Highwire, a putative RING finger E3 ubiquitin ligase, negatively regulates synaptic growth at the neuromuscular junction (NMJ) in Drosophila. hiw mutants have dramatically larger synaptic size and increased numbers of synaptic boutons. Hiw binds to the Smad protein Medea (Med). Med is part of a presynaptic bone morphogenetic protein (BMP) signaling cascade consisting of three receptor subunits, Wit, Tkv, and Sax, in addition to the Smad transcription factor Mad. When compared to wild-type, mutants of BMP signaling components have smaller NMJ size, reduced neurotransmitter release, and aberrant synaptic ultrastructure. BMP signaling mutants suppress the excessive synaptic growth in hiw mutants. Activation of BMP signaling, which in wild-type does not cause additional growth, in hiw mutants does lead to further synaptic expansion. These results reveal a balance between positive BMP signaling and negative regulation by Highwire, governing the growth of neuromuscular synapses (McCabe, 2004).
To search for proteins that interact with Hiw, a yeast two-hybrid screen of a Drosophila cDNA library was carried out using two regions of the Hiw protein as bait (HWB1: aa 2063–3461; and HWB2: aa 4082–5233). From a screen of 4 × 107 transformants, three candidate proteins were isolated that interacted positively with HWB1 and one candidate protein that showed positive interaction with HWB2. One of the three candidate proteins that interacted with HWB1 was identified as the C-terminal region of the second mad homology domain (MH2) of Med (aa 616-745). The Med clone did not self-activate, and when positive interaction was tested by cotransforming the Med clone together with either HWB1 or HWB2, a strong interaction was found with HWB1 but no interaction was seen with HWB2. Furthermore, GST-fused Med protein is able to bind to in vitro translated preparations of Hiw. The binding region for Med was between amino acids 2063 and 3461 of Hiw (included in HWB1), while binding was not observed with the region of Hiw found in HWB2. Interestingly, the Med binding domain of Hiw includes the region used to generate the partial Hiw transgene, Hiw-DN, that produces a dominant-negative hiw phenotype when expressed in the nervous system. The results of yeast two-hybrid experiments together with in vitro binding results show that Med and Hiw proteins can interact and that the MH2 region of Med is sufficient for this interaction (McCabe, 2004).
This study demonstrates that Hiw negatively regulates the BMP signaling cascade, required for the normal growth and function of neuromuscular synapses in Drosophila. hiw mutants have a dramatic overexpansion of synaptic structures, with many more synaptic boutons than wild-type. In contrast to hiw mutants, mutants of Med have smaller synapses and many fewer synaptic boutons compared to wild-type. Med is part of a presynaptic signaling cascade that includes three cell surface receptors (Wit, Tkv, and Sax) and two intracellular transcription factors (Med and Mad) that transmits a BMP signal from the NMJ to the motoneuron nucleus. Genetic removal of either Med or Wit can completely suppress the synaptic overgrowth in hiw mutants, while activation of BMP signaling in hiw mutants produces even more synaptic growth. These findings provide evidence for a functional link between the action of Hiw and BMP signaling to control synaptic growth at the Drosophila NMJ (McCabe, 2004).
The type II BMP receptor Wit and the BMP ligand Gbb are required for synaptic growth at the neuromuscular junction. While Wit is required in presynaptic neurons for normal NMJ growth, the requirement for Gbb in postsynaptic muscles is consistent with a retrograde BMP signal. Yet it was not clear from these studies how Wit carried out this function, whether by a local signaling mechanism within the synapse or via a signaling cascade. Evidence is provided that strongly suggests BMP signaling through Wit regulates synaptic growth primarily by altering transcription. Two lines of evidence support this conclusion. (1) Mutants of both of the Smad transcription factors Med and Mad have similar defects in synaptic growth, presynaptic ultrastructure, and function to mutants of the cell surface receptors wit, tkv, and sax. (2) Phosphorylated Mad is absent from the nucleus of motoneurons in wit, sax, and tkv mutants. The absence of P-Mad in the nuclei of motoneurons in BMP receptor mutants combined with the similarity of synaptic phenotypes between mutants in receptors and mutants of intracellular Smads argues that BMPs exert their influence on synapses primarily by signaling to the nucleus rather than having a local synaptic activity (McCabe, 2004).
