The mechanisms regulating the outgrowth of neurites during development, as well as after injury, are key to the understanding of the wiring and functioning of the brain under normal and pathological conditions. The amyloid precursor protein (APP) is involved in the pathogenesis of Alzheimer's disease (AD). However, its physiological role in the central nervous system is not known. Many physical interactions between APP and intracellular signalling molecules have been described, but their functional relevance remains unclear. This study shows that human APP and Drosophila APP-Like (APPL) can induce postdevelopmental axonal arborization, which depends critically on a conserved motif in the C-terminus and requires interaction with the Abelson (Abl) tyrosine kinase. Brain injury induces APPL upregulation in Drosophila neurons, correlating with increased post-traumatic mortality in appld mutant flies. Finally, this study shows interactions between APP and the JNK stress kinase cascade. These findings suggest a role for APP in axonal outgrowth after traumatic brain injury (Leyssen, 2005).
APP and its Drosophila homologue APPL promote de novo axonal arborization in the Drosophila brain. Interestingly, this axonal arborization phenotype can also be induced when high levels of APP are induced only in fully mature adult neurons, a situation similar to that after traumatic brain injury in adult organisms. In contrast to previous reports in cell culture, the effect of APP depends critically on its intracellular domain. To elucidate the mechanisms used by APP to induce axonal arborization, the importance of different conserved residues in the APP molecule was investigated and genetic interaction studies were performed. Deletions and point mutations in APP domains known to mediate physical interactions between APP and components of the Abl signalling pathway (Trommsdorff, 1998), including Abl itself (Zambrano, 2001), abolish the APP effect on axonal outgrowth. Furthermore, APP signalling depends critically on the levels and activity of the Abl tyrosine kinase. Consistently, activation of Abl is sufficient to induce axonal arborization of the Drosophila brain small lateral neurons ventral (sLNv), downstream of APPL. The actin-binding protein Profilin, itself known to mediate Abl effects on axons, is required for APP-dependent axonal arborization, suggesting a functional link between APP and the reorganization of the actin cytoskeleton. It is noteworthy that the Abl adaptor protein Dab is an important factor in neuronal migration. As such, it would be interesting to investigate whether the focal dysplasia seen in the triple APP knockout mice is also linked to the Abl tyrosine kinase cascade (Leyssen, 2005).
Since no axonal outgrowth defects were found in the CNS of appl mutant Drosophila and because APP is able to induce axonal outgrowth phenotypes in fully mature CNS neurons, it was reasoned that APPL might have a role in specific postdevelopmental contexts like brain injury. A model was therefore developed for the induction of brain damage in adult Drosophila. Expression of APPL is increased for several days, specifically in neurons surrounding damaged brain areas after injury, and flies mutant for appl show increased post-traumatic mortality, suggesting that APPL has an important physiological role under these conditions (Leyssen, 2005).
Many neurons in the injured brain areas also show activation of the JNK-signalling cascade. JNK signalling is essential for correct regeneration of axons after injury in mammals. This study found that APP-induced axonal arborization depends on intact JNK signalling. This functional link can be explained by the presence of a molecular link between the pathways provided by the physical binding of JIP/APLIP1 to APP, as well as JNK-signalling components. In contrast, downstream effectors like Profilin may be functionally controlled by APP/Abl, while being under the transcriptional control of JNK signalling (Leyssen, 2005).
Theses data combined suggest a model which brings together several of the proposed APP-binding partners in the context of axonal arborization and trauma response in vivo. In this model, it is proposed that acute trauma results in increased APPL expression and independent, but simultaneous, JNK signalling activation. JNK activity provides, via transcriptional activation of Profilin and other cytoskeletal regulators, a permissive environment for remodelling of the actin cytoskeleton. Regulation of Abl signalling by APP ensures that these components are controlled functionally, resulting in an appropriate neuronal response to axonal damage (Leyssen, 2005).
These findings thus provide the first in vivo evidence for a role of APP in axonal arborization in the central nervous system and propose an integrated explanation for a number of intriguing, but thus far separate, observations from biochemical and cell culture studies in various contexts. Interestingly, while expression of the membrane-bound C-terminal fragment of APP (APP-CTF) is sufficient to induce axonal arborization, expression of the APP intracellular domain (AICD), which results from the γ-secretase-mediated cleavage of APP-CTF, does not. It is therefore speculated that the cleavage of APP-CTF by γ-secretase may terminate APP activity and therefore regulate APP-Abl signalling (Leyssen, 2005).
These findings may also have implications on the understanding of some aspects of the pathophysiology of AD. It is hypothesized that during adult life stressful and/or traumatic events in the brain cause APP upregulation. As a side-effect of this, Aβ peptides are generated, which may cause further disruption of neuronal connection. Whether Aβ peptides have any function under trauma conditions in mammals or are just a toxic side product of the cleavage remains to be seen. This hypothesis would, in part, explain the strong epidemiological relationship between brain trauma and AD, as well as the reports of AD-like brain pathology after severe head trauma. Finally, it would be interesting to investigate whether the downstream components of APP signalling identified in this work are AD susceptibility loci, leading to an inefficient neuronal trauma response and thus longer-lasting APP upregulation after brain injury (Leyssen, 2005).
Abl tyrosine kinase (Abl) regulates axon guidance by modulating actin dynamics. Abelson interacting protein (Abi), originally identified as a kinase substrate of Abl, also plays a key role in actin dynamics, yet its role with respect to Abl in the developing nervous system remains unclear. This study shows that mutations in abi disrupt axonal patterning in the developing Drosophila central nervous system (CNS). However, reducing abi gene dosage by half substantially rescues Abl mutant phenotypes in pupal lethality, axonal guidance defects and locomotion deficits. Moreover, mutations in Abl increase synaptic growth and spontaneous synaptic transmission frequency at the neuromuscular junction. Double heterozygosity for abi and enabled (ena) also suppresses the synaptic overgrowth phenotypes of Abl mutants, suggesting that Abi acts cooperatively with Ena to antagonize Abl function in synaptogenesis. Intriguingly, overexpressing Abi or Ena alone in cultured cells dramatically redistributed peripheral F-actin to the cytoplasm, with aggregates colocalizing with Abi and/or Ena, and resulted in a reduction in neurite extension. However, co-expressing Abl with Abi or Ena redistributed cytoplasmic F-actin back to the cell periphery and restored bipolar cell morphology. These data suggest that abi and Abl have an antagonistic interaction in Drosophila axonogenesis and synaptogenesis, which possibly occurs through the modulation of F-actin reorganization (Lin, 2009).
The in vivo role of Abi with respect to Abl has remained enigmatic. Abi was first identified as an Abl kinase substrate, functioning in modulating the transformation activity of oncogenic Abl in human cancers. Intriguingly, Abi also functions as an activator of Abl kinase activity. Moreover, the interaction of Abl and Abi can trigger an array of biochemical and functional changes in Abi, including protein phosphorylation, stability and subcellular localization, which might ultimately lead to the control of a particular biological process in vivo. Although both Abi and Abl proteins are highly expressed in the mammalian and Drosophila nervous systems, the role of Abi in modulating the function of Abl in developing nervous systems has remained unclear. In this investigation, genetic and functional studies were conducted to advance the understanding of how abi and Abl interact in vivo. To do this, abi loss-of-function alleles were generated and characterized for genetic and functional studies in Drosophila. Immunohistochemical analysis revealed that Abi is primarily expressed in the developing CNS. Consistent with this finding, phenotypic analysis suggested that mutations in abi resulted in axonal guidance defects in the CNS. In an analysis of Abl mutants, it was found Abl to be crucial for restricting synaptic overgrowth in the larval NMJ. Importantly, further studies of the genetic interaction found a functional link between abi and Abl in axonogenesis and synaptogenesis. Moreover, Abi and Ena were found to cooperate in modulating the function of Abl in NMJ growth. Finally, based on additional cellular biology studies, it is proposed that the functional interactions between Abi, Ena and Abl might be mediated through the modulation of actin cytoskeleton reorganization (Lin, 2009).
Accumulated evidence suggests that the highly conserved actin-regulatory pathways are essential for synaptogenesis and synaptic plasticity. Abi, Ena and Abl proteins are all involved in actin dynamics. Using the Drosophila NMJ as a model system, it is proposed that the abi-ena-Abl interaction in synaptogenesis might be associated with actin cytoskeleton reorganization. In fact, several actin regulatory molecules associated with both Abl and Abi have been implicated in synaptic growth. For example, Wiskott-Aldrich Syndrome protein (Wasp) is a kinase substrate of Abl and also a binding partner for Abi. The mutations in wasp result in phenotypes very similar to those present in Abl mutants, with synaptic overgrowth and hyperbranching at the NMJ. Another example is that of Diaphanous (Dia), which also interacts with both Abl and Abi, and has recently been found to modulate synaptic growth of the Drosophila NMJ. dia mutant heterozygotes have been found to be able to enhance the cellularization phenotype of an Abl maternal-null mutant, suggesting that Dia might be involved in the regulation of Abl signaling for actin reorganization. Consistent with this idea, the interaction of Dia with Abi protein has been found to be important in regulating the formation and stability of cell-cell junctions in mammalian cells. Future studies investigating whether and how Wasp and/or Dia can participate in Abl-Abi signaling for the regulation of Drosophila synaptogenesis could be interesting (Lin, 2009).
Besides Wasp and Dia, other actin regulators might also contribute to Abl-Abi signaling during nervous system development, for, as another study has suggested, Abl may be a key regulator in modulating different types and sites of actin polymerization within the cells. Abi has been shown to play a key role in the activation of the SCAR/WAVE complex, which relays signaling from Rac1 to the Arp2/3 complex for actin cytoskeleton remodeling. Genetic studies have shown that the heterozygosity of scar, but not of kette, suppressed Abl NMJ phenotypes. A current model suggests that eliminating any component from the SCAR/WAVE complex induces the breakdown of other complex components and subsequently results in abnormal lamellipodia formation. Genetic study using the Drosophila NMJ as a model does not appear to fully support this idea. The results suggested that only a subcomplex of SCAR/WAVE might be involved in synaptogenesis. In fact, recent studies have demonstrated that some components of the SCAR/WAVE complex might work outside the complex to regulate various biological processes, including neutrophil chemotaxis, cell motility and adhesion, and the formation of cell-cell junctions. Thus, it is possible that Kette is not in a complex with Abi and Scar to modulate the function of Abl in NMJ growth. To explore this hypothesis, it will be important to examine the genetic interactions between abi and scar or kette in NMJ morphogenesis (Lin, 2009).
This study found strong in vivo evidence for an antagonistic relationship between Abl and Abi in axonogenesis and synaptogenesis. Supporting this model, one very recent study has demonstrated that Abl can inhibit the role of Abi in the engulfment of apoptotic cells in C. elegans (Hurwitz, 2009). Given that Abi is the Abl kinase substrate and that it also functions as an adaptor protein for Abl in regulating other downstream effectors, it is feasible that Abi might act downstream of Abl in modulating NMJ growth. If so, the removal of both copies of abi could conceivably further suppress Abl-/- NMJ phenotypes. Preliminary morphological and functional data both suggest that the minor NMJ defects of Abl-/- abi+/- are further rescued in Abl-/- abi-/- mutants. However, Abl-/- abi-/- double mutants showed early lethality and defects in axonal innervations, rendering the finding inconclusive. Further epistasis analysis combining abi and abl gain-of-function and loss-of-function mutations are needed to test this hypothesis (Lin, 2009).
Since the data suggest an antagonistic interaction between abi and Abl for the CNS and NMJ phenotypes, it is speculated that Abl heterozygosity would suppress the semilethal phenotype of abi mutants. Surprisingly, preliminary data showed that the lethality of abi hypomorphic mutants (abiP1/KO and abiP1/Df) is further increased by Abl+/-. This result does not seem to support a general bidirectional antagonistic relationship between Abl and abi for the biological processes involved during development. Thus, a complex genetic interaction network between Abl and abi might be present in development processes (Lin, 2009).
Another interesting issue is that the abi mutants did not display obvious defects in synaptic bouton number or synaptic transmission, although they exhibited midline crossing defects in the embryonic CNS. Because other members of SCAR complex, including Scar, Kette, Sra-1 and HSPC300, exhibit both CNS and NMJ phenotypes, it is still possible that abi mutants might show minor morphological or functional abnormalities if different phenotypic characteristics are studied. Detailed morphological assays are required to investigate other phenotypic traits of the NMJ in larval or later developmental stages. Alternatively, one could reason that the roles of Abi in synaptic growth and axonal guidance are not exactly identical. Results similar to this finding have been observed for the loss of spastin, a gene enriched in axons and synaptic connections, as spastin mutants only exhibit NMJ but not CNS defects (Lin, 2009).
This work also suggested that the synaptic overgrowth phenotypes in Abl mutants could be completely rescued by expressing Abl in the presynaptic nerve cells but not in the postsynaptic muscles, suggesting that the presynaptic Abl is more crucial than the postsynaptic population for Drosophila larval NMJ formation. However, studies in mammalian Abl and Arg (also known as Abl2) have shown that both proteins localize to the presynaptic terminals and dendritic spines of synapses in the hippocampal CA1 area. Abl and Arg have also been shown to be essential for the agrin-induced clustering of acetylcholine receptors (AChRs) on the postsynaptic membrane of the mammalian NMJ, suggesting that Abl function is required in the postsynaptic region of the mammalian NMJ. However, these reports do not exclude the possibility that Drosophila Abl might also function in postsynaptic regions of the developing brain. The reason for this speculation is that the mammalian NMJ uses acetylcholine as the neurotransmitter, unlike the Drosophila NMJ, which uses glutamate as a transmitter. Since acetylcholine receptors also function in the developing brain of Drosophila, it would be important to investigate whether Drosophila Abl also plays a role in the postsynaptic region of the neurons, where acetylcholine receptors are expressed (Lin, 2009).
In conclusion, these genetic studies in Drosophila suggest that Abi and Abl play opposing roles in axonogenesis and synaptogenesis. This conclusion is further supported by a series of biochemical, immunocytochemical and morphological studies in cultured cells. These findings offer new insights into the functional interaction between Abl, Abi and Ena in nervous system development (Lin, 2009).
The Abelson (Abl) family of non-receptor tyrosine kinases has an important role in cell morphogenesis, motility, and proliferation. Although the function of Abl has been extensively studied in leukemia, its role in epithelial cell invasion remains obscure. Using the Drosophila wing epithelium as an in vivo model system, this study shows that overexpression (activation) of Drosophila Abl (dAbl) causes loss of epithelial apical/basal cell polarity and secretion of matrix metalloproteinases, resulting in a cellular invasion and apoptosis. The in vivo data indicate that dAbl acts downstream of the Src kinases, which are known regulators of cell adhesion and invasion. Downstream of dAbl, Rac GTPases activate two distinct MAPK pathways: c-Jun N-terminal kinase signaling (required for cell invasion and apoptosis) and ERK signaling (inducing cell proliferation). Activated Abl also increases the activity of Src members through a positive feedback loop leading to signal amplification. Thus, targeting Src-Abl, using available dual inhibitors, could be of therapeutic importance in tumor cell metastasis (Singh, 2010a).
