction of the Wg induced gene senseless, the repression of neurogenic ectoderm, and segmental patterning. It is proposed that Mad serves as an integrator of patterning signals, which determine embryonic positional information. The finding that three major signaling pathways - MAPK, Wnt/GSK3 and BMP - are integrated at the level of Mad/Smad1/5/8 both in Drosophila and in the vertebrates has interesting implications for the evolution of animal forms through variations on an ancestral gene tool-kit (Eivers, 2009).

Electron transport chain remodeling by GSK3 during oogenesis connects nutrient state to reproduction

Reproduction is heavily influenced by nutrition and metabolic state. Many common reproductive disorders in humans are associated with diabetes and metabolic syndrome. This study characterized the metabolic mechanisms that support oogenesis and found that mitochondria in mature Drosophila oocytes enter a low-activity state of respiratory quiescence by remodeling the electron transport chain (ETC). This shift in mitochondrial function leads to extensive glycogen accumulation late in oogenesis and is required for the developmental competence of the oocyte. Decreased insulin signaling initiates ETC remodeling and mitochondrial respiratory quiescence through glycogen synthase kinase 3 (GSK3). Intriguingly, similar ETC remodeling and glycogen uptake was observed in maturing Xenopus oocytes, suggesting that these processes are evolutionarily conserved aspects of oocyte development. These studies reveal an important link between metabolism and oocyte maturation (Sieber, 2016).

SCF-Slimb is critical for Glycogen synthase kinase-3beta-mediated suppression of TAF15-induced neurotoxicity in Drosophila

Amyotrophic lateral sclerosis (ALS) is a severe neurodegenerative disorder characterized pathologically by motor neuron degeneration and associated with aggregation of RNA-binding proteins. TATA-binding protein-associated factor 15 (TAF15) accumulates as cytoplasmic aggregates in neuronal cells, and clearance of these aggregates is considered a potential therapeutic strategy for ALS. However, the exact pathogenic mechanism of TAF15-induced neurotoxicity remains to be elucidated. Glycogen synthase kinase-3 (GSK-3) plays a critical role in the protection of ALS pathology. In this study, a transgenic fly model over-expressing human TAF15 was used to study the protective effects of Shaggy/GSK3β on TAF15-induced neuronal toxicity in Drosophila brain. Transgenic flies were examined for locomotor activity and lithium treatment. The expression level and solubility of TAF15 were assessed with western blotting, whereas immunohistochemistry was used to assess TAF15 aggregation in Drosophila brain. This study has revealed that Shaggy/GSK3β was abnormally activated in neurons of TAF15-expressing flies and its inhibition can suppress the defective phenotypes, thereby preventing retinal degeneration and locomotive activity caused by TAF15. It was also found that Shaggy/GSK3β inhibition in neuronal cells leads to a reduction in TAF15 levels. Indeed, the F-box proteins Slimb and archipelago genetically interact with TAF15 and control TAF15 protein level in Drosophila. Importantly, SCF(slimb) is a critical regulator for Shaggy/GSK3β-mediated suppression of TAF15-induced toxicity in Drosophila. The present study has provided an in vivo evidence supporting the molecular mechanism of GSK3β inhibition for protection against TAF15-linked proteinopathies (Choi, 2020).

Protein interactions

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).

Control of ß-Catenin phosphorylation/degradation by a dual-kinase mechanism

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).

Combinatorial signaling by an unconventional Wg pathway and the Dpp pathway requires Nejire (CBP/p300) to regulate dpp expression in posterior tracheal branches

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).

A Neurogenic role for Shaggy

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).

Shaggy targets Cubitus interruptus, a transcription factor in the Hedgehog pathway

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).

Shaggy/GSK3 antagonizes Hedgehog signaling by regulating Cubitus interruptus

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-regulated Costal2-kinase complexes control phosphorylation and proteolytic processing of Cubitus interruptus

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).

PAR-1 phosphorylates tau at S262 and S356 as a prerequisite for the action of downstream kinases, including glycogen synthase kinase 3 and cyclin-dependent kinase-5

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).

Drosophila Twins regulates Armadillo levels in response to Wg/Wnt signal: Protein Phosphatase 2A targets Shaggy

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).

A resetting signal between Drosophila pacemakers synchronizes morning and evening activity; Shaggy function as a Timeless kinase

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).

The Drosophila circadian network is a seasonal timer

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).

Processing of the Drosophila hedgehog signaling effector Ci-155 to the repressor Ci-75 is mediated by direct binding to the SCF component Slimb: Requirement for modification of Ci by PKA, CK1, and GSK3

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).

Serotonin modulates circadian entrainment in Drosophila

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).

AKT and TOR signaling set the pace of the circadian pacemaker

The circadian clock coordinates cellular and organismal energy metabolism. The importance of this circadian timing system is underscored by findings that defects in the clock cause deregulation of metabolic physiology and result in metabolic disorders. On the other hand, metabolism also influences the circadian clock, such that circadian gene expression in peripheral tissues is affected in mammalian models of obesity and diabetes. However, to date there is little to no information on the effect of metabolic genes on the central brain pacemaker which drives behavioral rhythms. This study found that the AKT and TOR-S6K pathways, which are major regulators of nutrient metabolism, cell growth, and senescence, impact the brain circadian clock that drives behavioral rhythms in Drosophila. Elevated AKT or TOR activity lengthens circadian period, whereas reduced AKT signaling shortens it. Effects of TOR-S6K appear to be mediated by SGG/GSK3beta, a known kinase involved in clock regulation. Like SGG, TOR signaling affects the timing of nuclear accumulation of the circadian clock protein Timeless. Given that activities of AKT and TOR pathways are affected by nutrient/energy levels and endocrine signaling, these data suggest that metabolic disorders caused by nutrient and energy imbalance are associated with altered rest:activity behavior (Zheng, 2010).