The specificity of synaptic BMP signaling seems to be maintained by Wit, which is expressed only in the nervous system, while Med, Mad, Tkv, and Sax are found in many tissues. This conclusion is supported by the finding of developmental defects in many tissues such as the fat body in Med, Mad, tkv, and sax mutants that are not found in wit mutants. Recently, an independent study found synaptic defects in sax and Mad mutants (Rawson, 2003). These studies have been extended, demonstrating that Med and Tkv, in addition to Mad and Sax, are required in presynaptic neurons for normal NMJ growth, and mutants of all these molecules have similar characteristic defects in presynaptic active zone ultrastructure. This study further shows, by rescuing wit mutants with a pair of Tkv::Wit chimeric receptors, that Tkv and Wit function together in vivo, and that Tkv is localized at the NMJ. All these data therefore suggest that Wit, Tkv, and Sax receive a retrograde BMP signal from muscles by Gbb (and possibly other BMP ligands) at the NMJ and transmit this signal to the nucleus via Mad and Med to induce transcriptional change. This neurotrophic signal is essential for the coordination of presynaptic NMJ expansion with postsynaptic muscle growth (McCabe, 2004).
While BMP signaling plays an essential role in neuromuscular junction expansion, BMP signaling mutants also have dramatic reductions in the levels of neurotransmitter release and aberrant presynaptic ultrastructure at active zones. Several pieces of data suggest the role of BMP signaling in the regulation of neurotransmitter release may be separable from its role in synaptic growth. Restoration of Gbb in the nervous system of gbb mutants can rescue neurotransmitter release to wild-type levels while not restoring normal synaptic size. This result is reminiscent of Fasciclin II mutations, which also have reduced synaptic size but normal neurotransmitter release. Furthermore, Wit is necessary for the homeostatic regulation of neurotransmitter release. It may be that the involvement of BMP signaling in this process is independent of, but complementary to, its role in regulating synaptic structural growth (McCabe, 2004).
Despite the central requirement for BMP signaling in synaptic growth, when attempts were made to increase BMP signaling in motoneurons, no synaptic overgrowth beyond wild-type levels was seen. These observations are explained by proposing the presence of a negative regulatory process that tightly controls the levels of synaptic BMP signaling. Hiw is a key and necessary component of this regulatory process (McCabe, 2004).
Hiw is an extremely large protein, making in vitro confirmation of its ubiquitination activity difficult. Despite the absence of direct biochemical data, several lines of evidence suggest that Hiw does function as an E3 ubiquitin ligase. Hiw has a signature RING-H2 finger, a domain that has a general function in ubiquitin-mediated protein degradation. RING fingers can function as modules that interact with E2 ubiquitin-conjugating enzymes to catalyze ubiquitination. Futhermore, hiw mutants have a strong genetic interaction with the deubiquitinating enzyme Fat Facets. Overexpression of either Fat Facets or the yeast deubiquitinating protease UBP2 in presynaptic neurons produces a synaptic overgrowth phenotype very similar to the hiw mutant phenotype. Given this evidence, a model is proposed whereby Hiw negatively regulates BMP signaling at the NMJ by a ubiquitination-dependant mechanism, antagonizing BMP signaling and controlling synaptic growth (McCabe, 2004).
In support of this model, Hiw has been shown to specifically binds Med protein in both yeast two-hybrid and in vitro binding assays. This is consistent with a function for Hiw as an E3 ubiquitin ligase, since these proteins specifically bind to their substrates before targeting them for proteolysis. Unfortunately, several antibodies against mammalian Smad4 failed to detect Med, precluding assays of Med's ubiquitination status. Interestingly, however, the region in Hiw that interacts with Med is included in the sequence of a partial Hiw transgene, Hiw-DN, that causes synaptic overgrowth when expressed in the nervous system. Since this transgene does not include the RING finger domain, its dominant-negative effect could be mediated by its ability to inhibit the binding of Med by endogenous Hiw. In addition to physical interaction between Hiw and Med, genetic removal of Med has been demonstrated to suppress the increase in the number of synaptic boutons in hiw mutants to Med mutant levels. This implies that the dramatic increase in the number of synaptic boutons in hiw mutants is completely dependant upon the presence of Med. This increase is also suppressed by wit mutants, showing that it is Med's role as part of a BMP signaling cascade that mediates its suppression of hiw. Furthermore, it was shown that synaptic overgrowth due to the overexpression of the deubiquitinating enzymes Faf or UBP2 is also suppressed by disrupting BMP signaling. These results together support the model whereby Hiw regulates BMP signaling via a ubiquitin-dependent mechanism (McCabe, 2004).
