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 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.     

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.     

Profilin and the Abl tyrosine kinase are required for motor axon outgrowth in the Drosophila embryo

The ability of neuronal growth cones to be guided by extracellular cues requires intimate communication between signal transduction systems and the dynamic actin-based cytoskeleton at the leading edge. Profilin, a small, actin-binding protein, has been proposed to be a regulator of the cell motility machinery at leading edge membranes. However, its requirement in the developing nervous system has been unknown. Profilin associates with members of the Enabled family of proteins, suggesting that Profilin might link Abl function to the cytoskeleton. Here, genetic analysis in Drosophila is used to demonstrate that mutations in Profilin (chickadee) and Abl (abl) display an identical growth cone arrest phenotype for axons of intersegmental nerve b (ISNb). Moreover, the phenotype of a double mutant suggests that these components function together to control axonal outgrowth.     

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.     

Interactions between the secreted protein Amalgam,its transmembrane receptor Neurotactin and the Abelson tyrosine kinase affect axon pathfinding

Two novel dosage-sensitive modifiers of the Abelson tyrosine kinase (Abl) mutant phenotype have been identified. Amalgam (Ama) is a secreted protein that interacts with the transmembrane protein Neurotactin (Nrt) to promote cell:cell adhesion. We have identified an unusual missense ama allele, ama(M109), which dominantly enhances the Abl mutant phenotype, affecting axon pathfinding. Heterozygous null alleles of ama do not show this dominant enhancement, but animals homozygous mutant for both ama and Abl show abnormal axon outgrowth. Cell culture experiments demonstrate the Ama(M109) mutant protein binds to Nrt, but is defective in mediating Ama/Nrt cell adhesion. Heterozygous null alleles of nrt dominantly enhance the Abl mutant phenotype, also affecting axon pathfinding. Furthermore, we have found that all five mutations originally attributed to disabled are in fact alleles of nrt. These results suggest Ama/Nrt-mediated adhesion may be part of signaling networks involving the Abl tyrosine kinase in the growth cone.     

Deletion of the Src homology 3 domain and C-terminal proline-rich sequences in Bcr-Abl prevents Abl interactor 2 degradation and spontaneous cell migration and impairs leukemogenesis

The hematopoietic cells from patients with Bcr-Abl-positive chronic myelogenous leukemia exhibit multiple abnormalities of cytoskeletal function. The molecular events leading to these abnormalities are not fully understood. Previously we showed that Bcr-Abl elicits ubiquitin-dependent degradation of Abl interactor proteins. Because recent studies have suggested a role of Abl interactor proteins in the pathway that regulates cytoskeletal function, we investigated whether mutations in Bcr-Abl that interfere with the signaling to Abl interactor proteins affect its leukemogenic activity. We report here that the Src homology 3 domain and C-terminal proline-rich sequences of Bcr-Abl are required for its binding to Abl interactor 2 as well as for the induction of Abl interactor 2 degradation. Although the deletion of these regions did not affect the ability of the mutant Bcr-Abl to transform hematopoietic cells to growth factor independence, it abrogated its ability to stimulate spontaneous cell migration on fibronectin-coated surfaces. Furthermore, the mutant Bcr-Abl, defective in binding to Abl interactor 2 and inducing its degradation, failed to induce chronic myelogenous leukemia-like disease in mouse. These results are consistent with a role of Abl interactor proteins in the regulation of cytoskeletal function as well as in the pathogenesis of Bcr-Abl-induced leukemogenesis.     

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 beta-amyloid precursor protein APP is tyrosine-phosphorylated in cells expressing a constitutively active form of the Abl protoncogene

The cytosolic domain of the beta-amyloid precursor protein APP interacts with three PTB (phosphotyrosine binding domain)-containing adaptor proteins, Fe65, X11, and mDab1. Through these adaptors, other molecules can be recruited at the cytodomain of APP; one of them is Mena, that binds to the WW domain (a protein module with two conserved tryptophans) of Fe65. The enabled and disabled genes of Drosophila, homologues of the mammalian Mena and mDab1 genes, respectively, are genetic modulators of the phenotype observed in flies null for the Abl tyrosine kinase gene. The involvement of Mena and mDab1 in the APP-centered protein-protein interaction network suggests the possibility that Abl plays a role in APP biology. We show that Fe65, through its WW domain, binds in vitro and in vivo the active form of Abl. Furthermore, in cells expressing the active form of Abl, APP is tyrosine-phosphorylated. Phosphopeptide analysis and site-directed mutagenesis support the hypothesis that Tyr(682) of APP(695) is the target of this phosphorylation. Co-immunoprecipitation experiments demonstrate that active Abl and tyrosine-phosphorylated APP also form a stable complex, which could result from the interaction of the pYENP motif of the APP cytodomain with the SH2 domain of Abl. These results suggest that Abl, Mena, and mDab1 are involved in a common molecular machinery and that APP can play a role in tyrosine kinase-mediated signaling.     

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.     

