InteractiveFly: GeneBrief

Abelson Interacting Protein: Biological Overview | References

One of the central regulators coupling tyrosine phosphorylation with cytoskeletal dynamics is the Abelson interactor (Abi). Its activity regulates WASP-/WAVE mediated F-actin formation and in addition modulates the activity of the Abelson tyrosine kinase (Abl). Drosophila Abi is capable of promoting bristle development in a wasp dependent fashion. This study reports that Drosophila Abi induces sensory organ development by modulating EGFR signaling. Expression of a membrane-tethered activated Abi protein (Abi^Myr) leads to an increase in MAPK activity. Additionally, suppression of EGFR activity inhibits the induction of extra-sensory organs by Abi^Myr, whereas co-expression of activated Abi^Myr and EGFR dramatically enhances the neurogenic phenotype. In agreement with this observation Abi is able to associate with the EGFR in a common complex. Furthermore, Abi binds the Abl tyrosine kinase. A block of Abl kinase-activity reduces Abi protein stability and strongly abrogates ectopic sensory organ formation induced by Abi^Myr. Concomitantly, changes were noted in tyrosine phosphorylation supporting previous reports that Abi protein stability is linked to tyrosine phosphorylation mediated by Abl (Stephan, 2008).

A variety of morphogenetic events during metazoan development such as cell fate determination, proliferation and differentiation require a highly regulated network of signaling pathways resulting in distinct cellular responses. Cell-surface receptors and tyrosine kinases often play an important role in the control of these processes. Among the growth factor receptors, the best understood example is the epidermal growth factor receptor (EGF receptor), which plays important roles in physiological and pathological processes. Unlike the vertebrate homologues the Drosophila genome contains only a single gene encoding a member of the EGF receptor family, called DER/EGFR. In Drosophila, the EGFR pathway is required at multiple times during fly development such as oogenesis, axis determination and sensory organ formation during embryonic and imaginal disc development (Stephan, 2008).

The emergence of Drosophila sensory organs depends on a selection process called lateral inhibition where only one cell is singled out of a group of equivalent proneural cells to become a sensory organ precursor (SOP). The Drosophila notum bears many mechano-sensory bristles, 22 large macrochaetes and about 200 smaller microchaetes. In both cases, bristle formation and spacing requires cell–cell communication mediated by lateral inhibition. The microchaetes on the notum are arranged in rows and are variable in number and position within the rows. Unlike the microchaetes, the large macrochaetes are not arranged in rows, their number is constant and they are formed from proneural clusters at fixed stereotypic positions (Stephan, 2008 and references therein).

Once determined, the SOP undergoes a series of stereotyped asymmetric cell divisions giving rise to the different cells comprising an individual sensory organ. In Drosophila, two major antagonistic signaling pathways regulate the formation of SOPs, the Notch and the EGFR signaling pathways. Notch is pivotal for the process of lateral inhibition which restricts the formation of neural cells. Alternatively, activation of the EGF receptor (EGFR) positively regulates SOP formation. Loss of EGFR function leads to a loss of sensory macrochaetes whereas gain of EGFR-function evokes the formation of additional macrochaetes by stimulating proneural gene autoregulation (Stephan, 2008).

Upon EGFR activation in Drosophila a membrane-localized protein complex is formed acting through the Ras-signaling. The activation of Ras results in the translocation of di-phosphorylated MAPK from the cell surface to the nucleus, where it leads to the expression of the proneural gene scute, which in turn promotes sensory organ development (Stephan, 2008).

This study shows that Abi regulates sensory organ development by modulating EGFR signaling. Following expression of an activated Abi (Abi^Myr) in proneural clusters, an increase was noted in MAPK activity. Inhibition of the EGFR pathway suppresses the formation of ectopic sensory organs evoked by Abi^Myr. Furthermore, co-expression of activated Abi^Myr and EGFR dramatically enhances the induction of extra-sensory organs. This effect also depends on the presence of active Abl tyrosine kinase, which stabilizes and further stimulates Abi by tyrosine phosphorylation (Juang, 1999). The physical interaction of Abi and EGFR depends on the membrane recruitment whereas the ability of the complex to induce neural development depends on both the membrane localization and the C-terminal SH3 domain of Abi, which can bind Wasp as well as the Abl kinase. These data suggest that once Abi is recruited to the membrane, EGFR signaling becomes activated to promote SOP development (Stephan, 2008).

Thus, overexpression of the Abelson kinase and its activator Abi is sufficient to promote the formation of ectopic sensory organs. Previous work has shown a partial loss of sensory organs in adult flies expressing a double-stranded abi RNA (Bogdan, 2005). However, expression of double-stranded RNA can lead to off target effects and confirmation of the mutant phenotypes by genetic analysis is needed. To address this question abi null alleles were generated. However, animals lacking zygotic abi function die relatively late during development as early pupae and no defects can be noted in the SOP pattern. This suggests that either abi is not required for SOP development or that its function is obscured by maternal contributions. In support for this notion, residual Abi protein can be detected in homozygous abi mutant larvae. In addition as previously reported for the abl kinase gene, abi might be integrated into a complex genetic network that masks direct requirements. The role of Abl is only exposed by the simultaneous loss of interaction partners such as disabled or failed axon connections (Stephan, 2008).

The Abi proteins are versatile molecules comprised of multiple protein-protein interaction domains allowing the formation of different protein complexes. Abi does not only function as an activator of the Abelson kinase but in turn Abl positively regulates Abi stability through phosphorylation (Juang, 1999; Huang, 2007). Moreover Abi plays an important role in regulating F-actin dynamics by activating Wave and Wasp proteins, two main activators of Arp2/3-dependent actin polymerization (Bogdan, 2005; Dai, 1995; Innocenti, 2005; Innocenti, 2004; Kunda, 2003; Stephan, 2008 and references therein).

How can alterations in actin dynamics modulate EGFR signaling? Previous studies have shown that cytoskeletal rearrangements are likely to induce the asymmetric distribution and clustering of the EGFR in neuronal stem cells to generate diverse CNS progenitor cells. Since Abi functions as a potent activator of Wasp (Bogdan, 2005; Innocenti, 2005), the enhanced formation of F-actin may lead to clustering of the EGF receptor at the membrane to stimulate its sustained activation (Stephan, 2008).

The enhancement of actin polymerization may also affect endocytosis of the EGFR, which is F-actin dependent and as recently shown can be promoted by Abi and Wasp activity (Benesch, 2005; Innocenti, 2005; Sirotkin, 2005). Although it has long been assumed that endocytosis is the main pathway involved in down-regulation of the EGFR, endocytosis is also directly linked to EGFR activation. Block of endocytosis of the EGFR specifically abrogates MAPK activity. Thus Abi could be involved establishing active signaling compartments. Unfortunately, at present the molecular tools to determine the subcellular localization of the activated EGFR in the developing fly are unavailable (Stephan, 2008).

In addition, activation of the Abi complex may recruit kinases such as Abl to the EGF receptor. Abi acts as a positive regulator of the Abl kinase activity promoting the phosphorylation of several substrates including Mena/Ena, Wave, N-Wasp, the B-cell adaptor for phosphoinositide 3-kinase (BCAP) or Cdc2 (Burton, 2005; Leng, 2005; Lin, 2004; Maruoka, 2005; Tani, 2003). Interestingly, Abi is also a substrate of Abl resulting in increased protein stability (Juang, 1999; Huang, 2007). This positive regulatory loop could hint to an important mechanism for regulating the level of Abl kinase activity during sensory organ development. Indeed, co-expression of Abi^Myr with a kinase-defective Abl suppresses tyrosine hyperphosphorylation as well as reduces the level of the Abi^Myr protein expression, corresponding to a strong suppression of the number of ectopic sensory organs induced by Abi^Myr (Stephan, 2008).