There are several possible mechanisms by which nutrient and energy metabolism could affect peripheral clocks. Local physiological factors dependent on metabolic activity could influence the expression of core clock components and of nuclear receptors that regulate clock gene expression. Indeed, cellular redox state, AMPK activity, NAD+ levels, and SIRT1 activities appear to feed into the circadian clock in peripheral tissues such as the liver. AMPK, which acts upstream of TSC in mammals, directly phosphorylates Cryptochrome in peripheral tissues. However, prior to this work, there was no known mechanism for the modulation of the central pacemaker by nutrient-sensing pathways. This study identifies such a mechanism by demonstrating that metabolic genes such as AKT and TOR-S6K act in the central pacemaker cells in the brain. The lengthened circadian period caused by high-fat diet in mammals is likely mediated by these molecules. This conclusion is further supported by a recent cell-culture-based genome-wide RNAi study that implicated the PI3K-TOR pathway in the regulation of circadian period. In addition, another ribosomal S6 kinase (S6KII) was found to influence the circadian clock through its interaction with casein kinase 2β. Importantly, daily fasting:feeding cycles driven by the central clock regulate circadian gene transcription in the liver, whereas clock function in the liver contributes to energy homeostasis. It is speculated that metabolic stress or energy imbalance affects AKT and TOR-S6K signaling, resulting in general circadian disruption, which in turn exacerbates metabolic deregulation and, consequently, facilitates the development of metabolic syndromes prevalent in modern society (Zheng, 2010).

Drosophila caspase transduces Shaggy/GSK-3beta kinase activity in neural precursor development

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 encoded by the sgg gene and essential for the negative regulation of Wingless signaling, is cleaved by the Dark-dependent caspase. This cleavage converts it to an active kinase, which contributes to the formation of neural precursor [sensory organ precursor (SOP)] cells. This evidence suggests that caspase regulation of the wingless pathway is not associated with apoptotic cell death. These results imply a novel role for caspases in modulating cell signaling pathways through substrate cleavage in neural precursor development (Kanuka, 2005; full text of article).

Previous genetic studies of sgg mutant flies showed the interesting observation that some phenotypes of sgg mutants can be rescued by the expression of sgg10 or sgg39 (the other sgg isoform similar to sgg10), but not sgg46, suggesting that Sgg46 might be an inactive form. The Sgg10 kinase phosphorylates the Arm protein and induces its degradation. Various forms of Sgg protein were tested for this activity. Expression of Sgg10 induces Arm phosphorylation and degradation in a kinase-dependent manner. In contrast, full-length Sgg46 did not produce the same effects on the Arm protein. Interestingly, expression of a putative cleaved form of Sgg46, containing the kinase domain (myc-Sgg46 DeltaN235 and myc-Sgg46 DeltaN300), led to Arm phosphorylation and degradation in a manner similar to that of Sgg10. These results suggest that full-length Sgg46 is an inactive form that can be converted into an active kinase via caspase-dependent cleavage (Kanuka, 2005).

Whether these findings would be applicable to macrochaete and SOP cell development in vivo was tested by using transgenic flies expressing Sgg proteins. The ectopic expression of Sgg10 by sca-GAL4 caused the loss of macrochaetes and SOP cells. No apoptotic cells in the myc-Sgg10 protein-expressing region of the wing disc could be detected, indicating that this disappearance did not result from the death of SOP cells. Consistent with the immunoblotting results, full-length Sgg46 did not influence macrochaete and SOP cell formation, whereas the cleaved form of Sgg46 (Sgg46 DeltaN300) worked in a manner similar to that of Sgg10. After crossing sca-GAL4+UAS-DRONC DN to UAS-sgg10, most F1 progeny showed a clear loss of macrochaetes in the scutellum, indicating that Sgg kinase activation might be downstream of caspases. These observations suggest that the processing of Sgg46 by caspases leads to the formation of an active kinase that can negatively regulate SOP cell development (Kanuka, 2005).

Finally, whether Sgg46 contributes significantly to macrochaete and SOP cell formation in vivo was investigated. The ectopic expression of Sgg46 D235G/D300G by sca-GAL4 significantly induced extra macrochaetes and SOP cells. Since Sgg46 D235G/D300G could not be cleaved by caspases, this noncleaved Sgg46 might act as dominant-negative form against endogenous Sgg function. Furthermore, an ectopic knockdown of Sgg protein expression by dsRNA-expressing constructs revealed that the specific reduction of the Sgg46 protein induced extra macrochaetes. However, inhibition of Sgg46 is less effective at producing extra macrochaetes than inhibiting Dark or DRONC, suggesting that modulation of Sgg kinase activity may not be the only mechanism contributing to SOP formation. It still remains to be examined whether or not Sgg46 is actually cleaved and converted into an active form in proneural clusters, and will require further examination in vivo. Based on the findings that loss of Sgg function or inhibition of caspase activity resulted in extra macrochaetes mainly in the scutellum of the adult notum (pSC and aSC), where Wingless is highly expressed, and that caspases are activated in scabrous-expressing cluster, it could be considered that scabrous-expressing SOP cells that will produce pSC and aSC macrochaetes are located in specific region, where precise formation of each set of macrochaetes might require both (1) Wg expression (to increase bristle) and (2) caspase activation (to decrease bristle). Thus, it appears that Dark-dependent caspase signaling mediates the total Sgg kinase activity by processing Sgg46 into an active form, thereby negatively regulating Wingless-sensitive macrochaete development (Kanuka, 2005).

A genome-wide Drosophila RNAi screen identifies DYRK-family kinases as regulators of NFAT

Precise regulation of the NFAT (nuclear factor of activated T cells) family of transcription factors (NFAT1-4) is essential for vertebrate development and function. In resting cells, NFAT proteins are heavily phosphorylated and reside in the cytoplasm; in cells exposed to stimuli that raise intracellular free Ca2+ levels, they are dephosphorylated by the calmodulin-dependent phosphatase calcineurin and translocate to the nucleus. NFAT dephosphorylation by calcineurin is countered by distinct NFAT kinases, among them casein kinase 1 (CK1) and glycogen synthase kinase 3 (GSK3). This study used a genome-wide RNA interference (RNAi) screen in Drosophila to identify additional regulators of the signalling pathway leading from Ca2+-calcineurin to NFAT. This screen was successful because the pathways regulating NFAT subcellular localization (Ca2+ influx, Ca2+-calmodulin-calcineurin signalling and NFAT kinases) are conserved across species, even though Ca2+-regulated NFAT proteins are not themselves represented in invertebrates. Using the screen, DYRKs (dual-specificity tyrosine-phosphorylation regulated kinases) has been identified as novel regulators of NFAT. DYRK1A and DYRK2 counter calcineurin-mediated dephosphorylation of NFAT1 by directly phosphorylating the conserved serine-proline repeat 3 (SP-3) motif of the NFAT regulatory domain, thus priming further phosphorylation of the SP-2 and serine-rich region 1 (SRR-1) motifs by GSK3 and CK1, respectively. Thus, genetic screening in Drosophila can be successfully applied to cross evolutionary boundaries and identify new regulators of a transcription factor that is expressed only in vertebrates (Gwack, 2006).

