The Abl pathway bifurcates to balance Enabled and Rac signaling in axon patterning in Drosophila

Abstract

The Abl tyrosine kinase signaling network controls cell migration, epithelial organization, axon patterning and other aspects of development. Although individual components are known, the relationships among them remain unresolved. We now use FRET measurements of pathway activity, analysis of protein localization and genetic epistasis to dissect the structure of this network in Drosophila. We find that the adaptor protein Disabled stimulates Abl kinase activity. Abl suppresses the actin-regulatory factor Enabled, and we find that Abl also acts through the GEF Trio to stimulate the signaling activity of Rac GTPase: Abl gates the activity of the spectrin repeats of Trio, allowing them to relieve intramolecular repression of Trio GEF activity by the Trio N-terminal domain. Finally, we show that a key target of Abl signaling in axons is the WAVE complex that promotes the formation of branched actin networks. Thus, we show that Abl constitutes a bifurcating network, suppressing Ena activity in parallel with stimulation of WAVE. We suggest that the balancing of linear and branched actin networks by Abl is likely to be central to its regulation of axon patterning.

New perspectives on the roles of Abl tyrosine kinase in axon patterning

“…24,33 For some time, the role of Dab in Abl signaling was controversial, although several studies have now convincingly established the central role of Dab in the Abl network. 27,34,35 Dab interacts synergistically with Abl and trio mutations and antagonistically to ena, demonstrating that Dab is a positive component of the Abl pathway, and Dab protein co-immunoprecipitates with Abl. In addition, it was found that Dab regulates Abl localization in the retinal neuroepithelium and embryonic epithelia of Drosophila, suggesting it may be upstream of Abl.…”

“…This makes immediate readouts of pathway activity essential for drawing reliable conclusions. 27 Finally, the dynamic processes in the cytoskeleton are typically not linear pathways; instead they are cyclical. Manipulations that simply lock a piece of the machine into an ON or OFF state generally are not an effective way to study the mechanism of the cycle phenotypically since either condition just breaks the cycle.…”

“…32 Recently, using a combination of direct FRET measurements of the biochemical outputs of pathway activity and analysis of protein localization, in addition to genetic epistasis, we explored the architecture of the Abl pathway in Drosophila neurons. 27 This 3-pronged approach allowed us to establish that Abl defines a bifurcating pathway that achieves a balance between the 2 major classes of actin structures in the cell, linear actin filaments vs. branched actin networks 27 ( Fig. 1).…”

“…35 This has now been confirmed by FRET measurements of Abl kinase activity that showed Dab stimulates the kinase activity of Abl, presumably by physical binding. 27 It has long been known that Abl interacts genetically with Trio/Rac signaling in neurons. [36][37][38] Genetic experiments showed that axonal phenotypes of trio mutants mimic those of Abl mutations and heterozygous mutations of Trio enhance Abl phenotypes and vice versa.…”

“…We further verified this conclusion by demonstrating that UASTrio suppresses the phenotype of an Abl mutant, but not vice versa, confirming that Abl acts linearly upstream of Trio in axon growth and guidance. 27 Mechanisms by which Abl regulates Trio to stimulate Rac GTPase If Abl regulates Trio, how does it do so? At a molecular level, we ruled out the hypothesis that Abl acts by directly stimulating the Trio GEF1 domain, by showing that the activity of the isolated Trio GEF1 is not altered in an Abl mutant.…”