Abl tyrosine kinase: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

Gene name - Abl tyrosine kinase

Synonyms -

Cytological map position - 73B3--73B4

Function - signaling protein

Keyword(s) - cytoskeleton, axonogenesis, oncogene

Symbol - Abl

FlyBase ID: FBgn0000017

Genetic map position - 3-[44]

Classification - Protein tyrosine kinases

Cellular location - cytoplasmic



NCBI link: Entrez Gene
Abl orthologs: Biolitmine

Recent literature
Jodoin, J. N. and Martin, A. C. (2016). Abl suppresses cell extrusion and intercalation during epithelium folding. Mol Biol Cell [Epub ahead of print]. PubMed ID: 27440923
Summary:
Tissue morphogenesis requires control over cell shape changes and rearrangements. In the Drosophila mesoderm, linked epithelial cells apically constrict, without cell extrusion or intercalation, to fold the epithelium into a tube that will then undergo a epithelial-to-mesenchymal transition (EMT). Apical constriction drives tissue folding or cell extrusion in different contexts, but the mechanisms that dictate the specific outcomes are poorly understood. Using live-imaging, this study found that Abelson (Abl) tyrosine kinase depletion during gastrulation causes apically constricting cells to undergo aberrant basal cell extrusion and cell intercalation. abl depletion disrupted apical-basal polarity and adherens junction organization in mesoderm cells, suggesting that extruding cells undergo premature EMT. The polarity loss was associated with abnormal basolateral contractile actomyosin and Enabled (Ena) accumulation. Depletion of the Abl effector Enabled (Ena) in abl depleted embryos suppressed the abl phenotype, consistent with cell extrusion resulting from misregulated abl. This work provides new insight as to how Abl loss and Ena misregulation promote cell extrusion and EMT.
Rogers, E.M, Spracklen, A.J., Bilancia, C.G., Sumigray, K.D., Allred, S.C., Nowotarski, S.H., Schaefer, K.N., Ritchie, B.J. and Peifer, M. (2016). Abelson kinase acts as a robust, multifunctional scaffold in regulating embryonic morphogenesis. Mol Biol Cell [Epub ahead of print]. PubMed ID: 27385341
Summary:
Abelson family kinases (Abl) are key regulators of cell behavior and the cytoskeleton during development and in leukemia. Abl's SH3, SH2, and tyrosine kinase domains are joined via a linker to an F-actin-binding domain (FABD). Research on Abl's roles in cell culture have led to several hypotheses for its mechanism of action: 1) Abl phosphorylates other proteins, modulating their activity. 2) Abl directly regulates the cytoskeleton via its cytoskeletal interaction domains, and/or 3) Abl is a scaffold for a signaling complex. The importance of these roles during normal development remains untested. This study tested these mechanistic hypotheses during Drosophila morphogenesis using a series of mutants to examine Abl's many cell biological roles. Strikingly, Abl lacking the FABD fully rescues morphogenesis, cell shape change, actin regulation, and viability, while kinase dead Abl, though reduced in function, retains substantial rescuing ability in some but not all Abl functions. The function of four conserved motifs in the linker region was also tested, revealing a key role for a conserved PXXP motif known to bind Crk and Abi. The study proposes that Abl acts as a robust multi-domain scaffold with different protein motifs and activities contributing differentially to diverse cellular behaviors.