This is the first study to provide in vivo evidence for the role of Abl in cell invasion. Cells expressing dAbl (in the dpp-domain) become invasive and migrate into the area of the posterior compartment, where they are located basally to the basement membrane. Although during this process many cells die, those that 'resist' cell death can be visualized by the presence of GFP at the base of the epithelium in either compartment. Furthermore, mechanistic evidence is provided for an Src-Abl signaling cascade and an Abl/Src signal amplification loop in epithelial cell invasion. Targeting both kinase types using dual Abl/Src inhibitors in cancer patients could thus be of clinical significance. It was also shown that increased cell proliferation associated with Abl can be separated from its cell invasion function by distinct downstream effectors. Different MAPKs are activated downstream of dAbl and Rac, and mediate the cell proliferation and cell invasion phenotypes, respectively (Singh, 2010a).
Loss of cell polarity has been linked to tumor growth and cell invasion. The mechanism(s) by which dAbl downregulates cell adhesion/polarity genes like DE-Cadherin, β-Catenin/Armadillo and Dlg are not known. This could be a direct effect of dAbl on junctional complexes or a consequence of the cell invasive behavior. Downregulation of E-cadherin has been linked to several types of tumors. Furthermore, Src family members have been shown to increase the turnover of AJs, which in turn would cause an increase in cell mobility, a possible mechanism by which Abl can mediate loss of cell polarity. This hypothesis is further supported by the observation that overexpression of DE-cadherin suppresses the effects induced by Src upregulation (through Csk reduction, using UAS-dCsk-RNAi) in the retina. Consistent with this notion, overexpression of DE-Cadherin rescues the dAbl-induced cell invasion phenotype. Moreover, removing a genomic copy of mmp1 and mmp2 results in suppression of the dAbl cell invasion phenotype. On the basis of these data it is concluded that loss of cell polarity and MMP secretion are the key factors in contributing to cell invasive behavior of dAbl-expressing cells. However, the possibility of minor contributions of unknown factors in this process cannot be completely ruled out (Singh, 2010a).
A complicated question is how dAbl causes cell proliferation in epithelial cells (dAbl lacks nuclear localization signals). The effect of dAbl expression results in cell-autonomous and non-autonomous cell proliferation. In Drosophila, cells destined to undergo apoptosis express specific growth factors (Wingless and Dpp; their upregulation is mediated by JNK activation), inducing non-autonomous compensatory proliferation in neighboring cells. This compensatory proliferation is important for maintaining proper tissue homeostasis and may also be relevant for the induction of tumor cell proliferation. As dAbl activation results in cell death in migrating cells, one argument could be that cell proliferation associated with dAbl activation is a consequence of compensatory proliferation. Interestingly, dAbl expression results in an increase in Wg expression, suggesting that compensatory proliferation takes place in response to dAbl. Taken together, these data suggest that at least some aspects of dAbl-mediated cell proliferation (mediated by activation of ERK) are cell-autonomous independent of such compensatory proliferation, as Bsk-DN co-expression in a dAbl overexpression background (which blocks JNK signaling and thus induction of Wg and Dpp expression), does not block excessive proliferation within the dAbl expression domain (Singh, 2010a).
The cell invasion phenotype of dAbl overexpression is similar to Csk reduction (dCsk-RNAi) and the data indicate that dAbl acts downstream of dCsk. As Csk negatively regulates Abl/Src family kinases (SFKs), this suggested that Src mediates the effect of dCsk on dAbl. Previous studies have shown that Abl can act as a substrate of SFKs, though other studies have shown that the opposite can also be true. The data indicate that Src acts upstream of Abl and that Abl can feed back and amplify the signal through its positive effect on Src. How is the dAbl-Src feedback loop working mechanistically? From the in vivo experiments it is not possible to conclude whether dAbl acts directly on Src, dCsk, or unknown upstream components. dAbl does not co-immunoprecipitate either dCsk or the Src kinases in Drosophila S2 cells. Since binding between kinases can be of very transient nature, it is possible that even if dAbl would bind Src or dCsk in vivo, it may not be possible to detect it. However, in vivo data suggest that dCsk does not mediate the dAbl effect: if dAbl would act through dCsk (by inhibiting it), phospho-Src (pSrc) levels should be similar with dCsk-IR or dAbl expression, which is not the case. dAbl expression results in a much more robust activation Src with pSrc detected in all dAbl/GFP-positive cells, whereas dCsk-IR does not result in such strong activation. This observation suggests that dAbl does not act through dCsk in this process. Although the possibility cannot be excluded that dAbl could modulate an unknown component upstream of dCsk, the fact that co-expression of dCsk-IR and dAbl (dCsk-IR; UAS-Abl at 18°C) shows a synergistic effect (even at 18°C, where neither individual transgene has a phenotype on its own) suggests that dAbl and Csk act in parallel on Src. As Abl can phosphorylate Src kinases, a direct effect of dAbl on the Src kinases is favored (Singh, 2010a).
JNK signaling is activated in response to environmental stress and by several classes of cell surface receptors, including cytokine receptors and receptor tyrosine kinases. In mammalian cells, JNK has been implicated in oncogenic transformation in fibroblasts and hematopoietic cells, and in cell invasion. In oncogenic transformation, JNK signaling can promote tumor growth, while it can also act as a tumor suppressor. It also functions in basement membrane remodeling during imaginal disc eversion and tumor invasion. This study provides evidence for a link between Src and JNK during cell invasion, mediated through dAbl. The cell invasion and apoptosis phenotypes induced by dAbl require JNK activity, whereas the cell proliferation function of dAbl appears to be mediated by ERK signaling. dAbl does not affect expression levels of JNK but instead causes an increase in active JNK (phospho-JNK). It is worth noting that removing a genomic copy of each of the Drosophila Rac genes suppresses all phenotypes associated with dAbl overexpression (cell invasion, death, and proliferation). These data are consistent with the study of BCR-Abl-mediated cell growth, which requires Rac function, suggesting a general relevance of Rac GTPases as Abl effectors (Singh, 2010a).
It is not established how Abl mediates Rac activation. A possible link can be Crk, which primarily consists of SH2 and SH3 domains, serving as an adaptor. Crk-I can associate with and be phosphorylated by c-Abl. Furthermore, ectopic expression of Crk can result in JNK activation. As overexpression/activation of dAbl results in JNK activation, Crk may provide a missing link between dAbl and Rac for JNK activation. Another candidate to mediate an interaction between dAbl and Rac GTPases can be Trio, a guanine exchange factor. Trio has two putative Rac and Rho-binding domains. In Drosophila, Trio function has been studied extensively in the context of axon guidance where it has been shown to interact with dAbl. Interestingly, a recent report has identified Trio as one of the guanine exchange factors responsible for invasive behavior of glioblastoma. Thus, a potential role of Trio in the context of Abl-mediated cell invasion warrants further investigation (Singh, 2010a).
Abelson (Abl) family tyrosine kinases have been implicated in cell morphogenesis, adhesion, motility, and oncogenesis. Using a candidate approach for genes involved in planar cell polarity (PCP) signaling, Drosophila Abl (dAbl) was identified as a modulator of Frizzled(Fz)/PCP signaling. dAbl positively regulates the Fz/Dishevelled (Dsh) PCP pathway without affecting canonical Wnt/Wg-Fz signaling. Genetic dissection suggests that Abl functions via Fz/Dsh signaling in photoreceptor R3 specification, a well-established Fz-PCP signaling readout. Molecular analysis shows that dAbl binds and phosphorylates Dsh on Tyr473 within the DEP domain. This phosphorylation event on Dsh is functionally critical, as the equivalent DshY473F mutant is nonfunctional in PCP signaling and stable membrane association, although it rescues canonical Wnt signaling. Strikingly, mouse embryonic fibroblasts (MEFs) deficient for Abl1 and Abl2/Arg genes also show reduced Dvl2 phosphorylation as compared with control MEFs, and this correlates with a change in subcellular localization of endogenous Dvl2. As in Drosophila, such Abl-deficient MEFs show no change in canonical Wnt signaling. Taken together, these results argue for a conserved role of Abl family members in the positive regulation of Dsh activity toward Fz-Dsh/PCP signaling by Dsh phosphorylation (Singh, 2010b).
Evidence is provided for a specific role of tyrosine phosphorylation of Dsh by Abl family kinases in Fz/Dsh-PCP signaling. dAbl is required for R3/R4 fate specification. dAbl interacts with fz and dsh genetically in PCP signaling. Biochemical experiments indicate that dAbl binds Dsh and phosphorylates it on Tyr473 within the DEP domain, which has been specifically implicated in PCP signaling and is largely dispensable for canonical Wnt/Wg signaling. The data further show that Abl kinases do not affect canonical Wnt signaling in either Drosophila or MEFs. Taken together, these data indicate that Abl family kinases positively regulate PCP signaling by affecting Dsh/Dvl family members via phosphorylation of Tyr473. Abl family kinases appear to provide a molecular gating mechanism to increase the capability of Dsh/Dvl proteins to signal via the Fz/Dsh-PCP pathway. These data suggest the possibility that this function of Abl family kinases might be conserved from flies to mammals, as similar effects were observed with mammalian Abl and Dvl family members (Singh, 2010b).
Most Abl studies in mammalian cell culture have focused on their role in tumor formation. Little is known about Abls normal physiological roles during development, except in the context of junctional stability and cytoskeletal events. Previous studies in the Drosophila eye established that dAbl is expressed dynamically in all photoreceptors, and dAbl mutant flies have a rough eye phenotype with significant photoreceptor loss. Its potential role in cell fate specification has not been addressed. A detailed analysis of the dAbl eye phenotype was performed in the context of PCP establishment and R3/R4 specification. It was demonstrated that dAbl is required for specification of both R3-R4 cells. Differential activation of Fz/PCP signaling specifies R3 and leads to the activation of Notch signaling in the neighboring R4 to induce its proper fate. The data suggest that dAbl is required in R3 for fate specification via its interaction with Dsh and positive input into Fz/PCP signaling. This Abl function appears to be common to Fz/PCP signaling in general, as Fz and dAbl also synergize in PCP establishment in the wing. Moreover, dAbl phosphorylation of DshTyr473 is essential for Dsh PCP function in general. In vertebrates, Abl kinases affect Dvl localization in MEFs, and Abl1-/-,Abl2-/- mice display similar phenotypes as dvl1-/-,dvl2-/- mice, with severe open neural tube defects in 9.5-day embryos. The requirement of dAbl in R4 specification remains obscure, as Fz/PCP signaling is not required in R4 (Singh, 2010b).
A likely explanation derives from studies of dAbl in noncanonical Notch signaling, where dAbl has been suggested to act downstream from Notch. Although the role of Notch in the developing eye has focused on canonical Su(H)-dependent Notch activity, it is quite likely that Abl could modulate Notch signaling activity in R4. Further work will be needed to explain the role of dAbl in R4 fate specification and associated Notch signaling (Singh, 2010b).
Dsh contains three highly conserved domains and a stretch of basic residues and several serine/threonine-rich regions between the DIX and PDZ domains, as well as a proline-rich region downstream from the PDZ domain, which encodes a class I consensus sequence for an SH3-binding protein. Many proteins that have been shown to bind Dsh/Dvl bind to Dsh in the PDZ domain. Abl binding, however, maps to the proline-rich region of Dsh just C-terminal to the PDZ domain, while the PDZ domain alone showed no binding. Different Dsh domain requirements are known for canonical and PCP signaling. While the DIX domain functions exclusively in canonical Wnt signaling, the DEP domain is required for PCP signaling and, in particular, stable membrane association. The results indicate that dAbl phosphorylates Dsh at Tyr473 (and possibly other Tyr residues in the DEP/C-term region). Phosphorylation of Dsh-Tyr473 is unique, as dAbl is a tyrosine kinase and all previously analyzed Dsh kinases have been serine/threonine kinases (Singh, 2010b).
Wnt signaling is important in diverse physiological processes and, when deregulated, often leads to disease states. Studies in model organisms have unraveled two conserved pathways, now referred to as canonical Wnt/Wg signaling and Wnt-Fz/PCP signaling. The canonical Wnt signal is transduced via Fz family receptors (along with the LRP5/6 coreceptors), leading to Dsh-Axin complex formation, which in turn causes the stabilization of cytoplasmic β-catenin and allows gene transcription. In Fz/PCP signaling, Dsh is recruited to the membrane in an Axin- and LRP5/6-independent manner and acts on different downstream effectors, depending on the cellular context. The mechanism of pathway-specific Dsh 'activation' is poorly understood, and, similarly, the question as to how the individual pathways are specifically activated at the level of either Fz or Dsh remains unresolved. This study highlights the importance of Abl in the context of Fz/PCP signaling at the level of Dsh/Dvl. At the level of both Abl mutants as well as the DshY473F phosphorylation mutant, canonical Wnt/Wg signaling remains unaffected, while PCP signaling is defective. As the signal that activates dAbl in this context is not known, it is possible that Abl acts in a permissive manner in PCP signaling. In conclusion, this study provides evidence for Dsh tyrosine phosphorylation and a role of Abl in PCP signaling; further studies will be needed to establish a full framework for the regulation of Abl in PCP signaling and in the biology of Dsh (Singh, 2010b).
Wnt Planar Cell Polarity (PCP) signaling is a universal regulator of polarity in epithelial cells, but it regulates axon outgrowth in neurons, suggesting the existence of axonal modulators of Wnt-PCP activity. The Amyloid precursor proteins (APPs) are intensely investigated because of their link to Alzheimer's disease (AD). APP's in vivo function in the brain and the mechanisms underlying it remain unclear and controversial. Drosophila possesses a single APP homologue called APP Like, or APPL. APPL is expressed in all neurons throughout development, but has no established function in neuronal development. This study therefore investigated the role of Drosophila APPL during brain development. APPL was found to be involved in the development of the Mushroom Body αβ neurons and, in particular, is required cell-autonomously for the β-axons and non-cell autonomously for the α-axons growth. Moreover, APPL was found to be a modulator of the Wnt-PCP pathway required for axonal outgrowth, but not cell polarity. Molecularly, both human APP and fly APPL form complexes with PCP receptors, thus suggesting that APPs are part of the membrane protein complex upstream of PCP signaling. Moreover,APPL regulates PCP pathway activation by modulating the phosphorylation of the Wnt adaptor protein Dishevelled (Dsh) by Abelson kinase (Abl). Taken together these data suggest that APPL is the first example of a modulator of the Wnt-PCP pathway specifically required for axon outgrowth (Soldano, 2013).