To validate the use of genome-wide RNAi screening in Drosophila to identify regulators of the Ca2+-calcineurin-NFAT signalling pathway, an NFAT-GFP (green fluorescent protein) fusion protein containing the entire regulatory domain of NFAT1 was used. This domain bears >14 phosphorylated serines, 13 of which are dephosphorylated by calcineurin. Five of the thirteen serines are located in the SRR-1 motif, which controls exposure of the nuclear localization sequence (NLS) and is a target for phosphorylation by CK1; three are located in the SP-2 motif, which can be phosphorylated by GSK3 after a priming phosphorylation by protein kinase A (PKA); and four are located in the SP-3 motif, for which a relevant kinase had yet to be identified at the time this study was initiated. The SP-2 and SP-3 motifs do not directly regulate the subcellular localization of NFAT1, but their dephosphorylation increases both the probability of NLS exposure and the affinity of NFAT for DNA. NFAT-GFP was correctly regulated in Drosophila S2R+ cells: it was phosphorylated and localized to the cytoplasm under resting conditions; it became dephosphorylated and translocated to the nucleus with appropriate kinetics in response to Ca2+ store depletion with the sarcoplasmic/endoplasmic reticulum ATPase (SERCA) inhibitor thapsigargin; and its dephosphorylation and nuclear translocation were both sensitive to the calcineurin inhibitor cyclosporin A (CsA). S2R+ cells treated with limiting amounts of thapsigargin displayed intermediate phosphorylated forms of NFAT-GFP, most likely reflecting progressive dephosphorylation of serines within individual conserved motifs of the regulatory domain. Depletion of the primary NFAT regulator, calcineurin, by RNAi in S2R+ cells inhibited thapsigargin-dependent dephosphorylation and nuclear import of NFAT-GFP. Thus, the major pathways regulating NFAT phosphorylation and subcellular localization (store-operated Ca2+ influx, calcineurin activation and NFAT phosphorylation/dephosphorylation) are conserved in Drosophila and appropriately regulate vertebrate NFAT (Gwack, 2006).

A genome-wide RNAi screen on unstimulated S2R+ cells was performed, and aberrant nuclear localization of NFAT-GFP was scored. Positive candidates obtained in the screen include (1) Na+/Ca2+ exchangers and SERCA Ca2+ ATPases, the knockdown of which would be expected to increase basal levels of intracellular free Ca2+ ([Ca2+]i); (2) the scaffold protein Homer, which has been linked to Ca2+ influx and Ca2+ homeostasis; (3) stromal interaction molecule (STIM), a recently identified regulator of store-operated Ca2+ influx, and (4) several protein kinases that control NFAT function either directly via phosphorylation or indirectly via basal [Ca2+]i levels, calcineurin activity, or other kinases. To identify kinases that directly phosphorylate the NFAT regulatory domain, Flag-tagged human homologues of selected Drosophila kinases were expressed in HEK293 cells, and anti-Flag immunoprecipitates were tested in an in vitro kinase assay for their ability to phosphorylate a GST-NFAT1(1-415) fusion protein. Three kinases -- protein kinase cGMP-dependent (PRKG1), DYRK2 and interleukin (IL)-1 receptor-associated kinase 4 (IRAK4) -- showed strong activity in this assay. In cells, only DYRK2 countered the dephosphorylation of NFAT-GFP by calcineurin, even though both PRKG1 and DYRK2 were expressed at high levels. CD4+ TH1 cells isolated from Irak4-/- mice showed normal NFAT1 dephosphorylation, re-phosphorylation and nuclear transport compared to control TH1 cells. Therefore focus was placed on DYRK-family kinases as potential direct regulators of NFAT (Gwack, 2006).

DYRKs constitute an evolutionarily conserved family of proline- or arginine-directed protein kinases belonging to the CMGC family of cyclin-dependent kinases (CDKs), mitogen-activated protein kinases (MAPKs), GSK and CDK-like kinases (CLKs). The DYRK family has multiple members that can be predominantly nuclear (DYRK1A and DYRK1B) or cytoplasmic (DYRK2-4 and homeodomain interacting protein kinase 3 (HIPK3)/DYRK6). RT-PCR and western blotting suggested that DYRK1A and DYRK2 were major representatives of nuclear and cytoplasmic DYRKs, respectively, in Jurkat T cells, a conclusion supported by several additional observations. (1) Overexpression of DYRK2 prevented dephosphorylation of NFAT-GFP after ionomycin treatment; overexpression of wild-type DYRK2, but not a kinase-dead mutant of DYRK2, prevented NFAT nuclear localization in thapsigargin-treated cells. The slower-migrating form of NFAT might indicate that DYRK2, a proline-directed kinase, acts in part by phosphorylating the SPRIEITP docking sequence on NFAT1 and thereby blocking the calcineurin-NFAT interaction. However, DYRK was still effective when the potential DYRK phosphorylation sites in the docking sequence were eliminated by substituting HPVIVITGP for SPRIEITPS. (2) Both wild-type and kinase-dead DYRK co-immunoprecipitated with NFAT1. (3) Depletion of the DYRK-family candidate CG40478 in S2R+ cells did not affect (and DYRK2 overexpression in Jurkat T cells only slightly diminished) Ca2+ mobilization in response to thapsigargin. (4) Most importantly, depletion of endogenous DYRK1A with DYRK1A-specific short interfering RNAs (siRNA) in HeLa cells stably expressing NFAT-GFP increased the rate and extent of NFAT1 dephosphorylation and nuclear import while slowing re-phosphorylation and nuclear export. These results show that DYRK1A and DYRK2 are physiological negative regulators of NFAT activation in cells. The absence of basal NFAT dephosphorylation in DYRK1A-depleted cells may reflect both the expression of other DYRK family members in human cells and the predominantly nuclear localization of DYRK1A (Gwack, 2006).