Cheong, H. S. J. and VanBerkum, M. F. A. (2017). Long disordered regions of the C-terminal domain of Abelson tyrosine kinase have specific and additive functions in regulation and axon localization. PLoS One 12(12): e0189338. PubMed ID: 29232713
Summary:
Abelson tyrosine kinase (Abl) is a key regulator of actin-related morphogenetic processes including axon guidance, where it functions downstream of several guidance receptors. While the long C-terminal domain (CTD) of Abl is required for function, its role is poorly understood. In this study, a battery of mutants of Drosophila Abl was created that systematically deleted large segments of the CTD from Abl or added them back to the N-terminus alone. The functionality of these Abl transgenes was assessed through rescue of axon guidance defects and adult lethality in Abl loss-of-function, as well as through gain-of-function effects in sensitized slit or frazzled backgrounds that perturb midline guidance in the Drosophila embryonic nerve cord. Two regions of the CTD play important and distinct roles, but additive effects for other regions were also detected. The first quarter of the CTD, including a conserved PxxP motif and its surrounding sequence, regulates Abl function while the third quarter localizes Abl to axons. These regions feature long stretches of intrinsically disordered sequence typically found in hub proteins and are associated with diverse protein-protein interactions. Thus, the CTD of Abl appears to use these disordered regions to establish a variety of different signaling complexes required during formation of axon tracts.
Kannan, R., Cox, E., Wang, L., Kuzina, I., Gu, Q. and Giniger, E. (2018). Tyrosine phosphorylation and proteolytic cleavage of Notch are required for non-canonical Notch/Abl signaling in Drosophila axon guidance. Development 145(2). PubMed ID: 29343637
Summary:
Notch signaling is required for the development and physiology of nearly every tissue in metazoans. Much of Notch signaling is mediated by transcriptional regulation of downstream target genes, but Notch controls axon patterning in Drosophila by local modulation of Abl tyrosine kinase signaling, via direct interactions with the Abl co-factors Disabled and Trio. This study shows that Notch-Abl axonal signaling requires both of the proteolytic cleavage events that initiate canonical Notch signaling. It furthers show that some Notch protein is tyrosine phosphorylated in Drosophila, that this form of the protein is selectively associated with Disabled and Trio, and that relevant tyrosines are essential for Notch-dependent axon patterning but not for canonical Notch-dependent regulation of cell fate. Based on these data, a model is proposed for the molecular mechanism by which Notch controls Abl signaling in Drosophila axons.
Bernardoni, R., et al. (2019). A new BCR-ABL1 Drosophila model as a powerful tool to elucidate the pathogenesis and progression of chronic myeloid leukemia. Haematologica 104(4): 717-728. PubMed ID: 30409797
Summary:
Chronic myeloid leukemia relies on constitutive BCR-ABL1 kinase activity, but not all the interactors and regulators of the oncoprotein are known. This paper describes and validates a Drosophila leukemia model based on inducible human BCR-ABL1 expression controlled by tissue-specific promoters. The model was conceived to be a versatile tool for performing genetic screens. BCR-ABL1 expression in the developing eye interferes with ommatidia differentiation and expression in the hematopoietic precursors increases the number of circulating blood cells. This study shows that BCR-ABL1 interferes with the pathway of endogenous dAbl with which it shares the target protein Ena. Loss of function of ena or Dab, an upstream regulator of dAbl, respectively suppresses or enhances both the BCR-ABL1-dependent phenotypes. Importantly, in patients with leukemia decreased human Dab1 and Dab2 expression correlates with more severe disease and Dab1 expression reduces the proliferation of leukemia cells. This Drosophila model promises to be an excellent system for performing unbiased genetic screens aimed at identifying new BCR-ABL1 interactors.
Clarke, A., McQueen, P. G., Fang, H. Y., Kannan, R., Wang, V., McCreedy, E., Wincovitch, S. and Giniger, E. (2020). Abl signaling directs growth of a pioneer axon in Drosophila by shaping the intrinsic fluctuations of actin. Mol Biol Cell: mbcE19100564. PubMed ID: 31967946
Summary:
The fundamental problem in axon growth and guidance is to understand how cytoplasmic signaling modulates the cytoskeleton to produce directed growth cone motility. In an accompanying paper, live imaging of the TSM1 axon of the developing Drosophila wing was use to show that the essential role of the core guidance signaling molecule, Abl tyrosine kinase, is to modulate the organization and spatial localization of actin in the advancing growth cone. This study dissects in detail the properties of that actin organization, and its consequences for growth cone morphogenesis and motility. Sdvance of the actin mass in the distal axon is shown to drive the forward motion of the dynamic filopodial domain that defines the growth cone. It was further shown that Abl regulates both the width of the actin mass and its internal organization, spatially biasing the intrinsic fluctuations of actin to achieve net advance of the actin, and thus of the dynamic filopodial domain of the growth cone, while maintaining the essential coherence of the actin mass itself. These data suggest a model whereby guidance signaling systematically shapes the intrinsic, stochastic fluctuations of actin in the growth cone to produce axon growth and guidance.
Cheong, H. S. J., Nona, M., Guerra, S. B. and VanBerkum, M. F. (2020). The first quarter of the C-terminal domain of Abelson regulates the WAVE regulatory complex and Enabled in axon guidance. Neural Dev 15(1): 7. PubMed ID: 32359359
Summary:
Abelson tyrosine kinase (Abl) plays a key role in axon guidance in linking guidance receptors to actin dynamics. The long C-terminal domain (CTD) of Drosophila Abl is important for this role, and previous work identified the 'first quarter' (1Q) of the CTD as essential. This study links the physical interactions of 1Q binding partners to Abl's function in axon guidance. Protein binding partners of 1Q were identified by GST pulldown and mass spectrometry and validated using axon guidance assays in the embryonic nerve cord and motoneurons. The role of 1Q was assessed genetically, utilizing a battery of Abl transgenes in combination with mutation or overexpression of the genes of pulled down proteins, and their partners in actin dynamics. The set of Abl transgenes had the following regions deleted: all of 1Q, each half of 1Q ('eighths', 1E and 2E) or a PxxP motif in 2E, which may bind SH3 domains. GST pulldown identified Hem and Sra-1 as binding partners of 1Q, and genetic analyses show that both proteins function with Abl in axon guidance, with Sra-1 likely interacting with 1Q. As Hem and Sra-1 are part of the actin-polymerizing WAVE regulatory complex (WRC), the analyses was extended to Abi and Trio, which interact with Abl and WRC members. Overall, the 1Q region (and especially 2E and its PxxP motif) are important for Abl's ability to work with WRC in axon guidance. These areas are also important for Abl's ability to function with the actin regulator Enabled. In comparison, 1E contributes to Abl function with the WRC at the midline, but less so with Enabled. It is concluded that the 1Q region, and especially the 2E region with its PxxP motif, links Abl with the WRC, its regulators Trio and Abi, and the actin regulator Ena. Removing 1E has specific effects suggesting it may help modulate Abl's interaction with the WRC or Ena. Thus, the 1Q region of Abl plays a key role in regulating actin dynamics during axon guidance.
Yu, H. H. and Zallen, J. A. (2020). Abl and Canoe/Afadin mediate mechanotransduction at tricellular junctions. Science 370(6520). PubMed ID: 33243859
Summary:
Epithelial structure is generated by the dynamic reorganization of cells in response to mechanical forces. Adherens junctions transmit forces between cells, but how cells sense and respond to these forces in vivo is not well understood. This study identified a mechanotransduction pathway involving the Abl tyrosine kinase and Canoe/Afadin that stabilizes cell adhesion under tension at tricellular junctions in the Drosophila embryo. Canoe is recruited to tricellular junctions in response to actomyosin contractility, and this mechanosensitivity requires Abl-dependent phosphorylation of a conserved tyrosine in the Canoe actin-binding domain. Preventing Canoe tyrosine phosphorylation destabilizes tricellular adhesion, and anchoring Canoe at tricellular junctions independently of mechanical inputs aberrantly stabilizes adhesion, arresting cell rearrangement. These results identify a force-responsive mechanism that stabilizes tricellular adhesion under tension during epithelial remodeling.
Rogers, E. M., Allred, S. C. and Peifer, M. (2021). Abelson kinase's intrinsically disordered region plays essential roles in protein function and protein stability. Cell Commun Signal 19(1): 27. PubMed ID: 33627133
Summary:
The non-receptor tyrosine kinase Abelson (Abl) is a key player in oncogenesis. Drosophila offers a superb model for studying Abl's normal function, because, unlike mammals, there is only a single fly Abl family member. Attention turned to one of Abl's least understood features-the long intrinsically-disordered region (IDR) linking Abl's kinase and F-actin binding domains. The past decade revealed unexpected, important roles for IDRs in diverse cell functions, as sites of posttranslational modifications, mediating multivalent interactions and enabling assembly of biomolecular condensates via phase separation. Previous work deleting conserved regions in Abl's IDR revealed an important role for a PXXP motif, but did not identify any other essential regions. This study extended this analysis by deleting the entire IDR, and asking whether AblΔIDR rescues the diverse roles of Abl in viability and embryonic morphogenesis in Drosophila. This revealed that the IDR is essential for embryonic and adult viability for cell shape changes and cytoskeletal regulation during embryonic morphogenesis, and, most surprisingly, revealed a role in modulating protein stability. These data provide new insights into the role of the IDR in an important signaling protein, the non-receptor kinase Abl, suggesting that it is essential for all aspects of protein function during embryogenesis, and revealing a role in protein stability. These data will stimulate new explorations of the mechanisms by which the IDR regulates Abl stability and function, both in Drosophila and also in mammals. They also will stimulate further interest in the broader roles IDRs play in diverse signaling proteins.
Marquilly, C., Busto, G. U., Leger, B. S., Boulanger, A., Giniger, E., Walker, J. A., Fradkin, L. G. and Dura, J. M. (2021). Htt is a repressor of Abl activity required for APP-induced axonal growth. PLoS Genet 17(1): e1009287. PubMed ID: 33465062
Summary:
Huntington's disease is a progressive autosomal dominant neurodegenerative disorder caused by the expansion of a polyglutamine tract at the N-terminus of a large cytoplasmic protein. The Drosophila huntingtin (htt) gene is widely expressed during all developmental stages from embryos to adults. However, Drosophila htt mutant individuals are viable with no obvious developmental defects. This study asked if such defects could be detected in htt mutants in a background that had been genetically sensitized to reveal cryptic developmental functions. Amyloid precursor protein (APP) is linked to Alzheimer's disease. Appl is the Drosophila APP ortholog and Appl signaling modulates axon outgrowth in the mushroom bodies (MBs), the learning and memory center in the fly, in part by recruiting Abl tyrosine kinase. This study finds that htt mutations suppress axon outgrowth defects of αβ neurons in Appl mutant MB by derepressing the activity of Abl. Abl was shown to be required in MB αβ neurons for their axon outgrowth. Importantly, both Abl overexpression and lack of expression produce similar phenotypes in the MBs, indicating the necessity of tightly regulating Abl activity. Htt was found to behave genetically as a repressor of Abl activity, and consistent with this, in vivo FRET-based measurements reveal a significant increase in Abl kinase activity in the MBs when Htt levels are reduced. Thus, Appl and Htt have essential but opposing roles in MB development, promoting and suppressing Abl kinase activity, respectively, to maintain the appropriate intermediate level necessary for axon growth.

BIOLOGICAL OVERVIEW

The proto-oncogenic kinase Abelson (Abl) regulates actin in response to cell signaling. Drosophila Abl is required in the nervous system, and also in epithelial cells, where it regulates adherens junction stability and actin organization. Abl acts at least in part via the actin regulator Enabled (Ena), but the mechanism by which Abl regulates Ena is unknown. A novel role is described for Abl in early Drosophila development, where it regulates the site and type of actin structures produced. In Abl's absence, excess actin is polymerized in apical microvilli, whereas too little actin is assembled into pseudocleavage and cellularization furrows. These effects involve Ena misregulation. In abl mutants, Ena accumulates ectopically at the apical cortex where excess actin is observed, suggesting that Abl regulates Ena's subcellular localization. Other actin regulators were also examined. Loss of Abl leads to changes in the localization of the Arp2/3 complex and the formin Diaphanous, and mutations in diaphanous or capping protein beta enhance abl phenotypes (Grevengoed, 2003).

Genetic analysis suggests that in the early Drosophila embryo, the primary means by which Abl influences the cytoskeleton is through Ena. Reducing the Ena dose by half profoundly suppresses ablM; it is as effective as adding back a wild-type abl transgene. Ena is also a critical target of Abl during embryonic morphogenesis. Although the data suggest that the primary effect of loss of Abl is Ena deregulation, they do not rule out Abl acting on the cytoskeleton by other mechanisms (Grevengoed, 2003).

The mechanism by which Abl regulates Ena has remained mysterious. This study demonstrates that Abl regulates Ena by regulating its intracellular localization. In the absence of Abl, Ena localizes to ectopic sites. Alterations in Ena and actin localization have been observed at the leading edge of migrating epidermal cells in abl mutants during dorsal closure. This suggests that regulation of Ena localization by Abl may be a more general mechanism. It is hypothesized that Abl targets Ena to places where it is needed to modulate actin dynamics, perhaps by excluding it from other sites where Ena activity would be detrimental (Grevengoed, 2003).