However, the induction of ectopic sensory bristles by Abl does not depend on an intact EGFR signaling or on an activation of the Ras/Raf/MAPK pathway. Co-expression of Abl and EGFR results in an increase of sensory organs but inhibition of EGFR by co-expression of Kekkon1 or heterozygous loss of pole hole function are not sufficient to suppress the Abl-induced neurogenic phenotype suggesting that Abi and Abl act in parallel. In conclusion, it is proposed that the complex of Abi and the EGFR is joined by Abl, which stabilizes not only Abi but also stimulates Wasp function, highlighting the intricate interaction between cytoskeletal dynamics and signaling during sensory organ development (Stephan, 2008).

Abi plays an opposing role to Abl in Drosophila axonogenesis and 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).

Abi activates WASP to promote sensory organ development

Actin polymerization is a key process for many cellular events during development. To a large extent, the formation of filamentous actin is controlled by the WASP and WAVE proteins that activate the Arp2/3 complex in different developmental processes. WAVE function is regulated through a protein complex containing Sra1, Kette and Abi. Using biochemical, cell biological and genetic tools, this study shows that the Abi protein also has a central role in activating WASP-mediated processes. Abi binds WASP through its carboxy-terminal domain and acts as a potent stimulator of WASP-dependent F-actin formation. To elucidate the biological function of abi in Drosophila, bristle development, a process known to require wasp function, was studied. Reduction of abi function leads to a loss of bristles similar to that observed in wasp mutants. Activation of Abi results in the formation of ectopic bristles, a phenotype that is suppressed by a reduction of wasp activity but is not affected by the reduction of wave function. Thus, in vivo Abi may set the balance between WASP and WAVE in different actin-based developmental processes (Bogdan, 2005). In a mammalian system Abi also binds and activates N-WASP, suggesting a functional conservation of this central player (Innocenti, 2005).

Developmental processes involving movement and cell shape changes are based on a highly dynamic reorganization of the actin cytoskeleton. The Arp2/3 complex is an important regulator of actin polymerization. Its activation is mediated by members of the Wiskott-Aldrich syndrome proteins (N-WASP/WAVE). WASP and N-WASP (neural WASP) are specific effectors of Cdc42 that are characterized by a CRIB (Cdc42/Rac interactive binding) domain and an NH₂-terminal WASP homology domain. By contrast, WAVE proteins lack these sequence motifs but possess a highly specific WAVE homology domain (WHD) and transduce Rac signalling to the Arp2/3 complex (Bogdan, 2005).

The in vivo roles of WASP and WAVE proteins are as different as their molecular architectures. This has been clearly established both in vertebrates and Drosophila. Whereas WAVE acts as the main Arp2/3 regulator during axonal growth, oogenesis and eye development, WASP is required for cell fate decisions during sensory organ development. This last function is independent of Cdc42 but is mediated through Arp2/3 (Bogdan, 2005).

To activate WASP, an intramolecular inhibition has to be released by factors such as Cdc42, phosphatidylinositol-4,5-bisphosphate, or several SH3-domain-containing proteins. In contrast, WAVE proteins are constitutively active in vitro. Activity is regulated by a multi-protein complex comprising Kette (NAP1, HEM), Sra-1 (CYFIP, PIR121), Abi (E3b1) and HSPC300. Initial work had suggested that WAVE1 is trans-inhibited and cannot stimulate Arp2/3 nucleation activity in vitro. This finding was supported by genetic data, showing that Kette or PIR121 antagonize WAVE function. However, new data clearly show that a reconstituted pentameric WAVE complex is constitutively active in stimulating Arp2/3 function and remains stable, even after Rac stimulation. This argues against a trans-inhibition model of WAVE function and raises the question as to whether in vivo additional proteins or post-translational modifications are required to control WAVE activity (Bogdan, 2005).

Surprisingly, Sra1 and Kette are also involved in the regulation of WASP. These unforeseen genetic interactions prompted an attempt to elucidate the functional relationship of these genes. Biochemical, cell biological and genetic data presented in this study clearly identify Abi as a central player in orchestrating the regulation of WASP, and thus define a new key component in directing cytoskeletal dynamics (Bogdan, 2005).

The Abi protein has a relative molecular mass of 52,000, and was first identified as a substrate of the Abelson kinase. During embryonic development, Abi shows a prominent coexpression with known members of the WAVE complex, Kette and Sra-1. It is maternally expressed and later in development becomes concentrated in the nervous system. Abi contains a C-terminal SH3 domain, which can interact with the Abelson kinase and the amino-terminal WAVE-interacting domain (WAB domain), which is required for binding and recruiting WAVE (Bogdan, 2005).

In addition, Abi binds Kette and WASP. Yeast two-hybrid experiments show that a deletion of the N-terminal 85 amino acids of the Drosophila Abi protein (AbiDeltaN) abrogates its interaction with WAVE but does not affect the interaction with Kette or WASP. Conversely, the C-terminal SH3 domain of Abi is necessary and sufficient for WASP interaction, and deletion of this domain blocks the interaction with WASP but not with WAVE. These deletion constructs also show that Abi binds Kette through its central domain (Bogdan, 2005).

To further test the interaction between Abi and WASP, glutathione S-transferase (GST) pull-down experiments were performed. Affinity purification of Abi from S2R+ cells using GST-WASP coupled to glutathione-Sepharose beads results in the copurification of Kette, which directly binds Abi, and Sra-1, which binds Kette. Significant amounts of WAVE were also found to be present in the protein complex bound to GST-WASP. Disruption of F-actin by Latrunculin A does not affect this coprecipitation. Furthermore, in WAVE pull-down experiments, some WASP protein could be detected in the precipitate. As expected, Abi is able to coprecipitate WAVE as well as WASP. Since WASP cannot bind directly to WAVE in a yeast two-hybrid assay, these findings suggest that Abi is in a position to coordinate the activity of two main regulators of the Arp2/3 complex (Bogdan, 2005).

As described for mammalian cells, endogenous WAVE is distributed relatively uniformly at the leading edge in Drosophila S2R+ cells. Abi and WASP show a more dotted expression pattern. Thus, Abi, WASP and WAVE are all localized at the leading edge of the cell, as reported for N-WASP and WAVE2 in lamellipodia of mouse myoblasts (Bogdan, 2005).

To determine the functional relevance of the proteins, RNA interference (RNAi) experiments were performed. RNAi-mediated silencing of abi, kette or sra-1 in S2 cells leads to a collapse of all lamellipodia-like structures and induces a starfish-like morphology. Reduction of WASP expression results in a more complex phenotype and the most sensitive phenotypic trait appeared to be the cell size. Twenty per cent of the cells showed a normal F-actin pattern but were consistently smaller than control cells. In 55% of the cells, lamellipodia appeared to collapse, leaving isolated F-actin-rich cell extensions. A requirement of the mammalian N-WASP for lamellipodia formation has also been observed in mammalian cell lines. Twenty-five per cent of the cells showed a starfish-like phenotype, which is similar to the abi knockdown phenotype. These effects are dependent on the wasp RNAi dose and require efficient protein silencing (Bogdan, 2005).

In contrast to the loss-of-function phenotype, even high levels of Abi expression did not affect cell morphology and F-actin organization. Ectopically expressed Abi is distributed similarly to the endogenous protein, but increased levels are associated with F-actin. It has been shown that membrane recruitment of Kette or Sra-1 results in their activation and induces WASP-dependent phenotypes in vivo. Expression of membrane-tethered Abi (Abi^Myr) or of a N-terminal deletion variant lacking the WAVE interaction domain Abi (Abi^DeltaN) results in extensive filopodia formation in about 50% of the transfected cells. However, deletion of the C-terminal SH3 domain from Abi^Myr, which mediates binding to WASP, abrogates this effect. If Abi^Myr acts through WASP, then a membrane-tethered WASP should also induce the formation of filopodia-like structures. Indeed, WASP^Myr expression induces a regularly spaced array of filopodia-like structures in about 80% of the cells. Whereas wild-type S2R+ cells are characterized by membrane ruffling, both Abi^Myr and WASP^Myr induce small and dynamic cell extensions. In contrast, reduction of abi function by RNAi leads to less dynamic structures and finally cell collapse. To test whether the Abi^Myr phenotype depends on WASP or WAVE, RNAi experiments were performed in cells expressing Abi^Myr. In both cases the knockdown phenotype of WAVE or WASP is epistatic to the Abi^Myr-induced phenotype, indicating that in S2R+ cells WAVE either performs vital functions, or that WAVE-dependent formation of lamellipodia is a prerequisite for Abi^Myr-induced filopodia formation (Bogdan, 2005).

