The Abl/Enabled signaling pathway regulates Golgi architecture in Drosophila photoreceptor neurons

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

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.     

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.     

Balancing different types of actin polymerization at distinct sites: roles for Abelson kinase and Enabled

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

Abelson kinase (Abl) and RhoGEF2 regulate actin organization during cell constriction in Drosophila

Morphogenesis involves the interplay of different cytoskeletal regulators. Investigating how they interact during a given morphogenetic event will help us understand animal development. Studies of ventral furrow formation, a morphogenetic event during Drosophila gastrulation, have identified a signaling pathway involving the G-protein Concertina (Cta) and the Rho activator RhoGEF2. Although these regulators act to promote stable myosin accumulation and apical cell constriction, loss-of-function phenotypes for each of these pathway members is not equivalent, suggesting the existence of additional ventral furrow regulators. Here, we report the identification of Abelson kinase (Abl) as a novel ventral furrow regulator. We find that Abl acts apically to suppress the accumulation of both Enabled (Ena) and actin in mesodermal cells during ventral furrow formation. Further, RhoGEF2 also regulates ordered actin localization during ventral furrow formation, whereas its activator, Cta, does not. Taken together, our data suggest that there are two crucial preconditions for apical constriction in the ventral furrow: myosin stabilization/activation, regulated by Cta and RhoGEF2; and the organization of apical actin, regulated by Abl and RhoGEF2. These observations identify an important morphogenetic role for Abl and suggest a conserved mechanism for this kinase during apical cell constriction.     

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.     

Actin-based motility: stop and go with Ena/VASP proteins

Proteins of the Ena/VASP (Enabled/vasodilator-stimulated phosphoprotein) family are involved in Abl and/or cyclic nucleotide-dependent protein kinase signaling pathways. These proteins are also crucial factors in regulating actin dynamics and associated processes such as cell-cell adhesion, platelet function and actin-based motility of both cytopathogenic Listeria and their eukaryotic host cells. Although biochemical mechanisms have emerged depicting Ena/VASP proteins as enhancers of actin filament formation, increasing evidence also suggests that these proteins have inhibitory functions in integrin regulation, cell motility and axon guidance.     

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.     

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.     

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.     

Mutations in Drosophila Enabled and Rescue by Human Vasodilator-stimulated Phosphoprotein (VASP) Indicate Important Functional Roles for Ena/VASP Homology Domain 1 (EVH1) and EVH2 Domains

Drosophila Enabled (Ena) was initially identified as a dominant genetic suppressor of mutations in the Abelson tyrosine kinase and, more recently, as a member of the Ena/human vasodilator-stimulated phosphoprotein (VASP) family of proteins. We have used genetic, biochemical, and cell biological approaches to demonstrate the functional relationship between Ena and human VASP. In addition, we have defined the roles of Ena domains identified as essential for its activity in vivo. We have demonstrated that VASP rescues the embryonic lethality associated with loss of Ena function in Drosophila and have shown that Ena, like VASP, is associated with actin filaments and focal adhesions when expressed in cultured cells. To define sequences that are central to Ena function, we have characterized the molecular lesions present in two lethal ena mutant alleles that affected the Ena/VASP homology domain 1 (EVH1) and EVH2. A missense mutation that resulted in an amino acid substitution in the EVH1 domain eliminated in vitro binding of Ena to the cytoskeletal protein zyxin, a previously reported binding partner of VASP. A nonsense mutation that resulted in a C-terminally truncated Ena protein lacking the EVH2 domain failed to form multimeric complexes and exhibited reduced binding to zyxin and the Abelson Src homology 3 domain. Our analysis demonstrates that Ena and VASP are functionally homologous and defines the conserved EVH1 and EVH2 domains as central to the physiological activity of Ena.     

Prostaglandins temporally regulate cytoplasmic actin bundle formation during Drosophila oogenesis

Prostaglandins (PGs)—lipid signals produced downstream of cyclooxygenase (COX) enzymes—regulate actin dynamics in cell culture and platelets, but their roles during development are largely unknown. Here we define a new role for Pxt, the Drosophila COX-like enzyme, in regulating the actin cytoskeleton—temporal restriction of actin remodeling during oogenesis. PGs are required for actin filament bundle formation during stage 10B (S10B). In addition, loss of Pxt results in extensive early actin remodeling, including actin filaments and aggregates, within the posterior nurse cells of S9 follicles; wild-type follicles exhibit similar structures at a low frequency. Hu li tai shao (Hts-RC) and Villin (Quail), an actin bundler, localize to all early actin structures, whereas Enabled (Ena), an actin elongation factor, preferentially localizes to those in pxt mutants. Reduced Ena levels strongly suppress early actin remodeling in pxt mutants. Furthermore, loss of Pxt results in reduced Ena localization to the sites of bundle formation during S10B. Together these data lead to a model in which PGs temporally regulate actin remodeling during Drosophila oogenesis by controlling Ena localization/activity, such that in S9, PG signaling inhibits, whereas at S10B, it promotes Ena-dependent actin remodeling.     