There are many ways in which Abl could restrict Ena localization. Abl's kinase activity is essential, and thus Abl phosphorylation of Ena may restrict its localization by preventing Ena binding to partners that localize to particular cortical sites, or by promoting Ena binding to partners that sequester it in the cytoplasm. Phosphorylation of Ena by Abl in vitro inhibits binding of Ena to SH3 domains, whereas Mena/VASP phosphorylation by PKA alters binding to SH3 domains and actin. However, if direct phosphorylation were the only mechanism by which Abl regulated Ena, mutating Ena's phosphorylation sites should create a protein that can no longer be regulated and thus would localize to ectopic sites. Instead, mutation of all of the Abl phosphorylation sites in Ena modestly reduced Ena function, rather than making it ectopically active as is seen in abl mutants (Grevengoed, 2003).

Thus, Abl may regulate Ena by additional mechanisms. Abl may modulate Ena localization and restrict Ena activity by direct binding (this could still require Abl kinase activity, since auto-phosphorylation or phosphorylation of other partners may regulate protein-protein interactions). Abl might sequester Ena in the cytoplasm in an inactive state, or it might recruit Ena to appropriate sites. Alternately, binding of Abl's SH3 domain to the Ena proline-rich region might prevent Ena from binding to other partners, such as profilin, which might in turn modulate both Ena localization and activity. In thinking about these different possible mechanisms, it is interesting to note that Abl localizes to the actin caps and apical pseudocleavage furrows during syncytial stages and the apical portion of the cellularization furrow, the precise places where ectopic actin accumulation occurs in its absence. Thus, it is poised to act at this location. Working out the details of the mechanism by which Abl regulates Ena localization will be one of the next challenges (Grevengoed, 2003).

This work provides an in vivo test of the current model for Ena function, and allows extension of this model. The excess growth of microvilli seen when Ena is ectopically localized in early embryos fits well with work on Ena/VASP function in mammalian fibroblasts, where forced localization of Ena/VASP proteins to the leading edge promotes the formation of long, unbranched filaments. Ena also localizes to the ends of filopodia and microspikes, suggesting that Ena's role in promoting long unbranched actin structures is broadly conserved. Earlier experiments in fibroblasts artificially altered Ena localization. This study demonstrates that Ena localization is a normal regulatory point in vivo, and that Abl is a critical player in this process. Finally, in vitro experiments have suggested that Ena promotes filament elongation by antagonizing capping protein. Mutations in cpb enhance the effects of mutations in abl in the CNS and probably during oogenesis. These data are consistent with Ena and capping protein playing antagonistic roles in vivo, with Abl potentially influencing the outcome of this antagonism. However, Abl and capping protein may also work together independently of Ena in the regulation of actin dynamics (Grevengoed, 2003).

Different actin regulators play fundamentally different biochemical roles. Models often picture all of these regulators modulating actin assembly and disassembly at a single site, but of course individual cells target different actin regulators to distinct sites, creating actin structures with diverse functions. Syncytial embryos provide an excellent example. During interphase, they assemble actin-based microvillar caps above each nucleus. As they enter prophase, caps are disassembled and actin polymerization is redirected to the pseudocleavage furrows. This is likely to require new machinery: both Arp2/3 and the formin Dia are required for pseudocleavage furrow formation, but not for actin caps. Cellularization also requires distinct machinery to polymerize/disassemble apical microvilli and to recruit and modulate actin at the cellularization front. For transitions to occur smoothly, two fundamental changes have to occur: the location at which actin polymerization occurs must change, and a different constellation of actin regulators must be deployed to produce the distinct actin structures observed (Grevengoed, 2003).

The data support a hypothesis in which the balance of activity of different actin regulators at distinct sites is tightly regulated, influencing the nature of the actin structures produced. One regulator is Abl. In its absence, Ena localizes ectopically to the cortical region, upsetting the temporal and spatial balance of actin regulators. This leads to a change in both the location and nature of actin polymerization during mitosis. Excess actin is polymerized into microvillar projections that extend from the apical region of the furrows, whereas insufficient actin is directed to the pseudocleavage furrows. Similarly, during cellularization in ablM mutants, actin polymerization continues to be directed to apical microvilli, whereas in a wild-type embryo this ceases early in cellularization (Grevengoed, 2003).

The data also suggest that there is cross-talk between different modulators of actin polymerization, and that the balance of their activities determines the outcome. Although many actin modulators are unaffected in ablM mutants, both the Arp2/3 complex and Dia are recruited to sites of ectopic actin polymerization. However, genetic analysis suggests that although Ena mislocalization plays a critical role in the actin alterations seen in ablM mutants, Dia and Arp2/3 mislocalization may not. In fact, reduction of the dose of Dia enhanced the ablM phenotype. Dia normally promotes actin polymerization lining the furrows. In ablM mutants, the balance of actin polymerization is already shifted to the apical microvilli because of ectopic Ena localization. Reduction in the dose of Dia might further reduce actin polymerization in pseudocleavage furrows, resulting in the observed enhancement of the ablM phenotype. The abnormal recruitment of Dia to the apical regions in ablM mutants may also reduce pseudocleavage furrow formation (Grevengoed, 2003).

It will now be important to investigate how the cell regulates the distinct types of actin polymerization required for distinct cellular and developmental processes. One mechanism of cross-talk may involve direct or indirect recruitment of one type of actin modulator by another. Abl's ability to interact with both Ena and the Arp2/3 regulator WAVE1 is interesting in this regard. However, the recruitment of Arp3 and Dia to ectopic actin structures observed in ablM mutants may have a more simple explanation. Both are thought to have a higher affinity for newly polymerized, ATP-bound actin, which is likely to be increased where ectopic actin polymerization appears to occur (Grevengoed, 2003).

Drosophila Abl also functions in other contexts. It has a role in embryonic morphogenesis, where it also acts, at least in part, via Ena. However, in this context Abl also affects AJ stability. Since Ena is normally highly enriched in AJs, it is hypothesized that Abl helps restrict Ena localization to AJs, and thus helps initiate the proper organization of actin underlying AJs. In Abl's absence, Ena may localize to sites other than AJs, leading to ectopic actin polymerization at those sites and reduction in actin polymerization at AJs (analogous to the divergent effects on apical actin and pseudocleavage/cellularization furrows). Since the cortical actin belt underlying the AJ plays an important role in its stability, this could explain the phenotype of abl mutants. A similar model may help explain the roles of Abl and Ena in axon outgrowth. The network of actin filaments in the growth cone is complex, with different types of actin in filopodia and in the body of the growth cone. By regulating Ena localization, Abl may influence the balance of the different types of actin, thus influencing growth cone motility. Likewise, in fibroblasts, where Ena/VASP proteins regulate motility, the Arp2/3 regulators N-WASP and WAVE localize to sites at the leading edge distinct from those where Mena is found. Whether Abl or Arg regulate the localization of Ena/VASP family proteins in mammals remains to be determined. Likewise, it is possible that deregulation of Ena/VASP proteins underlie some of the alterations in cell behavior in Bcr-Abl–transformed lymphocytes. Experiments to test whether Ena/VASP activity is important for either mammalian Abl's normal function or for the pathogenic effects of Bcr-Abl will help answer these questions (Grevengoed, 2003).

The Hem protein mediates neuronal migration by inhibiting WAVE degradation and functions opposite of Abelson tyrosine kinase

In the nervous system, neurons form in different regions, then they migrate and occupy specific positions. RP2/sib, a well-studied neuronal pair in the Drosophila ventral nerve cord (VNC), has a complex migration route. This study shows that the Hem protein, via the WAVE complex, regulates migration of GMC-1 and its progeny RP2 neuron. In Hem or WAVE mutants, RP2 neuron either abnormally migrates, crossing the midline from one hemisegment to the contralateral hemisegment, or does not migrate at all and fails to send out its axon projection. Hem regulates neuronal migration through stabilizing WAVE. Since Hem and WAVE normally form a complex, the data argues that in the absence of Hem, WAVE, which is presumably no longer in a complex, becomes susceptible to degradation. It was also found that Abelson tyrosine kinase affects RP2 migration in a similar manner as Hem and WAVE, and appears to operate via WAVE. However, while Abl negatively regulates the levels of WAVE, it regulates migration via regulating the activity of WAVE. The results also show that during the degradation of WAVE, Hem function is opposite to that of and downstream of Abl (Zhu, 2011).

Several studies have suggested that Hem dynamically regulates polymerization of F-actin. Hem can play a crucial role in linking extracellular signals to the cytoskeleton. On the other hand, Hem is also part of the WAVE complex and it may regulate the activity of the WAVE complex to promote polymerization of F-actin. The result that the migration defect in Hem mutants can be completely rescued by expression of WAVE from a transgene indicates that Hem regulates neuronal migration via WAVE (Zhu, 2011).