Because Abi binds WASP and can induce WASP-dependent phenotypes, whether the regulation of F-actin formation can be reconstituted in vitro was tested. Drosophila WASP and Abi proteins expressed in Escherichia coli were purified and their ability to activate mammalian Arp2/3 was tested in an actin polymerization assay. Drosophila WASP has only a small effect on Arp2/3-dependent actin polymerization kinetics by itself. The addition of Abi significantly increases this basal activity in a dose-dependent manner (Bogdan, 2005).

These findings prompted similar experiments in vivo. The expression of different Abi transgenes were induced using the Gal4-UAS system. Overexpression of Abi^Myr in wing imaginal discs results in a significant elevation in the level of F-actin. The en-Gal4 driver allows the anterior compartment of the wing imaginal disc to be used as a negative control. Upon expression of high levels of Abi^Myr, F-actin fibres are rearranged and the free G-actin pool appears to be reduced. When WASP function is present, F-actin fibres extend into Abi^Myr-expressing processes. The elevation in actin polymerization was neither observed upon overexpression of wild-type Abi nor upon overexpression of Abi^Myr lacking the C-terminal SH3 domain, suggesting that both membrane localization and interaction with WASP are required for F-actin formation. To further test the requirement for WASP, a C-terminal truncation (WASP^DeltaCA) was used that still contains the proline-rich sequences binding to Abi but lacks sequences required for Arp2/3 complex binding. Expression of WASP^DeltaCA results in a wasp-like mutant phenotype. Coexpression of membrane-tethered Abi and the dominant-negative WASP^DeltaCA construct clearly suppressed ectopic F-actin formation, indicating that Abi^Myr stimulates actin polymerization via WASP. Similarly, the effects caused by Abi^Myr expression could be suppressed by reducing the wasp gene dose, clearly showing that Abi positively regulates WASP activity in vitro and in vivo. Expression of WASP^DeltaCA alone does not have any effect on F-actin or G-actin distribution. The specificity of F-actin and G-actin detection was demonstrated by Latrunculin A treatment (Bogdan, 2005).

The functional relevance of abi was examined in the developing fly, focusing on the development of the sensory organs. Drosophila harbours only one gene encoding a WASP-like protein. Homozygous zygotic wasp mutants survive until adulthood and show a loss of bristles. Further analyses showed that WASP governs cell-fate decisions of specific external sensory organs that develop from proneural clusters. Expression of the dominant-negative WASP^DeltaCA by the sca-GAL4 driver, which activates expression in the proneural clusters, leads to a loss of thoracic bristles similar to but not as extreme as is seen in wasp mutant flies. Likewise, reduction of abi function by in vivo expression of double-stranded abi RNA results in a similar bristle phenotype. In addition, the bristles are shorter and thinner compared with the wild type. In contrast, expression of Abi^Myr resulted in the formation of additional bristles, independent of the GAL4 driver used (ptc-GAL4) and the wave gene dosage. Expression of an Abi^Myr protein lacking the N-terminal WAVE interaction domain still induces the formation of extra bristles. These experiments show that Abi does not act through WAVE during Drosophila bristle development. Moreover, heterozygous loss of wasp function or coexpression of the dominant-negative wasp^DeltaCA transgene partially suppresses the formation of ectopic bristles induced by Abi^Myr, which is most clearly seen for the notopleural bristles. By contrast, deletion of the C-terminal WASP-binding domain of Abi abrogates the ability of Abi^Myr to induce bristles. Thus, in vivo Abi acts through WASP (Bogdan, 2005).

During development of Drosophila sensory organs, a single cell is selected out of a group of equivalent proneural cells (proneural cluster) by lateral inhibition to become a sensory organ precursor (SOP). The SOP undergoes a series of stereotyped asymmetric cell divisions giving rise to the different cells comprising an individual sensory organ. Zygotic wasp mutants show defects in asymmetric SOP division and lack bristles. Because a WASP protein lacking the CA domain is not able to rescue this phenotype, Arp2/3-dependent processes are involved in establishing cell fate (Bogdan, 2005).

To test directly whether Abi affects asymmetric cell division or promotes the allocation of neural fate the number of SOPs was determined using neuralized-lacZ or hindsight expression. Wild-type wing imaginal discs are characterized by a stereotypic patterning of SOPs. Expression of abi RNAi in proneural clusters results in a loss of many SOPs, which is similar to the wasp mutant phenotype. In contrast, expression of Abi^Myr in proneural clusters leads to the formation of additional hindsight-expressing cells, indicating the formation of additional SOPs; all dorsocentral, scutelar and notopleural SOPS are affected; on average, three (instead of one) SOPs are found (Bogdan, 2005).

In summary, these findings place the Kette-Sra-1-Abi complex at a key position coordinating the formation of F-actin by regulating WAVE and WASP proteins. Furthermore, Abi-WASP is involved in SOP specification. The formation of SOPs is regulated by two main antagonistic pathways. Whereas Notch restricts the formation of neural cells, activation of the epidermal growth factor receptor (EGFR) promotes SOP formation. WASP forms a complex with EGFR and transmits Nck-dependent signals from EGFR to polarize cortical actin filaments. In turn, cytoskeletal rearrangements lead to receptor clustering and their subsequent activation. These experiments highlight the possibility that reverse signalling may occur in vivo and point to the interwoven relationship between receptor signalling and actin dynamics that is mediated by Abi and WASP. Thus, Abi functions as a central regulator that directs WASP- or WAVE-dependent developmental processes, presumably with the help of additional factors (Bogdan, 2005).

The involvement of Abl and PTP61F in the regulation of Abi protein localization and stability and lamella formation in Drosophila S2 cells.

Most aspects of cellular events are regulated by a series of protein phosphorylation and dephosphorylation processes. Abi (Abl interactor protein) functions as a substrate adaptor protein for Abl and a core member of the WAVE complex, relaying signals from Rac to Arp2/3 complex and regulating actin dynamics. It is known that the recruitment of Abi into the lamella promotes polymerization of actin, although how it does this is unclear. PTP61F, a Drosophila homolog of mammalian PTP1B, can reverse the Abl phosphorylation of Abi and colocalizes with Abi in Drosophila S2 cells. Abi can be translocalized from the cytosol to the cell membrane by either increasing Abl or reducing endogenous PTP61F. This reciprocal regulation of Abi phosphorylation is also involved in modulating Abi protein level, which is thought to affect the stability of the WAVE complex. Using mass spectrometry, several important tyrosine phosphorylation sites were identified in Abi. The translocalization and protein half-life of wild type (wt) and phosphomutant Abi and their abilities to restore the lamellipodia structure of the Abi-reduced cells were compared. The phosphomutant was found to have reduced ability to translocalize and to have a protein half-life shorter than that of wt Abi. Although the wt Abi could fully restore the lamellipodia structure, the phosphomutant could not. Together, these findings suggest that the reciprocal regulation of Abi phosphorylation by Abl and PTP61F may regulate the localization and stability of Abi and may regulate the formation of lamella (Huang, 2007).

Tyrosine phosphorylation/dephosphorylation is a common and important post-translational modification of key signaling proteins. Although Abi was originally identified as a kinase substrate of Abl, little is known about how phosphorylation contributes to its biological significance. This study demonstrates that the level of Abi tyrosine phosphorylation is balanced between phosphorylation by Abl and dephosphorylation by PTP61F. This conclusion is based on several lines of evidence. First, RNAi knockdown of PTP61F expression greatly elevates the tyrosine phosphorylation level of endogenous Abi in the Drosophila S2 cells. PTP61F was shown to be a major physiological phosphatase of Abi by substrate trapping, GST pulldown, and colocalization experiments. Second, RNAi experiments also suggested that Abl is the major kinase mediator of tyrosine phosphorylation of Abi. Finally and most importantly, Abl-mediated tyrosine phosphorylation of Abi was shown to be counteracted by the dephosphorylation of a wild-type but not the catalytically inactive PTP61F proteins. Taken together, these results indicate that the coordinated phosphorylating action of Abl and dephosphorylating action of PTP61F together coordinate to regulate the tyrosine phosphorylation level of Abi (Huang, 2007).