Overexpression of a constitutively active Tkv type I receptor transgene in neurons does not cause any overgrowth at the NMJ. Similarly, loss-of-function mutants of the inhibitory Smad, Dad, does not show any synaptic overgrowth. Consistent with the model that Hiw regulates BMP signaling, in hiw mutants, activation of BMP signaling can now lead to further synaptic overgrowth. This suggests that while hiw mutants may have elevated levels of BMP signaling, further activation of the BMP pathway can induce yet more synaptic growth. By activating BMP signaling using transgenic constitutively active type I receptors, other factors that could conceivably limit the signal can presumably be bypassed, such as the availability of Wit or Gbb (McCabe, 2004).
In contrast to the current findings, dad mutants have previously been reported to produce large numbers of extra synaptic boutons. While the current study examined only one homoallelic mutant combination of dad, several homo and heteroallelic mutant combinations were examined, to eliminate the possibility of second site mutations, and this prior result was not confirmed. Thus the discrepancy remains unresolved. Consistent with the current data, it has been shown that overexpression of Wit cannot induce synapse overgrowth, despite the ability of overexpressed type II receptors to activate signaling in the absence of ligand. The current results are also supported by findings of Rawson (2003) (McCabe, 2004).
While the current data indicate that synaptic structural growth can be controlled by Hiw regulating BMP signaling, neurotransmitter release at the NMJ does not seem to be governed by an identical mechanism. BMP mutants have decreased neurotransmitter release, in addition to reduced numbers of synaptic boutons, when compared to wild-type. In contrast, hiw mutants have many more synaptic boutons than wild-type, but despite this, neurotransmitter release in hiw mutants is also reduced to levels similar to that of BMP mutants. Interestingly, double mutants of hiw;wit or hiw;Med have levels of neurotransmitter release similar to those of wit or Med mutants alone. This result indicates that the role of Hiw in controlling neurotransmitter release is distinct from its role as a negative regulator of synaptic structural growth. Previous results support this idea; the unknown retrograde signal that controls the homeostasis of neurotransmission at the NMJ is disrupted in wit mutants but remains functional in hiw mutants. Other aspects of the hiw mutant phenotype also appear to be independent of BMP signaling. Individual synaptic bouton size is reduced in hiw mutants, a phenotype not observed in BMP mutants and not suppressed by the inhibition of BMP signaling in hiw mutants. Also, the excessive degree of synaptic branching and arborization observed in hiw mutants is only partially suppressed by the disruption of BMP signaling. It is likely therefore that Hiw regulates other molecules responsible for these aspects of synaptic development (McCabe, 2004).
How is synaptic growth maintained? A model is proposed whereby a positive BMP signaling cascade is negatively regulated by the ubiquitin-protein ligase action of Hiw on the Smad Med. This model, however, leaves an important question unanswered: how is the balance between these two opposing forces maintained? This question cannot be answered with current knowledge, but some scenarios can be suggested. One possibility is that the level of phosphorylated Mad competes for binding of Med with Hiw. Once phosphorylated by type I receptors, Mad forms a complex with Med, and the formation of this complex is required for efficient signaling to the nucleus. It is conceivable that an equilibrium exists between the binding of phosphorylated Mad to Med and the binding of Med to Hiw. This equilibrium could potentially act to set a consistent level of BMP signaling and thus normal synaptic growth at the NMJ. Another alternative is that the ability of Hiw to block BMP signaling could be regulated by a third protein that is itself under the transcriptional control of BMP signaling. In this scenario, activation of BMP signaling leads to increased levels of this third protein that in turn activate Hiw's ability to target Med for ubiquitination, completing a negative feedback loop. Future studies will allow these and other possibilities to be tested to further dissect the opposing molecular forces that govern synaptic growth and function (McCabe, 2004).
Highwire is an extremely large, evolutionarily conserved E3 ubiquitin ligase that negatively regulates synaptic growth at the Drosophila NMJ. Highwire has been proposed to restrain synaptic growth by downregulating a synaptogenic signal. This study identifies such a downstream signaling pathway. A screen for suppressors of the highwire synaptic overgrowth phenotype yielded mutations in wallenda, a MAP kinase kinase kinase (MAPKKK) homologous to vertebrate DLK and LZK. wallenda is both necessary for highwire synaptic overgrowth and sufficient to promote synaptic overgrowth, and synaptic levels of Wallenda protein are controlled by Highwire and ubiquitin hydrolases. highwire synaptic overgrowth requires the MAP kinase JNK and the transcription factor Fos. These results suggest that Highwire controls structural plasticity of the synapse by regulating gene expression through a MAP kinase signaling pathway. In addition to controlling synaptic growth, Highwire promotes synaptic function through a separate pathway that does not require Wallenda (Collins, 2006).