AD is a neurodegenerative disorder characterized by progressive loss of neurons in specific regions of the brain that correlates with progressive impairment of higher cognitive functions. A growing body of evidence identifies the APP and its metabolite the Aβ peptide as main players in the pathogenesis of AD. In particular, the accumulation of Aβ peptides in the brain seems to be the trigger of the pathological cascade that eventually results in neuronal loss and degeneration. Despite efforts to characterize the molecular mechanisms underlying Aβ's toxic function, it is still not clear what triggers the accumulation of the peptide and how this is correlated with the pathogenesis of the disease and the dementia. In fact, most of the work done to unveil the pathogenesis of the disease has focused on the analysis of Aβ-peptide and the search for its receptors and downstream effectors. Even though the numerous in vitro studies performed in cell culture identified several molecules that interact with Aβ peptide, the in vivo biological relevance of these interactions remains to be clarified. The amyloid cascade hypothesis has also dominated the search for AD treatments, but the promising molecular candidates developed to modulate the Aβ peptide and reached clinical trials failed. Finally, over the last few years many studies indicated that there is no linear correlation between the accumulation of the peptide and the cognitive decline, leading to a revision of the amyloidogenic hypothesis. Taken together, these observations suggest that the accumulation of the peptide is not the only cause of the pathology and that other factors are involved. Interestingly, under physiological conditions APP is mainly found in its uncleaved or α-cleaved form, suggesting that the shift towards amyloidogenic processing not only increases the production of Aβ peptide but also depletes the pool of APP that undergoes non-amyloidogenic processing, with hitherto unknown consequences. It is therefore of paramount importance to understand the physiological role of APP and how perturbing this role could contribute to the pathogenesis of the disease. An important contribution to the study of the function of a protein comes from the analysis of the knock-out (KO) animals. In the case of APP, several KO models have been generated and analyzed in detail both from the morphological and behavioral point of view. Despite these efforts, the normal physiological function of APP in vivo in the nervous system remains largely elusive and highly controversial. This is due to the lack of consensus over the neuronal phenotypes in null mutant animals and the mechanism of action in vivo. The data collected by different labs confirmed the involvement of APPs in development and function of the nervous system, but these studies do not provide an in-depth analysis of the development of the brain during the pre-natal stages or the molecular mechanism underlying APPs' putative functions. Therefore this study took advantage of Drosophila melanogaster to further analyze the consequence of loss of APP Like (APPL) during brain development (Soldano, 2013).
The present study demonstrates that APPL is involved in brain development of Drosophila melanogaster, particularly in the Mushroom Body (MB) neurons. APPL is required for the development of αβ neurons. In Appl-/- flies, MB neurons fail to project the α lobe in 14% of the cases and the β-lobe in 12% of the cases. Further analysis of the phenotype reveals that APPL is required cell-autonomously for the development of the β lobe and non-cell autonomously for the development of the α lobe. In fact, single cell Appl-/- clones display only β-lobe loss and no α loss. The re-introduction of a full-length, membrane-tethered form of APPL, but not a soluble form, rescues β-lobe los. This is of particular interest because it confirms that, similar to mammalian APPs, the physiological role of APPL is mediated both by its full-length form, required in the neurons to achieve the correct β-lobe pattern, and by its soluble form (sAPPL) that regulates the extension of the α lobe. Moreover, the rescue data indicate that, at least in this context, the function of sAPPL is mediated not by homo-dimerization with the full-length form but by some other receptor, hitherto unknown. Further experiments are required to clarify the sAPPL non-cell autonomous effect, but it is hypothesized that it might be involved in modulating signaling mediated by the cells that surround the MB axons. Taken together, the analysis of the Appl-/- animals confirmed the important role of APPs during brain development but reinforced the idea that the phenotypes are present with incomplete penetrance and might be subtle. It would therefore be of interest to analyze the phenotype of the KO mice in greater detail and, in particular, to better characterize the APP's downstream pathway leading to these defects (Soldano, 2013).
Moreover, the results described clearly support a model of APPL as a novel, neuronal-specific positive modulator of the Wnt-PCP pathway. The PCP pathway was initially described because of its role in tissue polarity establishment and, in particular, of its regulation of cell orientation in plane of an epithelium. Among the different processes regulated by PCP signaling, axon growth and guidance is of particular interest. Mice null for Fzd3/Ceslr3-/- genes show severe defects in several major axon tracts like thalamocortical, corticothalamic, and nigrostriatal tracts, defects of the anterior commissure, and similarly to APP KO mice, the variable loss of the corpus callosum (Soldano, 2013).
The molecular mechanism underlying the function of PCP-signaling in regulating tissue polarity has been broadly studied. The current model suggests that, upon polarized expression of the different core proteins, Dsh is recruited to the membrane via Fz and leads to the activation of a cascade of small GTPases finally resulting in cytoskeleton rearrangements. In the case of regulation of axon growth and guidance, it is less clear how the signaling is regulated and transmitted to the cytoskeleton. A recent publication suggested that during axon growth the transmembrane PCP receptor-like Vang and Fzd are localized at the growth cone area on the tip of the fillopodia, thus suggesting that in this context the asymmetric localization is not needed (Soldano, 2013).
Furthermore, Dsh needs to relocalize from the cytoplasm to the membrane to ensure the proper activation of PCP signaling, and this is dependent on its phosphorylation status. Abelson has been shown to be one kinase responsible for this modification, but the receptor upstream of the kinase was not identified (Singh, 2010b). Based on current evidence, it is proposed that APPL is a novel regulator of Wnt-PCP pathway involved in axon growth and guidance. This is of interest because while the PCP core proteins are ubiquitously expressed, APPL is restricted to the nervous system, suggesting that it could be the first described tissue-specific modulator of the pathway (Soldano, 2013).
Mechanistically, it is proposed that APPL-Abl complex modulates Dsh via dual protein-protein interactions. First, Abl might have an intrinsic affinity for its substrate Dsh (Singh, 2010b). Secondly, this interaction is strengthened or stabilized by the inclusion of APPL in a PCP receptor complex. This dual affinity complex leads to increased PCP signaling efficiency at the developing growth cone. Both biochemical and physiological data show that this function is highly conserved in mammalian APP, suggesting that it may play a similar role in the mammalian brain. The canonical-Wnt signaling pathway has already been connected to AD pathogenesis because of its link to the tau-kinase GSK-3β. Interestingly, no clear link between the Wnt-PCP pathway and this neurodegenerative disorder has been made. Previous reports have show that, in flies and mice, Jun N-terminal Kinase (JNK) is the final effector of PCP in axon outgrowth and JNK was shown to be required for the effect of APP overexpression in the fly. Interestingly, JNK signaling has also been linked to the neuronal loss observed in AD. It is therefore worth investigating whether the physiological function of APP as a neuronal PCP modulator explains the JNK-AD connection (Soldano, 2013).
The ability of neuronal growth cones to be guided by extracellular cues requires intimate communication between signal
transduction systems and the dynamic actin-based cytoskeleton at the leading edge. Profilin (chickadee), a small, actin-binding protein,
has been proposed to be a regulator of the cell motility machinery at leading edge membranes. However, any requirement it may have in
the developing nervous system has been unknown. Profilin associates with members of the Enabled family of proteins,
suggesting that Profilin might link Abl function to the cytoskeleton.
In a genetic screen in Drosophila to identify genes required for the correct navigation and
outgrowth of motoneuron growth cones two alleles of a
stranded (sand) mutation were recovered in which motor growth cones arrest before reaching their final targets. The molecular genetic analysis reveals that stranded alleles are zygotic lethal mutations in Profilin. In vitro experiments
confirm that axon extension is impaired in Profilin mutants. Moreover, phenotypic comparisons and genetic interactions
between chic and abl mutants support the notion that Profilin and Abl cooperate to promote axon
extension. Genetic analysis in Drosophila has been used to
demonstrate that mutations in Profilin (chickadee) and Abl (abl) display an identical growth cone arrest phenotype for axons
of intersegmental nerve b (ISNb). Moreover, the phenotype of a double mutant suggests that these components function
together to control axonal outgrowth (Wills, 1999a).
Genetic analysis of growth cone guidance choice points in Drosophila has identified neuronal receptor protein tyrosine
phosphatases (RPTPs) as key determinants of axon pathfinding behavior. The Drosophila Abl
tyrosine kinase functions in the intersegmental nerve b (ISNb) motor choice point pathway as an antagonist of the RPTP
Dlar. The function of Abl in this pathway is dependent on an intact catalytic domain. The Abl
phosphoprotein substrate Enabled (Ena) is required for choice point navigation. Both Abl and Ena proteins associate with
the Dlar cytoplasmic domain and serve as substrates for Dlar in vitro, suggesting that they play a direct role in the Dlar
pathway. These data suggest that Dlar, Abl, and Ena define a phosphorylation state-dependent switch that controls growth
cone behavior by transmitting signals at the cell surface to the actin cytoskeleton (Wills, 1999b).
The reciprocal catalytic activities of a tyrosine kinase and phosphatase predict that a reduction in kinase activity within the Dlar pathway might suppress the Dlar motor axon phenotype. In Dlar mutant embryos, subsets of axons derived from the intersegmental nerve route (ISN), called ISNb and ISNd, fail to enter adjacent muscle target domains just outside the ventral nerve cord. Instead, Dlar mutant ISNb and ISNd axons follow the ISN toward dorsal targets (the bypass phenotype. Since abl loss of function is known to disrupt the outgrowth of ISNb, the Abl tyrosine kinase is an excellent candidate for a role in Dlar signaling. Therefore, various genetic backgrounds were examined in which homozygous Dlar mutations were combined with mutations in a single allele of abl. Reduction of abl of up to half the normal gene dose has a profound effect on the penetrance of the Dlar motor axon guidance phenotype, suppressing the Dlar phenotype up to 10-fold; for example, ISNb bypass in Dlar mutants is reduced from 38% to 4% in abl heterozygote mutants (Wills, 1999b).
Western blot analysis shows that endogenous Abl protein binds specifically to the full-length Dlar cytoplasmic domain (GST-Dlar D1-D2). The association of Dlar and Abl in cell extracts is consistent with a direct functional relationship between the two proteins. However, the binding could depend on other factors present in the crude extract. Therefore, the association of purified recombinant Abl protein with Dlar fusion proteins was examined in the absence of other Drosophila proteins. Recombinant Abl binds to Dlar with somewhat less specificity than does the Abl endogenous to S2 cells. Purified mammalian v-Abl binds to Dlar under the same conditions, with a profile of specificity very similar to that of Drosophila Abl. Since v-Abl represents only the kinase and SH2 domains of Abl, these domains appear sufficient to mediate Dlar binding. As further evidence of direct physical interactions between Abl and the Dlar D2 domain, kinase assays reveal that Drosophila Abl phosphorylates GST-Dlar D2 in vitro. In addition to the Dlar D2 domain, Drosophila Abl can weakly phosphorylate the D2 domain of another receptor tyrosine kinase, Protein tyrosine phosphatase 69D (Ptp69D); this is interesting, since Ptp69D is tyrosine phosphorylated in S2 cells. The physical interactions between Abl and Dlar support a model whereby both proteins function in the same signaling pathway. Furthermore, the phosphorylation of the D2 domain in vitro raises the intriguing possibility that d-Abl activity regulates Dlar function in vivo (Wills, 1999b).
The contrast between the abl and Dlar phenotypes and the suppression of the Dlar phenotype by abl alleles suggest that Abl and Dlar play functionally antagonistic roles in ISNb development. This hypothesis makes a simple prediction: gain of function in Abl should result in a phenotype similar to loss of Dlar. Therefore, the GAL4 expression system was used to target high-level expression of wild-type Abl to postmitotic neurons and then the development of motor axon pathways was examined. With three independent neural specific GAL4 drivers, in combination with an abl cDNA under the control of the GAL4 upstream activator sequence (UAS), GAL4-dependent phenotypes were observed. When wild-type Abl is overexpressed, ISNb axons bypass their ventral target muscles in a manner indistinguishable from that of the ISNb phenotype observed in Dlar mutants. The kinase activity of Abl has been shown to be necessary for its role in ISNb neurons (Wills, 1999b).
Since Ena acts as a genetic antagonist of Abl, it was reasoned that loss of Ena should resemble gain of Abl. ISNb bypass phenotypes are seen in all ena mutant combinations. Two types of ISNb phenotypes are observed in ena mutants: (1) failure of ISNb to enter the ventral muscles after a successful defasciculation (characteristic of embryos lacking Dlar alone), and (2) failure of ISNb axons to defasciculate from the ISN pathway (characteristic of embryos lacking multiple phosphatases. In addition, the frequency of ISNb bypass in strong ena mutants is twice that observed in the strongest Dlar alleles. These observations may indicate that Ena acts as a point of convergence for multiple inputs in the ISNb guidance mechanism. Ena family members share a conserved domain structure, including an N-terminal EVH1 domain that mediates binding to Zyxin and Listeria ActA, a proline-rich region that supports associations with Profilin and SH3 domains, and a C-terminal EVH2 domain that promotes multimerization. Mutations are available that specifically disrupt either the EVH1 or the EVH2 domains of Ena. Mutations in either domain display highly penetrant ISNb bypass, demonstrating a requirement for both domains in the guidance mechanism. Although Ena is restricted to axons in the developing nervous system late in embryogenesis, it is expressed broadly prior to germ band retraction. To confirm that neuronal Ena function is necessary for ISNb choice point navigation, wild-type ena cDNA was expressed under neuronal GAL4 control in an ena mutant background. Neural specific ena expression attenuates the ISNb phenotype significantly. If the quantity of Ena protein is rate limiting in wild-type ISNb axons, one might expect Ena overexpression to disrupt ISNb guidance. However, no ISNb phenotypes are observed, even when UAS-ena is combined with the strongest neural driver P[elav-GAL4] (Wills, 1999b).
The genetic relationship between Abl and Dlar and the requirement of Ena function for ISNb target entry suggest that Ena might act in the Dlar signaling pathway. To test this model, it was asked whether Ena associates with the cytoplasmic domain of Dlar. Endogenous Ena protein associates with a Dlar full-length cytoplasmic domain (GST-Dlar D1-D2) or with D2 alone but not comparably with wild-type D1. Since Abl is known to associate with Ena, and since binding between Abl and Dlar has been demonstrated, it is possible that Ena binding to Dlar requires Abl or additional proteins. Purified Ena has been shown to bind to the Dlar cytoplasmic domain. In both extract and recombinant protein binding assays, Ena shows only weak association with DPTP10D. However, Ena binds effectively to the D2 domain of Ptp69D. The preferential binding of Ena to the D2 domains of Dlar and Ptp69D, as compared with the D1 domains of the same RPTPs, suggests that these interactions are specific. The parallel between Dlar and Ptp69D binding is interesting, given the published observation that Ptp69D is required for ISNb guidance and can partially substitute for Dlar in vivo. Furthermore, the nature and penetrance of ISNb defects in ena mutants suggest that Ena may function downstream of multiple inputs (Wills, 1999b).
The relationships between Abl, Ena, and Dlar in motor axon guidance suggest a model whereby Abl and Dlar compete for shared substrates to regulate growth cone behavior. Although the Dlar cytoplasmic domain was previously shown to encode an active PTP domain, using artificial phospho-peptide substrates in vitro, no physiological substrates have been identified. Since nearly all of the tyrosine phosphatase activity of LAR family RPTPs resides in the D1 domain, the ability of the GST-Dlar D1 fusion protein to dephosphorylate purified Drosophila Abl or Ena proteins after these proteins have been phosphorylated with recombinant d-Abl was examined. Incorporated 32P is rapidly released from both Abl and Ena after addition of wild-type GST-Dlar D1 but not after addition of the catalytically inactive C-to-S mutant GST-Dlar D1 fusion protein. These results suggest that the bacterially expressed GST-Dlar protein is correctly folded and that Drosophila Abl and Ena are both potential Dlar substrates. However, because PTPs are known to be promiscuous in vitro, additional experiments will be necessary to determine whether Abl and/or Ena are targets for Dlar activity in vivo (Wills, 1999b).