Because many adaptor proteins undergo protein relocalization in response to tyrosine phosphorylation and protein-protein interaction, whether the balance of tyrosine phosphorylation in Abi affected its localization subcellularly was investigated. In cells ectopically expressing Abl and Abi, both Abl and Abi proteins relocalize to the same cortex cell regions, where active actin polymerization occurs. The relocalization appears to be dependent, as least in part, on the kinase activity of Abl because the kinase inactive Abl would exhibit markedly reduced ability to relocalize Abi from a cytosolic punctate position to a plasma membrane position. This finding was further supported by additional experiments showing that a reduction in endogenous PTP61F could increase the relocalization of GFP-Abi from cytosol to the plasma membrane, and when Abi^4YF phosphomutant protein was expressed, there was a decrease in this translocation activity. It is thought that the Abl/PTP61F-mediated phosphorylation/dephosphorylation of Abi is critical in regulating Abi functions by relocalizing Abi from the cytoplasm to the cell membrane. The importance of Abi phosphorylation is also evident in a study of Abi protein stability. Using overexpression or RNAi knockdown approach to control Abl or PTP61F protein levels, it was demonstrated that both protein level and activity of these two enzymes are essential in the control of Abi protein stability. Furthermore, it was shown that the protein turnover rate of Abi^4YF is much faster than that of wt Abi, further supporting the idea that the tyrosine phosphorylation of Abi is critical in modulating its protein stability. Together, these results highlight the importance of the Abi phosphorylation/dephosphorylation in modulating its localization and protein stability (Huang, 2007).

Although the data indicate that Abl phosphorylation of Abi is critical in modulating Abi subcellular localization, the underlying mechanism behind this process remains elusive. Nevertheless, the Abl protein is known to contain a myristoyl group, a common module for anchoring a protein to a cell membrane. Moreover, recent structural studies, which found that removal of the myristoyl group activates c-Abl kinase activity, suggest that by binding myristoyl group to the kinase pocket of Abl, an inactive conformation may be promoted. Because Abi functions as a positive regulator of Abl kinase, the Abl-Abi interaction might induce a conformational change through which the buried region that bears an N-myristoylation signal is exposed. This would result in targeting the Abl·Abi complex to cell membrane. In fact, the data showed that the interaction of a kinase-defective Abl with Abi was also somewhat able to trigger the Abi relocalization from the cytosol to the cell membrane. It was also found that Abl phosphorylation of Abi in the complex further promoted the relocalization of Abi to cell membrane. One recent structural and biochemical study has shown that a phosphotyrosine ligand can further convert the Abl protein into a more 'open' conformation. It might be possible that the phosphotyrosine residues of Abi further opened the semi-unfolded position of myristoyl group in Abl, further promoting the relocalization of Abl·Abi complex (Huang, 2007).

Another intriguing issue is the unique subcellular localization of PTP61F. Immunocytochemistry experiments showed that most PTP61F colocalizes with Abi in the perinuclear region, presumably where the endoplasmic reticulum (ER) is situated, but not at the leading edge of the spreading cells, where Abi is preferentially localized. These finds regarding the subcellular localization of PTP61F in S2 cells is similar to those reported for its mammalian counterpart, PTP1B. PTP1B largely resides on the external face of the ER. Given the finding that PTP61F counteracts Abl phosphorylation of Abi, the spatial separation of PTP61F from Abi in the leading edge might suggest that the phosphorylation of Abi is less affected by PTP61F in this region and that Abi residing at the leading edge might exert a higher tyrosine phosphorylation signal than Abi in the perinuclear region. In fact, several groups have reported that a number of proteins localized to the leading edge of motile cells are tyrosine-phosphorylated. Nevertheless, the substrates of PTP targeting the cell periphery ultimately have to be dephosphorylated to terminate its role in signaling. To elucidate the mechanism underlying this process, three models have been proposed. First, the receptor tyrosine kinases, such as epidermal growth factor receptor and platelet-derived growth factor receptor, can be directed from plasma membrane to ER by vesicle trafficking for dephosphorylation by PTP1B. Second, the ER membrane-anchored PTP1B can be cleaved by the calcium-dependent neutral protease calpain to relocate its catalytic domain from membranes to the cytosol. Last, by reaching the ER network to the cell periphery, PTP1B at the local attachment site can interact with its substrates in the plasma membrane. These models together with the current findings suggest that there is a possible link between PTP61F and Abl signaling in the regulation of Abi phosphorylation at the leading edge of the plasma membrane (Huang, 2007).

Protein stability is also a critical factor for regulating Abi functions in actin dynamics. When Abi expression is lost in S2 cells, there are severe defects in the formation of lamella. However, Abi alone does not affect the formulation of lamella. Abi forms a tight complex with WAVE, Hem, and Sra-1 to regulate the formation of lamellipodia (Steffen, 2004; Stradal, 2001). Moreover, the lamellipodia formation is dependent on the integrity of a functioning WAVE macromolecular complex (WAVE·Abi·Hem·Sra-1) (Kunda, 2003; Rogers, 2003), but the down-regulation of Abi expression impairs the stability of WAVE complex in both mammalian and Drosophila cells (Stradal, 2006). Thus, the protein stability of Abi is highly correlated with its regulation of the formation of lamella. A previous studies demonstrated that the interaction between Abl and Abi leads to an increased protein level of Abi (Juang, 1999). This study has extended this finding by showing that the impaired Abl-mediated phosphorylation of Abi results in protein instability of Abi in S2 cells. It was also demonstrated that the ectopic expression of a wild type, but not an inactive, PTP61F also dramatically diminishes the Abi protein stability. Thus, these results indicate that tyrosine phosphorylation/dephosphorylation by Abl/PTP61F is critical in controlling Abi protein turnover. Because it has been suggested that Abi undergoes an ubiquitin-mediated proteolysis (Dai, 1998), it might be possible that that the Abl phosphorylation of Abi represses the proteolysis degradation. One recent study has demonstrated that the Abl phosphorylation of c-Jun blocks the access of E3 ubiquitin ligase, Itch, to its binding epitope of c-Jun and thus prevents c-Jun from proteosome-mediated proteolysis (Huang, 2007).

Although tyrosine phosphorylation of Abi is important in stabilizing Abi protein level, the protein interaction between Abi and Abl might also attribute to Abi protein stability. This study showed that the coexpression of a kinase-defective c-Abl and Abi resulted in a 1.8-fold increase of Abi protein level. Moreover, this protective effect probably is a common feature of both the Abi·Abl and Abi·WAVE·Hem·Sra-1 complexes. Preliminary data showed that the ectopic coexpression of Abi, Hem, and WAVE could enhance Abi protein level and that the reduction of Hem or WAVE markedly decreased the endogenous Abi protein level, suggesting that a constitutive complex of Abi with WAVE, Hem, and Sra-1 could protect Abi from post-translational degradation. This finding is also comparable with what was documented in the scenario of WAVE/Scar stability (Huang, 2007).

Following this logic, it is reasonable to speculate that a sizeable fraction of the ectopically expressed Abi proteins would be short of match protein members to form the protein complex. Under this circumstance, the Abi would be more dependent on the phosphorylation regulation mechanism to modulate its protein stability. Supporting this view, it was shown that the protein stability of exogenous Abi was particularly sensitive to the phosphorylation regulation. Although component proteins of the WAVE/Scar complex are thought to form a constitutive multiprotein complex in cells, a recent study showed that a small population of WAVE2 exists as a monomeric form without forming a complex with other component proteins in A431 cells. In addition to WAVE2, Hem/Nap1 was also reported to regulate cell motility through a WAVE/Scar-independent pathway in Dictyostelium and human neutrophil. These studies suggest that the component proteins of WAVE/Scar complex may present as a 'noncomplex' form of protein in regulating cell motility. Accordingly, although exogenous Abi is relatively independent of the physiological state, it is more amenable to the study of protein stability (Huang, 2007).