JNK signaling affects many cellular processes, often by regulating transcription factor activity that leads to changes in gene expression. A common downstream effector of JNK-mediated changes in gene expression is the AP-1 complex of Fos and Jun transcription factors, which can regulate synaptic growth at the Drosophila NMJ. To investigate whether Drosophila Fos or Jun (known as D-fos and D-jun, respectively) are required for highwire-dependent synaptic overgrowth, each was inhibited by expressing dominant-negative transgenes that contain the DNA binding and dimerization domains of Fos and Jun but lack the transcriptional activation domains. Expression of these dominant-negative transgenes in postmitotic neurons allowed circumvention of early embryonic requirements for D-fos and D-jun (Collins, 2006).
When FosDN and JunDN are neuronally expressed in a wild-type background, there is a modest trend toward inhibition of synaptic growth. When expressed in a highwire mutant background, the FosDN transgene confers dramatic suppression of the highwire synaptic phenotype, reducing bouton number and branching (42%) and increasing the intensity of staining for synaptic vesicle markers at the synapse. The reduction in highwire-dependent synaptic overgrowth is much greater than the reduction of growth in a wild-type background. In contrast, JunDN does not suppress the highwire phenotype. This suggests the existence of a pathway that is separate from AP-1, consistent with results in Drosophila demonstrating that D-Fos can act independently of D-Jun. The requirement for D-Fos in highwire synaptic overgrowth suggests that the highwire phenotype involves changes in gene expression rather than exclusively local changes to the synapse (Collins, 2006).
If FosDN acts downstream of Wallenda to inhibit synaptic overgrowth, it should also suppress the synaptic overgrowth caused by overexpressing wallenda. Indeed, when FosDN was coexpressed with UAS-wnd in neurons, FosDN could suppress the wallenda gain-of-function phenotype, leading to a 38% reduction in synaptic bouton number, a 52% reduction in synaptic branching, a 54% increase in bouton size, and a 3.8-fold increase in the intensity of staining of synaptic vesicle markers. This is consistent with D-Fos acting downstream of Wallenda to promote synaptic growth. Therefore, the synaptic overgrowth phenotypes caused by loss of highwire and by overexpression of wallenda are similar in their requirements for the transcription factor D-Fos (Collins, 2006).
Current models suggest that Highwire functions as an E3 ubiquitin ligase to downregulate a signaling pathway that promotes synaptic growth. This study identified a MAPKKK, Wallenda, whose protein levels are controlled by Highwire and the activity of ubiquitin hydrolases. Wallenda is both necessary for highwire-dependent synaptic overgrowth and sufficient to promote synaptic growth. Downstream of Wallenda, the MAP kinase JNK and transcription factor Fos are required for highwire-dependent synaptic overgrowth. It is proposed that Highwire restrains synaptic growth by downregulating the MAPKKK Wallenda, thereby inhibiting signaling through the JNK MAP kinase and the Fos transcription factor. In the absence of highwire, this signaling pathway is overactive, leading to changes in gene expression that result in excessive synaptic growth (Collins, 2006).
The regulation of the MAPKKK Wallenda is conserved in Drosophila and C. elegans (Nakata, 2005). In both organisms, the synaptic phenotype of highwire/rpm-1 requires the Wallenda/DLK-1 MAPKKK and downstream MAPK signaling. However, the downstream MAPK pathways diverge: in C. elegans, the rpm-1 phenotype requires a p38 MAP kinase (Nakata, 2005), while the highwire phenotype requires JNK signaling. This suggests that regulation of the specific MAPKKK Wallenda/DLK-1, rather than a particular downstream MAP kinase pathway, is a fundamental activity of Highwire and its orthologs (Collins, 2006).
Since Highwire functions as an E3 ubiquitin ligase to restrain synaptic growth, Wallenda is a compelling candidate target for the following reasons: (1) wallenda functions downstream of highwire and is essential for the synaptic overgrowth in highwire mutants; (2) increasing the levels of Wallenda by overexpression is sufficient to confer synaptic overgrowth; (3) Highwire regulates Wallenda protein levels through a posttranscriptional and most likely posttranslational mechanism. Each of the points above is conserved in C. elegans (Nakata, 2005 ). (4) Wallenda protein levels are regulated by ubiquitination in vivo, since inhibiting ubiquitination by overexpressing ubiquitin hydrolases increases the levels of Wallenda protein. (5) The RING domain of the C. elegans homolog rpm-1 can interact with the Wallenda homolog DLK-1 (Nakata, 2005) and stimulate its ubiquitination when both are overexpressed in 293T cells (Collins, 2006).