How Hem regulates WAVE is controversial. It has been argued that Hem (together with PIP212) inhibits WAVE in the WAVE complex. Upon activation by Rac1 or Nck, the WAVE complex dissociates releasing an active WAVE-HSPC300 to mediate actin nucleation. This conclusion was also supported by the findings that loss-of-function for Hem leads to an excess of F-actin in the cytosol. Moreover, a reduction in the WAVE gene dosage suppressed axon guidance defects in Hem mutant embryos. But, in vitro studies using Drosophila tissue culture cells argue that Hem protects WAVE from proteasome-mediated degradation. The current in vivo results are consistent with these studies and show that WAVE is protected by Hem and the above alternate model may be incorrect (Zhu, 2011).

The WAVE protein was first identified in Dictyostelium discoideum as a suppressor of mutations in the cAMP receptor (SCAR) but it is present in flies to humans. All WAVEs contain a N-terminal WHD/SHD (WAVE/SCAR homologue domain), a central proline-rich region and a C-terminal VCA domain. WAVE protein regulates actin polymerization by mediating the signal of Rac to Arp2/3 in lamellipodia. It is involved in forming branched and cross-linked actin networks. Unlike WASp proteins, which are intrinsically inactive by autoinhibition and activated by directly binding to Cdc42, PIP2 etc., WAVE appears to be intrinsically active, at least in vitro.However, the majority of WAVE is in the 'WAVE complex' with four other proteins: Hem, Sra-1/PIR121/CYFIP, Abi and HSPC300/Brk1 (Zhu, 2011).

In the WAVE-complex, direct association between WAVE, Abi and HSPC300 represents the backbone of the complex. Hem binds to Sra-1 forming a sub-complex, which is able to bind to Rac through Sra-1. The interaction between Abi and Hem is what binds Hem and Sra-1 into the complex. Hem and Sra-1 are sequentially recruited to the WAVE complex. Free subunits and assembly intermediates of the WAVE-complex are usually not detected but supposedly degraded. Also, previous studies suggest that depletion of one component leads to degradation of others. Indeed, the current results, that in Hem mutants, the level of WAVE protein, but not the WAVE gene transcription, is drastically reduced supports this contention. Perhaps in the absence of Hem, WAVE complex is either not formed or partially formed, resulting in the degradation of WAVE and phenotypes such as mis-migration of neurons. When the levels of WAVE are supplemented using a WAVE transgene (UAS-WAVE), the migration defect in Hem mutants is promptly rescued (Zhu, 2011).

While a complete lack of WAVE (or Hem) function causes an arrest in the migration of RP2, a reduction in the levels of WAVE due to a reduction in the levels of Hem causes abnormal migration. For example, the lowest level of WAVE is seen in the Hem allele that has the strongest penetrance. Moreover, since this mis-migration defect is rescued by expressing WAVE from a transgene, it can be concluded that this mis-migration is also due to an effect on WAVE. It has been suggested that the WAVE-complex exists cytoplasmically and in membrane-bound forms. Through an interaction with Rac, WAVE gets recruited to the lamellipodia where actin polymerization required for membrane protrusion is initiated and regulated. The integrity of the complex is critical for its proper localization since removal of either WAVE or Abi prevents its translocation to the leading edge of the lamellipodia. It is possible that a reduction in the levels of WAVE in Hem mutant embryos causes non-translocation of the WAVE complex to the membrane, causing a non/mis-migration of RP2 (Zhu, 2011).

These results show that WAVE protein exists as three different molecular weight forms. Treatment of the extract with phosphatase collapses these three forms into a single band, indicating that WAVE protein is phosphorylated, with varying degrees of phosphorylation to yield different molecular weight species. Whether there are any changes in the three different forms with respect to their relative contributions in Hem and Abl mutants was examined. However, no changes were found in their relative contributions and the levels of all the forms were reduced in Hem mutants. Therefore, it may be that the reduction in all the forms, or that the reduction in one or two of the forms is responsible for the migration defect. In Abl mutants, the level of WAVE is modestly increased, which is the opposite to that of the effect of Hem on WAVE. Thus, it seems more likely that the activity of WAVE is affected in Abl mutants. Being a protein kinase, it was possible that Abl phosphorylates WAVE, thus affecting either its activity or level. However, no significant changes in were seen in the relative levels of the different phosphorylated forms of WAVE in Abl mutants. It has been shown in vitro that Abl is recruited to WAVE by Abi following cell stimulation, triggering the translocation of Abl together with the WAVE complex to the leading edge of the membrane. Thus, Abl might affect WAVE activity, either directly or indirectly, via the translocation of the WAVE complex to the membrane of an actively migrating RP2. It is also possible that Abl affects migration in a pathway that does not involve WAVE (Zhu, 2011).

In contrast, the effect of loss-of-function for Abl on WAVE levels is more pronounced in older embryos. These results indicate that Abl directly or indirectly regulates the levels of WAVE. Furthermore, though modest, ectopic expression of Abl does down-regulate WAVE. Interestingly, the results also show that Hem regulation of WAVE levels is downstream of the Abl regulation of WAVE since the Hem; Abl double mutants had the same levels of WAVE as Hem single mutants. It seems likely that in the absence of Hem, WAVE protein gets degraded, resulting in the loss of migration or abnormal migration. Whereas in Abl mutants, the most likely scenario is that the activity of WAVE is affected, resulting in the same migration defect (Zhu, 2011).

Enabled signaling pathway regulates Golgi architecture in Drosophila photoreceptor neurons

The golgi apparatus is optimized separately in different tissues for efficient protein trafficking, little is known of how cell signaling shapes this organelle. This study finds that the Abl tyrosine kinase signaling pathway controls the architecture of the golgi complex in Drosophila photoreceptor (PR) neurons. The Abl effector, Enabled (Ena), selectively labels the cis-golgi in developing PRs. Overexpression or loss-of-function of Ena increases the number of cis and trans-golgi cisternae per cell, and Ena overexpression also redistributes golgi to the most basal portion of the cell soma. Loss of Abl, or of its upstream regulator, the adaptor protein Disabled, lead to the same alterations of golgi as does overexpression of Ena. The increase in golgi number in Abl mutants arises in part from increased frequency of golgi fission events and a decrease in fusions, as revealed by live imaging. Finally, it was demonstrated that the effects of Abl signaling on golgi are mediated via regulation of the actin cytoskeleton. Together, these data reveal a direct link between cell signaling and golgi architecture. Moreover, they raise the possibility that some of the effects of Abl signaling may arise, in part, from alterations of protein trafficking and secretion (Kannan, 2014).

The Abl tyrosine kinase signaling pathway controls golgi morphology and localization in Drosophila photoreceptors through its regulation of the actin cytoskeleton. Ena, the main effector of Abl in morphogenesis, is associated with the cis-golgi compartment, and it regulates golgi localization and dynamics under the control of Abl and its interacting adaptor protein, Dab. Reducing the levels of Abl or Dab, or overexpressing Ena, led to similar defects in golgi fragmentation state and subcellular distribution. During golgi biogenesis, Abl increases the frequency of fusion of golgi cisternae, and decreases fission events. Abl evidently controls golgi organization through its regulation of actin structure, as the effect of Abl signaling on golgi could be blocked by modulating actin structure genetically or pharmacologically. Collectively, these data reveal an unexpected link between a fundamental tyrosine kinase signaling pathway in neuronal cells and the structure of the golgi compartment (Kannan, 2014).

The data reported here suggest that the Abl signaling pathway controls golgi morphology and localization through its control of actin structure. This is consistent with previous reports that altering the levels of actin modulators perturbs the structure and function of the golgi apparatus. A variety of proteins that modulate actin dynamics have been localized to golgi. Ultra-structural studies established the association of actin filaments with golgi membranes and the association of β and γ actin with the golgi. In cultured cell models, including neurons, actin depolymerization leads to golgi compactness, fragmentation and altered subcellular distribution. It is noted, moreover, that the reported golgi-associated signaling proteins include several that have been linked to Abl signaling, including the Abl target Abi, the Abi binding partner WAVE, and various effectors of Rac GTPase including ADF/cofilin, WASH and Arp2/3. Thus, for example, Abi and WAVE have been implicated in actin dependent golgi stack reorganization and in scission of the golgi at cell division to allow faithful inheritance of golgi complex to daughter cells in Drosophila S2 cell cycles (Kondylis, 2007). These data reinforce the importance of actin-regulating signaling pathways for controlling golgi biogenesis (Kannan, 2014).