Human and mouse Abelson interacting proteins (Abi) are SH3-domain containing proteins that bind to the proline-rich motifs of the Abelson protein tyrosine kinase. A new member of this gene family, a Drosophila Abi (dAbi), is a substrate for Abl kinase and co-immunoprecipitates with Abl if the Abi SH3 domain is intact. A new function has been identified for both dAbi and human Abi-2 (hAbi-2). Both proteins activate the kinase activity of Abl as assayed by phosphorylation of the Drosophila Enabled (Ena) protein. Removal of the dAbi SH3 domain eliminates dAbi's activation of Abl kinase activity. dAbi is an unstable protein in cells and is present at low steady state levels but its protein level is increased coincident with phosphorylation by Abl kinase. Expression of the antisense strand of dAbi reduces dAbi protein levels and abolishes activation of Abl kinase activity. Modulation of Abi protein levels may be an important mechanism for regulating the level of Abl kinase activity in the cell (Juang, 1999).
Many critical cellular functions, such as cell growth and differentiation, are regulated by the kinase activity of protein tyrosine kinases (PTKs) and deregulated kinase activity of PTKs is associated with oncogenic activity. Relatively little is known about the normal regulation of the kinase activity of the Abelson (Abl) non-receptor PTK. Regulation of Abl activity is important as suggested by the consequences of improper regulation. Oncogenic forms of Abl such as BcrAbl in chronic myelogenous leukemia (CML) and v-Abl in Abelson murine leukemia virus have increased, unregulated kinase activity that phosphorylate several cellular substrates. This increased phosphorylation is believed to activate signal transduction pathways that participate in the uncontrolled cell growth of the tumor cells. Mutation of the SH3 domain of c-Abl also activates the oncogenic potential of Abl, producing elevated kinase activity, predominantly cytoplasmic localization, and cell transformation. It has been proposed that the SH3 domain of c-Abl may have a function in the negative regulation of Abl's activity. Overexpression of c-Abl in cells results in autophosphorylation of c-Abl, suggesting that there might be a limited concentration of a cellular modulator that suppresses c-Abl activity in cells. Thus, one of the models which has been proposed for the inhibition of the c-Abl tyrosine kinase invokes a trans-acting cellular inhibitor interacting with the Abl SH3 domain (Juang, 1999).
Proteins that interact with the SH3 domain of Abl and that might thereby modulate kinase activity of Abl have recently been identified. Initial reports identified three mammalian Abi proteins; murine Abi-1, hAbi-2 and human ArgBP1 (Dai, 1995; Shi, 1995; Wang, 1996). The Abi proteins physically associate with Abl and Abelson-related (Arg) kinases. The sequence motifs of Abi family members include an SH3 domain, proline-rich regions, PEST regions, and a homeodomain homologous region (Dai, 1995; Shi, 1995). Abi proteins are phosphorylated by the Abl PTKs. Overexpressing full length mouse Abi-1 in NIH3T3 cells suppresses v-Abl transforming activity (Shi, 1995). Stable expression of the full length hAbi-2 is toxic in NIH3T3 cells, but expression of a truncated form of hAbi-2, which lacks the first 157 amino acids including a proline-rich domain required for binding the Abl-SH3 domain, activates c-Abl transforming activity in NIH3T3 cells (Dai, 1995). It was proposed (Dai, 1995; Shi, 1995) that Abi is a tumor suppressor in mammalian cells. Hypothesized regulatory functions of Abi include stabilization of the inactive form of c-Abl or, alternatively, inhibiting access of Abl to protein substrates and thereby negatively regulating Abl activity (Juang, 1999).
The Abl gene has been conserved through evolution and related genes have been found in human, cat, mouse, Drosophila and C. elegans. This study used Drosophila genetics to understand the regulatory molecules of the conserved Abl pathway. In order to determine if Abi regulation of Abl is a mechanism that has been conserved through evolution, attempts were made to isolate a Drosophila member of the Abi family. This paper reports the identification of dAbi. Analysis of the dAbi protein shows very similar structural features to that of mammalian proteins. dAbi behaves similarly to mammalian Abi proteins by associating with Abl kinase and serving as a substrate for the Abl kinase. Two new properties were revealed by the biochemical analysis of both dAbi and hAbi-2 proteins. First, dAbi and hAbi-2 act as potent activators of Abl kinase activity. Second, dAbi is an unstable protein that is stabilized coincident with phosphorylation. It is proposed that regulation of Abi protein levels may be a mechanism for modulating Abl kinase activity that has been well conserved during evolution (Juang, 1999).
Degenerate oligonucleotides were designed based on the conserved SH3 domain peptide sequences of mammalian Abi proteins and used for PCR of Drosophila embryonic cDNA libraries. A fragment of 146 bp, the expected size of the SH3 domain, was isolated, cloned and sequenced. The predicted open reading frame was most similar in sequence to the mammalian Abi SH3 domain. The DNA fragment was used as a probe for high stringency screening of the library and hybridized to a 1.7 Kb Drosophila cDNA. The cDNA encodes a predicted open reading frame of 473 amino acids. A BLAST search of the GenBank non-redundant peptide sequence database (nr) identified a number of Abi and Abi-related proteins. dAbi appeared most similar to human e3B1/hssh3bp1, sharing 42% amino acid sequence identity and 54% amino acid similarity. Human e3B1 was originally identified in a screen for proteins that bound the SH3 domain of eps8, a substrate of receptor tyrosine kinases including the epidermal growth factor receptor (EGFR) tyrosine kinase (Biesova, 1997). In vitro assays with GST fusion proteins showed that e3B1 also bound the SH3 domain Abl. Subsequently, one isoform of a candidate spectrin SH3 domain-binding protein was found to be identical to e3B1. The Drosophila sequence shows similar levels of amino acid identity when compared to other human Abi proteins. Pairwise sequence comparisons showed 40% amino-acid identity and 54% amino-acid similarity between dAbi and human ArgBP1A whereas comparison of dAbi and hAbi-2 gave similar results showing 39% amino-acid identity and 52% amino-acid similarity. The Drosophila sequence has motifs similar to those found in the human Abi proteins including a carboxy-terminal SH3 domain with 76% identity to hAbi-2. The amino-terminal domain includes a homeo-domain similarity, and the middle portion includes three polyproline motifs and a PEST domain. It is proposed that this sequence be called Drosophila Abi (dAbi) (Juang, 1999).
Abi proteins were identified in two hybrid screens using the carboxy-terminal domain of Abl or the SH3 domain of Abl. Abi and Abl can potentially interact at two sites, the SH3 domain of Abl with the proline motifs of Abi and the SH3 domain of Abi with the proline motifs of Abl. In addition to binding to Abl, Abi proteins appear to be excellent substrates for the kinase, raising a question of whether Abi proteins are upstream regulators or downstream effectors of the kinase. Abi-1 has been proposed to be a negative regulator of Abl because its full length protein suppresses v-Abl transformation (Shi, 1995). The full length hAbi-2 was also suggested to function as a potential tumor suppressor gene through negative regulation of Abl kinase activity (Dai, 1995). One hypothesis put forth to explain Abi's suppression of Abl's transforming activity is that binding of Abi to the Abl SH3 domain negatively regulates Abl kinase (Dai, 1995). This hypothesis was based in part on observations that structural alterations of Abl that attenuate binding to Abi, e.g. mutation of the Abl SH3, enhance the transforming activity of c-Abl. However, the observation that Abi-1 did not inhibit the overall kinase activity of v-Abl (Shi, 1995) suggests that Abi-1 suppression of v-Abl transforming activity may be due to other mechanisms of action (Juang, 1999).
The identification of a Drosophila Abi protein indicates that the Abl-Abi interaction may have been conserved during evolution. dAbi and hAbi-2 are 39% identical and 52% similar in amino acid sequence and share similar protein motifs. dAbi associates with Abl, as determined by co-immunoprecipitation, and is a substrate for the Abl PTK. The dAbi SH3 domain is required for a detectable interaction with the Abl kinase. Surprisingly, transient coexpression of Abl kinase with either dAbi or hAbi-2 proteins in both Drosophila S2 cells and mammalian COS cells leads to activation of Abl kinase that is dependent upon the presence of an intact Abi SH3 domain (Juang, 1999).
One model to explain how dAbi and hAbi-2 might activate Abl kinase would be that interaction of Abi with Abl generates a conformational change in Abl that results in exposure of its catalytic domain. Data to support this model comes from a recent report on site directed mutations in the Abl catalytic domain, SH3 domain and linker region between the SH2 and catalytic domain (SH2-CD). Mutations in all three domains were found to activate c-Abl kinase implicating intramolecular inhibitory interactions of the Abl SH3 domain with the catalytic domain and with the SH2-CD linker region. In addition, mutation of the SH2-CD linker region caused a conformational change in the protein resulting in altered and increased protease sensitivity of the mutated protein compared with c-Abl (Barila, 1998). Such a model could also reconcile the conflicting observations that dAbi and hAbi-2 activate Abl kinase activity yet Abi proteins are reported to suppress Abl transforming activity since binding of Abi to Abl may also have a role in regulating other biological activities of Abl such as Abl transforming activity (Dai, 1995; Shi, 1995; Juang, 1999 and references therein).
Although the activation of Abl kinase by dAbi implicates dAbi as an upstream regulator of Abl, Abl also has effects on dAbi. Phosphorylation of dAbi leads to a significant increase in the steady state level of dAbi protein. The rapid turnover of dAbi in S2 cells suggests that a potential mechanism for the increase in dAbi level is through a phosphorylation-dependent increase in dAbi protein stability. Although dAbi may have other binding partners than Abl, one possible effect of the increased dAbi protein levels would be to provide an amplified or sustained activation of the Abl kinase. In this role, an increase in dAbi level would result in dAbi binding to Abl, activation of Abl kinase activity, increased phosphorylation of Abl substrates, including dAbi, and an additional increase in dAbi steady state levels that would maintain activation of Abl kinase. Clearly some additional mechanism would be needed to terminate this hypothetical positive regulatory loop (Juang, 1999).
If dAbi is a key regulator of Abl kinase in vivo, it will be important to understand the regulation of dAbi protein turnover. Proteolysis is critical for controlling protein concentrations in other key processes, such as signal cascades and the cell cycle. Reversible protein phosphorylation has also been linked to the regulation of protein degradation. Phosphorylation of proteins, such as the cyclins, leads to ubiquitination and degradation of proteins. However, in other proteins, e.g. c-jun, phosphorylation results in a reduction in ubiquitination and stabilization. Analyses of dAbi and hAbi-2/ArgBP1 sequences have revealed PEST sequences, regions rich in proline (P), glutamic acid (E), serine (S) and threonine (T). Such sequences have been suggested to target rapid intracellular degradation of proteins. Many PEST-containing proteins are important regulatory molecules, such as components of signal transduction pathways, homeotic proteins, and key enzymes. Wang (1996) has reported that a human Abi isoform, ArgBP1A, with three PEST regions, has a consistently lower expression level than another isoform, ArgBP1B, with only two PEST regions, consistent with a role of the PEST motifs in Abi protein stability (Juang, 1999).
Regulation of protein stability is thought to be important in how cells respond to DNA damage. In response to DNA damage, p53 protein turnover is reduced and its level rises. The increased p53 levels are important to cause G1 arrest and provide cells an opportunity to repair the DNA damage, or to prevent further proliferation of the damaged cell by causing apoptosis. Regulation of c-Abl has also been implicated in the DNA damage-induced apoptosis. In response to DNA-damaging agents, c-Abl protein tyrosine kinase down-regulates cdk2 and causes G1 arrest by a p53-dependent mechanism and may also be involved in a p53-independent induction of apoptosis. In response to DNA damage, the ataxia telangiectasis mutant (ATM) protein is reported to phosphorylate and activate c-Abl. It will be interesting to determine whether DNA damage also alters the protein stability of Abi, as this would provide an additional mechanism for activating Abl kinase activity in response to DNA damage (Juang, 1999).
The Abl kinase has been implicated in cancer, regulation of the cell cycle, regulation of cell adhesion and normal developmental processes in Drosophila and mammalian systems. Further study of Abi proteins may provide new insights into how Abl kinase activity is regulated in one or more of these processes. The identification of a dAbi will also permit genetic analysis of Abi in normal development (Juang, 1999).
Drosophila Roundabout (Robo) is the founding member of a conserved family of repulsive axon guidance receptors that respond to secreted Slit proteins. Little is known about the signaling mechanisms that function downstream of Robo to mediate repulsion. Genetic and biochemical evidence is presented that the Abelson (Abl) tyrosine kinase and its substrate Enabled (Ena) play direct and opposing roles in Robo signal transduction. Genetic interactions support a model in which Abl functions to antagonize Robo signaling, while Ena is required in part for Robo's repulsive output. Both Abl and Ena can directly bind to Robo's cytoplasmic domain. A mutant form of Robo that interferes with Ena binding is partially impaired in Robo function, while a mutation in a conserved cytoplasmic tyrosine that can be phosphorylated by Abl generates a hyperactive Robo receptor (Bashaw, 2000).
Abl and Ena are complementary components of the signaling machinery downstream of the Robo repulsive axon guidance receptor. Genetic interactions indicate that loss of ena function partially disrupts Slit- and Robo-mediated repulsion from the midline. Limiting or removing ena function enhances partial loss-of-function robo phenotypes and suppresses robo gain-of-function phenotypes. In contrast, reduction of abl has the opposite consequence, suppressing the effects of a partial loss of robo function, while panneural overexpression of Abl antagonizes Robo function, leading to a phenotype resembling that of robo mutants (Bashaw, 2000).
Both Abl and Ena bind directly to Robo's cytoplasmic domain in vitro and Robo can act as a substrate for Abl kinase activity in vitro and in cell culture. Robo and Ena also show in vivo physical interactions. Furthermore, cytoplasmic domain mutants that reduce Ena binding to Robo result in impaired ability to rescue robo loss of function, while a Y-F mutation in a conserved tyrosine that can be phosphorylated by Abl in vitro has the opposite consequence, generating a hyperactive Robo receptor. These genetic and biochemical data support a model in which Abl and Ena play direct and opposing roles in the transmission of Robo's repulsive signal (Bashaw, 2000).
The implication of Ena in repulsive axon guidance is somewhat surprising in light of the previous results from the pathogen Listeria monocytogenes indicating that Mena is required for Listeria's actin-polymerization dependent motility. The Listeria data, together with the in vitro effects on actin of the Ena/VASP proteins has frequently been interpreted to suggest that Ena/VASP proteins function to promote actin polymerization, thereby promoting motility. On the contrary, the results presented here indicate that Ena is partially required for axon repulsion from the midline. These data suggest that Ena may have the opposite function, namely, to inhibit forward growth cone motility at sites where Robo encounters Slit (Bashaw, 2000).
In a companion paper (Bear, 2000), an independent study in mammalian cell culture has reached a similar conclusion. By expressing a multimerized EVH1 domain binding site attached to specific subcellular localization sequences, Ena/VASP family members can be efficiently targeted to different areas of cultured fibroblasts. This system has allowed a direct examination of the role of Ena/VASP proteins in cell motility. Surprisingly, when Ena/VASP proteins are directed away from the cell membrane, using a mitochondrial targeting sequence, the cells actually migrate more quickly. Conversely, targeting Ena/VASP proteins to the membrane, or overexpressing Mena, leads to a dose-dependent decrease in the rate of cell migration. A major conclusion of this study is that Ena/VASP proteins function in part to decrease the rate of whole cell motility. Whether Ena/VASP proteins achieve the observed in vivo effects on whole cell and growth cone motility by stimulating or inhibiting actin polymerization awaits future investigation (Bashaw, 2000).