DYRKs are direct NFAT1 kinases that selectively phosphorylate the SP-3 motif, but nevertheless control the overall phosphorylation of NFAT1. Flag-tagged DYRK2 expressed in HEK cells, as well as bacterially expressed recombinant DYRK1A and DYRK2, phosphorylated peptides corresponding to the SP-3 motif of the NFAT1 regulatory domain in vitro, but did not phosphorylate SRR-1 or SP-2 peptides or an SP-3 peptide with serine to alanine substitutions in the known phosphoserine residues. Two serine residues (underlined) in the SP-3 motif (SPQRSRSPSPQPSPHVAPQDD) fit the known sequence preference of DYRK kinases [R(x)xx(S/T)(P/V)], and both are known to be phosphorylated in cells. DYRK is reported to prime for GSK3-mediated phosphorylation of eukaryotic initiation factor 2B-epsilon (eIF2B-epsilon) and the microtubule-associated protein tau, as well as for GSK3- and CK1-mediated phosphorylation of OMA-1. Therefore whether DYRK kinases could also prime for GSK3- and CK1-mediated phosphorylation of NFAT1 was investigated. Pre-phosphorylation of the NFAT1 regulatory domain by DYRK2 led to robust phosphorylation by GSK3 and induced the mobility shift characteristic of phosphorylation of the SP-2 and SP-3 motifs; this shift was not observed after pre-phosphorylation by PKA. Furthermore, pre-phosphorylation by DYRK2 accelerated CK1-mediated phosphorylation of GST-NFAT1(1-415) by at least twofold. In contrast, DYRK2 did not prime phosphorylation of the SP-2 peptide by GSK3 nor the SRR-1 peptide by CK1, consistent with the fact that neither motif is a substrate for DYRK. This 'discontiguous' priming mechanism is distinct from conventional priming, which requires phosphorylation at +4 and -3 for GSK3 and CK1, respectively. A less likely interpretation is that the conventional priming sites for CK1 and GSK3 are efficiently phosphorylated by DYRK in the context of the GST-NFAT1(1-415) protein, although they are not phosphorylated in the peptide context (Gwack, 2006).

The kinase-dead mutant of DYRK2 shows that DYRK regulates the transcriptional activity of NFAT. Wild-type DYRK2 strongly diminished NFAT-dependent activity, whereas the kinase-dead mutant increased NFAT-dependent luciferase activity of the IL2 promoter, of an NFAT-activating protein 1 (AP-1) reporter, or of an AP-1-independent promoter (the kappa3 site of the tumour-necrosis factor-alpha (TNF-alpha) promoter). Similarly, wild-type DYRK2 diminished production of endogenous IL-2 by stimulated Jurkat T cells, whereas kinase-dead DYRK2 had the opposite effect (Gwack, 2006).

These data indicate that DYRK is a key kinase that regulates NFAT1 phosphorylation. DYRK, GSK3 and CK1 target completely distinct motifs of the NFAT1 regulatory domain, but DYRK-mediated phosphorylation of the SP-3 motif primes for further phosphorylation of the distinct SRR-1 and SP-2 motifs by CK1 and GSK3, respectively, thus facilitating complete phosphorylation and deactivation of NFAT1. This mechanism, which has been termed 'discontiguous priming', is reminiscent of that recently proposed for C. elegans oocyte maturation protein 1 (OMA-1), in which phosphorylation of Thr 239 by the DYRK-family kinase minibrain kinase 2 (MBK-2) potentiates GSK3-mediated phosphorylation of Thr 339. It is likely that DYRK2, DYRK3 and DYRK4, which are localized to the cytoplasm, function primarily as 'maintenance' kinases that sustain the phosphorylation status of cytoplasmic NFAT in resting cells, whereas DYRK1A and DYRK1B, which are localized to the nucleus, re-phosphorylate nuclear NFAT and promote its nuclear export. Notably, NFAT dephosphorylation may also proceed through a sequential mechanism, with dephosphorylation of the SRR-1 motif promoting dephosphorylation of the SP-2 and SP-3 motifs by increasing their accessibility to calcineurin. DYRK1A and the endogenous calcineurin regulator DSCR1/RCN/calcipressin-1 are both localized to the Down's syndrome critical region on human chromosome 21; thus, overexpression of these negative regulators of NFAT could contribute (by inhibiting NFAT activation) to the neurological and immunological developmental anomalies observed in individuals with chromosome 21 trisomy (Gwack, 2006).

Genome-wide RNAi screening in Drosophila is a valid and powerful strategy for exploring novel aspects of signal transduction in mammalian cells, provided that key members of the signalling pathway are evolutionarily conserved and represented in the Drosophila genome. This study used the method to identify conserved regulators of the purely vertebrate transcription factor NFAT; this is the first example of a genome-wide RNAi screen that crosses evolutionary boundaries in this manner. It is likely that conserved aspects of the regulation of other mammalian processes will also be successfully defined by developing assays in Drosophila cells (Gwack, 2006).

GSK-3β-regulated interaction of BICD with dynein is involved in microtubule anchorage at centrosome

Microtubule arrays direct intracellular organization and define cellular polarity. This study shows a novel function of glycogen synthase kinase-3β (GSK-3β) in the organization of microtubule arrays through the interaction with Bicaudal-D (BICD). BICD is known to form a complex with dynein-dynactin and to function in the intracellular vesicle trafficking. The data revealed that GSK-3β is required for the binding of BICD to dynein but not to dynactin. Knockdown of GSK-3β or BICD reduced centrosomally focused microtubules and induced the mislocalization of centrosomal proteins. The unfocused microtubules in GSK-3β knockdown cells were rescued by the expression of the dynein intermediate chain-BICD fusion protein. Microtubule regrowth assays showed that GSK-3β and BICD are required for the anchoring of microtubules to the centrosome. These results imply that GSK-3beta may function in transporting centrosomal proteins to the centrosome by stabilizing the BICD1 and dynein complex, resulting in the regulation of a focused microtubule organization (Fumoto, 2006).

