Drosophila abl and genetic redundancy in signal transduction.

Genetic studies on Drosophila Abl and, more recently, on mouse c-Abl and c-Src indicate that the functions of these non-receptor tyrosine kinases may duplicate activities of other molecules within signal transduction pathways. In Drosophila, second-site mutations have been recovered that disrupt the redundant functions so that the Abl tyrosine kinase is essential to the formation of axonal connections in the embryonic central nervous system and for attachment of embryonic muscles to the body wall. Molecular isolation and analysis of the genes identified by these second-site mutations should define the molecular basis for the genetic redundancy.     

Roles of receptor tyrosine kinases in Drosophila development.

Communication between cells is a fundamental component of development and morphogenesis. Identification of the molecules mediating cell-cell communication is crucial for elucidation of the molecular basis of these processes. Receptor tyrosine kinases (RTKs) appear to play a central role in this context by transmitting into cells information dictating their fate. The functions of RTKs in Drosophila are extremely diverse, and include maternal determination of embryonic polarity (torso and torpedo), determination of neuroblast identity (faint little ball), and guidance of tracheal cell migration in the embryo (breathless). During compound eye development, RTKs affect the number of photoreceptor clusters (Ellipse) and the determination of photoreceptor R7 identity (sevenless). The phenotypes of mutations in RTK loci serve as a starting point for understanding processes dictating cell identity at the level of the whole organism. Recently, they have also begun to provide a basis for selection of second-site suppressor mutations, encoding additional elements in their signal transduction pathway. Common themes between the functions, regulation, and signal transduction pathways of Drosophila RTKs are drawn.     

Abelson tyrosine kinase is required to transduce midline repulsive cues

Tyrosine phosphorylation-dependent signaling cascades play key roles in determining the formation of an axon pathway. The cytoplasmic Abelson tyrosine kinase participate in several signaling pathways that orchestrate both growth cone advance and steering in response to guidance cues. Here, a genetic approach is used to evaluate the role for Abelson in growth cones during a decision to cross or not to cross the Drosophila embryonic midline. Our data indicate that both loss- and gain-of-function conditions for Abl cause neurons within the pCC/MP2 pathway to project across the midline incorrectly. The frequency of abnormal crossovers is enhanced by mutations in the genes encoding the midline repellent, Slit, or its receptor, Roundabout. In comm mutants, where repulsive signals remain elevated, increasing or decreasing Abl activity partially rescues commissure formation. Thus, both too much and too little Abl activity causes axons to cross the midline inappropriately, indicating that Abl plays a critical role in transducing midline repulsive cues. How Abl functions in this role is not yet clear, but we suggest that Abl may help regulate cytoskeletal dynamics underlying a growth cone's response to midline cues.     

Disabled is a bona fide component of the Abl signaling network

Abl is an essential regulator of cell migration and morphogenesis in both vertebrates and invertebrates. It has long been speculated that the adaptor protein Disabled (Dab), which is a key regulator of neuronal migration in the vertebrate brain, might be a component of this signaling pathway, but this idea has been controversial. We now demonstrate that null mutations of Drosophila Dab result in phenotypes that mimic Abl mutant phenotypes, both in axon guidance and epithelial morphogenesis. The Dab mutant interacts genetically with mutations in Abl, and with mutations in the Abl accessory factors trio and enabled (ena). Genetic epistasis tests show that Dab functions upstream of Abl and ena, and, consistent with this, we show that Dab is required for the subcellular localization of these two proteins. We therefore infer that Dab is a bona fide component of the core Abl signaling pathway in Drosophila.     