While the dosage-sensitive genetic interactions between ena and robo support a role for Ena in midline repulsion, Ena clearly can not explain all of Robo's repulsive output. Indeed, although mild midline crossing defects are observed in ena mutants, on the whole, Robo-mediated repulsion works fairly well in the absence of Ena. In this light, it is perhaps not surprising that the Robo DeltaCC2 mutant receptor (in which the Ena binding site is deleted) still provides some repulsive activity and can partially rescue robo loss-of-function mutants. These results indicate that there must be other proteins that function downstream of Robo to mediate repulsion. One would predict that simultaneously removing ena and the as yet unknown additional factors would reveal stronger disruptions of midline repulsion (Bashaw, 2000).
Thus, Ena is only part of what must be a more complex repulsive output from Robo. Ena helps strengthen the output (perhaps by locally putting the break on the actin-based motility machinery), but is only part of the output. In this light, it is interesting to note that Robo2 also binds Slit and mediates repulsion (albeit apparently more weakly than Robo), but Robo2 does not have the Ena binding site and does not bind Ena (J. Simpson, personal communication to Bashaw, 2000).
An important question for future studies concerns whether Ena is always docked on Robo, or alternatively, whether Slit binding to Robo leads to the recruitment of Ena to Robo's cytoplasmic domain. From what is known about other receptor systems, this second alternative seems more likely, but it remains an open question and needs to be directly tested (Bashaw, 2000).
Genetic analysis shows that Abl antagonizes Robo-mediated repulsion. The two most likely possibilities are that Abl functions to antagonize this pathway by phosphorylating Robo or by phosphorylating Ena. Three results argue in favor of a direct interaction with Robo. (1) Certain kinds of dose-dependent genetic interactions between abl and robo are observed that are not observed between abl and ena, suggesting that the Abl and Robo proteins might directly interact. (2) Biochemical experiments have shown that Abl can directly phosphorylate Robo's cytoplasmic domain at one or more tyrosine residues. (3) A Y-F mutation in a conserved tyrosine that can be phosphorylated by Abl in vitro generates a hyperactive Robo receptor. Taken together, these genetic and biochemical data suggest that it is the dephosphorylated form of Robo that is most active (Bashaw, 2000).
How might Abl normally regulate the output of Robo signaling? Abl-mediated phosphorylation might normally modulate the output of Robo signaling. Alternatively, this phosphorylation might participate more directly in the ligand-gated signal. It is interesting to speculate that it is the binding of Robo to its ligand Slit that triggers dephosphorylation, and that this in turn activates the repulsive response (Bashaw, 2000).
The CNS-specific receptor protein tyrosine phosphatases (RPTPs) RPTP10D and 69D are candidates to be additional factors that contribute to Robo repulsion. Simultaneous removal of these two RPTPs results in substantial ectopic midline crossing, and the double mutant shows dose-sensitive genetic interactions with slit. Whether these two phosphatases interact directly with Robo and whether their phosphatase activity is required for their observed roles in repulsion await future investigation (Bashaw, 2000 and references therein).
In the model presented above, it is attractive to speculate that these two RPTPs function in opposition to the Abl kinase activity by directly dephosphorylating Robo upon Robo's interaction with Slit. Interestingly, the other two Robo family members in Drosophila (Robo2 and Robo3; J. Simpson, personal communication to Bashaw, 2000) share the phosphorylation sites in Robo that are phosphorylated by Abl in vitro. In addition, genetic interactions are observed between the RPTPs and Robo2. Together these observations suggest that perhaps a common mechanism is employed to regulate the signaling output of the three Robo receptors. It will be of interest to determine the in vivo significance of the conserved tyrosine phosphorylation sites in the three Robo receptors. The future elucidation of the events set in motion by ligand binding will require the development of cell culture systems that will allow analysis of the phosphorylation state and cytoplasmic domain associations of the Robo receptors before and after Slit stimulation (Bashaw, 2000).
In addition to their function during Robo signaling shown here, it is clear that both Abl and Ena function in multiple guidance signaling pathways, and thus that they are not committed to repulsion downstream of Robo. In the nematode C. elegans, ena acts as a suppressor of the axon migration defects associated with ectopic expression of the UNC5 repulsive Netrin receptor. This raises the possibility that ena functions downstream of diverse repulsive guidance receptors. In Drosophila, during motor axon pathfinding, ena and abl play roles in ISNb choice point control. Overexpression of abl or loss of ena generates an ISNb 'bypass' phenotype, where the ISNb fails to defasciculate and branch off at the appropriate location to enter its muscle target region. This phenotype is also observed in mutations in Dlar, the gene encoding a receptor protein tyrosine phosphatase (RPTP). Mutations in all three of these genes (ena, abl, and Dlar) give rise to only partially penetrant ISNb guidance phenotypes and appear to modulate guidance decisions at this choice point. At the midline, mutations in the genes encoding the ligand (Slit) and a key receptor (Robo) have strong and highly penetrant midline guidance phenotypes. In contrast, mutations in the genes encoding Ena, Abl, and RPTP10D and RPTP69D on their own have weaker and less penetrant phenotypes. This is consistent with Abl and the RPTPs modulating Robo receptor output, and Ena mediating only part of Robo output (Bashaw, 2000 and references therein).
If this same logic is applied to the motor axon ISNb choice point, then it is likely that some of the key components are still missing. At present, the only gene with a nearly 100% penetrant bypass phenotype at this choice point is sidestep. Side is an Ig superfamily transmembrane protein that is expressed on muscle surfaces and appears to function as an attractive ligand for motor axons. The Side receptor is not known. Whether the key receptor is the Side receptor or not, it is likely that the major growth cone receptor for the ISNb choice point has not yet been identified (Bashaw, 2000 and references therein).
In this context, it is tempting to speculate, by analogy with the proposed model for Robo signaling, that at the ISNb motor axon choice point, DLAR and Abl play complementary roles in modulating the output activity of the hypothetical guidance receptor, while Ena functions as part of the receptor output. In this way, the two guidance decisions -- to cross or not to cross the midline, and to fasciculate or defasciculate from other motor axons -- use different signals on the outside of the growth cone, but similar signaling and regulatory mechanisms on the inside. It is suggested that once the signal crosses the membrane, in both cases the output is regulated in opposing directions by Abl vs. one or more RPTPs, and that the output is partially mediated by Ena. This model provides a unifying way of viewing signal transduction during these two different guidance decisions. It will be interesting in the future to see to what degree this model holds up in terms of both the role of phosphorylation in modulating receptor output, and the role of Ena in mediating part of repulsive signaling (Bashaw, 2000).
Drosophila capulet (capt), a homolog of the adenylyl cyclase-associated protein that binds and regulates actin in yeast, associates with Abl in Drosophila cells, suggesting a functional relationship in vivo. A robust and specific genetic interaction is found between between capt and Abl at the midline choice point where the growth cone repellent Slit functions to restrict axon crossing. Genetic interactions between capt and slit support a model where Capt and Abl collaborate as part of the repellent response. Further support for this model is provided by genetic interactions that both capt and Abl display with multiple members of the Roundabout receptor family. These studies identify Capulet as part of an emerging pathway linking guidance signals to regulation of cytoskeletal dynamics and suggest that the Abl pathway mediates signals downstream of multiple Roundabout receptors (Wills, 2002).
Previous studies in Drosophila have shown that although Abl, Ena, and Profilin proteins are expressed broadly during embryogenesis, at later embryonic stages they accumulate at highest levels within the developing nervous system. Capt is abundantly expressed in early stage embryos (e.g., stage 4), consistent with its documented role in oogenesis. In addition to expression in mesoderm and developing gut epithelia, Capt is abundant in the ventral nerve cord (VNC) at stages 12 and 13 when axon pathways are pioneered within the CNS. At late stages (stages 16 and 17), when the last axon pathways are maturing and synaptogenesis is beginning, Capt preferentially accumulates within the VNC. Therefore, Capt, Abl, Ena, and Profilin are coexpressed in neurons at stages important for axonal development (Wills, 2002).
Capt and Abl are able to associate in a physiological setting. Using monoclonal antibodies (mAbs) specific to Abl, it is possible to immunoprecipitate (IP) endogenous Abl from Drosophila S2 cell lysates. When IPs of Abl were analyzed by SDS-PAGE and subsequent Western blot with anti-Capt antisera, endogenous Capt was detected as a protein that coprecipitates with Abl. For further confirmation of Capt protein association, S2 cells were transfected with a cDNA construct encoding full-length capt with a hemoagglutinin (HA)-epitope tag. Anti-HA antibody Western blots of Abl IPs detected a protein of the molecular weight expected for the tagged version of Capt (Wills, 2002).
Genetic experiments suggest that Abl interacts with a number of actin regulatory proteins to control cytoskeletal assembly. Given the functional redundancy observed between CAP and Profilin in yeast, it was thought that Capt and Profilin might participate in some form of protein complex regulated by the Abl kinase. S2 cells were transfected with full-length Drosophila Abl (dAbl), Drosophila Src64 (dSrc), or the truncated mammalian v-Abl and then Capt immunoprecipitations were assayed with anti-Profilin (Chic) and anti-actin antibodies. No significant binding of Capt and Profilin were seen in cells transfected with dSrc or v-Abl or in untransfected controls where endogenous dAbl is expressed at very low levels. However, an association of Capt with Profilin and with actin was observed when dAbl was elevated, suggesting a model where Abl, Capt, and Profilin function together in a cytoskeletal protein complex (Wills, 2002).
The expression and interactions of Capt protein raised the question of whether Capt contributes to the function of the Abl pathway during nervous system development. However, examination of many independent capt allelic combinations that remove zygotic expression without affecting other genes nearby revealed no defects in the embryonic CNS. This is attributed to the large maternal supply of Capt protein visible in the early embryo. Unfortunately, like Profilin null mutations (chickadee), capulet null alleles completely block oogenesis, preventing the use of germline mosaics for the study of zygotic phenotypes in the absence of maternal expression. However, because strong zygotic phenotypes can be induced when mutations in various Abl pathway components are combined with mutations in Abl (e.g., disabled), it was reasoned that zygotic functions of capt might be revealed through genetic interactions (Wills, 2002).
Among the strongest genetic interactions are synthetic phenotypes that arise in transheterozygotes, which lack only one copy of each interacting locus. Heterozygotes that lack one copy of capt or Abl alone show no detectable CNS phenotypes when compared to wild-type strains. However, combination of one capt and one Abl allele results in a distinct axon pathfinding defect (Wills, 2002).
Axons in the Drosophila embryonic CNS are organized into two major groups: longitudinal pathways that extend along the anterior-posterior axis and commissural pathways that carry contralateral projections across the midline. The midline, composed of specialized glial cells, acts as an organizing center that provides secreted growth cone attractants (Netrins) to build commissural pathways and a secreted repellent (Slit) to prevent inappropriate midline crossing. Subsets of longitudinal axons that depend on Slit to maintain their ipsilateral trajectories can be visualized specifically at late embryonic stages (stage 17) with the anti-Fasciclin II (FasII) antibody mAb 1D4; these FasII-positive axons never cross the midline in wild-type embryos (Wills, 2002).
In contrast to wild-type, capt-Abl transheterozygotes display consistent axon guidance defects at the CNS midline. In these double mutants, ipsilateral axon fascicles now ectopically cross, primarily from the most dorsal-medial MP1 pathway. An allelic series of this capt-Abl synthetic phenotype is seen across many different transallelic combinations, showing that the effect is independent of genetic background. No gross defects in the number or fates of postmitotic neurons were detected in any capt-Abl mutants. Although temporal delays were sometimes observed, capt-Abl transheterozygotes did not show any lasting defects in embryonic motor axon pathways (Wills, 2002).
To test whether the midline axon guidance function for capulet is dependent on Capt expression in postmitotic neurons, a wild-type capt transgene was expressed under control of P[elav-GAL4] in a strong capt-Abl background; a 15-fold rescue of the capt-Abl phenotype was observed. Interestingly, a parallel rescue experiment using an N-terminal deletion removing the putative Capt adenylyl cyclase-associated domain provides only a 2.7-fold rescue under the same conditions, despite the fact that the same transgene fully rescues captacuE636 to viability. Thus, capulet and Abl cooperate specifically during midline axon guidance (Wills, 2002).
The failure of the midline gatekeeper function in capt-Abl transheterozygote embryos suggested that Capt might function in the repellent pathway downstream of Slit. To test this genetically, transheterozygotes lacking one allele of capt and one allele of slit were examined. These mutants show a significant increase in the number of midline crossing errors compared to controls. This genetic interaction is seen consistently with multiple alleles of capt. Thus, capt and slit cooperate during midline guidance (Wills, 2002).
To further test the model that capt acts in the repellent pathway, the system of receptors was examined. However, examination of single gene mutations might not be sufficient. This is because the response to Slit is mediated by multiple receptors: Robo, Robo2, and Robo3. Indeed, capt transheterozygotes lacking single alleles in robo, robo2, or robo3 alone show little if any midline phenotype. Yet, when capt alleles are combined with double mutations lacking one copy of robo and robo2 simultaneously, a phenotype almost 2-fold greater than that seen in the robo,robo2 heterozygous embryos is observed. Interestingly, capt/+ does not enhance the phenotype of robo,robo3 heterozygotes, which is already quite strong (Wills, 2002).
As capt activity is further reduced, the interaction with robo2 gets stronger; mutants lacking two copies of capt and one copy each of robo2 and robo3 display penetrant midline phenotypes. Since these allelic combinations are the most severe, they were used for more detailed phenotypic analysis. For example, since the repulsion of growth cones at the midline is dependent on the presence of the midline glia, which secrete the Slit repellent, the midline glia in these mutants were examined with anti-Wrapper antibody, which specifically stains the surface of these glial cells. Midline glia are present in capt-robo2,robo3 mutants, even where axons inappropriately crossed the midline. The first axons in the MP1 fascicle were examined just as they pioneer the ipsilateral pathway early in CNS development. At stage 12, the posterior corner cell (pCC) extends its axon along an anterior trajectory parallel to the midline in order to pioneer the most medial Fasciclin II-positive (MP1) pathway. In capt-robo2,robo3 mutants, pCC axons were sometimes found that had turned toward and crossed the midline at this early stage. This phenotype is similar to that seen in robo alleles (Wills, 2002).
Previous studies of Abl function during midline guidance led to a model where Abl acts to antagonize Robo signaling. However, analysis of capt-Abl transheterozygotes suggests that Abl might play a dual role and also be required for restriction of midline crossing. Consistent with this prediction, examination of several Abl homozygotes reveals an allelic series of midline crossing phenotypes identical to those seen in capt-Abl transheterozygotes. Expression of a wild-type Abl transgene under its endogenous promotor in a strong mutant background rescues the midline crossing phenotype, as does expression of Abl specifically in neurons; however, a kinase-dead transgene was unable to rescue the defect. Like other aspects of Abl function, the midline crossing defects in Abl mutants can be suppressed by dose reduction of its substrate protein Ena or by loss of the receptor protein tyrosine phosphatase Dlar. These observations demonstrate that Abl is required for inhibiting the passage of ipsilateral axons across the midline and suggest that the role of Abl is more complex than previously appreciated (Wills, 2002).
Since analysis of Abl loss-of-function would predict cooperation between Abl and other genes in the repellent pathway, genetic interactions in embryos transheterozygous for Abl and either slit or combinations of mutations in different roundabout genes (ie. slit/+;Abl/+ or robo,robo2/+,+;Abl/+) were assayed. Surprisingly, these embryos displayed striking midline phenotypes far stronger than control genotypes. For example, slit2/+;Abl2/+ transheterozygotes show a 24-fold enhancement of the slit2/+ phenotype. This experiment strongly supports the model that Abl acts positively in the Slit pathway, consistent with the phenotypes of Abl homozygotes and of all the capulet genetic interactions observed (Wills, 2002).