Another interesting issue related to the Abi stabilization by Abl is linked to a possibility that the stabilized Abi protein might in return further enhance the Abl kinase activity and Abi phosphorylation, thus establishing a positive feedback loop between Abl activity and Abi phosphorylation/stability. Because Abl kinase activity is tightly regulated in cells, this positive feedback loop should be eventually interrupted by a third molecule. PTP61F might be such a suppressor and function as a brake on the overactivated Abl-Abi signaling pathway by dephosphorylating Abi and facilitating protein degradation (Huang, 2007).

Finally, this study investigated whether a phosphomutant of Abi, which is not phosphorylated by Abl, could disrupt the lamella formation in motile cells. The experiments suggest that the expression of Abi^4YF was unable to rescue the lamella defects that were caused by treatment with Abi^3'-UTR RNAi, supporting the idea that Abl might act positively in promoting Abi regulation of the lamella formation. Two recent reports have demonstrated that the mutation on a critical Abl phosphorylation residue of WAVE can disrupt its role in actin-dependent cytoskeleton remodeling. The data might suggest that Abl phosphorylation on Abi promotes actin polymerization. Therefore, it was asked whether Abl also phosphorylates all the other WAVE members and whether that would in turn modulate their roles in actin dynamics. Preliminary data showed that Abl, via the linkage of Abi, could tyrosine phosphorylate each protein member of the WAVE complex and that process was also counteracted by PTP61F (Huang, 2007).

Although additional experiments are required to determine the physiological significance of the phosphorylation/dephosphorylation of WAVE complex proteins, it is possible that the opposing actions of Abl and PTP61F in all of the other WAVE members might play a role in fine-tuning actin polymerization in cells. To resolve this possibility, it will be important to investigate whether Abl/PTP61F phosphorylation/dephosphorylation of the WAVE complex is occurred in a consecutive or synchronized manner in actin polymerization (Huang, 2007).

This work has demonstrated that Abi undergoes protein modification by Abl and PTP61F via reversible tyrosine phosphorylation/dephosphorylation. This process regulates its relocalization to the cell membrane and protein stability, and ultimately it regulates the formation of lamella in motile cells. Therefore, the balance between Abl kinase and PTP61F phosphatase in regulating tyrosine phosphorylation of Abi may mediate the function of WAVE complex in cell periphery. This study furthers understanding of how and where the reciprocal regulatory processes of an actin-associated protein function within the cells (Huang, 2007).

The golgi comprises a paired stack that is separated at G2 by modulation of the actin cytoskeleton through Abi and Scar/WAVE

During the cell cycle, the Golgi, like other organelles, has to be duplicated in mass and number to ensure its correct segregation between the two daughter cells. It remains unclear, however, when and how this occurs. This study shows that in Drosophila S2 cells, the Golgi likely duplicates in mass to form a paired structure during G1/S phase and remains so until G2 when the two stacks separate, ready for entry into mitosis. Pairing requires an intact actin cytoskeleton which in turn depends on Abi/Scar but not WASP. This actin-dependent pairing is not limited to flies but also occurs in mammalian cells. It is further shown that preventing the Golgi stack separation at G2 blocks entry into mitosis, suggesting that this paired organization is part of the mitotic checkpoint, similar to what has been proposed in mammalian cells (Kondylis, 2007).

During the cell cycle, the Golgi, like other organelles, has to duplicate in mass and/or number to ensure its correct segregation between the two daughter cells. It remains unclear, however, when and how this occurs. The process of Golgi duplication and inheritance in mammalian cells is still debated, as different modes of Golgi biogenesis have been proposed. One reason why this issue is not yet settled could be due to the elaborate organization of the Golgi stacks, which are interconnected to form a single-copy organelle capping the nucleus, thus impeding clear visualization of organelle duplication and segregation. Therefore, this study has exploited the relatively small number of Golgi stacks in Drosophila tissue-cultured S2 cells to revisit this issue (Kondylis, 2007).

In S2 cells, the Golgi stacks are found in close proximity to transitional endoplasmic reticulum (tER) sites, forming tER-Golgi units (Kondylis, 2003; Herpers, 2004). Their number nearly doubles at G2 phase. In an effort to identify factors mediating this process, focus was placed on cytoskeletal elements that have been involved in the organization of the mammalian Golgi apparatus. Microtubules are involved in mammalian Golgi ribbon maintenance, as their depolymerization leads to its reorganization into individual Golgi stacks in close proximity to ER exit sites (Kondylis, 2007 and references therein).

F-actin has also been implicated in the organization of the mammalian Golgi apparatus; its depolymerization leads to a compact appearance of this organelle without disruption of cisternal stacking. A key regulator of actin polymerization is the Arp2/3 complex. Its F-actin nucleation activity is triggered both by Wiskott-Aldrich syndrome protein (WASP) and WASP family verprolin-homologous (WAVE/Scar) proteins, which are in turn regulated by Rho small GTPases. WASP exists in an autoinhibited state that is released by the cooperative action of Cdc42, PI(4,5)P, and other SH3-containing proteins. In contrast, WAVE/Scar proteins, together with Sra-1, Kette (Nap1), Abi, and HSPC300, form a stable complex, which is itself regulated by Rac (Kondylis, 2007 and references therein).

Rho GTPases have recently been implicated in maintaining Golgi architecture. Cdc42 has been localized on the Golgi membrane and shown to recruit the Arp2/3 complex around this organelle via ARHGAP10. Furthermore, suppression of the brain-specific Rho-binding protein Citron-N in neurons was shown to lead to fragmentation of the Golgi apparatus, and Rho1 was proposed to exert its local effect on F-actin by regulating ROCK and profilin activity (Kondylis, 2007 and references therin).

This study shows that drug-induced F-actin depolymerization in S2 cells leads to doubling of the number of tER-Golgi units independent of anterograde transport. Using live cell imaging, electron microscopy, and three-dimensional (3D) electron tomography, this study shows that each Golgi is organized as a pair of stacks held together by an actin-based mechanism, both in Drosophila and in human cells. In S2 cells, this is mediated by Abi and Scar, suggesting a novel role for the Rac signaling cascade in Golgi architecture. Last, it was shown that the Golgi stacks undergo separation at G2 through modulation of Abi and Scar, and that blocking this separation prevents cells from entering mitosis, supporting the existence of a G2/M checkpoint related to Golgi structural organization (Kondylis, 2007).

The two Golgi stacks could be physically linked without displaying membrane continuity or being interconnected, for instance through intercisternal tubular connections, either permanent or transient. Tubules connecting cisternae of adjacent stacks are involved in the formation of the Golgi ribbon in mammalian cells and, recently, GM130 and GRASP65 have been proposed to be required for their integrity. However, the putative tubules connecting the two stacks in the pair would have different molecular requirements, at least in Drosophila, since depletion of dGM130 or dGRASP does not lead to their separation (Kondylis, 2003; Kondylis, 2005; Kondylis, 2007 and references therein).

F-actin could provide a physical link holding the paired Golgi stacks together, or it could help in the formation/maintenance of intercisternal tubules. In addition, short actin filaments have been proposed to link spectrin mosaics leading to the formation of a skeleton that surrounds the Golgi complex. One of its functions could be to hold the two Golgi stacks close enough to allow the formation and fusion of the tubules. It could also surround the tubules themselves, thus providing membrane stability. The localization of Abi and Scar at the periphery of the tER-Golgi units and between the two stacks in a pair is consistent with both proposed functions. These tomography studies so far have not revealed clear membrane continuities between Golgi cisternae, though examples have been found of a tubular network which is shared by the paired stacks (Kondylis, 2007).

tER sites behave similarly to the Golgi, as they also separate at G2 and upon F-actin depolymerization. Because little is known about the mechanism regulating the biogenesis of tER sites, it is difficult to envisage how the two parts could be held together. The spectrin-actin mesh described above could be common to Golgi and tER sites, and Golgi and tER site scission could be achieved in a synchronized fashion. Alternatively, either of these organelles could split first and lead to the scission of the other, perhaps by providing positional information. Recently, the centrosome component centrin 2 that is also localized to tER sites in Trypanosoma has been shown to give such positioning information. A more in-depth study combining immunogold labeling and 3D tomography would be required to elucidate such fine details of tER-Golgi structural organization (Kondylis, 2007).