Two lines of evidence suggest that the increase observed in golgi number in Abl pathway mutants is due primarily to net fragmentation of pre-existing golgi cisternae and not to de novo synthesis of golgi. First, live imaging of golgi dynamics in neurons of the Drosophila eye disc reveals that the steady-state number of golgi cisternae reflects an ongoing balance of fusion and fission events, much as observed previously in yeast. Quantification of these events in wildtype vs Abl mutant tissue demonstrated directly that loss of Abl significantly increased the frequency of fission events, and reduced the frequency of fusions. Second, the absolute volume of cis-golgi in Abl mutant photoreceptors was not substantially greater than that in wildtype, as judged by direct measurement of the volume of GM130- immunoreactive material in deconvoluted image stacks of photoreceptor clusters. While a small apparent increase was observed in golgi volume in the mutants (~55%, based on pixel counts), it is noted that golgi cisternae are small on the length scale of the point spread function of visible light, such that the fluorescent signal from a single cisterna extends into the surrounding cytoplasm. The increase in apparent golgi volume is therefore within the range expected due simply to fluorescence 'spillover' from the three-fold greater number of separate golgi cisternae in the mutants (Kannan, 2014).

It is striking that both increase and decrease of Ena led to net fragmentation of golgi. Why might this be? It is known that both fission and fusion of membranes requires actin dynamics: at scission, polymerization provides force for separating membranes, while in fusion, actin polymerization is essential for bringing membranes together and for supplying membrane vesicles, among other things. As a result, altering actin dynamics is apt to change the probabilities of multiple aspects of both fission and fusion events, making it impossible to predict a priori how the balance will be altered by a given manipulation, just as either increase or decrease of Ena can inhibit cell or axon motility, depending on the details of the experiment, due to the non-linear nature of actin dynamics. Indeed, this study also observed net golgi fragmentation when actin was stabilized with jasplakinolide, just as was done from depolymerization with cytochalasin or latrunculin. More direct experiments will be necessary to fully understand this dynamic, however. deficits selectively disrupt dendritic morphogenesis but not axogenesis, and perhaps consistent with this, Abl/Ena function is essential for dendrite arborization in these cells but has not been reported to affect their axon patterning. Finally, in some contexts, neuronal development requires local translation of guidance molecules in the growth cone rather than translation in the cell soma. It is likely that the need for actin dynamics to target different subcellular compartments in different cell types will be reflected in different patterns of Abl/Ena protein localization (Kannan, 2014).

This study reports the role of Abl/Ena-dependent regulation of actin structure on overall golgi structure and localization but there may be more subtle effects on golgi function as well. For example, recent evidence supports a role for actin-dependent regulation of the specificity of protein sorting in the golgi complex. Preferential sorting of cargos is achieved by nucleation of distinct actin filaments at the golgi complex. In Hela cells, for example, Arp2/3 mediated nucleation of actin branches at cis-golgi regulates retrograde trafficking of the acid hydroxylase receptor CI-MPR, while Formin family mediated nucleation of linear actin filaments at golgi regulates selective trafficking of the lysosomal enzyme cathepsin D. Similarly, the actin-severing protein ADF/cofilin, the mammalian ortholog of Drosophila twinstar, sculpts an actin-based sorting domain at the trans-golgi network for selective cargo sorting. It will be important to investigate whether the effects of Abl/Ena on golgi morphology have functional consequences on bulk secretion or protein sorting (Kannan, 2014).

Protein trafficking and membrane addition in neurons need to be coordinated with the growth requirements of the axonal and dendritic plasma membranes, but the mechanisms that do so have been obscure. Abl pathway proteins associate with many of the ubiquitous guidance receptors that direct axon growth and guidance throughout phylogeny, including Netrin, Roundabout, the receptor tyrosine phosphatase DLAR, Notch and others. The data therefore suggest a potential link between the regulatory machinery that senses guidance information and the secretory machinery that helps execute those patterning choices. Indeed, preliminary experiments suggest that some of the axonal defects of Abl pathway mutants may arise from alterations in golgi function. Beyond this, Abl signaling is essential in neuronal migration, epithelial polarity and integrity, cell adhesion, hematopoiesis and oncogenesis, among other processes The data reported in this study now compel a reexamination of the many functions of Abl to ascertain whether some of these effects arise, at least in part, from regulation of secretory function (Kannan, 2014).

Abl and Canoe/Afadin mediate mechanotransduction at tricellular junctions

Epithelial structure is generated by the dynamic reorganization of cells in response to mechanical forces. Adherens junctions transmit forces between cells, but how cells sense and respond to these forces in vivo is not well understood. This study identified a mechanotransduction pathway involving the Abl tyrosine kinase and Canoe/Afadin that stabilizes cell adhesion under tension at tricellular junctions in the Drosophila embryo. Canoe is recruited to tricellular junctions in response to actomyosin contractility, and this mechanosensitivity requires Abl-dependent phosphorylation of a conserved tyrosine in the Canoe actin-binding domain. Preventing Canoe tyrosine phosphorylation destabilizes tricellular adhesion, and anchoring Canoe at tricellular junctions independently of mechanical inputs aberrantly stabilizes adhesion, arresting cell rearrangement. These results identify a force-responsive mechanism that stabilizes tricellular adhesion under tension during epithelial remodeling (Yu, 2020).

This study shows that Canoe and Abl function in a mechanotransduction pathway that regulates dynamic changes in cell adhesion at tricellular junctions during epithelial remodeling. Canoe localization to tricellular junctions is acutely disrupted by laser ablation, demonstrating that Canoe localization is rapidly modulated by mechanical perturbation. Canoe mechanosensitivity requires Abl-dependent phosphorylation of a conserved tyrosine (Y1987) in the Canoe actin-binding domain, and Canoe localization to tricellular junctions is required to stabilize tricellular adhesion. Conversely, constitutively anchoring Canoe at tricellular junctions arrests cell rearrangement, indicating that Canoe levels can tune the rate of junctional remodeling. These results demonstrate that the mechanosensitivity of this critical junctional regulator is modulated by phosphorylation of a single tyrosine residue and reveal an essential role of Canoe in coupling tricellular adhesion with mechanical forces during epithelial remodeling (Yu, 2020).

Mechanical forces trigger a cascade of molecular events in cells that translate biophysical signals into altered cellular behaviors. However, the mechanosensors that directly change conformation under tension in vivo are not well defined. Cell surface receptors are well positioned to detect forces generated by neighboring cells, but Canoe localization and function at tricellular junctions do not require the PDZ domain that mediates its interaction with known receptors. One possibility is that the Canoe protein itself could act as a mechanosensor. Canoe is predicted to be anchored to the membrane through interactions with Rap1 and the actin cytoskeleton. Thus, cytoskeletal tension could stretch the Canoe protein and expose tyrosine 1987 to phosphorylation by Abl. Alternatively, Abl or its upstream activators could be regulated by tension during this process, because the activity of Abl and other tyrosine kinases such as Src and FAK has been shown to be regulated by tension in vitro. In a third possibility, the regulation of Canoe by Abl could allow Canoe to detect force-induced changes in other molecules at tricellular junctions. Because tyrosine 1987 is in the Canoe actin-binding domain, phosphorylation at this site could allow Canoe to recognize distinct actin structures at tricellular junctions, force-induced conformational changes in the cadherin-catenin complex, or other specialized features of tricellular junction composition or geometry. Once recruited to tricellular junctions, Canoe could stabilize adhesion at these sites by reinforcing the connection between adherens junctions and the actomyosin cytoskeleton (Yu, 2020).

Abl tyrosine kinases influence many structural changes that are driven by mechanical forces in tissues, including epithelial remodeling, tissue invagination, axon guidance, and cell migration. Abl has been shown to regulate a number of proteins that act at tension-bearing structures in cells in addition to Canoe/Afadin recruitment to tricellular junctions, including vinculin localization and β-catenin recycling at bicellular junctions and regulation of membrane curvature by BAR-domain proteins. Together, these results raise the possibility that Abl could transduce mechanical forces into a wide range of structural changes within cells. For example, during cell rearrangement, Abl could simultaneously stabilize tricellular adhesion by recruiting Canoe and destabilize bicellular adhesion by enhancing β-catenin turnover, which could allow bicellular junctions to complete contraction before tricellular junctions are remodeled. Tricellular junctions serve many important roles in epithelial development and homeostasis, including modulating cell rearrangement, orienting mitotic spindles, balancing stem cell proliferation and differentiation, and maintaining epithelial barrier function. An understanding of how mechanical inputs affect the conformation, localization, activity, and interactions of proteins at tricellular junctions will provide insight into how these structures sense and integrate mechanical forces in epithelial tissues (Yu, 2020).