Lgl2 Executes Its Function as a Tumor Suppressor by Regulating ErbB Signaling in the Zebrafish Epidermis

Changes in tissue homeostasis, acquisition of invasive cell characteristics, and tumor formation can often be linked to the loss of epithelial cell polarity. In carcinogenesis, the grade of neoplasia correlates with impaired cell polarity. In Drosophila, lethal giant larvae (lgl), discs large (dlg), and scribble, which are components of the epithelial apico-basal cell polarity machinery, act as tumor suppressors, and orthologs of this evolutionary conserved pathway are lost in human carcinoma with high frequency. However, a mechanistic link between neoplasia and vertebrate orthologs of these tumor-suppressor genes remains to be fully explored at the organismal level. Here, we show that the pen/lgl2 mutant phenotype shares two key cellular and molecular features of mammalian malignancy: cell autonomous epidermal neoplasia and epithelial-to-mesenchymal-transition (EMT) of basal epidermal cells including the differential expression of several regulators of EMT. Further, we found that epidermal neoplasia and EMT in pen/lgl2 mutant epidermal cells is promoted by ErbB signalling, a pathway of high significance in human carcinomas. Intriguingly, EMT in the pen/lgl2 mutant is facilitated specifically by ErbB2 mediated E-cadherin mislocalization and not via canonical snail–dependent down-regulation of E-cadherin expression. Our data reveal that pen/lgl2 functions as a tumor suppressor gene in vertebrates, establishing zebrafish pen/lgl2 mutants as a valuable cancer model.     

Abl interactor 1 promotes tyrosine 296 phosphorylation of mammalian enabled (Mena) by c-Abl kinase

Mammalian Enabled (Mena) is a mammalian homologue of Drosophila Enabled (Ena), which genetically interacts with Drosophila Abl tyrosine kinase. The signaling pathway involving c-Abl and Mena (Ena) is not fully understood. To find molecules that participate in the c-Abl/Mena pathway, we searched for Mena-binding proteins using a yeast two-hybrid system. We identified Abl interactor 1 (Abi-1), which is known to interact with c-Abl, as a binding protein for Mena. Binding analysis revealed that the Ena/Vasp homology 1 domain of Mena and the polyproline structure of Abi-1 are necessary for the interaction. The interaction between Mena and Abi-1 was also observed in a mammalian expression system. Importantly, Abi-1 dramatically promoted c-Abl-mediated tyrosine phosphorylation of Mena but not other substrates such as c-Cbl. Mutational analysis demonstrated that the phosphorylation site of Mena is Tyr-296. Our results suggest that Abi-1 regulates c-Abl-mediated phosphorylation of Mena by interacting with both proteins.     

Abi-1-bridged tyrosine phosphorylation of VASP by Abelson kinase impairs association of VASP to focal adhesions and regulates leukaemic cell adhesion

Mena [mammalian Ena (Enabled)]/VASP (vasodilator-stimulated phosphoprotein) proteins are the homologues of Drosophila Ena. In Drosophila, Ena is a substrate of the tyrosine kinase DAbl (Drosophila Abl). However, the link between Abl and the Mena/VASP family is not fully understood in mammals. We previously reported that Abi-1 (Abl interactor 1) promotes phosphorylation of Mena and BCAP (B-cell adaptor for phosphoinositide 3-kinase) by bridging the interaction between c-Abl and the substrate. In the present study we have identified VASP, another member of the Mena/VASP family, as an Abi-1-bridged substrate of Abl. VASP is phosphorylated by Abl when Abi-1 is co-expressed. We also found that VASP interacted with Abi-1 both in vitro and in vivo. VASP was tyrosine-phosphorylated in Bcr-Abl-positive leukaemic cells in an Abi-1-dependent manner. Co-expression of c-Abl and Abi-1 or the phosphomimetic Y39D mutation in VASP resulted in less accumulation of VASP at focal adhesions. VASP Y39D had a reduced affinity to the proline-rich region of zyxin. Interestingly, overexpression of both phosphomimetic and unphosphorylated forms of VASP, but not wild-type VASP, impaired adhesion of K562 cells to fibronectin. These results suggest that the phosphorylation and dephosphorylation cycle of VASP by the Abi-1-bridged mechanism regulates association of VASP with focal adhesions, which may regulate adhesion of Bcr-Abl-transformed leukaemic cells.     