The network of genetic interactions observed suggests that the Abl pathway is involved in signaling downstream of multiple Robo-family receptors. However, previous studies have shown Abl binding to the Robo cytoplasmic domain in vitro is dependent on a peptide motif (CC3) that is not present in Robo2 or Robo3. It was necessary to have an in vivo test for Abl-Robo interactions to explore this issue. Since Abl appears to act in both positive and negative capacities at the embryonic midline, an alternative genetic assay was used to evaluate Abl interaction with the robo gene family. When wild-type Abl is overexpressed in the developing compound eye, under the control of a synthetic glass promotor (GMR-GAL4), a mild rough-eye phenotype was observed. Thus, this Abl phenotype was tested for interactions with various UAS-Robo transgenes (Wills, 2002).
As predicted from loss-of-function analysis, while expression of wild-type Robo alone has little, if any, effect on retinal patterning, the combination of Abl and Robo causes a striking increase in the severity of the Abl gain-of-function eye phenotype. Thus, Robo serves as an enhancer of Abl activity in this kinase-dependent assay. This is also true of Robo2 and of Robo3. These data support the hypothesis that all Robo receptors can engage the Abl signaling pathway. So, is this in vivo interaction dependent on the Robo domains previously shown to recruit Abl and Ena proteins? Interestingly, neither deletion of CC2 nor deletion of CC3 was found to attenuate the Abl-Robo interaction. A UAS-robo transgene lacking the motif CC1 did show a reduction in eye phenotype when combined with UAS-Abl, but the difference was slight (Wills, 2002).
To confirm that Abl can interact with Robo in a CC3 domain-independent fashion during axon guidance, embryos that overexpress Abl and either wild-type Robo(+) or mutant Robo(DeltaCC3) were examined in postmitotic neurons. Abl gain-of-function alone generates two axon guidance phenotypes: (1) ISNb motor axon bypass of ventral target muscles and (2) ectopic midline crossing. Interestingly, coexpression of Abl and either Robo(+) or Robo(DeltaCC3) dramatically enhances the ISNb axon phenotype; however, there was no effect on midline crossing in any of these genotypes. Thus, in vivo, Abl is capable of a functional interaction with all three Robo receptors via some novel mechanism. However, the midline guidance system is specifically refractory to a simultaneous increase in Abl and Robo activities, perhaps due to the dual role of Abl in this context (Wills, 2002).
This study provides compelling evidence that a member of the adenylyl cyclase-associated protein (CAP) family plays a role in the accurate navigation of developing axons. Phenotypic analysis of double mutant embryos demonstrates that Capt cooperates with Abl, Slit, and multiple Roundabout receptors to prevent the inappropriate traffic of axons across the midline choice point. Consistent with published data on the relative contribution of Robo2 and Robo3 to midline repulsion, it has been found that capt and Abl show stronger interactions with robo,robo2 double mutants; however, Abl does appear to interact with all three receptors. The genetic and biochemical interactions observed suggest both that Capt functions directly in the Abl pathway and that this cytoskeletal regulatory pathway is involved in the repellent response to Slit (Wills, 2002).
Detailed studies of the prototypical growth cone repellent CollapsinI/Semaphorin3A have shown that the repellent response involves a collapse of the leading edge structures supported by actin cytoskeleton. Similar results have been seen for members of the Ephrin and Slit protein families. The fact that repellents promote a net disassembly of actin polymer arrays favors the simple model that repellent signaling antagonizes the actin assembly process (Wills, 2002).
Studies of CAP homologs from yeast, Dictyostelium, mouse, pig, and human suggest that the C-terminal actin binding domain acts to sequester monomers to prevent actin polymerization. More recent studies also suggest that human CAP promotes actin disassembly and monomer recycling through interactions with the actin-depolymerizing factor Cofilin. Consistent with an inhibitory role for CAP-family members, studies of epithelial development and oogenesis in Drosophila demonstrate that Capt functions to suppress the hyperassembly of actin microfilaments. Interestingly, a similar function has been ascribed to Abl and Arg during neurogenesis in the mouse. Thus, a model is favored where Abl helps to recruit and regulate CAP activity to inhibit net actin assembly downstream of Robo family receptors (Wills, 2002).
Previous data supported a simple model where Robo recruits Abl and Ena as components in the repellent pathway. In this model, Ena acts as an effector molecule to link Robo to actin assembly and Abl acts purely to antagonize and/or downregulate Robo. While this study confirms that Abl gain-of-function creates ectopic midline crossing, the additional discovery that Capt and Abl cooperate to support the repellent response and that Abl loss-of-function generates ectopic midline crossing suggests that new models are necessary (Wills, 2002).
The fact that Abl is required for midline restriction suggests that Abl plays a dual role in the Robo pathway. There are different models to explain this. As a key enzymatic component in the signaling pathway, Abl may support repellent signaling (by recruiting the necessary actin binding proteins) and also feed back on the receptor (by downregulating through phosphorylation) to adjust the sensitivity of the pathway. This model is attractive because it may explain how growth cones can adapt to different regions within a gradient of Slit. In order for a growth cone to perceive an extracellular gradient (attractive or repellent) over an extended distance, the dynamic range of the response must be continually adjusted. If the receptor system becomes saturated at any point in the gradient, the growth cone will be blind to the extracellular asymmetry at higher concentrations. Conversely, if receptor output is too low, then the signaling differential across the leading edge may be too small to detect the gradient. It has therefore been postulated that gradient guidance will require some form of adaptation to keep the signaling threshold within the appropriate dynamic range as the growth cone moves toward or away from the source. If Abl is part of the repellent response, it would also be an effective source of feedback to help match receptor sensitivity to the gradient conditions. A similar role has been postulated for MAP kinase in the Netrin signaling pathway (Wills, 2002).
The question of exactly how Abl and its signaling partners interface with the Robo receptor family is still unclear. The biochemical data suggest that Abl, Capt, and Profilin may form a large protein complex. However, the genetic interactions between Abl and robo indicate that the CC3 motif is not necessary for a functional link between Abl and Robo. This makes sense because Abl and capulet also interact with robo2, a receptor that lacks both CC2 and CC3 sequences. It is interesting that deletion of motif CC1, which is conserved in all the Drosophila Robo family members, causes a slight attenuation of the robo-Abl interaction in the assay used in this study. CC1 is also the Robo sequence phosphorylated by Abl in vitro (Wills, 2002).
The emerging picture of axon guidance signaling pathways is highly complex. While this may be required to coordinate the many cell biological events that underlie directional specificity during cell motility, it is also possible that this property provides greater opportunity for signal integration. In this light, the potential link between Capulet and adenyyl-cyclase is intriguing. Cyclic nucleotides (cAMP and cGMP) have potent modulatory effects on axon guidance responses in vitro . Although the rescue experiments show that the N-terminal region of Capulet equivalent to the cyclase-interacting domain of other CAP family proteins is not absolutely required for axon guidance function, the reduced rescue activity of this mutant is consistent with cyclase playing a modulatory role in the repellent pathway (Wills, 2002).
The attractive Netrin receptor Frazzled (Fra), and the signaling molecules Abelson tyrosine kinase (Abl), the guanine nucleotide-exchange factor Trio, and the Abl substrate Enabled (Ena), all regulate axon pathfinding at the Drosophila embryonic CNS midline. Genetic and/or physical interactions between Fra and these effector molecules suggest that they act in concert to guide axons across the midline. Mutations in Abl and trio dominantly enhance fra and Netrin mutant CNS phenotypes, and fra;Abl and fra;trio double mutants display a dramatic loss of axons in a majority of commissures. Conversely, heterozygosity for ena reduces the severity of the CNS phenotype in fra, Netrin and trio,Abl mutants. Consistent with an in vivo role for these molecules as effectors of Fra signaling, heterozygosity for Abl, trio or ena reduces the number of axons that inappropriately cross the midline in embryos expressing the chimeric Robo-Fra receptor. Fra interacts physically with Abl and Trio in GST-pulldown assays and in co-immunoprecipitation experiments. In addition, tyrosine phosphorylation of Trio and Fra is elevated in S2 cells when Abl levels are increased. Together, these data suggest that Abl, Trio, Ena and Fra are integrated into a complex signaling network that regulates axon guidance at the CNS midline (Forsthoefel, 2005).
The interactions of Abl with Fra are intriguing, since they suggest that in Drosophila, as in other organisms, this evolutionarily conserved guidance receptor is regulated by tyrosine phosphorylation, and also that Fra may regulate Abl substrates. Other studies have demonstrated Netrin-dependent tyrosine phosphorylation of DCC, Netrin/DCC-dependent activation of the tyrosine kinases FAK, Src and Fyn, and the requirement of DCC tyrosine phosphorylation for Netrin-dependent Rac1 activation and growth cone turning. Interestingly, the tyrosine residue in DCC identified as the principal target of Fyn/Src kinases is not conserved in Drosophila Fra or C. elegans UNC-40, suggesting that the precise mechanisms by which Fra/DCC/UNC-40 signaling is regulated by tyrosine kinases may differ between organisms. Tyrosine phosphorylation of UNC-40 has also been observed, and although the kinase(s) responsible has not been identified, genetic interactions suggest that UNC-40 signaling is regulated by the RPTP CLR-1, supporting the idea that regulation of tyrosine phosphorylation is a consequence of UNC-6/Netrin signaling in C. elegans as well. In this study, more robust tyrosine phosphorylation of Fra was observed in cells with pervanadate stimulation than with Abl overexpression alone, raising the possibility that additional kinase(s) may function during Fra signaling. Further investigation will be needed to address this issue and to determine how Abl-mediated phosphorylation of Fra modulates commissural growth cone guidance (Forsthoefel, 2005).
Abl is thought to control actin dynamics in part through its ability to regulate other proteins through tyrosine phosphorylation. Thus, in addition to potential regulation of Fra, Fra may recruit Abl to regulate other Abl substrates. Abl interacts genetically with trio, and in this study, Trio was found to physically interact with Abl in vitro, and Trio tyrosine phosphorylation increases dramatically with co-expression of Abl. Phosphorylation of Trio may affect its activity, as observed for other GEFs. For example, Abl regulates phosphorylation and Rac-GEF activity of Sos1, and Lck, Fyn, Hck and Syk kinases tyrosine phosphorylate Vav GEF and stimulate its activity (Forsthoefel, 2005).
Trio physically interacts with Fra in vitro and in S2 cells, suggesting that Fra can recruit Trio directly. In addition, heterozygosity for trio dominantly modifies the Robo-Fra chimeric receptor phenotype, consistent with a positive role for Trio as a downstream effector of Fra signaling in vivo. As a Rac/Rho GEF, Trio may link Netrin-Fra signaling to the regulation of Rho-family GTPases in commissural axons. Rho-family GTPases have been rigorously studied with regard to their role in the regulation of cytoskeletal dynamics and axon guidance, outgrowth and branching. Although positive roles for GTPases in commissure formation in the Drosophila embryo have not been directly demonstrated, trio and GEF64C, a Rho GEF, interact genetically with fra leading to the dramatic disruption of commissures. Additionally, expression of constitutively active or dominantly negative isoforms of both Rac and Rho, as well as constitutively active Cdc42, causes axons to cross the CNS midline inappropriately. Recent studies have implicated Cdc42 and Rac1/CED-10 as effectors of DCC and UNC-40 signaling, but reaching an understanding of the biochemical mechanisms by which GTPases are regulated has been elusive. Future experiments must determine whether Netrin-Fra signaling modulates the GEF activity of Trio, and how this occurs (Forsthoefel, 2005).
Reducing the genetic dose of ena causes either more or fewer axons to cross the CNS midline, depending on the genetic background, suggesting that the role of Ena in the growth cone is complex. Heterozygosity for ena in embryos expressing the Robo-Fra chimeric receptor reduces the number of axon bundles that inappropriately cross the CNS midline, consistent with a role for Ena as a positive effector of Fra signaling. Ena/UNC-34 has been identified genetically as an effector of DCC/UNC-40 in C. elegans. In cultured mouse neurons, Ena/VASP proteins are required for Netrin-DCC-dependent filopodia formation, and Mena is phosphorylated at a PKA regulatory site in response to Netrin stimulation. In migrating fibroblasts, increasing Ena/VASP proteins at the leading edge leads to unstable lamellae and decreased motility; by contrast, increasing Ena/VASP levels at the leading edge in growth cones causes filopodia formation, possibly due to differences in the distribution of actin bundling or branching proteins. Although the role of Ena in actin reorganization in Drosophila has not been rigorously studied, Ena localizes to filopodia tips in cultured Drosophila cells, suggesting that the role of Ena in filopodia formation may be conserved (Forsthoefel, 2005).
No direct biochemical interaction was observed between Fra and Ena. However, Abl binds and phosphorylates Ena, and heterozygosity for both Abl and ena further suppresses the Robo-Fra phenotype, suggesting that Fra may recruit Abl to regulate filopodial extension through Ena. Alternatively, Fra may regulate Ena through other molecule(s), and the synergistic suppression of the Robo-Fra phenotype by Abl and ena is a result of the compromise of parallel pathway(s) regulated by Fra. It is important to note that the functional consequences of biochemical interactions between Abl and Ena are not yet understood. Therefore it will be of particular interest to determine whether Ena is tyrosine phosphorylated in response to Netrin-Fra signaling, and if Ena phosphorylation regulates its activity during filopodial extension (Forsthoefel, 2005).
In addition to suppressing the Robo-Fra chimeric receptor phenotype, mutations in ena also suppress the loss-of-commissure phenotype in fra, Netrin, trio and Abl mutant combinations. In Drosophila (as well as in C. elegans), Ena interacts genetically and biochemically with the repulsive receptor Robo, indicating that Ena may restrict axon crossing at the midline. Thus, the fact that mutations in ena dominantly suppress fra, Netrin, trio and Abl CNS phenotypes could simply reflect the compromise of a parallel, opposing signaling pathway. Consistent with this idea, some axons that cross the midline in ena heterozygous, trio,Abl homozygous embryos are Fas2 positive, indicating a partial reduction in repulsive signaling. However, ena also dominantly suppresses fra and Netrin commissural pathfinding defects, without causing longitudinal Fas2-positive axons to cross the midline. Reductions in Robo signaling therefore may not fully explain the ability of ena to suppress defects in fra, Netrin, Abl and trio mutants (Forsthoefel, 2005).
Based on the fact that mutations in ena suppress a number of Abl mutant phenotypes, it has been proposed that Abl antagonizes Ena function. In Abl mutant embryos, Ena and actin mislocalize during dorsal closure and cellularization, and apical microvilli are abnormally elongated, indicating that Abl regulates the localization of Ena. In migrating fibroblasts, increasing Ena/VASP levels at the leading edge results in long, unbranched actin filaments, unstable lamellae, and decreased motility due to increased antagonism of capping protein. Interestingly, mutations in the gene encoding Drosophila capping protein ß enhance CNS axon pathfinding defects in Abl mutants, including commissure formation. Therefore, if Fra and/or Abl regulate Ena localization in commissural axons, then in fra, Netrin or Abl mutants, Ena may be mislocalized in the growth cone, leading to inappropriate inhibition of capping protein and excessive F-actin filament elongation. Additionally, reducing regulation of Ena by Fra or Abl may also allow greater Ena regulation by Slit-Robo signaling. In either case, reducing the gene dose of ena in fra, Netrin and trio,Abl mutant embryos would partially relieve these effects, allowing axons to respond more efficiently to other cues and cross the midline, as was observed. Consistent with this idea, it was found that either increasing or decreasing Ena/VASP proteins at the leading edge impairs the elaboration of growth cone filopodia in response to Netrin-DCC signaling, suggesting that Ena/VASP levels must be tightly regulated in order for the growth cone to respond optimally to extracellular signals (Forsthoefel, 2005).