Soldano, A., Okray, Z., Janovska, P., Tmejova, K., Reynaud, E., Claeys, A., Yan, J., Atak, Z. K., De Strooper, B., Dura, J. M., Bryja, V. and Hassan, B. A. (2013). The Drosophila homologue of the amyloid precursor protein is a conserved modulator of Wnt PCP signaling. PLoS Biol 11: e1001562. PubMed ID: 23690751

Marquilly, C., Busto, G. U., Leger, B. S., Boulanger, A., Giniger, E., Walker, J. A., Fradkin, L. G. and Dura, J. M. (2021). Htt is a repressor of Abl activity required for APP-induced axonal growth. PLoS Genet 17(1): e1009287. PubMed ID: 33465062

Htt is a repressor of Abl activity required for APP-induced axonal growth

Huntington's disease is a progressive autosomal dominant neurodegenerative disorder caused by the expansion of a polyglutamine tract at the N-terminus of a large cytoplasmic protein. The Drosophila huntingtin (htt) gene is widely expressed during all developmental stages from embryos to adults. However, Drosophila htt mutant individuals are viable with no obvious developmental defects. This study asked if such defects could be detected in htt mutants in a background that had been genetically sensitized to reveal cryptic developmental functions. Amyloid precursor protein (APP) is linked to Alzheimer's disease. Appl is the Drosophila APP ortholog and Appl signaling modulates axon outgrowth in the mushroom bodies (MBs), the learning and memory center in the fly, in part by recruiting Abl tyrosine kinase. htt mutations were found suppress axon outgrowth defects of alphabeta neurons in Appl mutant MB by derepressing the activity of Abl. Abl is required in MB alphabeta neurons for their axon outgrowth. Importantly, both Abl overexpression and lack of expression produce similar phenotypes in the MBs, indicating the necessity of tightly regulating Abl activity. Htt behaves genetically as a repressor of Abl activity, and consistent with this, in vivo FRET-based measurements reveal a significant increase in Abl kinase activity in the MBs when Htt levels are reduced. Thus, Appl and Htt have essential but opposing roles in MB development, promoting and suppressing Abl kinase activity, respectively, to maintain the appropriate intermediate level necessary for axon growth (Marquilly, 2021).

Neurodegenerative disease (ND) encompasses a large and heterogeneous group of maladies, including many that are associated with accumulation of specific misfolded proteins. Despite their variety, however, these diseases share a number of cellular pathologies, raising the question of whether different ND-associated genes might function in shared genetic pathways. Several of these disease genes, moreover, have been implicated in neurodevelopmental processes, suggesting that studies of development may be an effective strategy to reveal initially cryptic connections among genes implicated in ND (Marquilly, 2021).

Huntington's disease (HD) is a progressive, autosomal dominant, neurodegenerative disorder. It is a monogenic disease caused by the expansion of a polyglutamine (polyQ) tract at the N-terminus of a large cytoplasmic protein (3144 a.a.), Huntingtin (Htt). Several studies indicate that an alteration of wild-type Htt function might also contribute to disease progression. Consistent with this, numerous biochemical and in vitro studies have suggested that Htt functions in mammalian neuronal development, synaptic function and axonal trafficking. While HD has been characterized as a neurodegenerative disease, a recent study indicates it is also required for normal human brain development (Marquilly, 2021).

Drosophila has been useful as a model to examine the effects of polyQ-expanded human huntingtin transgenes on neuronal form and function. The fly Huntingtin protein (Htt, 3583 a.a.), although lacking a polyQ tract, is similar to the human Htt protein, with four regions of high sequence homology clustered along the protein in the N-, central- and C-terminal regions. Fly Htt is expressed ubiquitously at low level in embryos, larval and adult tissues, with no specific pattern of expression. Fly Htt is found predominantly in the cytoplasm, even when overexpressed. Despite htt being highly conserved across all Drosophila species, indicating an essential role for biological fitness, null htt mutants display no gross developmental defects, although brains from Drosophila Htt mutants have reduced axon complexity. This suggests that it could be necessary to alter the expression of another gene (or genes) during development to be able to detect a htt loss of function phenotype. Therefore, it was asked whether mutant htt modifies a brain axon growth defect present when another neurodegeneration-related protein is lacking, i.e., in a sensitized genetic background (Marquilly, 2021).

In contrast to HD, Alzheimer's Disease (AD) is highly genetically complex. Like HD, AD is also viewed as a proteinopathy, since it is associated with accumulation of amyloid fibrils derived from the Amyloid Precursor Protein (APP). APPs have therefore been investigated intensely, however their normal function in the brain remains unclear and controversial. Drosophila encodes a single APP homologue, called Appl, that is expressed in all neurons throughout development. It has been shown that Appl is a conserved neuronal modulator of a Wnt planar cell polarity (Wnt/PCP) pathway. This signaling pathway is essential for proper axon outgrowth in the learning and memory center of the fly, a bilaterally symmetric pair of structures called the Mushroom Bodies (MB). In this context, it has been proposed that Appl is part of the membrane complex formed by the core PCP receptors, and further that it promotes phosphorylation of the Dishevelled (Dsh) cytoplasmic adaptor protein. Dsh is a core component required for all known Wnt pathways, including the Wnt/PCP pathway. Specifically, Appl recruits a non-receptor protein tyrosine kinase, called Abl, to the PCP receptor complex and positively modulates its phosphorylation of Dsh (Soldano, 2013). Consistent with this view, it has been shown that a 50% reduction of Abl leads to enhancement of the Appl mutant phenotype. Conversely, overexpression of wild-type Abl+, but not a kinase-dead version, in the MB neurons led to a strong rescue of the Appl mutant phenotype. Together with accompanying biochemical experiments, these data suggested that Appl promotes the phosphorylation of Dsh by Abl kinase, and further showed that this mechanism is conserved in mammals (Soldano, 2013). Thus, Abl is a key downstream effector of Appl required for it to stimulate MB axon outgrowth (Marquilly, 2021).

The Abl family of non-receptor tyrosine kinases includes human ABL1 and ABL2 as well as Drosophila Abl. Each Abl protein shares a conserved domain structure consisting of a SH3-SH2-TK (Src homology 3-Src homology 2-tyrosine kinase) domain cassette which confers autoregulated kinase activity. A carboxy-terminal F (F-actin-binding) domain ties Abl-dependent phosphoregulation to actin filament reorganization. ABL1 has been implicated in a range of cellular processes including actin dynamics and cell migration. Abl was discovered as a cellular proto-oncogene that is constitutively active in human chronic myelogenous leukemia and acute lymphocytic leukemia. Kinase activity of Abl in vivo is limited both by intramolecular interactions and by cellular inhibitors, such as Pag/Msp23. After removal of inhibition, Abl acquires substantial catalytic activity that is further enhanced by primary and secondary (auto)phosphorylation. The Abl kinases have also been shown to play a crucial role in the development of the nervous system. Overexpression of active Abl in adult mouse neurons results in neurodegeneration and neuroinflammation and activation of Abl has been shown to occur in human neurodegenerative disease. In contrast to mammalian Abl, Drosophila Abl has not been shown to directly cause tissue hyperplasia or cell fate transformation in vivo, but rather is essential for cell adhesion and morphogenetic processes such as axonogenesis and growth cone motility. Several studies have shown that the precise level of Abl activity is critical to its axonal function, with loss- and gain-of-function both leading to severe defects in neural patterning. Recent experiments revealed the cell biological and biophysical basis of this relationship, showing that either increased or decreased levels of Abl activity induce disorder in growth cone actin and thereby greatly augment the frequency of stochastic errors in growth and guidance. Of particular relevance to this study, Abl has been implicated in axonal arborization and growth in the Drosophila brain, including the MBs though the precise role of Abl in normal MB development has not been documented (Marquilly, 2021).