Cell cycle control of Wnt receptor activation; Regulation of LRP6/Arrow activity

Low-density lipoprotein receptor related proteins 5 and 6 (LRP5/6; Drosophila Arrow) are transmembrane receptors that initiate Wnt/β-catenin signaling. Phosphorylation of PPPSP motifs in the LRP6 cytoplasmic domain is crucial for signal transduction. Using a kinome-wide RNAi screen, it was shown that PPPSP phosphorylation requires the Drosophila Cyclin-dependent kinase (CDK) L63. L63 and its vertebrate homolog PFTK are regulated by the membrane tethered G2/M Cyclin, Cyclin Y, which mediates binding to and phosphorylation of LRP6. As a consequence, LRP6 phosphorylation and Wnt/β-catenin signaling are under cell cycle control and peak at G2/M phase; knockdown of the mitotic regulator CDC25/string, which results in G2/M arrest, enhances Wnt signaling in a Cyclin Y-dependent manner. In Xenopus embryos, Cyclin Y is required in vivo for LRP6 phosphorylation, maternal Wnt signaling, and Wnt-dependent anteroposterior embryonic patterning. G2/M priming of LRP6 by a Cyclin/CDK complex introduces an unexpected new layer of regulation of Wnt signaling (Davidson, 2009).

Wnt/β-catenin signaling regulates patterning and cell proliferation throughout embryonic development and is widely implicated in human disease, notably cancer. Two principal classes of transmembrane (TM) receptors function to transduce Wnt/β-catenin signaling; the seven pass TM Frizzled (Fz) proteins and the single pass TM low density lipoprotein receptor-related proteins 5 and 6 (LRP5/6; Drosophila Arrow). Frizzled receptors activate β-catenin-dependent (canonical) as well as β-catenin-independent (noncanonical, such as planar cell polarity) pathways, while LRP5/6 function more specifically in the Wnt/β-catenin pathway (Davidson, 2009).

LRP6 signaling requires Ser/Thr phosphorylation of its intracellular domain (ICD), which contains five PPPSPXS dual phosphorylation motifs comprising Pro-Pro-Pro-Ser-Pro (PPPSP) and directly adjacent casein kinase 1 (CK1) sites. Phosphorylation of the most N-terminal PPPSP (S1490) involves glycogen synthase kinase 3 (GSK3), while CK1g phosphorylates two Ser/Thr clusters near S1490. Phosphorylation of CK1 sites is downstream of, and requires, PPPSP phosphorylation; however, alternative epistasis models have also been proposed. Both PPPSP and CK1 site phosphorylation is necessary for Axin binding to LRP6 and Wnt/β-catenin pathway activation. Phosphorylated PPPSPXS motifs directly inhibit the ability of GSK3 to phosphorylate β-catenin, providing a potential mechanism linking LRP6 activation to β-catenin stabilization. Investigating how LRP6 phosphorylation is regulated is thus crucial for understanding Wnt receptor activation and downstream signaling. Constitutive, non-Wnt-induced S1490 phosphorylation has been observed, suggesting that additional proline-directed kinases may be involved, such as the ERK or Cyclin-dependent kinase (CDK) subgroups (Davidson, 2009).

CDKs are regulators of the cell cycle and require Cyclin partners, whose levels are precisely controlled during the cell cycle, endowing CDKs with both temporal activity and substrate specificity. Several less well-characterized CDK-like proteins exist, including the PFTAIRE kinase subfamily. This study reports on the identification of a Cyclin/PFTAIRE-CDK complex that phosphorylates LRP6 S1490 in a cell cycle-dependent manner, which brings Wnt/β-catenin signaling under G2/M control and introduces a surprising new principle in Wnt regulation (Davidson, 2009).

An important issue in the field of Wnt/β-catenin signaling concerns the regulation of LRP5/6/Arrow function via phosphorylation. This study has identified the unusual plasma membrane tethered Cyclin Y/PFTAIRE complex which functions predominantly at the G2/M phase of the cell cycle to phosphorylate the PPPSP motifs of LRP6. The results suggest a G2/M priming model of LRP5/6/Arrow phosphorylation, where the Cyclin Y/CDK complex phosphorylates LRP6 at PPPSP motifs, which then primes adjacent phosphorylation by CK1. However, PPPSP priming alone is not sufficient for phosphorylation by CK1, as Wnt-induced LRP6 aggregation is also required. Combined phosphorylation at PPPSP and CK1 sites then promotes Gsk3-Axin binding to LRP6 and signalosome formation. Since GSK3 and Cyclin Y/CDK are both essential for LRP6 priming they apparently act nonredundantly. So why is there a dual kinase input to PPPSP phosphorylation? The phosphorylation of LRP6 by GSK3 occurs in acute response to Wnt signaling and it was suggested that it serves to amplify receptor activation. Cyclin Y/CDK phosphorylates Wnt independently at G2/M, thereby gating signal transduction in proliferating cells. One possibility is that individually both kinases prime LRP6 substoichiometrically at the five PPPSP sites and that only their combined action is sufficient for full LRP6 signaling competence (Davidson, 2009).

These findings have important implications for the link between proliferation and Wnt signaling. It has been long known that there is cross talk between mitogenic growth factors and Wnt signaling. The current results may explain why mitogenic growth factors synergize with Wnt/β-catenin signaling, namely by G2/M priming of LRP6 through enhanced cell proliferation, which sensitizes LRP6 for incoming Wnt signals. Moreover, not only extracellular but also intracellular cell cycle check point regulators controlling G2/M entry are likely to affect Wnt signaling (Davidson, 2009).

