BIOLOGICAL OVERVIEW

Enabled is cytoskeletal regulator that facilitates continued actin polymerization at the barbed ends of actin filaments, induces cellular projections when overexpressed, and functions together with several different receptors (including Robo and UNC-40/DCC) implicated in axon guidance. This overview of Enabled starts with ABL, one of a number of mammalian cancer causing proteins (oncoproteins). ABL is a highly conserved nonreceptor tyrosine kinase containing SH3 and SH2 protein interaction domains. In less technical terms, ABL functions to modify other proteins by phosphorylation, and it attaches to other proteins by means of SH3 and SH2, two different protein interaction domains. One target of Drosophila ABL is Enabled, a protein that binds to ABL and other oncoproteins; Ena possess the Src homology 3 (SH3) protein interaction domain. SH2 and SH3 domains are found in many "adaptor" proteins that bind to other proteins in the progress of carrying out their function.

Recently a breakthrough in understanding Enabled and Abl has come from the cloning of a Mouse homolog of Enabled (Mena). Both Enabled and Mena are relatives of VASP (Vasodilator-Stimulated Phosphoprotein). A conserved domain of Mena targets it to proteins containing a specific proline-rich motif. The association of Mena with the surface of the intracellular pathogen Listeria monocytogenes and the G-actin binding protein Profilin suggests that Mena may participate in bacterial movement by facilitating actin polymerization. That is, Enabled and Mena, and indirectly ABL, serve to modify the actin based cytoplasm, an important agent in maintaining cell shape, regulating cell migration, and many other cell functions. In Drosophila, ABL and ENA act in the developmental processes of axon pathfinding and eye morphogenesis.

First the relationship of ABL to cancer will be examined because of the insight this provides to an understanding of ABL and Enabled function. Derivatives of ABL protein tyrosine kinase are involved in Philadelphia chromosome positive chronic myelogenous leukemia and acute lymphocytic leukemia in humans, and the pre-B-cell leukemia caused by Abelson murine leukemia virus in mice (from which ABL derives its name). The Philadelphia chromosome is generate by fusion between two normal human chromosomes. These translocations are not found at random, but in the middle of two genes, Abl and Bcr (named after the breakpoint cluster region). The product of this gene fusion is BCR-ABL protein, a hybrid protein able to confer a leukemia phenotype on cells in which it is expressed (Gertler, 1995 and references).

BCR is a multifunctional protein with a pseudo Kinase domain (apparently non-functional), a CDC24 domain and a GAP domain. CDC24 is a yeast gene that when mutated results in defective bud formation. It is believed that CDC24 plays a critical role in the establishment of cell polarity, development of normal cell shape, localization of secretion, and cell-surface deposition. The GAP (GTPase-activating protein) function targets p21 RAC, an important signaling protein in determining and modifying cell shape. Not only does BCR form a hybrid protein with ABL in the Philadelphia translocation, but BCR itself serves to target ABL, resulting in ABL activation (Arlinghaus, 1992 and references). When occuring on a composite protein, the interaction of BCR with ABL causes cell transformation. The chimeric protein products of these oncogenes exhibit elevated tyrosine kinase activity, oligomerization and association with the actin cytoskeleton, tyrosine phosphorylation of BCR sequences, association with signal transducing proteins GRB2, SHC, CBL, SYP and members of the 14-3-3 protein family (Gertler, 1995 and references).

The discovery that the bacterium Listeria recruits the cellular protein VASP, generated great intrigue in the scientific community. Using VASP, Listeria co-opts the cell's cytoskeleton to transport itself within the host cell it infects. VASP is a ligand for profilin, an actin-monomer binding protein that can stimulate the formation of filamentous actin, the non-muscle form of actin that serves as the basis for the actin based cytoskeleton of the cell. Listeria motility results from the rapid polymerization of F-actin at one pole of the bacterium, a process enhanced by host profilin. The properties of VASP make it a candidate host factor that can mediate the recruitment of profilin to the surface of Listeria, and thus promote F-actin assembly. Mena and Evl are two murine proteins highly related to Enabled as well as to VASP. A conserved domain in the N terminal portion of Mena and ENA targets these proteins to proline rich regions of other proteins. VASP interacts with zyxin, a LIM-domain protein localized in focal adhesions that shares a proline-rich motif with vinculin and ActA, two other cytoskeletal proteins. The proline rich region in VASP, which is shared with ENA and Mena, is most likely the profilin-binding site shared by these proteins. Mena itself has been shown to bind to the actin-binding protein Profilin. Localization of Mena to focal adhesions is mediated by its conserved N-terminus, and Mena, like VASP is recruited to the surface of Listera. Mena produces multiple isotypes. One is widely expressed, and a second is enriched in, or specific to, neural cells types. Thus Mena lends some understanding to the possible role of Drosophila ENA in axon pathfinding. (Gertler, 1996).

The well studied migration of neuronal growth cones serves as a paradigm for the actin-driven formation of membrane protrusions (Forscher, 1992). 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. The mechanism by which the growth cone advances is based on dynamic rearrangement of the actin based cytoskeleton, and it is in this process that Enabled and ABL affect axonogenesis.

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 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 have 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).

Abi acts cooperatively with Ena to antagonize Abl function in synaptogenesis

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 (abi^P1/KO and abi^P1/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).

GENE STRUCTURE

PROTEIN STRUCTURE

Amino Acids - 684

Structural Domains

The most notable feature of the predicted ENA protein is a proline-rich core, with 58 proline residues located between amino acids 340-480 and seven matches to the proline-rich consensus site for binding the ABl SH3 domain (Gertler, 1995).

The ActA protein of the intracellular pathogen Listeria monocytogenes induces a dramatic reorganization of the actin-based cytoskeleton. Two profilin binding proteins, VASP and Mena, are the only cellular proteins known so far to bind directly to ActA. This interaction is mediated by a conserved module, the EVH1 domain. E/DFPPPPXD/E, a motif repeated four times within the primary sequence of ActA, is identified as the core of the consensus ligand for EVH1 domains. This motif is also present and functional in at least two cellular proteins, zyxin and vinculin, which are in this respect major eukaryotic analogs of ActA. The functional importance of the novel protein-protein interaction was examined in the Listeria system. Removal of EVH1 binding sites on ActA reduces bacterial motility and strongly attenuates Listeria virulence. ActA-EVH1 binding is a paradigm for a novel class of eukaryotic protein-protein interactions involving a proline-rich ligand that is clearly different from those described for SH3 and WW/WWP domains. This class of interactions appears to be of general importance for processes dependent on rapid actin remodeling (Niebuhr, 1997).

The Enabled/VASP homology 1 (EVH1; also called WH1) domain is an interaction module found in several proteins implicated in actin-based cell motility. EVH1 domains bind the consensus proline-rich motif FPPPP and are required for targeting the actin assembly machinery to sites of cytoskeletal remodeling. The crystal structure of the mammalian Enabled (Mena) EVH1 domain complexed with a peptide ligand reveals a mechanism of recognition distinct from that used by other proline-binding modules. The EVH1 domain fold is unexpectedly similar to that of the pleckstrin homology domain, a membrane localization module. This finding demonstrates the functional plasticity of the pleckstrin homology fold as a binding scaffold and suggests that membrane association may play an auxiliary role in EVH1 targeting (Prehoda, 1999).

date revised: 8 dec 96 

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