Drosophila Rho1 is unlikely to have a role in holding the two Golgi stacks together. The overexpression of the Rho1 constitutively inactive mutant or treatment of S2 cells with ROCK or myosin light chain inhibitors (Y27632 and blebbistatin) did not affect the Golgi number. Cdc42 is also unlikely to participate as the depletion of its downstream effector WASP did not lead to Golgi separation, although the overexpression of the Cdc42^T17N dominant negative did. However, this effect could be due to nonspecific sequestration of the guanine nucleotide exchange factor involved in maintaining the paired Golgi stacks and may be shared with other small GTPases (Kondylis, 2007).

Interestingly, the results are consistent with a role for Rac GTPases in Drosophila Golgi architecture. Expression of the constitutively inactive form of Rac1 led to a near-doubling in the Golgi number, and depletion of Scar/WAVE or Abi, which are regulated by Rac GTPases, led to a similar phenotype. The identical results obtained in Scar and Abi RNAi suggest that this well-established Scar/WAVE pentameric complex is involved in holding the paired Golgi stacks together by promoting F-actin polymerization. These data indicate that the Rac signaling pathway is involved. However, the Scar/Abi complex has recently been shown to also stimulate Arp2/3 and F-actin polymerization independently of Rac. This would need to be investigated further (Kondylis, 2007).

This study shows that the separation of the paired Golgi stacks occurs at G2, prior to mitosis. A similar phenomenon has already been reported during cell division in Toxoplasma gondii, where a single Golgi stack grows as a duplicated organelle and is separated as the cell divides. However, the mechanism underlying this separation is not known (Kondylis, 2007).

The Golgi doubling in number at G2 phase resembles many aspects of this observed upon F-actin depolymerization. In both cases, a similar increase in Golgi number and decrease in their size are observed. Furthermore, this study has shown that it is the modulation of the F-actin cytoskeleton and the activity of Abi/Scar at G2 that lead to Golgi stack separation. (1) It was found that both Scar and Abi localized to the Golgi, strongly arguing for having a role in actin remodeling around this organelle. (2) The Golgi stacks in G2 cells remain insensitive to F-actin depolymerization. (3) Cells depleted of Abi and Scar that exhibit separated Golgi stacks do not split them further at G2. (4) The overexpression of Abi prevents Golgi separation at G2. This strongly suggests that the F-actin/Abi/Scar-mediated link of the two stacks has been severed in a G2-specific manner, perhaps by kinases such as Polo (Kondylis, 2007).

Because tER sites and the Golgi apparatus ultimately disperse later in mitosis, both in mammalian and Drosophila S2 cells, the Golgi stack separation prior to dispersion might be part of the proposed Golgi G2/M checkpoint. Indeed, reagents that interfere with the GRASP65/55 phosphorylation by Polo and ERK/MEK, respectively, arrest or delay the cell cycle at the G2/M transition. This study shows that blocking Golgi separation at G2 by overexpressing Abi also prevents S2 cells from entering mitosis. This strengthens the relationship between Golgi organization and mitotic entry, although it cannot formally be excluded that the mitotic block observed is partly due to additional effects of Abi overexpression, for instance at the plasma membrane (Kondylis, 2007).

It is proposed that at G2, the paired stacks are separated along with the adjacent tER sites. As the cell enters mitosis, the Golgi membrane and the tER sites disperse, and are segregated into the two daughter cells, where the tER-Golgi units are rebuilt. The Golgi could be rebuilt as a very small paired stack in close association with Scar, Abi, and F-actin, or as a single stack that will duplicate by a mechanism that still needs to be unraveled. Since G1 cells are all sensitive to F-actin depolymerization, this suggests that the formation of the paired Golgi stack starts just after the exit from mitosis and persists until S phase, when the Golgi seems to grow significantly. A more detailed understanding will come from EM study of S and G2 cells (Kondylis, 2007).

One of the remaining questions regards the impact of the Abi/Scar role on Golgi organization during development. Using Scar/WAVE, Abi, Kette, and Sra-1 mutants, as well as transgenic flies carrying inducible RNAi constructs, it will be possible to assess whether any of the observed phenotypes (defects in oogenesis, cell and organ morphology, neuroanatomical malformations, and failure in cell migration) is in part due to defects in Golgi organization (Kondylis, 2007).

Drosophila abelson interacting protein (dAbi) is a positive regulator of abelson tyrosine kinase activity

Human and mouse Abelson interacting proteins (Abi) are SH3-domain containing proteins that bind to the proline-rich motifs of the Abelson protein tyrosine kinase. A new member of this gene family, a Drosophila Abi (dAbi), is a substrate for Abl kinase and co-immunoprecipitates with Abl if the Abi SH3 domain is intact. A new function has been identified for both dAbi and human Abi-2 (hAbi-2). Both proteins activate the kinase activity of Abl as assayed by phosphorylation of the Drosophila Enabled (Ena) protein. Removal of the dAbi SH3 domain eliminates dAbi's activation of Abl kinase activity. dAbi is an unstable protein in cells and is present at low steady state levels but its protein level is increased coincident with phosphorylation by Abl kinase. Expression of the antisense strand of dAbi reduces dAbi protein levels and abolishes activation of Abl kinase activity. Modulation of Abi protein levels may be an important mechanism for regulating the level of Abl kinase activity in the cell (Juang, 1999).

Many critical cellular functions, such as cell growth and differentiation, are regulated by the kinase activity of protein tyrosine kinases (PTKs) and deregulated kinase activity of PTKs is associated with oncogenic activity. Relatively little is known about the normal regulation of the kinase activity of the Abelson (Abl) non-receptor PTK. Regulation of Abl activity is important as suggested by the consequences of improper regulation. Oncogenic forms of Abl such as BcrAbl in chronic myelogenous leukemia (CML) and v-Abl in Abelson murine leukemia virus have increased, unregulated kinase activity that phosphorylate several cellular substrates. This increased phosphorylation is believed to activate signal transduction pathways that participate in the uncontrolled cell growth of the tumor cells. Mutation of the SH3 domain of c-Abl also activates the oncogenic potential of Abl, producing elevated kinase activity, predominantly cytoplasmic localization, and cell transformation. It has been proposed that the SH3 domain of c-Abl may have a function in the negative regulation of Abl's activity. Overexpression of c-Abl in cells results in autophosphorylation of c-Abl, suggesting that there might be a limited concentration of a cellular modulator that suppresses c-Abl activity in cells. Thus, one of the models which has been proposed for the inhibition of the c-Abl tyrosine kinase invokes a trans-acting cellular inhibitor interacting with the Abl SH3 domain (Juang, 1999).

Proteins that interact with the SH3 domain of Abl and that might thereby modulate kinase activity of Abl have recently been identified. Initial reports identified three mammalian Abi proteins; murine Abi-1, hAbi-2 and human ArgBP1 (Dai, 1995; Shi, 1995; Wang, 1996). The Abi proteins physically associate with Abl and Abelson-related (Arg) kinases. The sequence motifs of Abi family members include an SH3 domain, proline-rich regions, PEST regions, and a homeodomain homologous region (Dai, 1995; Shi, 1995). Abi proteins are phosphorylated by the Abl PTKs. Overexpressing full length mouse Abi-1 in NIH3T3 cells suppresses v-Abl transforming activity (Shi, 1995). Stable expression of the full length hAbi-2 is toxic in NIH3T3 cells, but expression of a truncated form of hAbi-2, which lacks the first 157 amino acids including a proline-rich domain required for binding the Abl-SH3 domain, activates c-Abl transforming activity in NIH3T3 cells (Dai, 1995). It was proposed (Dai, 1995; Shi, 1995) that Abi is a tumor suppressor in mammalian cells. Hypothesized regulatory functions of Abi include stabilization of the inactive form of c-Abl or, alternatively, inhibiting access of Abl to protein substrates and thereby negatively regulating Abl activity (Juang, 1999).