Wnt/β-catenin signaling itself promotes G1 progression by inducing c-myc and cyclin D1. This suggests that Wnt/β-catenin signaling can entrain a positive feedback loop in proliferating cells by promoting cell cycle progression, which triggers LRP6 phosphorylation at G2/M. Simultaneous stimulation by Wnt and mitogenic growth factors could initiate such a loop. Indeed, the results may explain the previously noted G2/M enrichment of β-catenin and Wnt signaling. Likewise, protein levels of the direct Wnt target gene Axin2, considered a marker gene for Wnt/β-catenin signaling, also peak during mitosis (Davidson, 2009).

What may be the function of a Wnt positive feedback loop during the cell cycle? One of the many roles of Wnt/beta-catenin signaling is to promote cell proliferation and the positive feedback loop suggested by this study may enhance the systems' levels properties of the cell cycle. Specifically, the loop may promote synchrony of cell cycle regulated events or constitute a bistable switch between cell proliferation and cell cycle exit (Davidson, 2009).

One interesting question raised by this study concerns preferential transcription of Wnt target genes around G2/M. Most genes are transcriptionally silenced between late prophase and early telophase, yet TOPFLASH reporter and AXIN2 peak around G2/M. It will therefore be interesting to investigate whether Wnt target genes are transcribed during the more permissive stages G2, early prophase, or late telophase (Davidson, 2009).

Another important question raised by this study is whether G2/M priming is essential or only modulatory for Wnt/β-catenin signaling in general, in particular in light of Wnt signaling in nondividing cells. The fact that LRP6 signaling is promoted by G2/M phase does not exclude Wnt/β-catenin signaling in other cell cycle phases or in nondividing cells. Even though during interphase the levels of LRP6 signalosomes, Sp1490, β-catenin, and reporter activation are lower compared to G2/M, such Wnt/β-catenin signaling is likely physiologically relevant and may involve additional PPPSP kinases, such as GSK3. Surprisingly little is known about Wnt/β-catenin signaling in nondividing cells. In transgenic Wnt-reporter mice, Wnt activity is detected in apparently postmitotic cells in the adult brain, retina, and certain liver cells. In the adult liver, Wnt/β-catenin signaling controls perivenous gene expression. Furthermore, Wnts play a role in axon remodeling in postmitotic neurons and at least one study suggests that this can involve the β-catenin pathway. In light of the current results it will be interesting to examine more systematically Wnt/β-catenin signaling and in particular the LRP6 kinases involved in postmitotic cells (Davidson, 2009).

Traditionally it is thought that Wnt/β-catenin signaling acts to regulate gene expression of downstream targets. Why then should Wnt/β-catenin signaling peak at G2/M? One likely answer is that components of the Wnt/β-catenin pathway play a crucial role during mitosis beyond transcriptional activation. In C. elegans, Wnt signaling regulates the orientation of the mitotic spindle in early development. In mammalian cells, phosphorylated β-catenin itself binds to centrosomes and is involved in spindle separation during mitosis. Likewise, GSK3, Adenomatous polyposis coli protein (APC) and Axin2, which are components of the β-catenin destruction complex, also have direct functions in mitosis. Taken together these data suggest that Cyclin Y/CDK phosphorylates LRP6 at G2/M to induce Wnt/β-catenin signaling for orchestrating a mitotic program (Davidson, 2009).

Endogenous GSK-3/shaggy regulates bidirectional axonal transport of the amyloid precursor protein

Neurons rely on microtubule (MT) motor proteins such as kinesin-1 and dynein to transport essential cargos between the cell body and axon terminus. Defective axonal transport causes abnormal axonal cargo accumulations and is connected to neurodegenerative diseases, including Alzheimer's disease (AD). Glycogen synthase kinase 3 (GSK-3) has been proposed to be a central player in AD and to regulate axonal transport by the MT motor protein kinesin-1. Using genetic, biochemical and biophysical approaches in Drosophila melanogaster, this study found that endogenous GSK-3 is a required negative regulator of both kinesin-1-mediated and dynein-mediated axonal transport of the amyloid precursor protein (APP), a key contributor to AD pathology. GSK-3 also regulates transport of an unrelated cargo, embryonic lipid droplets. By measuring the forces motors generate in vivo, this study found that GSK-3 regulates transport by altering the activity of kinesin-1 motors but not their binding to the cargo. These findings reveal a new relationship between GSK-3 and APP, and demonstrate that endogenous GSK-3 is an essential in vivo regulator of bidirectional APP transport in axons and lipid droplets in embryos. Furthermore, they point to a new regulatory mechanism in which GSK-3 controls the number of active motors that are moving a cargo (Weaver, 2013).

Mad linker phosphorylations control the intensity and range of the BMP-activity gradient in developing Drosophila tissues

The BMP ligand Dpp, operates as a long range morphogen to control many important functions during Drosophila development from tissue patterning to growth. The BMP signal is transduced intracellularly via C-terminal phosphorylation of the BMP transcription factor Mad, which forms an activity gradient in developing embryonic tissues. This study shows that Cyclin dependent kinase 8 and Shaggy phosphorylate three Mad linker serines. Linker phosphorylations control the peak intensity and range of the BMP signal across rapidly developing embryonic tissues. Shaggy knockdown broadened the range of the BMP-activity gradient and increased high threshold target gene expression in the early embryo, while expression of a Mad linker mutant in the wing disc resulted in enhanced levels of C-terminally phosphorylated Mad, a 30% increase in wing tissue, and elevated BMP target genes. In conclusion, these results describe how Mad linker phosphorylations work to control the peak intensity and range of the BMP signal in rapidly developing Drosophila tissues (Aleman, 2014: PubMed).

TOG proteins are spatially regulated by Rac-GSK3β to control interphase microtubule dynamics

Microtubules are regulated by a diverse set of proteins that localize to microtubule plus ends (+TIPs) where they regulate dynamic instability and mediate interactions with the cell cortex, actin filaments, and organelles. Although individual +TIPs have been studied in depth and their basic contributions to microtubule dynamics are understood, there is a growing body of evidence that these proteins exhibit cross-talk and likely function to collectively integrate microtubule behavior and upstream signaling pathways. This study have identified a novel protein-protein interaction between the XMAP215 homologue in Drosophila, Mini spindles (Msps), and the CLASP homologue, Orbit. These proteins have been shown to promote and suppress microtubule dynamics, respectively. Microtubule dynamics are regionally controlled in cells by Rac acting to suppress GSK3β in the peripheral lamellae/lamellipodium. Phosphorylation of Orbit by GSK3β triggers a relocalization of Msps from the microtubule plus end to the lattice. Mutation of the Msps-Orbit binding site revealed that this interaction is required for regulating microtubule dynamic instability in the cell periphery. Based on these findings, it is proposed that Msps is a novel Rac effector that acts, in partnership with Orbit, to regionally regulate microtubule dynamics (Trogden, 2015).