The role of Abl in the growth cone is also likely to be complex. The observations implicate Abl as an effector of attractive Fra signaling. In addition, tyrosine phosphorylation of Robo by Abl is thought to negatively regulate repulsive signaling by Robo. Paradoxically though, loss-of-function mutations in Abl, robo and slit interact genetically, resulting in inappropriate axon crossing at the midline, and indicating that Abl may also promote repulsion in longitudinally migrating growth cones. Obviously, much remains to be understood about the molecular basis for genetic interactions of Abl, particularly how Abl and its various substrates cooperate with different growth cone receptors to yield specific cytoskeletal outputs (Forsthoefel, 2005).
In summary, genetic and biochemical interactions indicate that Abl, Trio and Ena are integrated into a complex signaling network with Fra and the Netrins during commissure formation. These observations identify another receptor that acts through these effectors, and provide a framework for further investigation of signaling by this key, evolutionarily conserved guidance receptor (Forsthoefel, 2005).
Most aspects of cellular events are regulated by a series of protein phosphorylation and dephosphorylation processes. Abi (Abl interactor protein) functions as a substrate adaptor protein for Abl and a core member of the WAVE complex, relaying signals from Rac to Arp2/3 complex and regulating actin dynamics. It is known that the recruitment of Abi into the lamella promotes polymerization of actin, although how it does this is unclear. PTP61F, a Drosophila homolog of mammalian PTP1B, can reverse the Abl phosphorylation of Abi and colocalizes with Abi in Drosophila S2 cells. Abi can be translocalized from the cytosol to the cell membrane by either increasing Abl or reducing endogenous PTP61F. This reciprocal regulation of Abi phosphorylation is also involved in modulating Abi protein level, which is thought to affect the stability of the WAVE complex. Using mass spectrometry, several important tyrosine phosphorylation sites were identified in Abi. The translocalization and protein half-life of wild type (wt) and phosphomutant Abi and their abilities to restore the lamellipodia structure of the Abi-reduced cells were compared. The phosphomutant was found to have reduced ability to translocalize and to have a protein half-life shorter than that of wt Abi. Although the wt Abi could fully restore the lamellipodia structure, the phosphomutant could not. Together, these findings suggest that the reciprocal regulation of Abi phosphorylation by Abl and PTP61F may regulate the localization and stability of Abi and may regulate the formation of lamella (Huang, 2007).
Tyrosine phosphorylation/dephosphorylation is a common and important post-translational modification of key signaling proteins. Although Abi was originally identified as a kinase substrate of Abl, little is known about how phosphorylation contributes to its biological significance. This study demonstrates that the level of Abi tyrosine phosphorylation is balanced between phosphorylation by Abl and dephosphorylation by PTP61F. This conclusion is based on several lines of evidence. First, RNAi knockdown of PTP61F expression greatly elevates the tyrosine phosphorylation level of endogenous Abi in the Drosophila S2 cells. PTP61F was shown to be a major physiological phosphatase of Abi by substrate trapping, GST pulldown, and colocalization experiments. Second, RNAi experiments also suggested that Abl is the major kinase mediator of tyrosine phosphorylation of Abi. Finally and most importantly, Abl-mediated tyrosine phosphorylation of Abi was shown to be counteracted by the dephosphorylation of a wild-type but not the catalytically inactive PTP61F proteins. Taken together, these results indicate that the coordinated phosphorylating action of Abl and dephosphorylating action of PTP61F together coordinate to regulate the tyrosine phosphorylation level of Abi (Huang, 2007).
Because many adaptor proteins undergo protein relocalization in response to tyrosine phosphorylation and protein-protein interaction, whether the balance of tyrosine phosphorylation in Abi affected its localization subcellularly was investigated. In cells ectopically expressing Abl and Abi, both Abl and Abi proteins relocalize to the same cortex cell regions, where active actin polymerization occurs. The relocalization appears to be dependent, as least in part, on the kinase activity of Abl because the kinase inactive Abl would exhibit markedly reduced ability to relocalize Abi from a cytosolic punctate position to a plasma membrane position. This finding was further supported by additional experiments showing that a reduction in endogenous PTP61F could increase the relocalization of GFP-Abi from cytosol to the plasma membrane, and when Abi4YF phosphomutant protein was expressed, there was a decrease in this translocation activity. It is thought that the Abl/PTP61F-mediated phosphorylation/dephosphorylation of Abi is critical in regulating Abi functions by relocalizing Abi from the cytoplasm to the cell membrane. The importance of Abi phosphorylation is also evident in a study of Abi protein stability. Using overexpression or RNAi knockdown approach to control Abl or PTP61F protein levels, it was demonstrated that both protein level and activity of these two enzymes are essential in the control of Abi protein stability. Furthermore, it was shown that the protein turnover rate of Abi4YF is much faster than that of wt Abi, further supporting the idea that the tyrosine phosphorylation of Abi is critical in modulating its protein stability. Together, these results highlight the importance of the Abi phosphorylation/dephosphorylation in modulating its localization and protein stability (Huang, 2007).
Although the data indicate that Abl phosphorylation of Abi is critical in modulating Abi subcellular localization, the underlying mechanism behind this process remains elusive. Nevertheless, the Abl protein is known to contain a myristoyl group, a common module for anchoring a protein to a cell membrane. Moreover, recent structural studies, which found that removal of the myristoyl group activates c-Abl kinase activity, suggest that by binding myristoyl group to the kinase pocket of Abl, an inactive conformation may be promoted. Because Abi functions as a positive regulator of Abl kinase, the Abl-Abi interaction might induce a conformational change through which the buried region that bears an N-myristoylation signal is exposed. This would result in targeting the Abl·Abi complex to cell membrane. In fact, the data showed that the interaction of a kinase-defective Abl with Abi was also somewhat able to trigger the Abi relocalization from the cytosol to the cell membrane. It was also found that Abl phosphorylation of Abi in the complex further promoted the relocalization of Abi to cell membrane. One recent structural and biochemical study has shown that a phosphotyrosine ligand can further convert the Abl protein into a more 'open' conformation. It might be possible that the phosphotyrosine residues of Abi further opened the semi-unfolded position of myristoyl group in Abl, further promoting the relocalization of Abl·Abi complex (Huang, 2007).
Another intriguing issue is the unique subcellular localization of PTP61F. Immunocytochemistry experiments showed that most PTP61F colocalizes with Abi in the perinuclear region, presumably where the endoplasmic reticulum (ER) is situated, but not at the leading edge of the spreading cells, where Abi is preferentially localized. These finds regarding the subcellular localization of PTP61F in S2 cells is similar to those reported for its mammalian counterpart, PTP1B. PTP1B largely resides on the external face of the ER. Given the finding that PTP61F counteracts Abl phosphorylation of Abi, the spatial separation of PTP61F from Abi in the leading edge might suggest that the phosphorylation of Abi is less affected by PTP61F in this region and that Abi residing at the leading edge might exert a higher tyrosine phosphorylation signal than Abi in the perinuclear region. In fact, several groups have reported that a number of proteins localized to the leading edge of motile cells are tyrosine-phosphorylated. Nevertheless, the substrates of PTP targeting the cell periphery ultimately have to be dephosphorylated to terminate its role in signaling. To elucidate the mechanism underlying this process, three models have been proposed. First, the receptor tyrosine kinases, such as epidermal growth factor receptor and platelet-derived growth factor receptor, can be directed from plasma membrane to ER by vesicle trafficking for dephosphorylation by PTP1B. Second, the ER membrane-anchored PTP1B can be cleaved by the calcium-dependent neutral protease calpain to relocate its catalytic domain from membranes to the cytosol. Last, by reaching the ER network to the cell periphery, PTP1B at the local attachment site can interact with its substrates in the plasma membrane. These models together with the current findings suggest that there is a possible link between PTP61F and Abl signaling in the regulation of Abi phosphorylation at the leading edge of the plasma membrane (Huang, 2007).
Protein stability is also a critical factor for regulating Abi functions in actin dynamics. When Abi expression is lost in S2 cells, there are severe defects in the formation of lamella. However, Abi alone does not affect the formulation of lamella. Abi forms a tight complex with WAVE, Hem, and Sra-1 to regulate the formation of lamellipodia. Moreover, the lamellipodia formation is dependent on the integrity of a functioning WAVE macromolecular complex (WAVE·Abi·Hem·Sra-1), but the down-regulation of Abi expression impairs the stability of WAVE complex in both mammalian and Drosophila cells (Stradal, 2006). Thus, the protein stability of Abi is highly correlated with its regulation of the formation of lamella. A previous studies demonstrated that the interaction between Abl and Abi leads to an increased protein level of Abi (Juang, 1999). This study has extended this finding by showing that the impaired Abl-mediated phosphorylation of Abi results in protein instability of Abi in S2 cells. It was also demonstrated that the ectopic expression of a wild type, but not an inactive, PTP61F also dramatically diminishes the Abi protein stability. Thus, these results indicate that tyrosine phosphorylation/dephosphorylation by Abl/PTP61F is critical in controlling Abi protein turnover. Because it has been suggested that Abi undergoes an ubiquitin-mediated proteolysis (Dai, 1998), it might be possible that that the Abl phosphorylation of Abi represses the proteolysis degradation. One recent study has demonstrated that the Abl phosphorylation of c-Jun blocks the access of E3 ubiquitin ligase, Itch, to its binding epitope of c-Jun and thus prevents c-Jun from proteosome-mediated proteolysis (Huang, 2007).
Although tyrosine phosphorylation of Abi is important in stabilizing Abi protein level, the protein interaction between Abi and Abl might also attribute to Abi protein stability. This study showed that the coexpression of a kinase-defective c-Abl and Abi resulted in a 1.8-fold increase of Abi protein level. Moreover, this protective effect probably is a common feature of both the Abi·Abl and Abi·WAVE·Hem·Sra-1 complexes. Preliminary data showed that the ectopic coexpression of Abi, Hem, and WAVE could enhance Abi protein level and that the reduction of Hem or WAVE markedly decreased the endogenous Abi protein level, suggesting that a constitutive complex of Abi with WAVE, Hem, and Sra-1 could protect Abi from post-translational degradation. This finding is also comparable with what was documented in the scenario of WAVE/Scar stability (Huang, 2007).
Following this logic, it is reasonable to speculate that a sizeable fraction of the ectopically expressed Abi proteins would be short of match protein members to form the protein complex. Under this circumstance, the Abi would be more dependent on the phosphorylation regulation mechanism to modulate its protein stability. Supporting this view, it was shown that the protein stability of exogenous Abi was particularly sensitive to the phosphorylation regulation. Although component proteins of the WAVE/Scar complex are thought to form a constitutive multiprotein complex in cells, a recent study showed that a small population of WAVE2 exists as a monomeric form without forming a complex with other component proteins in A431 cells. In addition to WAVE2, Hem/Nap1 was also reported to regulate cell motility through a WAVE/Scar-independent pathway in Dictyostelium and human neutrophil. These studies suggest that the component proteins of WAVE/Scar complex may present as a 'noncomplex' form of protein in regulating cell motility. Accordingly, although exogenous Abi is relatively independent of the physiological state, it is more amenable to the study of protein stability (Huang, 2007).
Another interesting issue related to the Abi stabilization by Abl is linked to a possibility that the stabilized Abi protein might in return further enhance the Abl kinase activity and Abi phosphorylation, thus establishing a positive feedback loop between Abl activity and Abi phosphorylation/stability. Because Abl kinase activity is tightly regulated in cells, this positive feedback loop should be eventually interrupted by a third molecule. PTP61F might be such a suppressor and function as a brake on the overactivated Abl-Abi signaling pathway by dephosphorylating Abi and facilitating protein degradation (Huang, 2007).
Finally, this study investigated whether a phosphomutant of Abi, which is not phosphorylated by Abl, could disrupt the lamella formation in motile cells. The experiments suggest that the expression of Abi4YF was unable to rescue the lamella defects that were caused by treatment with Abi3'-UTR RNAi, supporting the idea that Abl might act positively in promoting Abi regulation of the lamella formation. Two recent reports have demonstrated that the mutation on a critical Abl phosphorylation residue of WAVE can disrupt its role in actin-dependent cytoskeleton remodeling. The data might suggest that Abl phosphorylation on Abi promotes actin polymerization. Therefore, it was asked whether Abl also phosphorylates all the other WAVE members and whether that would in turn modulate their roles in actin dynamics. Preliminary data showed that Abl, via the linkage of Abi, could tyrosine phosphorylate each protein member of the WAVE complex and that process was also counteracted by PTP61F (Huang, 2007).
Although additional experiments are required to determine the physiological significance of the phosphorylation/dephosphorylation of WAVE complex proteins, it is possible that the opposing actions of Abl and PTP61F in all of the other WAVE members might play a role in fine-tuning actin polymerization in cells. To resolve this possibility, it will be important to investigate whether Abl/PTP61F phosphorylation/dephosphorylation of the WAVE complex is occurred in a consecutive or synchronized manner in actin polymerization (Huang, 2007).
This work has demonstrated that Abi undergoes protein modification by Abl and PTP61F via reversible tyrosine phosphorylation/dephosphorylation. This process regulates its relocalization to the cell membrane and protein stability, and ultimately it regulates the formation of lamella in motile cells. Therefore, the balance between Abl kinase and PTP61F phosphatase in regulating tyrosine phosphorylation of Abi may mediate the function of WAVE complex in cell periphery. This study furthers understanding of how and where the reciprocal regulatory processes of an actin-associated protein function within the cells (Huang, 2007).
Although Abl functions in mature neurons, work to date has not addressed Abl's role on Cdk5 in neurodegeneration. β-amyloid (Aβ42) initiates Abl kinase activity and blockade of Abl kinase rescues both Drosophila and mammalian neuronal cells from cell death. Activated Abl kinase is necessary for the binding, activation, and translocalization of Cdk5 in Drosophila neuronal cells. Conversion of p35 into p25 is not observed in Aβ42-triggered Drosophila neurodegeneration, suggesting that Cdk5 activation and protein translocalization can be p25-independent. These genetic studies also showed that abl mutations repress Aβ42-induced Cdk5 activity and neurodegeneration in Drosophila eyes. Although Aβ42 induces conversion of p35 to p25 in mammalian cells, it does not sufficiently induce Cdk5 activation when c-Abl kinase activity is suppressed. Therefore, it is proposed that Abl and p35/p25 cooperate in promoting Cdk5-pY15, which deregulates Cdk5 activity and subcellular localization in Aβ42-triggered neurodegeneration (Lin, 2007).