Phosphorylation of Enabled by the Drosophila Abelson Tyrosine Kinase Regulates the In Vivo Function and Protein-Protein Interactions of Enabled

Drosophila Enabled (Ena) is a member of a family of cytoskeleton-associated proteins including mammalian vasodilator-stimulated phosphoprotein and murine Enabled that regulate actin cytoskeleton assembly. Mutations in Drosophila ena were discovered as dominant genetic suppressors of mutations in the Abelson tyrosine kinase (Abl), suggesting that Ena and Abl function in the same pathway or process. We have identified six tyrosine residues on Ena that are phosphorylated by Abl in vitro and in vivo. Mutation of these phosphorylation sites to phenylalanine partially impaired the ability of Ena to restore viability to ena mutant animals, indicating that phosphorylation is required for optimal Ena function. Phosphorylation of Ena by Abl inhibited the binding of Ena to SH3 domains in vitro, suggesting that one effect of Ena phosphorylation may be to modulate its association with other proteins.     

Dosage-sensitive, reciprocal genetic interactions between the Abl tyrosine kinase and the putative GEF trio reveal trio's role in axon pathfinding

The Abelson tyrosine kinase (Abl) is integrated into signal transduction networks regulating axon outgrowth. We have identified the Drosophila trio gene through a mutation that exacerbates the Abl mutant phenotype. Drosophila Trio is an ortholog of mammalian Trio, a protein that contains multiple spectrin-like repeats and two Dbl homology (DH) domains that affect actin cytoskeletal dynamics via the small GTPases Rho and Rac. Phenotypic analysis demonstrates that trio and Abl cooperate in regulating axon outgrowth in the embryonic central nervous system (CNS). Dosage-sensitive interactions between trio and Abl, failed axon connections (fax), and enabled (ena) indicate that Trio is integrated into common signaling networks with these gene products. These observations suggest a mechanism by which Abl-mediated signaling networks influence the actin cytoskeleton in neuronal growth cones.     

The tyrosine kinase Abl and its substrate enabled collaborate with the receptor phosphatase Dlar to control motor axon guidance

Genetic analysis of growth cone guidance choice points in Drosophila identified neuronal receptor protein tyrosine phosphatases (RPTPs) as key determinants of axon pathfinding behavior. We now demonstrate that the Drosophila Abl tyrosine kinase functions in the intersegmental nerve b (ISNb) motor choice point pathway as an antagonist of the RPTP Dlar. The function of Abl in this pathway is dependent on an intact catalytic domain. We also show that the Abl phosphoprotein substrate Enabled (Ena) is required for choice point navigation. Both Abl and Ena proteins associate with the Dlar cytoplasmic domain and serve as substrates for Dlar in vitro, suggesting that they play a direct role in the Dlar pathway. These data suggest that Dlar, Abl, and Ena define a phosphorylation state-dependent switch that controls growth cone behavior by transmitting signals at the cell surface to the actin cytoskeleton.     

The Abelson tyrosine kinase, the Trio GEF and Enabled interact with the Netrin receptor Frazzled in Drosophila

The attractive Netrin receptor Frazzled (Fra), and the signaling molecules Abelson tyrosine kinase (Abl), the guanine nucleotide-exchange factor Trio, and the Abl substrate Enabled (Ena), all regulate axon pathfinding at the Drosophila embryonic CNS midline. We detect genetic and/or physical interactions between Fra and these effector molecules that suggest that they act in concert to guide axons across the midline. Mutations in Abl and trio dominantly enhance fra and Netrin mutant CNS phenotypes, and fra;Abl and fra;trio double mutants display a dramatic loss of axons in a majority of commissures. Conversely, heterozygosity for ena reduces the severity of the CNS phenotype in fra, Netrin and trio,Abl mutants. Consistent with an in vivo role for these molecules as effectors of Fra signaling, heterozygosity for Abl, trio or ena reduces the number of axons that inappropriately cross the midline in embryos expressing the chimeric Robo-Fra receptor. Fra interacts physically with Abl and Trio in GST-pulldown assays and in co-immunoprecipitation experiments. In addition, tyrosine phosphorylation of Trio and Fra is elevated in S2 cells when Abl levels are increased. Together, these data suggest that Abl, Trio, Ena and Fra are integrated into a complex signaling network that regulates axon guidance at the CNS midline.     