Microtubules interact with the small GTPase Rac in a complex pattern of cross-talk at the leading edge of motile cells. Growing microtubules induce cortical Rac activation by locally activating a guanine exchange factor (GEF) to induce protrusion and directional migration. In what is thought to be a positive feedback loop, active Rac promotes persistent microtubule growth in the lamellae and lamellipodia by locally regulating the activity of microtubule-associated proteins (MAPs). At least three MAPs have been implicated in regulation of microtubule dynamics downstream of Rac. The first is CLIP-170, a microtubule plus end interacting protein (+TIP) that interacts with the Rac effector IQGAP1 to capture microtubule plus ends at the plasma membrane. The second is Stathmin/OP18, a microtubule destabilizing factor that is locally inhibited at the leading edge due to phosphorylation by the Rac effector kinase Pak. The third is CLASP, another +TIP that suppresses dynamics, leading to increased stabilization of microtubules at the leading edge of polarized fibroblasts. CLASP binds directly to microtubules through a central lattice-binding domain and localizes to growing plus ends through an interaction with EB1. In the cell cortex, CLASP is phosphorylated by GSK3β, which blocks its ability to bind to the microtubule lattice, thus targeting it to growing plus ends. At the leading edge, GSK3β is locally inhibited by Rac and dephosphorylated CLASP binds along the microtubule lattice. Although local inhibition of Stathmin and activation of CLASP seem to be necessary for persistent microtubule growth at the leading edge, neither factor is sufficient, suggesting that other regulatory mechanisms remain to be discovered (Trogden, 2015 and references therein).

The present study identified the XMAP215 homolog, Msps, as a downstream effector of the Rac pathway and describes a novel regulatory mechanism for Msps through a protein-protein interaction with the microtubule stabilizer Orbit and the scaffolding protein Sentin. In S2 cells, the Drosophila CLASP homolog Orbit localizes to microtubule plus ends, but binds to the microtubule lattice upon expression of active Rac1 or depletion of GSK3β. These observations are similar to the dynamics of CLASP in mammalian cells and suggest that this mode of regulation is conserved. Activation of Rac or depletion of GSK3β promotes Msps binding to the microtubule lattice and this localization requires Orbit. The data suggests that, like CLASP, Orbit is directly phosphorylated by GSK3β which prevents it from interacting with and recruiting Msps to the microtubule lattice. The Orbit-Msps interaction further requires another +TIP, Sentin. As the localization of Sentin at the microtubule plus ends is not regulated by Rac-GSK3β, it is likely serving a scaffolding function to promote interactions between Msps and Orbit. This study mapped the protein-protein interaction sites on Msps and Orbit to their C-termini and found that mutations that block their interaction severely perturb microtubule dynamics. Both a non-phosophorylatable Orbit mutant and a mutant that prevented the Msps-Orbit interaction lead to more persistent growth, with the non-phosphorylatable Orbit mutant also causing an increase in microtubule pause. This may indicate that this interaction is important for persistent microtubule growth downstream of Rac-GSK3β (Trogden, 2015).

A growing body of evidence indicates that +TIPs exhibit cross-talk with one another to regulate microtubule dynamics in response to upstream regulatory cues. The current results indicate that Msps and Orbit function together during interphase to regulate dynamic instability in response to Rac and GSK3β activity. It is well established that members of the XMAP215 family promote microtubule dynamics by catalyzing microtubule polymerization and depolymerization. These activities are conserved in Drosophila Msps; microtubules in S2 cells lacking Msps are less dynamic, spending most of their lifetime in a pause state. In contrast, Orbit acts to suppress microtubule dynamics and promotes their stability. Thus, Msps and Orbit would be seem to regulate microtubule dynamics antagonistically, a functional relationship supported by recent genetic studies. However, the current results indicate that the two proteins share a more complex interaction (Trogden, 2015).

Msps exhibits two distinct localization patterns on microtubules in S2 cells- at the plus ends of microtubules and along the distal microtubule lattice in the periphery. The data support the model that these different modes of microtubule association represent functionally distinct pools of Msps. First, the Msps-Orbit interaction sites were identified, they were mutated to ablate the interaction, and these mutants were used to rescue cells depleted of either endogenous Msps or Orbit. Expression of either Msps-GFP 3A or 3K3A was able to rescue microtubule dynamics as compared to Msps-depleted cells. However, microtubules in these cells exhibited abnormally high frequencies of rescue and low frequencies of catastrophe, spending more time in growth and less in shrinkage compared to control cells. Cells expressing GFP-Orbit with the GSK3β phospho-acceptor sites mutated to alanine (5S->A) suppressed Orbit RNAi-induced increases in dynamic instability, but microtubules in these cells also exhibited higher frequencies of rescue, lower frequencies of catastrophe, and more time in the pause state compared to control cells. These results indicate that Msps must interact with Orbit in order to properly regulate microtubule dynamics in the cell periphery. Second, when the growth rates of microtubules were examined by tracking EB1-GFP, it was noted that EB1 comets in the cell periphery exhibit slower velocities than those in the cell cortex. These differences likely reflect interactions between growing microtubules and lamellipodial actin undergoing retrograde centripetal flow in the cell periphery. Recent work has also shown that EB1 comets are structurally different in the cortex versus the periphery, so the differences may be explained by changes in the microtubule as well. However, when EB1 comets on microtubules were compared with Msps at the plus end to those that had Msps localized along the distal lattice, it was discovered that the latter exhibited a significantly slower rate of growth. Collectively, the results indicate that the Msps-Orbit interaction 'tunes' microtubule dynamics in response to Rac activation in the cell periphery. It is suggested that Msps could be shunted onto the lattice to act as a localized 'sink' that attenuates its activity as a microtubule polymerase. This inactive pool may serve as a mechanism to partially suppress Msps activity so that microtubules grow at specific rates upon reaching the edge of the cell. The mechanism of how Msps regionally governs microtubule dynamics presents an intriguing problem; future studies employing biochemical reconstitution of microtubule dynamics with recombinant +TIPs and their regulators will likely be required to address these models (Trogden, 2015).