Targeting a MAPKKK, which sits at the top of a MAP kinase signaling pathway, is an attractive mechanism for spatially and temporally controlling a synaptogenic signal without affecting downstream components shared by multiple MAPK signaling cascades. Restraining MAP kinase signaling is essential for controlling diverse cellular processes, including cell proliferation, differentiation, and apoptosis. The targeting of MAPKKKs by specific ubiquitin ligases may be a powerful and general mechanism for regulating MAP kinase signals (Collins, 2006).
While Wallenda is an essential mediator of the highwire mutant phenotypes in both Drosophila and C. elegans, an endogenous synaptic function for Wallenda has not yet been identified in either organism: the wallenda mutants have surprisingly normal synapse morphology and function. This may be due to another pathway that compensates for the loss of wallenda function. Such redundancy would obscure the role of wallenda. A second possibility is that wallenda functions in an aspect of synaptic growth that is not detected or required under laboratory culture conditions. For instance, wallenda could promote synaptic growth as part of a structural plasticity program that responds to unknown experience-dependent stimuli. A third possibility is that Wallenda does not normally function at synapses, but its upregulation in highwire mutants causes a neomorphic phenotype. In this scenario, the regulation of Wallenda by Highwire is required for normal synaptic development, but endogenous Wallenda would not itself regulate the synapse. The neuropil and synaptic localization of Wallenda and the vertebrate homolog DLK (Hirai, 2005) is, however, consistent with a synaptic function (Collins, 2006).
As an activator of MAP kinase signaling, Wallenda and its homologs might also control other processes beyond the synapse. Functional studies in vertebrates suggest that DLK and JNK signaling regulate neuronal migration and axon outgrowth in the developing cortex (Hirai, 2002). Outside of the nervous system, DLK influences keratinocyte differentiation, and LZK is highly expressed in the pancreas, liver, and placenta. In Drosophila, wallenda mutants are female sterile. It is predicted that the regulation of DLK and LZK is conserved from worms and flies to vertebrates. Therefore, the vertebrate homologs of Highwire might regulate some of these neuronal and/or extraneuronal developmental processes (Collins, 2006 and references therein).
Highwire is a large, multidomain protein that, in addition to acting as an E3 ubiquitin ligase, has been shown to inhibit adenylate cyclase, influence TSC signaling and pteridine biosynthesis, and interact with the myc oncogene and the co-SMAD Medea. It is remarkable that throughout millions of years of evolution, members of the Highwire family have retained an exceptionally large size and complex domain structure. An attractive explanation for this conservation is that this molecule could serve as an intersection point for multiple signaling pathways, integrating MAP kinase and other signals during neural development (Collins, 2006).
The ubiquitin ligase activity alone could be responsible for regulating more than one downstream target. Interactions with components of TSC (tuberin/hamartin) and TGF-β signaling pathways suggest that Highwire might target either or both of these pathways. The model that Highwire regulates TGF-β signaling through interaction with the co-SMAD Medea has received considerable attention. Since the TGF-β pathway regulates synaptic growth at the NMJ, it has been proposed that synaptic overgrowth of highwire mutants is caused by overactivity of this pathway. Null alleles of wit, which completely disrupt TGF-β signaling at the NMJ, can partially suppress the highwire phenotypes: they partially suppress the increase in bouton number, but show little or no suppression of the reduced bouton size and the reduced intensity for synaptic vesicle markers. This partial suppression of highwire by wit is consistent with the model that overactive TGF-β signaling contributes to the highwire phenotype. However, the data are also consistent with the alternate model that TGF-β signaling and Highwire act in parallel pathways. An assay for the activity of TGF-β signaling is to stain for phosphorylated-MAD (phospho-MAD), the major transducer of BMP signals in Drosophila, in motoneuron nuclei. No change was detected in the levels of phospho-MAD staining in highwire mutants compared to wild-type. This assay is sensitive to changes in pathway activity—neuronal expression of the constitutively active type I receptor thick veins leads to a 40% increase in phospho-MAD staining. Interestingly, this increase in TGF-β signaling does not lead to excess synaptic growth. Combining a highwire mutant with expression of constitutively active thick veins does cause excess growth, but it does not lead to any further increase in phospho-MAD staining. These data are consistent with highwire and TGF-β signaling acting in parallel pathways (Collins, 2006).