The Abl gene has been conserved through evolution and related genes have been found in human, cat, mouse, Drosophila and C. elegans. This study used Drosophila genetics to understand the regulatory molecules of the conserved Abl pathway. In order to determine if Abi regulation of Abl is a mechanism that has been conserved through evolution, attempts were made to isolate a Drosophila member of the Abi family. This paper reports the identification of dAbi. Analysis of the dAbi protein shows very similar structural features to that of mammalian proteins. dAbi behaves similarly to mammalian Abi proteins by associating with Abl kinase and serving as a substrate for the Abl kinase. Two new properties were revealed by the biochemical analysis of both dAbi and hAbi-2 proteins. First, dAbi and hAbi-2 act as potent activators of Abl kinase activity. Second, dAbi is an unstable protein that is stabilized coincident with phosphorylation. It is proposed that regulation of Abi protein levels may be a mechanism for modulating Abl kinase activity that has been well conserved during evolution (Juang, 1999).

Degenerate oligonucleotides were designed based on the conserved SH3 domain peptide sequences of mammalian Abi proteins and used for PCR of Drosophila embryonic cDNA libraries. A fragment of 146 bp, the expected size of the SH3 domain, was isolated, cloned and sequenced. The predicted open reading frame was most similar in sequence to the mammalian Abi SH3 domain. The DNA fragment was used as a probe for high stringency screening of the library and hybridized to a 1.7 Kb Drosophila cDNA. The cDNA encodes a predicted open reading frame of 473 amino acids. A BLAST search of the GenBank non-redundant peptide sequence database (nr) identified a number of Abi and Abi-related proteins. dAbi appeared most similar to human e3B1/hssh3bp1, sharing 42% amino acid sequence identity and 54% amino acid similarity. Human e3B1 was originally identified in a screen for proteins that bound the SH3 domain of eps8, a substrate of receptor tyrosine kinases including the epidermal growth factor receptor (EGFR) tyrosine kinase (Biesova, 1997). In vitro assays with GST fusion proteins showed that e3B1 also bound the SH3 domain Abl (Biesova, 1997). Subsequently, one isoform of a candidate spectrin SH3 domain-binding protein was found to be identical to e3B1. The Drosophila sequence shows similar levels of amino acid identity when compared to other human Abi proteins. Pairwise sequence comparisons showed 40% amino-acid identity and 54% amino-acid similarity between dAbi and human ArgBP1A whereas comparison of dAbi and hAbi-2 gave similar results showing 39% amino-acid identity and 52% amino-acid similarity. The Drosophila sequence has motifs similar to those found in the human Abi proteins including a carboxy-terminal SH3 domain with 76% identity to hAbi-2. The amino-terminal domain includes a homeo-domain similarity, and the middle portion includes three polyproline motifs and a PEST domain. It is proposed that this sequence be called Drosophila Abi (dAbi) (Juang, 1999).

Abi proteins were identified in two hybrid screens using the carboxy-terminal domain of Abl or the SH3 domain of Abl. Abi and Abl can potentially interact at two sites, the SH3 domain of Abl with the proline motifs of Abi and the SH3 domain of Abi with the proline motifs of Abl. In addition to binding to Abl, Abi proteins appear to be excellent substrates for the kinase, raising a question of whether Abi proteins are upstream regulators or downstream effectors of the kinase. Abi-1 has been proposed to be a negative regulator of Abl because its full length protein suppresses v-Abl transformation (Shi, 1995). The full length hAbi-2 was also suggested to function as a potential tumor suppressor gene through negative regulation of Abl kinase activity (Dai, 1995). One hypothesis put forth to explain Abi's suppression of Abl's transforming activity is that binding of Abi to the Abl SH3 domain negatively regulates Abl kinase (Dai, 1995). This hypothesis was based in part on observations that structural alterations of Abl that attenuate binding to Abi, e.g. mutation of the Abl SH3, enhance the transforming activity of c-Abl. However, the observation that Abi-1 did not inhibit the overall kinase activity of v-Abl (Shi, 1995) suggests that Abi-1 suppression of v-Abl transforming activity may be due to other mechanisms of action (Juang, 1999).

The identification of a Drosophila Abi protein indicates that the Abl-Abi interaction may have been conserved during evolution. dAbi and hAbi-2 are 39% identical and 52% similar in amino acid sequence and share similar protein motifs. dAbi associates with Abl, as determined by co-immunoprecipitation, and is a substrate for the Abl PTK. The dAbi SH3 domain is required for a detectable interaction with the Abl kinase. Surprisingly, transient coexpression of Abl kinase with either dAbi or hAbi-2 proteins in both Drosophila S2 cells and mammalian COS cells leads to activation of Abl kinase that is dependent upon the presence of an intact Abi SH3 domain (Juang, 1999).

One model to explain how dAbi and hAbi-2 might activate Abl kinase would be that interaction of Abi with Abl generates a conformational change in Abl that results in exposure of its catalytic domain. Data to support this model comes from a recent report on site directed mutations in the Abl catalytic domain, SH3 domain and linker region between the SH2 and catalytic domain (SH2-CD) (Barila, 1998). Mutations in all three domains were found to activate c-Abl kinase implicating intramolecular inhibitory interactions of the Abl SH3 domain with the catalytic domain and with the SH2-CD linker region. In addition, mutation of the SH2-CD linker region caused a conformational change in the protein resulting in altered and increased protease sensitivity of the mutated protein compared with c-Abl (Barila, 1998). Such a model could also reconcile the conflicting observations that dAbi and hAbi-2 activate Abl kinase activity yet Abi proteins are reported to suppress Abl transforming activity since binding of Abi to Abl may also have a role in regulating other biological activities of Abl such as Abl transforming activity (Dai, 1995; Shi, 1995; Juang, 1999 and references therein).

Although the activation of Abl kinase by dAbi implicates dAbi as an upstream regulator of Abl, Abl also has effects on dAbi. Phosphorylation of dAbi leads to a significant increase in the steady state level of dAbi protein. The rapid turnover of dAbi in S2 cells suggests that a potential mechanism for the increase in dAbi level is through a phosphorylation-dependent increase in dAbi protein stability. Although dAbi may have other binding partners than Abl, one possible effect of the increased dAbi protein levels would be to provide an amplified or sustained activation of the Abl kinase. In this role, an increase in dAbi level would result in dAbi binding to Abl, activation of Abl kinase activity, increased phosphorylation of Abl substrates, including dAbi, and an additional increase in dAbi steady state levels that would maintain activation of Abl kinase. Clearly some additional mechanism would be needed to terminate this hypothetical positive regulatory loop (Juang, 1999).

If dAbi is a key regulator of Abl kinase in vivo, it will be important to understand the regulation of dAbi protein turnover. Proteolysis is critical for controlling protein concentrations in other key processes, such as signal cascades and the cell cycle. Reversible protein phosphorylation has also been linked to the regulation of protein degradation. Phosphorylation of proteins, such as the cyclins, leads to ubiquitination and degradation of proteins. However, in other proteins, e.g. c-jun, phosphorylation results in a reduction in ubiquitination and stabilization. Analyses of dAbi and hAbi-2/ArgBP1 sequences have revealed PEST sequences, regions rich in proline (P), glutamic acid (E), serine (S) and threonine (T). Such sequences have been suggested to target rapid intracellular degradation of proteins. Many PEST-containing proteins are important regulatory molecules, such as components of signal transduction pathways, homeotic proteins, and key enzymes. Wang (1996) has reported that a human Abi isoform, ArgBP1A, with three PEST regions, has a consistently lower expression level than another isoform, ArgBP1B, with only two PEST regions, consistent with a role of the PEST motifs in Abi protein stability (Juang, 1999).