One puzzling aspect of this study is that, despite the biochemical and functional evidence for the Msps-Orbit interaction, colocalization of Msps and Orbit on the microtubule lattice in the cell periphery was not detected under unperturbed conditions. It is speculated that this protein-protein interaction is transient, occurring at the plus end, but is required for some conformational change in Msps that unmasks its microtubule lattice-binding activity. Two lattice-binding sites have been detected in the inter-TOG linker regions that seem to be inactive while the protein is localized to plus ends. This results were also confirmed using in vitro reconstitution assays. It is possible, however, that Orbit does localize to the lattice in the cell periphery, but at levels so low it was not possible to detect in living cells using the available probes. A third possibility is that Orbit is able to alter the structure of the microtubule lattice proximal to the plus end in order to promote lattice binding of Msps. This alteration could represent a change in the local nucleotide state of the polymer as a recent study indicated that mammalian CLASPs are able to promote GTP hydrolysis by polymerized tubulin. Alternatively, it has been shown that EB1 family members promote structural transitions within the microtubule lattice that favor GTP hydrolysis and compaction of the lattice itself. Perhaps similar localized changes in microtubule structure signal to Msps to transition from tip-association to lattice binding. Further work will be required to understand how these proteins interact to regulate their respective functions and it is expected that in vitro reconstitution assays will prove valuable to advance understanding of this protein-protein interaction (Trogden, 2015).

It is interesting to note that the localization patterns for Msps and Orbit observed in Drosophila cells seem to exhibit the converse relationships to those described for XMAP215/CH-TOG and CLASP in mammalian cells. Msps also differs from XMAP215 and ch-TOG through its lack of ability to either bind directly to EB1 or independently recognize growing microtubule ends. It is possible that the interaction with Orbit developed to increase Msps' ability to target the microtubule plus end. This interaction may also be present in mammalian cells, where it may serve to modulate growth rates of microtubules. Although Msps and XMAP215/CH-TOG exhibit high degrees of identity overall, it will be interesting to compare their relative activities in living cells using Msps to replace CH-TOG, and vice-versa, using heterologous systems (Trogden, 2015).

The data point to an outstanding question about how this localized regulation of dynamic instability impact behavior at the level of the cell. Dynamic microtubules exhibit a complex, bidirectional cross-talk with the Rho family of small G proteins. It is suggested that Msps and other XMAP215 family members are critical components of these pathways. In migrating cells, for example, Rac activity promotes processive microtubule growth while microtubule dynamics also promote Rac activation. It is predicted that Msps/XMAP215 family members are likely to participate in this positive feedback look and are, therefore, likely to play crucial roles in cell motility. Microtubules are also essential for directed membrane traffic to the leading edge. Msps-induced microtubule growth may also contribute to this polarized delivery of cargo to the front of motile cells. In order to address these fascinating questions, the Msps-Orbit interaction will have to be addressed in the context of migratory cell lines or, better still, within the developing embryo (Trogden, 2015).

In the current model in the cortex of the cell, Rac activity is low and therefore GSK3β is active, leading to phosphorylation of Orbit on 5 serine residues. Both Orbit and Msps are at the plus end, but cannot interact with each other. Msps is localized to the plus end through its interaction with Sentin. Orbit can bind either Sentin or EB1 to target the plus end. In the periphery of an S2 cell (or the leading edge of a migrating cell), Rac is active, which leads to the local inactivation of GSK3β and dephosphorylation of Orbit. Orbit is still on the plus end, but is now able to interact with Msps, allowing Msps to bind to the lattice. How this interaction allows Msps to bind the lattice with Orbit remaining on the plus end remains to be determined. It is hypothesized that when Msps is at the plus end it is in a closed conformation where the C-terminus covers the Linker4-TOG5 region that can bind the microtubule lattice. When Msps and Orbit bind to one another, this causes Msps to adopt an open conformation, exposing the lattice binding region which allows Msps to diffuse along to lattice (Trogden, 2015).

The insulin signaling pathway in Drosophila melanogaster: A nexus revealing an "Achilles' heel" in DDT resistance

Insecticide resistance is an ongoing challenge in agriculture and disease vector control. This study demonstrates a novel strategy to attenuate resistance. This study used genomics tools to target fundamental energy-associated pathways and identified a potential "Achilles' heel" for resistance, a resistance-associated protein that, upon inhibition, results in a substantial loss in the resistance phenotype. Specifically, the gene expression profiles and structural variations of the insulin/insulin-like growth factor signaling (IIS) pathway genes were compared in DDT-susceptible (91-C) and -resistant (91-R) Drosophila melanogaster (Drosophila) strains. A total of eight and seven IIS transcripts were up- and down-regulated, respectively, in 91-R compared to 91-C. A total of 114 nonsynonymous mutations were observed between 91-C and 91-R, of which 51.8% were fixed. Among the differentially expressed transcripts, phosphoenolpyruvate carboxykinase (PEPCK), down-regulated in 91-R, encoded the greatest number of amino acid changes, prompting the performance of PEPCK inhibitor-pesticide exposure bioassays. The inhibitor of PEPCK, hydrazine sulfate, resulted in a 161- to 218-fold decrease in the DDT resistance phenotype (91-R) and more than a 4- to 5-fold increase in susceptibility in 91-C. A second target protein, Glycogen synthase kinase 3β (GSK3β-PO), had one amino acid difference between 91-C and 91-R, and the corresponding transcript was also down-regulated in 91-R. A GSK3β-PO inhibitor, lithium chloride, likewise reduced the resistance but to a lesser extent than did hydrazine sulfate for PEPCK. This study has demonstrated the potential role of IIS genes in DDT resistance and the potential discovery of an "Achilles' heel" against pesticide resistance in this pathway (Zhang, 2021).

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

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