FGF signalling through RAS/MAPK and PI3K pathways regulates cell movement and gene expression in the chicken primitive streak without affecting E-cadherin expression

Background  

FGF signalling regulates numerous aspects of early embryo development. During gastrulation in amniotes, epiblast cells undergo an epithelial to mesenchymal transition (EMT) in the primitive streak to form the mesoderm and endoderm. In mice lacking FGFR1, epiblast cells in the primitive streak fail to downregulate E-cadherin and undergo EMT, and cell migration is inhibited. This study investigated how FGF signalling regulates cell movement and gene expression in the primitive streak of chicken embryos.     

Myosin-dependent remodeling of adherens junctions protects junctions from Snail-dependent disassembly

Although Snail is essential for disassembly of adherens junctions during epithelial–mesenchymal transitions (EMTs), loss of adherens junctions in Drosophila melanogaster gastrula is delayed until mesoderm is internalized, despite the early expression of Snail in that primordium. By combining live imaging and quantitative image analysis, we track the behavior of E-cadherin–rich junction clusters, demonstrating that in the early stages of gastrulation most subapical clusters in mesoderm not only persist, but move apically and enhance in density and total intensity. All three phenomena depend on myosin II and are temporally correlated with the pulses of actomyosin accumulation that drive initial cell shape changes during gastrulation. When contractile myosin is absent, the normal Snail expression in mesoderm, or ectopic Snail expression in ectoderm, is sufficient to drive early disassembly of junctions. In both cases, junctional disassembly can be blocked by simultaneous induction of myosin contractility. Our findings provide in vivo evidence for mechanosensitivity of cell–cell junctions and imply that myosin-mediated tension can prevent Snail-driven EMT.     

P115 RhoGEF and microtubules decide the direction apoptotic cells extrude from an epithelium

To preserve epithelial barrier function, dying cells are squeezed out of an epithelium by “apoptotic cell extrusion.” Specifically, a cell destined for apoptosis signals its live neighboring epithelial cells to form and contract a ring of actin and myosin II that squeezes the dying cell out of the epithelial sheet. Although most apoptotic cells extrude apically, we find that some exit basally. Localization of actin and myosin IIA contraction dictates the extrusion direction: basal extrusion requires circumferential contraction of neighboring cells at their apices, whereas apical extrusion also requires downward contraction along the basolateral surfaces. To activate actin/myosin basolaterally, microtubules in neighboring cells reorient and target p115 RhoGEF to this site. Preventing microtubule reorientation restricts contraction to the apex, driving extrusion basally. Extrusion polarity has important implications for tumors where apoptosis is blocked but extrusion is not, as basal extrusion could enable these cells to initiate metastasis.     

In the Absence of Frazzled Over-Expression of Abelson Tyrosine Kinase Disrupts Commissure Formation and Causes Axons to Leave the Embryonic CNS

Background

In the Drosophila embryonic nerve cord, the formation of commissures require both the chemoattractive Netrin receptor Frazzled (Fra) and the Abelson (Abl) cytoplasmic tyrosine kinase. Abl binds to the cytoplasmic domain of Fra and loss-of-function mutations in abl enhance fra-dependent commissural defects. To further test Abl''s role in attractive signaling, we over-expressed Abl in Fra mutants anticipating rescue of commissures.

Methodology/Principal Findings

The Gal4-UAS system was used to pan-neurally over-express Abl in homozygous fra embryos. Surprisingly, this led to a significant decrease in both posterior and anterior commissure formation and induced some commissural and longitudinal axons to project beyond the CNS/PNS border. Re-expressing wild-type Fra, or Fra mutants with a P-motif deleted, revert both commissural and exiting phenotypes, indicating that Fra is required but not a specific P-motif. This is supported by S2 cell experiments demonstrating that Abl binds to Fra independent of any specific P-motif and that Fra continues to be phosphorylated when individual P-motifs are removed. Decreasing midline repulsion by reducing Robo signaling had no effect on the Abl phenotype and the phenotypes still occur in a Netrin mutant. Pan-neural over-expression of activated Rac or Cdc42 in a fra mutant also induced a significant loss in commissures, but axons did not exit the CNS.

Conclusion/Significance

Taken together, these data suggest that Fra activity is required to correctly regulate Abl-dependent cytoskeletal dynamics underlying commissure formation. In the absence of Fra, increased Abl activity appears to be incorrectly utilized downstream of other guidance receptors resulting in a loss of commissures and the abnormal projections of some axons beyond the CNS/PNS border.