The receptor protein tyrosine phosphatase PTP69D antagonizes Abl tyrosine kinase to guide axons in Drosophila

During Drosophila embryogenesis, both the cytoplasmic Abelson tyrosine kinase (Abl) and the membrane bound tyrosine phosphatase PTP69D are required for proper guidance of CNS and motor axons. We provide evidence that PTP69D modulates signaling by Abl and its antagonist, Ena. An Abl loss-of function mutation dominantly suppresses most Ptp69D mutant phenotypes including larval/pupal lethality and CNS and motor axon defects, while increased Abl and decreased Ena expression dramatically increase the expressivity of Ptp69D axonal defects. In contrast, Ptp69D mutations do not affect Abl mutant phenotypes. These results support the hypothesis that PTP69D antagonizes the Abl/Ena genetic pathway, perhaps as an upstream regulator. We also find that mutation of the gene encoding the cytoplasmic Src64B tyrosine kinase exacerbates Ptp69D phenotypes, suggesting that two different cytoplasmic tyrosine kinases, Abl and Src64B, modify PTP69D-mediated axon patterning in quite different ways.     

Abl tyrosine kinase and its substrate Ena/VASP have functional interactions with kinesin-1

Relatively little is known about how microtubule motors are controlled or about how the functions of different cytoskeletal systems are integrated. A yeast two-hybrid screen for proteins that bind to Drosophila Enabled (Ena), an actin polymerization factor that is negatively regulated by Abl tyrosine kinase, identified kinesin heavy chain (Khc), a member of the kinesin-1 subfamily of microtubule motors. Coimmunoprecipitation from Drosophila cytosol confirmed a physical interaction between Khc and Ena. Kinesin-1 motors can carry organelles and other macromolecular cargoes from neuronal cell bodies toward terminals in fast-axonal-transport. Ena distribution in larval axons was not affected by mutations in the Khc gene, suggesting that Ena is not itself a fast transport cargo of Drosophila kinesin-1. Genetic interaction tests showed that in a background sensitized by reduced Khc gene dosage, a reduction in Abl gene dosage caused distal paralysis and axonal swellings. A concomitant reduction in ena dosage rescued those defects. These results suggest that Ena/VASP, when not inhibited by the Abl pathway, can bind Khc and reduce its transport activity in axons.     

Abl deregulates Cdk5 kinase activity and subcellular localization in Drosophila neurodegeneration

Although Abl functions in mature neurons, work to date has not addressed Abl's role on Cdk5 in neurodegeneration. We found that beta-amyloid (Abeta42) initiated Abl kinase activity and that blockade of Abl kinase rescued both Drosophila and mammalian neuronal cells from cell death. We also found activated Abl kinase to be necessary for the binding, activation, and translocalization of Cdk5 in Drosophila neuronal cells. Conversion of p35 into p25 was not observed in Abeta42-triggered Drosophila neurodegeneration, suggesting that Cdk5 activation and protein translocalization can be p25-independent. Our genetic studies also showed that abl mutations repressed Abeta42-induced Cdk5 activity and neurodegeneration in Drosophila eyes. Although Abeta42 induced conversion of p35 to p25 in mammalian cells, it did not sufficiently induce Cdk5 activation when c-Abl kinase activity was suppressed. Therefore, we propose that Abl and p35/p25 cooperate in promoting Cdk5-pY15, which deregulates Cdk5 activity and subcellular localization in Abeta42-triggered neurodegeneration.     

Abl regulates planar polarized junctional dynamics through β-catenin tyrosine phosphorylation

Interactions between epithelial cells are mediated by adherens junctions that are dynamically regulated during development. Here we show that the turnover of β-catenin is increased at cell interfaces that are targeted for disassembly during Drosophila axis elongation. The Abl tyrosine kinase is concentrated at specific planar junctions and is necessary for polarized β-catenin localization and dynamics. abl mutant embryos have decreased β-catenin turnover at shrinking edges, and these defects are accompanied by a reduction in multicellular rosette formation and axis elongation. Abl promotes β-catenin phosphorylation on the conserved tyrosine 667 and expression of an unphosphorylatable β-catenin mutant recapitulates the defects of abl mutants. Notably, a phosphomimetic β-catenin(Y667E) mutation is sufficient to increase β-catenin turnover and rescue axis elongation in abl deficient embryos. These results demonstrate that the asymmetrically localized Abl tyrosine kinase directs planar polarized junctional remodeling during Drosophila axis elongation through the tyrosine phosphorylation of β-catenin.     