The MBs, together with the central complex, form the core of the adult central brain of Drosophila. Due to extensive study, they offer an exceptionally powerful system for analyses of genetic and molecular mechanisms of development and function. The MBs are two bilaterally symmetric structures that are required for learning and memory. Each MB is comprised of 2000 neurons that arise from 4 identified neuroblasts. Three types of neurons appear sequentially during development: the embryonic/early larval β', the larval α'β' and the late larval/pupal αβ. Each αβ neuron projects an axon that branches to send an α branch dorsally, which contributes to the formation of the α lobe, and a β branch medially, which contributes to the formation of the β lobe. Both lobes require the PCP mechanism for efficient axon extension. The PCP genes, however, do not act cell-autonomously to promote MB axon growth. Rather, PCP produces a 'community effect' that coordinates the growth decisions of large groups of MB axons, and that overrides the effect of mutations in single PCP components in any single axon or cluster of axons. Appl is required for this PCP-dependent 'community effect'. In β-branches, Appl is evidently also required for some other mechanism that acts cell-autonomously, in addition to its contribution to the non-cell-autonomous PCP mechanism. The molecular nature of this second autonomous Appl function remains unknown (Marquilly, 2021).

This study investigated the genetic and functional interactions of Htt, Appl, Abl and the core PCP gene dsh in Drosophila. Whtt mutations suppress the MB axonal outgrowth defects observed in Appl mutants. Since Abl is known to act downstream of Appl it seemed a potential target for htt mutant-induced suppression of the Appl phenotype. Therefore this study characterized the role of Abl in normal MB development. Using analysis of Abl loss-of function (LOF) alleles in single-cell MARCM clones it was shown that Abl is required for axonal growth in the developing αβ neurons of the MBs and that it is expressed in these neurons. Importantly, the overexpression of Abl in these neurons also leads to axonal growth defects, suggesting the possible existence of cellular proteins that negatively control neuronal Abl activity. Finally, this study demonstrates that Htt acts as a cellular inhibitor of Abl activity, both genetically, as it functions antagonistically to Abl in MB axon growth, and biochemically, as FRET measurement of Abl kinase activity in vivo reveals that is derepressed by reducing Htt expression. These results indicate that Appl and htt, whose human homologs are central players in neurodegeneration, regulate Abl kinase in opposite directions to maintain its activity in the narrow range necessary for normal axon outgrowth in MB αβ neurons (Marquilly, 2021).

Tumorigenesis and neurodegeneration may be two sides of the same coin. Indeed, defining the overlap of molecular pathways implicated in cancer and neurodegeneration may open the door to novel therapeutic approaches for both groups of disorders. Correlative studies have highlighted a decreased cancer incidence in the population with the neurodegenerative disorder Huntington's disease and both wild-type and mutant huntingtin (Htt) have been implicated in tumor progression. Interestingly, it has been proposed that, in the normal physiological situation, the neurodegeneration-related Amyloid precursor protein (APP) recruits the oncogenic Abelson (Abl) kinase in order to promote axonal outgrowth (Soldano, 2013). It is, therefore, tempting to propose that different neurodegenerative diseases (ND) may share components and mechanisms and that Abl may also have a role in ND (Marquilly, 2021).

This study has shown that Abl is required for axonal growth in the MBs, a brain structure that is involved in memory. Furthermore, both Abl overexpression and lack of expression in the MBs result in similar phenotypes, indicating the need to tightly regulate Abl activity during MB axon outgrowth. This raises the question of how Abl activity is normally negatively regulated during MB axon outgrowth. This study confirmed the previous observation that overexpression of Abl rescues the Appld MB phenotypes (Soldano, 2013). Furthermore, Abl overexpression also rescues the dsh1 MB phenotype. These two results support the model that Appl activates Abl, which in turn phosphorylates Dsh. At the genetic level, it was expected that an increase of Abl activity in an individual bearing a loss-of-function mutation of a putative Abl repressor. It was therefore hypothesized that reducing the levels of an Abl repressor would result in suppression of the Appl and dsh mutant MB phenotype. Htt is such an inhibitor of Abl activity in the MBs. The loss of one dose of htt increased the activity of the wild-type Abl still present in Abl2/+ individuals and therefore prevented the enhancement of the MB mutant phenotype. While a number of studies have concluded that Htt deficiency results in significant alterations to kinase signaling pathways, this study is the first to implicate the crucial tyrosine kinase, Abl. Together, these data demonstrate the power that neurodevelopmental studies have to reveal close functional relationships between genes implicated in different forms of neurodegenerative disease (Marquilly, 2021).

It was a surprise that null htt mutants show no obvious developmental defects in Drosophila although strong defects could have been expected from the lack of such a conserved protein. One possible hypothesis is that, due to its fundamental importance, some functional redundancy has been selected to buffer against variation in the production of the Htt protein. It was reasoned that altering the levels of another protein, particularly another protein known to be implicated in neurodegeneration, could reveal cryptic phenotypes of htt mutation during brain development. Following this reasoning, a null Appl mutant was combined with heterozygous null htt mutations in double mutant individuals. Unexpectedly, it was found that mutant htt suppressed the Appl MB axonal outgrowth defect (Marquilly, 2021).

Abl is a key component of the Appl signaling pathway required for axonal arborization and growth in the fly brain and the functional relationship between these two proteins is likely conserved in mammals. While the role of APP-mediated signaling has been shown most clearly in Drosophila, a number of lines of evidence suggest that mammalian APP also fulfills a signaling role. Importantly, and in line with its proto-oncogenic role, Abl tyrosine kinase activity is tightly regulated by intramolecular inhibition. Although Abl is clearly required as a downstream effector of Appl in the MB axon growth, its precise role and regulation in the MBs has not been described previously (Marquilly, 2021).

The three Abl alleles used in this study have all been shown to result in truncated proteins. While Abl1 retains the SH3, SH2 and TK domains of Abl, Abl2 is mutated within the TK domain and only retains the SH3 and SH2 domains, while Abl4 is mutated in the SH2 domain, and only retains an intact SH3 domain. It therefore seems likely that very little or no residual Abl function remains in Abl2/Abl4 and Abl4/Abl1 individuals and may explain why no rescue was observed when one dose of htt was removed in these genetic backgrounds. In contrast, the Abl1/Abl2 allelic combination is likely less severe than the other two genotypes and Abl1/Abl2 animals do accumulate truncated Abl proteins. Indeed, while significant amounts of truncated Abl1 and Abl2 mutant proteins are detectable, only faint protein bands are observed in Abl4 pupae. Therefore, some functionally significant kinase activity could remain in Abl2/Abl1 individuals and the loss of one dose of htt might increase the activity of the remaining kinase activity. Finally, removing one dose of htt in animals overexpressing Abl would result in even more Abl activity, which in turn would exacerbate the mutant phenotype. Contrarily, over-expressing Htt, as in the UAS-Abl, UAS-htt doubly overexpressing individuals, would inhibit Abl function when compared to the UAS-Abl overexpression alone which thus might explain the observed rescue (Marquilly, 2021).

There are three different levels at which Htt might act to modulate Abl activity. First, Htt could either directly or indirectly affect Abl mRNA levels. Although fly Htt has been described as a cytoplasmic protein, htt has been shown to be a suppressor of position-effect variegation, suggesting a possible role in chromatin organization. This hypothesis is unlikely for the MB phenotype described in this study since qRT-PCR analysis of third instar brains did not reveal a significant differences in Abl mRNA levels between httint/+ and control individuals. Second, Htt could play a role regulating Abl protein level in the MBs. This is also considered unlikely since the quantity of the endogenous Abl is unchanged in httint/+ relative to control individuals. Third, Htt could influence the kinase activity of Abl itself. Taking advantage of a FRET biosensor enabling Abl kinase activity to be assayed directly in the MBs, a significant increase in active Abl was seen in httint/+ versus control individuals. Therefore, a model is favored of Htt acting as an inhibitor of Abl kinase activity during normal MB axonal growth. Abl activity in axons needs to be maintained within rather narrow limits. These two recent studies show that either increase or decrease of Abl activity cause disorganization of actin structure in the growth cone and prevent the orderly oscillation of growth cone actin that is the motor for growth cone advance and thus axon extension. Those papers also explain why Abl gain and loss can result in superficially similar mutant axon patterning phenotypes even though the molecular effects of Abl increase versus decrease are opposite (Marquilly, 2021).