Like Cdk5, cellular Abl functions in neural development and its kinase activity and subcellular localization are tightly regulated. This study shows that Abl appears to be essential for Aβ42-triggered Drosophila neurodegeneration both in vivo and in vitro. It is of interest in this regard that Abl may serve as a putative molecular target to stop the progress of neurodegeneration. Interestingly, the anti-leukemic agent Abl kinase inhibitor, STI571, has been shown to rescue the Aβ42-induced neurodegeneration in both Drosophila and mammalian cells. However, STI571 is probably not an ideal reagent for testing this idea in vivo because of its low penetration capability through the blood-brain barrier. Another previous link between Aβ42 and Abl inhibition by STI571 has been reported. Aβ42 production is reduced by STI571 in neuronal cultures and in guinea-pig brain. Therefore, it is reasonable to speculate that Abl kinases might affect amyloid signaling at various points including Aβ42 production (Lin, 2007).
One of the central regulators coupling tyrosine phosphorylation with cytoskeletal dynamics is the Abelson interactor (Abi). Its activity regulates WASP-/WAVE mediated F-actin formation and in addition modulates the activity of the Abelson tyrosine kinase (Abl). Drosophila Abi is capable of promoting bristle development in a wasp dependent fashion. This study reports that Drosophila Abi induces sensory organ development by modulating EGFR signaling. Expression of a membrane-tethered activated Abi protein (AbiMyr) leads to an increase in MAPK activity. Additionally, suppression of EGFR activity inhibits the induction of extra-sensory organs by AbiMyr, whereas co-expression of activated AbiMyr and EGFR dramatically enhances the neurogenic phenotype. In agreement with this observation Abi is able to associate with the EGFR in a common complex. Furthermore, Abi binds the Abl tyrosine kinase. A block of Abl kinase-activity reduces Abi protein stability and strongly abrogates ectopic sensory organ formation induced by AbiMyr. Concomitantly, changes were noted in tyrosine phosphorylation supporting previous reports that Abi protein stability is linked to tyrosine phosphorylation mediated by Abl (Stephan, 2008).
A variety of morphogenetic events during metazoan development such as cell fate determination, proliferation and differentiation require a highly regulated network of signaling pathways resulting in distinct cellular responses. Cell-surface receptors and tyrosine kinases often play an important role in the control of these processes. Among the growth factor receptors, the best understood example is the epidermal growth factor receptor (EGF receptor), which plays important roles in physiological and pathological processes. Unlike the vertebrate homologues the Drosophila genome contains only a single gene encoding a member of the EGF receptor family, called DER/EGFR. In Drosophila, the EGFR pathway is required at multiple times during fly development such as oogenesis, axis determination and sensory organ formation during embryonic and imaginal disc development (Stephan, 2008).
The emergence of Drosophila sensory organs depends on a selection process called lateral inhibition where only one cell is singled out of a group of equivalent proneural cells to become a sensory organ precursor (SOP). The Drosophila notum bears many mechano-sensory bristles, 22 large macrochaetes and about 200 smaller microchaetes. In both cases, bristle formation and spacing requires cell–cell communication mediated by lateral inhibition. The microchaetes on the notum are arranged in rows and are variable in number and position within the rows. Unlike the microchaetes, the large macrochaetes are not arranged in rows, their number is constant and they are formed from proneural clusters at fixed stereotypic positions (Stephan, 2008 and references therein).
Once determined, the SOP undergoes a series of stereotyped asymmetric cell divisions giving rise to the different cells comprising an individual sensory organ. In Drosophila, two major antagonistic signaling pathways regulate the formation of SOPs, the Notch and the EGFR signaling pathways. Notch is pivotal for the process of lateral inhibition which restricts the formation of neural cells. Alternatively, activation of the EGF receptor (EGFR) positively regulates SOP formation. Loss of EGFR function leads to a loss of sensory macrochaetes whereas gain of EGFR-function evokes the formation of additional macrochaetes by stimulating proneural gene autoregulation (Stephan, 2008).
Upon EGFR activation in Drosophila a membrane-localized protein complex is formed acting through the Ras-signaling. The activation of Ras results in the translocation of di-phosphorylated MAPK from the cell surface to the nucleus, where it leads to the expression of the proneural gene scute, which in turn promotes sensory organ development (Stephan, 2008).
This study shows that Abi regulates sensory organ development by modulating EGFR signaling. Following expression of an activated Abi (AbiMyr) in proneural clusters, an increase was noted in MAPK activity. Inhibition of the EGFR pathway suppresses the formation of ectopic sensory organs evoked by AbiMyr. Furthermore, co-expression of activated AbiMyr and EGFR dramatically enhances the induction of extra-sensory organs. This effect also depends on the presence of active Abl tyrosine kinase, which stabilizes and further stimulates Abi by tyrosine phosphorylation (Juang, 1999). The physical interaction of Abi and EGFR depends on the membrane recruitment whereas the ability of the complex to induce neural development depends on both the membrane localization and the C-terminal SH3 domain of Abi, which can bind Wasp as well as the Abl kinase. These data suggest that once Abi is recruited to the membrane, EGFR signaling becomes activated to promote SOP development (Stephan, 2008).
Thus, overexpression of the Abelson kinase and its activator Abi is sufficient to promote the formation of ectopic sensory organs. Previous work has shown a partial loss of sensory organs in adult flies expressing a double-stranded abi RNA (Bogdan, 2005). However, expression of double-stranded RNA can lead to off target effects and confirmation of the mutant phenotypes by genetic analysis is needed. To address this question abi null alleles were generated. However, animals lacking zygotic abi function die relatively late during development as early pupae and no defects can be noted in the SOP pattern. This suggests that either abi is not required for SOP development or that its function is obscured by maternal contributions. In support for this notion, residual Abi protein can be detected in homozygous abi mutant larvae. In addition as previously reported for the abl kinase gene, abi might be integrated into a complex genetic network that masks direct requirements. The role of Abl is only exposed by the simultaneous loss of interaction partners such as disabled or failed axon connections (Stephan, 2008).
The Abi proteins are versatile molecules comprised of multiple protein-protein interaction domains allowing the formation of different protein complexes. Abi does not only function as an activator of the Abelson kinase but in turn Abl positively regulates Abi stability through phosphorylation (Juang, 1999; Huang, 2007). Moreover Abi plays an important role in regulating F-actin dynamics by activating Wave and Wasp proteins, two main activators of Arp2/3-dependent actin polymerization (Stephan, 2008 and references therein).
How can alterations in actin dynamics modulate EGFR signaling? Previous studies have shown that cytoskeletal rearrangements are likely to induce the asymmetric distribution and clustering of the EGFR in neuronal stem cells to generate diverse CNS progenitor cells. Since Abi functions as a potent activator of Wasp (Bogdan, 2005; Innocenti, 2005), the enhanced formation of F-actin may lead to clustering of the EGF receptor at the membrane to stimulate its sustained activation (Stephan, 2008).
The enhancement of actin polymerization may also affect endocytosis of the EGFR, which is F-actin dependent and as recently shown can be promoted by Abi and Wasp activity. Although it has long been assumed that endocytosis is the main pathway involved in down-regulation of the EGFR, endocytosis is also directly linked to EGFR activation. Block of endocytosis of the EGFR specifically abrogates MAPK activity. Thus Abi could be involved establishing active signaling compartments. Unfortunately, at present the molecular tools to determine the subcellular localization of the activated EGFR in the developing fly are unavailable (Stephan, 2008).
In addition, activation of the Abi complex may recruit kinases such as Abl to the EGF receptor. Abi acts as a positive regulator of the Abl kinase activity promoting the phosphorylation of several substrates including Mena/Ena, Wave, N-Wasp, the B-cell adaptor for phosphoinositide 3-kinase (BCAP) or Cdc2. Interestingly, Abi is also a substrate of Abl resulting in increased protein stability. This positive regulatory loop could hint to an important mechanism for regulating the level of Abl kinase activity during sensory organ development. Indeed, co-expression of AbiMyr with a kinase-defective Abl suppresses tyrosine hyperphosphorylation as well as reduces the level of the AbiMyr protein expression, corresponding to a strong suppression of the number of ectopic sensory organs induced by AbiMyr (Stephan, 2008).
However, the induction of ectopic sensory bristles by Abl does not depend on an intact EGFR signaling or on an activation of the Ras/Raf/MAPK pathway. Co-expression of Abl and EGFR results in an increase of sensory organs but inhibition of EGFR by co-expression of Kekkon1 or heterozygous loss of pole hole function are not sufficient to suppress the Abl-induced neurogenic phenotype suggesting that Abi and Abl act in parallel. In conclusion, it is proposed that the complex of Abi and the EGFR is joined by Abl, which stabilizes not only Abi but also stimulates Wasp function, highlighting the intricate interaction between cytoskeletal dynamics and signaling during sensory organ development (Stephan, 2008).
Eyes absent (Eya), named for its role in Drosophila eye development but broadly conserved in metazoa, possesses dual functions as a transcriptional coactivator and protein tyrosine phosphatase. Although Eya's transcriptional activity has been extensively characterized, the physiological requirements for its phosphatase activity remain obscure. This study provides insight into Eya's participation in phosphotyrosine-mediated signaling networks by demonstrating cooperative interactions between Eya and the Abelson (Abl) tyrosine kinase during development of the Drosophila larval visual system. Mechanistically, Abl-mediated phosphorylation recruits Eya to the cytoplasm, where in vivo studies reveal a requirement for its phosphatase function. Thus, a model is proposed in which, in addition to its role as a transcription factor, Eya functions as a cytoplasmic protein tyrosine phosphatase (Xiong, 2009).
This analysis of the subcellular compartmentalization of Eya function has revealed a requirement for Eya activity in the cytoplasm. Specifically, although nuclearly restricted NLS-Eya, with an inserted nuclear localization sequence, appears to be fully competent as a coactivator, as judged by cultured cell transcriptional reporter assays, it exhibits a reduced ability to induce eye tissue in either wild-type or eya loss-of-function backgrounds. Coexpression of cytoplasmically restricted Myr-Eya (containing a Src myristoylation tag) restores a wild-type level of eye inducing activity to the NLS-Eya background, supporting the interpretation that NLS-Eya is fully competent with respect to transcription, but cannot perform the essential function normally provided by cytoplasmic Eya. Eya phosphatase activity appears to contribute to cytosolic function, since phosphatase-dead versions of cytoplasmically restricted Eya transgenes fail to complement the NLS-Eya background effectively. Thus, it is proposed that whereas regulation of gene expression by the core retinal determination (RD) network relies primarily on nuclear Eya function, other signaling events important for retinal development may rely on transcription-independent functions of the cytoplasmic Eya phosphatase (Xiong, 2009).
Mechanistically, it is proposed that Eya traffics dynamically between nuclear and cytoplasmic compartments, with its final localization determined by its phosphorylation state and interactions with specific signaling partners. Thus, in contexts in which Abl signaling is activated, Abl-mediated phosphorylation may provide a cytoplasmic retention signal that targets Eya to its appropriate site of action, presumably through interactions with specific phosphotyrosine-binding proteins. Autocatalytic Eya phosphatase activity would play a critical positive role with respect to overall Eya function by returning Eya to the nucleus to prevent depletion of the nuclear pool needed to carry out essential transcriptional programs. Although cytosolic Eya substrates have not yet been identified, the fact that phosphatase-dead cytoplasmically restricted Eya was less active than the wild-type version in an assay in which dynamic shuttling between nuclear and cytoplasmic compartments was not relevant suggests that Eya-mediated dephosphorylation of substrates other than itself is likely important (Xiong, 2009).
Although Eya has been primarily characterized as a nuclear protein, two previous observations are consistent with the proposed model of extranuclear function: (1) in mammalian cultured cells, Eya nuclear localization and/or retention requires the presence of its binding partner Six, such that in its absence Eya localizes to the cytosol; (2) protein-protein interactions with several membrane-associated and cytoplasmic proteins have been demonstrated in two-hybrid screens, although only one interaction has been further investigated. In this example, interactions between Eya and the G protein Gαi can recruit Eya to the cytoplasm of cultured cells, and a balance between binding to G proteins and Six has been proposed to regulate Eya distribution and function (Xiong, 2009 and references therein).
To what aspects of eye development might cytosolic Eya activity contribute? Although identification of Eya substrates and elucidation of the specific signaling events regulated by cytoplasmic Eya activity will be required to answer this question definitively, several intriguing models are worth considering. (1) The ectopic eye induction and genetic rescue assays used to characterize the complementation between Myr-Eya and NLS-Eya transgenes imply a requirement for extranuclear Eya in retinal specification. In considering this context, it is important to note that whereas a great deal is understood about how transcriptional hierarchies such as the RD network drive retinal induction, much less is known about how specific differentiation programs are coordinated with the morphogenetic events that pattern the tissue. In Drosophila, specification of retinal fates is immediately preceded by adhesive and morphological changes in and posterior to the morphogenetic furrow. Although phosphotyrosine signaling at the morphogenetic furrow has not been extensively studied, its importance to cell adhesion and epithelial morphogenesis in other contexts is well documented. For example, recent work studying the invagination of the ventral furrow during Drosophila gastrulation demonstrated that Abl signaling acting in parallel to the Rho activator RhoGEF2 regulates actin organization to drive apical cell constriction. Given the importance of cell constriction in the retinal furrow, it will be interesting to investigate whether similar signaling mechanisms operate in this context and whether cytoplasmic Eya phosphatase activity is involved. Encouragingly, loss of eya impairs morphogenetic furrow propagation, suggesting that investigation of defects in epithelial remodeling and reorganization of cell-cell contacts at the furrow in eya mutants could be fruitful (Xiong, 2009).
(2) Another possibility is that cytoplasmic Eya phosphatase function might provide critical feedback regulation on other signaling pathways during retinal specification. Indeed, a complex web of interactions between multiple signaling networks including the Wingless, Notch, Hedgehog, and EGFR pathways has been shown to be critical for RD network function and retinal induction. Thus, if cytosolic Eya phosphatase activity were absent or mislocalized, the resulting signaling imbalances could potentially compromise eye specification and development (Xiong, 2009).
(3) Finally, cytoplasmic Eya function could be important for neuronal morphogenesis, perhaps through involvement in Abl-mediated signaling events. Because Abl signaling has not yet been explored in the retina, determining which downstream branches of the pathway operate in this developmental context will be important for elucidating the molecular and cellular defects underlying the phenotypes and interactions that have been reported. For example, the photoreceptor axon targeting defects observed in eya or abl mutants, or upon Myr-EyaWT expression, could reflect impaired receiving or processing of attractive signals from either brain cells or adjacent retinal neurons, weakening of repulsive signaling between axons, or strengthening of adhesive properties between the axons such that they fail to spread properly as they exit the optic stalk (Xiong, 2009).
In considering the mechanistic possibilities whereby Eya might interact with the Abl signaling network, it is important to reiterate that although genetic analyses indicate eya and abl function cooperatively, the two genes encode proteins with opposing catalytic functions. Thus, a simple relationship whereby Eya dephosphorylates Abl or its substrates is unlikely to offer a suitable explanation since this would most likely be reflected as antagonism rather than synergy. Instead, Abl phosphorylation and recruitment of Eya to the cytoplasm may facilitate formation of protein complexes important for Abl signaling and/or promote interactions with components of other phosphotyrosine signaling pathways, which together would target Eya phosphatase activity toward appropriate substrates. Finally, these results do not preclude Eya's nuclear transcriptional activities from also contributing to Abl signaling; thus, it will be important to investigate further the complex spatiotemporal requirements for Eya, other members of the RD network, and the Abl signaling pathway during retinal development (Xiong, 2009).
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