Whether or not Highwire regulates TGF-β signaling, it is likely to target an additional pathway. Highwire not only restrains synaptic growth, but also promotes synaptic function. Synaptic function requires the ubiquitin ligase activity of Highwire and is sensitive to the levels of the ubiquitin hydrolase fat facets. This study demonstrates that this regulation of neurotransmitter release does not require Wallenda. Therefore, Highwire must regulate at least two distinct molecular pathways. If Wallenda is a substrate whose downregulation is essential for restraining synaptic growth, there is likely another substrate for Highwire whose downregulation promotes neurotransmitter release (Collins, 2006).
Downstream of Wallenda, the JNK MAP kinase and Fos transcription factor are required for the highwire synaptic morphology phenotype. Therefore, Highwire attenuates a JNK signaling pathway that presumably controls gene expression to regulate synaptic growth. Previous studies have implicated JNK-dependent transcriptional control in activity-dependent growth of the Drosophila NMJ. However, this previously described pathway is probably distinct from the JNK signal that is controlled by Highwire and activated by Wallenda. The previously described role for JNK requires AP-1, a heterodimer of Fos and Jun transcription factors; inhibiting either D-Fos or D-Jun disrupts this pathway. In contrast, highwire-induced overgrowth requires D-Fos, but not D-Jun. The Wallenda pathway could therefore involve a homodimer of D-Fos or another transcription factor that interacts with Fos. Such D-Jun-independent functions of D-Fos have been described previously in Drosophila. The differential requirement for transcription factors suggests that the output of Wallenda signaling cannot simply be activation of JNK, but instead activation of JNK in a particular spatial or temporal context, such as in the presence of cofactors that influence downstream signaling (Collins, 2006).
In addition to transcription factors, substrates for activated JNK include components of the cytoskeleton. Because the NMJ is distant from the motoneuron nucleus, and because vertebrate DLK colocalizes with tubulin in axonal regions of the brain, it was initially expected that the Highwire/Wallenda/JNK pathway would influence synaptic morphology through local action upon the synaptic cytoskeleton. Instead, a requirement was identified for a transcription factor and presumably changes in gene expression. However, this does not exclude an interaction with the cytoskeleton or local changes at the synapse. It is possible that Highwire regulates the Wallenda signal in the cell body. However, the observation that Wallenda accumulates in the synapse-rich neuropil and at the NMJ when Highwire is absent suggests that Wallenda could become activated at the synapse. This would imply the need for a mechanism to transport the activated JNK signal back to the nucleus. In addition, cell-wide changes in gene expression must then be translated into localized growth at the synapse. Activated Wallenda at the synapse is an attractive candidate to integrate changes in gene expression with regulation of the synaptic cytoskeleton to control synaptic growth (Collins, 2006).
The growth of new synapses shapes the initial formation and subsequent rearrangement of neural circuitry. Genetic studies have demonstrated that the ubiquitin ligase Highwire restrains synaptic terminal growth by down-regulating the MAP kinase kinase kinase Wallenda/dual leucine zipper kinase (DLK). To investigate the mechanism of Highwire action, DFsn has been identified as a binding partner of Highwire and characterized the roles of DFsn (CG4643) in synapse development, synaptic transmission, and the regulation of Wallenda/DLK kinase abundance. This study identified DFsn as an F-box protein that binds to the RING-domain ubiquitin ligase Highwire and that can localize to the Drosophila neuromuscular junction. Loss-of-function mutants for DFsn have a phenotype that is very similar to highwire mutants -- there is a dramatic overgrowth of synaptic termini, with a large increase in the number of synaptic boutons and branches. In addition, synaptic transmission is impaired in DFsn mutants. Genetic interactions between DFsn and highwire mutants indicate that DFsn and Highwire collaborate to restrain synaptic terminal growth. Finally, DFsn regulates the levels of the Wallenda/DLK kinase, and wallenda is necessary for DFsn-dependent synaptic terminal overgrowth. In conclusion, the F-box protein DFsn binds the ubiquitin ligase Highwire and is required to down-regulate the levels of the Wallenda/DLK kinase and restrain synaptic terminal growth. It is proposed that DFsn and Highwire participate in an evolutionarily conserved ubiquitin ligase complex whose substrates regulate the structure and function of synapses (Wu, 2007; full text of article).
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