Repulsive axon guidance: Abelson and Enabled play opposing roles downstream of the roundabout receptor

Drosophila Roundabout (Robo) is the founding member of a conserved family of repulsive axon guidance receptors that respond to secreted Slit proteins. Little is known about the signaling mechanisms which function downstream of Robo to mediate repulsion. Here, we present genetic and biochemical evidence that the Abelson (Abl) tyrosine kinase and its substrate Enabled (Ena) play direct and opposing roles in Robo signal transduction. Genetic interactions support a model in which Abl functions to antagonize Robo signaling, while Ena is required in part for Robo's repulsive output. Both Abl and Ena can directly bind to Robo's cytoplasmic domain. A mutant form of Robo that interferes with Ena binding is partially impaired in Robo function, while a mutation in a conserved cytoplasmic tyrosine that can be phosphorylated by Abl generates a hyperactive Robo receptor.     

Genetic Interactions between the Drosophila Abelson (Abl) Tyrosine Kinase and Failed Axon Connections (Fax), a Novel Protein in Axon Bundles

Mutations in the failed axon connections (fax) gene have been identified as dominant genetic enhancers of the Abl mutant phenotype. These mutations in fax all result in defective or absent protein product. In a genetic background with wild-type Abl function, the fax loss-of-function alleles are homozygous viable, demonstrating that fax is not an essential gene unless the animal is also mutant for Abl. The fax gene encodes a novel 47-kD protein expressed in a developmental pattern similar to that of Abl in the embryonic mesoderm and axons of the central nervous system. The conditional, extragenic noncomplementation between fax and another Abl modifier gene, disabled, reveal that the two proteins are likely to function together in a process downstream or parallel to the Abl protein tyrosine kinase.     

Abelson kinase regulates epithelial morphogenesis in Drosophila.

Activation of the nonreceptor tyrosine kinase Abelson (Abl) contributes to the development of leukemia, but the complex roles of Abl in normal development are not fully understood. Drosophila Abl links neural axon guidance receptors to the cytoskeleton. Here we report a novel role for Drosophila Abl in epithelial cells, where it is critical for morphogenesis. Embryos completely lacking both maternal and zygotic Abl die with defects in several morphogenetic processes requiring cell shape changes and cell migration. We describe the cellular defects that underlie these problems, focusing on dorsal closure as an example. Further, we show that the Abl target Enabled (Ena), a modulator of actin dynamics, is involved with Abl in morphogenesis. We find that Ena localizes to adherens junctions of most epithelial cells, and that it genetically interacts with the adherens junction protein Armadillo (Arm) during morphogenesis. The defects of abl mutants are strongly enhanced by heterozygosity for shotgun, which encodes DE-cadherin. Finally, loss of Abl reduces Arm and alpha-catenin accumulation in adherens junctions, while having little or no effect on other components of the cytoskeleton or cell polarity machinery. We discuss possible models for Abl function during epithelial morphogenesis in light of these data.     