The HEAT repeat domains of Htt are thought to function as a solenoid-like structure that acts as a scaffold and mediates inter- and intra-molecular interactions. It is therefore tempting to propose that a scaffolding role of Htt could elicit repression of Abl kinase activity. At least in the MBs, a balance seems to exist in the activity of Abl, positively regulated by the membrane complex formed by the core PCP proteins and Appl, and suppressed by Htt and possibly other proteins, as well. In one case, if Appl is absent, Abl is not optimally activated leading to defects in MB axon growth. Conversely, decrease of Htt to 50% of wild type levels leads to de-repression of Abl kinase activity, which in turn compensates for its sub-optimal level of activation in the absence of Appl. This unexpected apparent balance of activation and inhibition of Abl by Appl and htt, whose mutant orthologs are central players in human neurological disease, may define a conserved functional interaction to maintain Abl activity in the relatively narrow window to appropriately effect axon outgrowth (Marquilly, 2021).

Previous treatment of Abl in The Interactive Fly

The Philadelphia chromosomal translocation is responsible for the fusion of two genes, ABL and BCR. Recognized as a frequently occuring aberrant chromosome in acute lymphoblastic leukemia, it was understood more than a decade ago that the Philadelphia chromosome occurs as a result of a recombination between two genes: the cellular ABL gene on chromosome 9, and BCR (breakpoint cluster region) gene located on chromosome 22. The Drosophila Abelson (Abl) protein is a homolog of mammalian c-Abl the wild-type gene coding for the ABL sequence found in the BRC/ABL hybrid protein. The BCR/ABL oncogene product, derived from this specific chromosomal translocation is present in Philadelphia chromosome-postive acute lymphoblastic leukemia. Despite its homology to c-Abl, the wild type mammalian protein, Drosophila ABL protein shows distinct properties and functional differences from c-Abl that suggest the two proteins are not strictly comparable. Notably, the Drosophila protein shows no nuclear localization; this suggests that the functions listed below, found for the mammalian protein, are absent in Drosophila:

Assuming that no other Abl type proteins are found in Drosophila, and no nuclear function can be found for Drosophila Abl, Abl can be used to illustrate the extent of an evolutionary divergence of nuclear function.

Nevertheless, the cytoplasmic functions of Abl appear to be conserved. Both fly and mammalian proteins have a conserved actin interaction domain and both proteins show genetic or physical interaction with a conserved protein known as Disabled (Dab). The Drosophila Disabled protein colocalizes with Abl in cell bodies and axons of embryonic CNS neurons. Dab is essential for normal CNS development, even in the presence of Drosophila Abl. Dab is tyrosine phosphorylated in insect cells and given its co-localization with Abl in the CNS, it has been suggested that Dab may be a physiological substrate of Abl. Identification of Abl substrates is important in building a picture of the role of Abl in the CNS. Drosophila Abl is required in disabled heterozygotes after the time of cell fate specification and during the time of axonogenesis in the embryonic CNS (Gertler, 1993). Murine Disabled is expressed in certain neuronal and hematopoietic cell lines and is localized to the growing nerves of embryonic mice. During mouse embryogenesis, murine Disabled is tyrosine phosphorylated when the nervous system is undergoing dramatic expansion, but when nerve tracts are established, murine Disabled lacks detectable phosphotyrosine. The properties of murine Disabled and genetic analysis of Disabled in Drosophila suggest that these molecules function in key signal transduction pathways involved in a function downstream of Abl in axonogenesis (Howell, 1997). The involvement of Abl in axonogenesis does not require Abl's kinase domain (Henkemeyer, 1990).

The importance of Abl in neurogenesis is also evidenced by Abl's interaction with another conserved protein, Enabled. Enabled is a member of the rapidly expanding VASP family of cytoskeletal associated proteins. The well studied migration of neuronal growth cones serves as a model for the actin-driven formation of membrane protrusions. Establishment of proper connections in the central nervous system depends on the ability of neuronal growth cones to guide neurites to their final targets. The ABL-ENA-Profilin pathway is implicated in the process of axonal outgrowth and fasciculation. Genetic screens for dominant second-site mutations that suppress the lethality of Abl mutations in Drosophila identify alleles of only one gene, enabled. The ENA protein contains proline-rich motifs and binds to ABL and Src SH3 domains. ENA is (as Disabled has been suggested to be) also a substrate for the Abl kinase. Tyrosine phosphorylation of ENA is increased when it is coexpressed in cells with human or Drosophila Abl, and endogenous ENA tyrosine phosphorylation is reduced in Abl mutant animals. Like Abl, ena is expressed at highest levels in the axons of the embryonic nervous system and ena mutant embryos show defects in axonal architecture. Therefore, it has been concluded that a critical function of Drosophila ABL is to phosphorylate and negatively regulate ENA protein during neural development (Gertler, 1995).

Drosophia Abl interacts with four other fly genes: prospero, failed axon connections, and Fasciclin 1 and Fasciclin 2, two genes coding for cell adhesion proteins. The interaction with prospero has not been explored further since the non-uniform cytoplasmic distribution of Prospero protein was discovered. The action of Inscuteable, a cytoskeleton associated protein, suggests an involvement of cytoskeleton in providing cues for Prospero and Numb subcellular localization. Cytochalasin D, which disrupts Actin filaments, eliminates Inscuteable crescents and results in incorrect Prospero crescent positioning (Kraut, 1996). Since the highest levels of Abl are not in neuroblasts but in postmitiotic neurons (Bennett, 1992), the relation between Prospero and Abl is currently unknown.

The interaction of Abl with Fas1, Fas2, and failed axon connections suggests the involvement of Abl in a feedback mechanism in which the cytoskeleton of the cell is given information about cell adherence events occurring during axonogenesis. This might be the Drosophila function of Abl with greatest relevance to vertebrate neurobiology. Abl also seems to be involved in signaling in early embryonic development both in Drosophila and Xenopus. The nature of this involvement has not yet been documented.


GENE STRUCTURE

Bases in 5' UTR - 96

Exons - 10

Bases in 3' UTR - 1611


PROTEIN STRUCTURE

Amino Acids - 1520

Structural Domains

Abelson (Abl) gene consists of 10 exons extending over 26 kilobase pairs of genomic DNA. The DNA sequence encodes a protein of 1,520 amino acids with strong sequence similarity to the human c-abl proto-oncogene beginning in the type lb 5' exon and extending through the region essential for tyrosine kinase activity. When the tyrosine kinase homologous region is expressed in Escherichia coli, phosphorylation of proteins on tyrosine residues is observed. These results show that the abl gene is highly conserved through evolution and encodes a functional tyrosine protein kinase required for Drosophila development (Henkemeyer, 1988). There are five different 3' ends to ABL mRNA sequences, due to multiple polyadenylation sites separated from one another by as much as one kb (Telford, 1985).

The amino-terminal region of Abl tyrosine kinase related proteins shares several common features with Src-family members. These include a myristoylation site, and the arrangement of Src-homology domains in the primary sequence. Characteristic of the Abl-family members, however, is a large C-terminal extention beyond the kinase domain. The C-terminal segments are not well conserved among Abl-family members. For example, Drosophila Abl and the mammalian c-Abl are between 16-32% identical in this region. The divergent C-terminal segments are not interchangeable between Drosophila Abl and c-Abl, while the kinase domains are interchangeable. These results indicate that the C-terminal segments are important in specifying the biological functions of the Abl tyrosine kinases, and suggest that Drosophila Abl and c-Abl may perform different functions. Nevertheless, a C-terminal actin binding domain appears to be conserved. A presumed DNA binding domain and nuclear transport sequence is present in a region in c-Abl that is not conserved in Drosophila Abl (Wang, 1993 and references).

The Drosophila melanogaster abl and the murine v-abl genes encode tyrosine protein kinases (TPKs) whose amino acid sequences are highly conserved. To assess functional conservation between the two gene products, Drosophila abl/v-abl-chimeric Abelson murine leukemia viruses were constructed. In these chimeric Abelson murine leukemia viruses, the TPK and carboxy-terminal regions of v-Abl were replaced with the corresponding regions of Drosophila Abl. The chimeric Abelson murine leukemia viruses are able to mediate morphological and oncogenic transformation of NIH 3T3 cells and are able to abrogate the interleukin-3 dependence of a lymphoid cell line. A virus that contains both TPK and carboxy-terminal Drosophila Abl regions has no in vitro transforming activity for primary bone marrow cells and lacks the ability to induce tumors in susceptible mice. A virus that replaces only a portion of the v-Abl TPK region with that of Drosophila Abl has low activity in in vitro bone marrow transformation and tumorigenesis assays. These results indicate that the transforming functions of Abl TPKs are only partially conserved through evolution. These results also imply that the TPK region of v-Abl is a major determinant of its efficient lymphoid cell-transforming activity (Holland, 1990).


Abl tyrosine kinase: Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

date revised: 15 October 2005 

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