Regulation of protein stability is thought to be important in how cells respond to DNA damage. In response to DNA damage, p53 protein turnover is reduced and its level rises. The increased p53 levels are important to cause G1 arrest and provide cells an opportunity to repair the DNA damage, or to prevent further proliferation of the damaged cell by causing apoptosis. Regulation of c-Abl has also been implicated in the DNA damage-induced apoptosis. In response to DNA-damaging agents, c-Abl protein tyrosine kinase down-regulates cdk2 and causes G1 arrest by a p53-dependent mechanism and may also be involved in a p53-independent induction of apoptosis. In response to DNA damage, the ataxia telangiectasis mutant (ATM) protein is reported to phosphorylate and activate c-Abl. It will be interesting to determine whether DNA damage also alters the protein stability of Abi, as this would provide an additional mechanism for activating Abl kinase activity in response to DNA damage (Juang, 1999).

The Abl kinase has been implicated in cancer, regulation of the cell cycle, regulation of cell adhesion and normal developmental processes in Drosophila and mammalian systems. Further study of Abi proteins may provide new insights into how Abl kinase activity is regulated in one or more of these processes. The identification of a dAbi will also permit genetic analysis of Abi in normal development (Juang, 1999).

Search PubMed for articles about Drosophila Abi

Barila, D. and Superti-Furga, G. (1998). An intramolecular SH3-domain interaction regulates c-Abl activity. Nature Genetics 18: 280-282. PubMed Citation: 9500553

Benesch, S. et al. (2005). N-WASP deficiency impairs EGF internalization and actin assembly at clathrin-coated pits. J. Cell Sci. 118: 3103-3115. PubMed Citation: 15985465

Biesova, Z., Piccoli, C. and Wong, W. T. (1997). Isolation and characterization of e3B1, an eps8 binding protein that regulates cell growth. Oncogene 14: 233-241. PubMed Citation: 9010225

Bogdan, S., Stephan, R., Lobke, C., Mertens, A. and Klambt, C. (2005). Abi activates WASP to promote sensory organ development. Nat. Cell Biol. 7: 977-984. PubMed Citation: 16155589

Burton, E. A., Oliver, T. N. and Pendergast, A. M. (2005). Abl kinases regulate actin comet tail elongation via an N-WASP-dependent pathway. Mol. Cell Biol. 25: 8834-8843. PubMed Citation: 16199863

Dai, Z. and Pendergast, A.M. (1995). Abi-2, a novel SH3-containing protein interacts with the c-Abl tyrosine kinase and modulates c-Abl transforming activity. Genes Dev. 9: 2569-2582. PubMed Citation: 9010225

Dai, Z., Quackenbush, R. C., Courtney, K. D., Grove, M., Cortez, D., Reuther, G. W., and Pendergast, A. M. (1998). Oncogenic Abl and Src tyrosine kinases elicit the ubiquitin-dependent degradation of target proteins through a Ras-independent pathway. Genes Dev. 12: 1415-1424. PubMed Citation: 9585502

Herpers, B. and Rabouille, C. (2004). mRNA localization and ER-based protein sorting mechanisms dictate the use of transitional endoplasmic reticulum-golgi units involved in gurken transport in Drosophila oocytes. Mol. Biol. Cell 15(12): 5306-17. PubMed Citation: 15385627

Huang, C. H., Lin, T. Y., Pan, R. L. and Juang, J. L. (2007). The involvement of Abl and PTP61F in the regulation of Abi protein localization and stability and lamella formation in Drosophila S2 cells. J. Biol. Chem. 282(44): 32442-32452. PubMed Citation: 17804420

Innocenti, M., et al. (2004). Abi1 is essential for the formation and activation of a WAVE2 signalling complex. Nat. Cell Biol. 6: 319-327. PubMed Citation: 15048123

Innocenti, M., et al. (2005). Abi1 regulates the activity of N-WASP and WAVE in distinct actin-based processes. Nat. Cell Biol. 7: 969-976. PubMed Citation: 16155590

Juang, J. L. and Hoffmann, F. M. (1999). Drosophila abelson interacting protein (dAbi) is a positive regulator of abelson tyrosine kinase activity. Oncogene 18: 5138-5147. PubMed Citation: 10498863

Kondylis, V. and Rabouille, C. (2003). A novel role for dp115 in the organization of tER sites in Drosophila. J. Cell Biol. 162(2): 185-98. PubMed Citation: 12876273

Kondylis, V., et al. (2007). The golgi comprises a paired stack that is separated at G2 by modulation of the actin cytoskeleton through Abi and Scar/WAVE. Dev. Cell 12(6): 901-15. PubMed citation: 17543863

Kunda, P., et al. (2003). Abi, Sra1, and Kette control the stability and localization of SCAR/WAVE to regulate the formation of actin-based protrusions. Curr. Biol. 13: 1867-1875. PubMed Citation: 14588242

Leng, Y., et al. (2005). Abelson-interactor-1 promotes WAVE2 membrane translocation and abelson-mediated tyrosine phosphorylation required for WAVE2 activation. Proc. Natl. Acad. Sci. 102: 1098-1103. PubMed Citation: 15657136

Lin, Y. T., et al. (2004). Abi enhances Abl-mediated CDC2 phosphorylation and inactivation. J. Biomed. Sci. 11: 902-910. PubMed Citation: 15591787

Lin, T.-Y., et al. (2009). Abi plays an opposing role to Abl in Drosophila axonogenesis and synaptogenesis. Development 136: 3099-3107. PubMed Citation: 19675132

Maruoka, M., et al. (2005). Identification of B cell adaptor for PI3-kinase (BCAP) as an Abl interactor 1-regulated substrate of Abl kinases. FEBS Lett. 579: 2986-2990. PubMed Citation: 15893754

Rogers, S. L., Wiedemann, U., Stuurman, N. and Vale, R. D. (2003). Molecular requirements for actin-based lamella formation in Drosophila S2 cells. J. Cell Biol. 162: 1079-1088. PubMed Citation: 12975351

Shi, Y., Kimona, A. and Goff, S. P. (1995). Abl-interactor-1, a novel SH3 protein binding to the carboxy-terminal portion of the Abl protein, suppresses v-abl transforming activity. Genes Dev. 9: 2583-2597. PubMed Citation: 7590237

Sirotkin, V., et al. (2005). Interactions of WASp, myosin-I, and verprolin with Arp2/3 complex during actin patch assembly in fission yeast. J. Cell Biol. 170: 637-648. PubMed Citation: 16087707

Steffen, A., Rottner, K., Ehinger, J., Innocenti, M., Scita, G., Wehland, J. and Stradal, T. E. (2004). Sra-1 and Nap1 link Rac to actin assembly driving lamellipodia formation. EMBO J. 23: 749-759. PubMed Citation: 14765121

Stephan, R., Grevelhörster, A., Wenderdel, S., Klämbt, C. and Bogdan, S. (2008). Abi induces ectopic sensory organ formation by stimulating EGFR signaling. Mech. Dev. 125(3-4): 183-95. PubMed Citation: 18221859

Stradal, T., Courtney, K. D., Rottner, K., Hahne, P., Small, J. V. and Pendergast, A. M. (2001). Curr. Biol. 11: 891-895. PubMed Citation: 11516653

Stradal, T. E., and Scita, G. (2006). Protein complexes regulating Arp2/3-mediated actin assembly. Curr. Opin. Cell Biol. 18: 4-10. PubMed Citation: 16343889

Tani, K., et al. (2003). Abl interactor 1 promotes tyrosine 296 phosphorylation of mammalian enabled (Mena) by c-Abl kinase. J. Biol. Chem. 278: 21685-21692. PubMed Citation: 12672821

Wang, B., et al. (1996). Identification of ArgBP1, an Arg protein tyrosine kinase binding protein that is the human homologue of a CNS-specific Xenopus gene. Oncogene 12: 1921-1929. PubMed Citation: 8649853

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date revised: 20 December 2009

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