Targeted mutagenesis and genetic analysis of a Drosophila receptor-linked protein tyrosine phosphatase gene

Several Drosophila receptor-linked protein tyrosine phosphatases (R-PTPs) are selectively expressed on axons of the developing embryonic central nervous system. The extracellular domains of these axonal R-PTPs are homologous to neural adhesion molecules. Thus, R-PTPs may directly couple cell recognition to signal transduction via control of tyrosine phosphorylation. To examine the function of these molecules during nervous system development, we wished to generate mutations in R-PTP genes. It was unclear whether a mutation in a single R-PTP gene would confer lethality, however, because the similarities in sequence and expression pattern between the axonal R-PTPs suggest that they may have partially redundant functions. To circumvent this problem, we developed a directed mutagenesis strategy based on local transposition of P elements, and used this approach to isolate a null mutation in the DPTP99A gene. This strategy, which we describe in detail here, should be applicable to any Drosophila gene within a lettered division of an appropriately marked P element. Flies lacking DPTP99A expression are viable and fertile, and we have been unable to detect any alterations in the embryonic nervous system of DPTP99A embryos using a variety of antibody markers.     

Abi induces ectopic sensory organ formation by stimulating EGFR signaling

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). We have recently shown that the Drosophila Abi is capable of promoting bristle development in a wasp dependent fashion. Here, we report 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, we noted changes in tyrosine phosphorylation supporting previous reports that Abi protein stability is linked to tyrosine phosphorylation mediated by Abl.     

How do Abl family kinases regulate cell shape and movement?

Genetic analysis and studies of normal and leukemia cells in culture have shown that Abl family nonreceptor tyrosine kinases regulate cell morphogenesis and motility. Abl family kinases, which include Drosophila (D-) Abl and the vertebrate Abl and Arg proteins, relay signals from cell surface growth-factor and adhesion receptors to promote cytoskeletal rearrangements. Recent biochemical and crystallographic analyses have clarified the mechanisms by which growth-factor and adhesion receptors might regulate the activity of Abl family kinases. When activated, Abl family kinases can regulate cytoskeletal dynamics by phosphorylating several known cytoskeletal regulatory proteins. In addition, the C-terminal half of Abl family kinases has several domains that bind to cytoskeletal components. Emerging evidence suggests that Abl family kinases can use these domains to directly organize cytoskeletal structure in vivo.     

Using Bcr-Abl to examine mechanisms by which abl kinase regulates morphogenesis in Drosophila

Signaling by the nonreceptor tyrosine kinase Abelson (Abl) plays key roles in normal development, whereas its inappropriate activation helps trigger the development of several forms of leukemia. Abl is best known for its roles in axon guidance, but Abl and its relatives also help regulate embryonic morphogenesis in epithelial tissues. Here, we explore the role of regulation of Abl kinase activity during development. We first compare the subcellular localization of Abl protein and of active Abl, by using a phosphospecific antibody, providing a catalog of places where Abl is activated. Next, we explore the consequences for morphogenesis of overexpressing wild-type Abl or expressing the activated form found in leukemia, Bcr-Abl. We find dose-dependent effects of elevating Abl activity on morphogenetic movements such as head involution and dorsal closure, on cell shape changes, on cell protrusive behavior, and on the organization of the actin cytoskeleton. Most of the effects of Abl activation parallel those caused by reduction in function of its target Enabled. Abl activation leads to changes in Enabled phosphorylation and localization, suggesting a mechanism of action. These data provide new insight into how regulated Abl activity helps direct normal development and into possible biological functions of Bcr-Abl.     

Cyclin D-Cdk4 and cyclin E-Cdk2 regulate the Jak/STAT signal transduction pathway in Drosophila

The JAK/STAT signal transduction pathway regulates many developmental processes in Drosophila. However, the functional mechanism of this pathway is poorly understood. In this report, we identify the Drosophila cyclin-dependent kinase 4 (Cdk4), which exhibits embryonic mutant phenotypes identical to those in the Hopscotch/JAK kinase and stat92E/STAT mutations. Specific genetic interactions between Cdk4 and hop mutations suggest that Cdk4 functions downstream of the HOP tyrosine kinase. We further show that Cyclin D-Cdk4 (as well as Cyclin E-Cdk2) binds and regulates STAT92E protein stability. STAT92E regulates gene expression for various biological processes, including the endocycle S phase. These data suggest that Cyclin D-Cdk4 and Cyclin E-Cdk2 play more versatile roles in Drosophila development.