Inhibitors of the Abl kinase directed at either the ATP- or myristate-binding site

The ATP-competitive inhibitors dasatinib and nilotinib, which bind to catalytically different conformations of the Abl kinase domain, have recently been approved for the treatment of imatinib-resistant CML. These two new drugs, albeit very efficient against most of the imatinib-resistant mutants of Bcr–Abl, fail to effectively suppress the Bcr–Abl activity of the T315I (or gatekeeper) mutation. Generating new ATP site-binding drugs that target the T315I in Abl has been hampered, amongst others, by target selectivity, which is frequently an issue when developing ATP-competitive inhibitors. Recently, using an unbiased cellular screening approach, GNF-2, a non-ATP-competitive inhibitor, has been identified that demonstrates cellular activity against Bcr–Abl transformed cells. The exquisite selectivity of GNF-2 is due to the finding that it targets the myristate binding site located near the C-terminus of the Abl kinase domain, as demonstrated by genetic approaches, solution NMR and X-ray crystallography. GNF-2, like myristate, is able to induce and/or stabilize the clamped inactive conformation of Abl analogous to the SH2-Y527 interaction of Src. The molecular mechanism for allosteric inhibition by the GNF-2 inhibitor class, and the combined effects with ATP-competitive inhibitors such as nilotinib and imatinib on wild-type Abl and imatinib-resistant mutants, in particular the T315I gatekeeper mutant, are reviewed.     

The substrate specificity of the catalytic domain of Abl plays an important role in directing phosphorylation of the adaptor protein Crk

c-Abl preferentially phosphorylates peptide substrates that contain proline at the P+3 site (relative to the phosphorylated tyrosine). We previously described a mutant form of the Abl catalytic domain (Y569W) with altered substrate specificity at the P+3 position, as measured using synthetic peptides. In this study, we examine the phosphorylation of Crk, a protein substrate of Abl that is phosphorylated in the sequence Tyr221-Ala-Gln-Pro. In vitro, phosphorylation of Crk by Y569W Abl is greatly reduced relative to wild-type Abl. Overexpression of Y569W mutant Abl in 293T kidney cells produces a similar overall pattern of tyrosine phosphorylation as wild-type Abl, indicating that not all cellular proteins depend on Pro at P+3 for Abl recognition. However, phosphorylation of Crk by Y569W Abl in these cells is markedly reduced relative to wild-type Abl. A truncated form of Abl lacking the C-terminal polyproline region is not able to phosphorylate Crk in these assay conditions. Thus, proper phosphorylation of Crk by Abl depends not only on the interaction of the Crk SH3 domain with the Abl polyproline region, but also on the recognition of amino acids surrounding tyrosine by the Abl catalytic domain.     

An intracellular conformational sensor assay for Abl T315I

Conformational change is a common molecular mechanism for the regulation of kinase activities. Small molecule modulators of protein conformations, including allosteric kinase inhibitors, are highly wanted as tools for the interrogation of kinase biology and as selective therapeutic agents. However, straightforward cellular assays monitoring kinase conformations in a manner conducive to high-throughput screening (HTS) are not readily available. Here we describe such an HTS-compatible conformational sensor assay for Abl based on a split luciferase construct. The Abl sensor responds to intramolecular structural rearrangements associated with intracellular Abl deactivation and small molecule inhibition. The intact regulatory CAP-SH3-SH2 domain is required for the full functionality of the sensor. Moreover, a T334I Abl mutant (T315I in Abl1a) was found to be particularly well suited for HTS purposes and mechanistic intracellular studies of T334I mutant inhibitors. We expect that the split luciferase-based conformational sensor approach might be more broadly useful to probe the intracellular activation of other kinases and enzymes in general.     

Abl tyrosine kinases are required for infection by Shigella flexneri

Infection by the opportunistic bacterial pathogen Shigella flexneri stimulates tyrosine phosphorylation of host cell proteins, but the kinases involved and their effects on the regulation of cell signaling pathways during bacterial entry remain largely undefined. Here, we demonstrate a requirement for the Abl family of tyrosine kinases during Shigella internalization. Family members Abl and Arg are catalytically activated upon Shigella infection, accumulate at the site of bacterial entry, and are required for efficient bacterial uptake, as internalization is blocked upon targeted deletion of these kinases or treatment with a specific pharmacological inhibitor. We identify the adapter protein Crk as a target for Abl kinases during Shigella uptake, and show that a phosphorylation-deficient Crk mutant significantly inhibits bacterial uptake. Moreover, we define a novel signaling pathway activated during Shigella entry that links Abl kinase phosphorylation of Crk to activation of the Rho family GTPases Rac and Cdc42. Together, these findings reveal a new role for the Abl kinases, and suggest a novel approach to treatment of Shigella infections through inhibition of host cell signaling pathways.     

Abi-1 forms an epidermal growth factor-inducible complex with Cbl: role in receptor endocytosis

The Abl-interactor (Abi) proteins are involved in the regulation of actin polymerization and have recently been shown to modulate epidermal growth factor receptor (EGFR) endocytosis. Here we describe the identification of a novel complex between Abi-1 and the Cbl ubiquitin ligase that is induced by stimulation with EGF. Notably, an Abi-1 mutant lacking the SH3 domain (DeltaSH3) fails to interact with Cbl and inhibits EGFR internalization. We show that expression of the Abi-1DeltaSH3 mutant inhibits Cbl accumulation at the plasma membrane after EGF treatment. We have previously shown that the oncogenic Abl tyrosine kinase inhibits EGFR internalization. Here we report that the oncogenic Abl kinase disrupts the EGF-inducible Abi-1/Cbl complex, highlighting the importance of Abl kinases and downstream effectors in the regulation of EGFR internalization. Thus, our work reveals a new role for oncogenic Abl tyrosine kinases in the regulation of the Abi-1/Cbl protein complex and uncovers a role for the Abi-1/Cbl complex in the regulation of EGFR endocytosis.     

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.     

Use of a chemical genetic technique to identify myosin IIb as a substrate of the Abl-related gene (Arg) tyrosine kinase

Abl family kinases have been implicated in the regulation of cell morphogenesis and migration, but the molecular mechanisms through which they operate are not fully elucidated. We applied the bump-hole technique, pioneered by Shokat and colleagues, to identify direct substrates of Abl and the Abl-related gene (Arg) kinases. This technique required the engineering of Abl/Arg to utilize an unnatural ATP analogue as a phospho-donor. Mutation of T334A and T361A in Abl and Arg, respectively, altered their nucleotide specificity and allowed them to utilize N6-benzyl-ATP as a phospho-donor. These mutations did not affect the catalytic activity or protein substrate specificity of Abl and Arg. An unexpected high level of background labeling necessitated further optimization of this approach. Dialysis, pretreatment with a broad-spectrum Ser/Thr kinase inhibitor, K-252a, and purification of phosphotyrosine-containing proteins allowed for definitive identification of putative substrates. Using mass spectrometry, we identified eight putative substrates. One of these putative substrates, myosin IIB, can be phosphorylated in vivo by Arg. Our results indicate that the bump-hole technique can be used to identify Abl family kinase substrates and suggests that myosin IIB may be regulated by tyrosine phosphorylation.     

Nonmyristoylated Abl proteins transform a factor-dependent hematopoietic cell line.

N-terminal myristoylation can promote the association of proteins with the plasma membrane, a property that is required for oncogenic variants of Src and Abl to transform fibroblastic cell types. The P210bcr/abl protein of chronic myelogenous leukemia cells is not myristoylated and does not stably transform NIH 3T3 fibroblasts; however, it will transform lymphoid and myeloid cell types in vitro and in vivo, suggesting that myristoylation is not required for Abl variants to transform hematopoietic cells. To test this hypothesis, we introduced point mutations that disrupt myristoylation into two activated Abl proteins, v-Abl and a deletion mutant of c-Abl (delta XB), and examined their ability to transform an interleukin-3-dependent lymphoblastoid cell line, Ba/F3. Neither of the nonmyristoylated Abl proteins transformed NIH 3T3 fibroblasts, but like P210bcr/abl, both were capable of transforming the Ba/F3 cells to factor independence and tumorigenicity. Nonmyristoylated Abl variants did not associate with the plasma membrane in the transformed Ba/F3 cells. These results demonstrate that Abl proteins can transform hematopoietic cells in the absence of membrane association and suggest that distinct functions of Abl are required for transformation of fibroblast and hematopoietic cell types.     

Disruption of the gene encoding subunit VI of yeast cytochrome bc1 complex causes respiratory deficiency of cells with reduced cytochrome c levels

A double mutant of Saccharomyces cerevisiae, in which CYCL gene is deleted and the chromosomal copy of the 17 kDa protein gene is disrupted, has been constructed. This mutant cannot grow on nonfermentable carbon sources, but normal growth can be restored by complementation of either mutation with a yeast vector containing either the wild-type 17 kDa protein gene or the CYCl gene. These results show that although the 17 kDa protein, subunit VI of yeast cytochrome bc1 complex is dispensable for yeast mitochondrial respiration in cells with the wild-type levels of cytochrome c, the 17 kDa protein is essential for respiration when the level of cytochrome c is limited, indicating that is plays a role in electron transport. This glycerol- phenotype of the double mutant can serve as the basis for further genetic studies on the function of the 17 kDa protein in yeast mitochondria and may provide insight into the physiological function of the hinge protein, the counterpart of the yeast 17 kDa protein, in beef heart mitochondria.     

A Drosophila homolog of cyclase-associated proteins collaborates with the Abl tyrosine kinase to control midline axon pathfinding

We demonstrate that Drosophila capulet (capt), a homolog of the adenylyl cyclase-associated protein that binds and regulates actin in yeast, associates with Abl in Drosophila cells, suggesting a functional relationship in vivo. We find a robust and specific genetic interaction between capt and Abl at the midline choice point where the growth cone repellent Slit functions to restrict axon crossing. Genetic interactions between capt and slit support a model where Capt and Abl collaborate as part of the repellent response. Further support for this model is provided by genetic interactions that both capt and Abl display with multiple members of the Roundabout receptor family. These studies identify Capulet as part of an emerging pathway linking guidance signals to regulation of cytoskeletal dynamics and suggest that the Abl pathway mediates signals downstream of multiple Roundabout receptors.