Slit Stimulation Recruits Dock and Pak to the Roundabout Receptor and Increases Rac Activity to Regulate Axon Repulsion at the CNS Midline

Abstract

Drosophila Roundabout (Robo) is the founding member of a conserved family of repulsive axon guidance receptors that respond to secreted Slit proteins. Here we present evidence that the SH3-SH2 adaptor protein Dreadlocks (Dock), the p21-activated serine-threonine kinase (Pak), and the Rac1/Rac2/Mtl small GTPases can function during Robo repulsion. Loss-of-function and genetic interaction experiments suggest that limiting the function of Dock, Pak, or Rac partially disrupts Robo repulsion. In addition, Dock can directly bind to Robo's cytoplasmic domain, and the association of Dock and Robo is enhanced by stimulation with Slit. Furthermore, Slit stimulation can recruit a complex of Dock and Pak to the Robo receptor and trigger an increase in Rac1 activity. These results provide a direct physical link between the Robo receptor and an important cytoskeletal regulatory protein complex and suggest that Rac can function in both attractive and repulsive axon guidance.

Cross GTPase-activating protein (CrossGAP)/Vilse links the Roundabout receptor to Rac to regulate midline repulsion

“…Although CrGAP overexpression does not appear to result in a reciprocal phenotype to that observed when CrGAP function is reduced, the apparent antagonism of Robo repulsion is consistent with the observed positive role for rac in regulating midline crossing (14,15,24). To test directly whether CrGAP antagonizes rac, we increased CrGAP levels in embryos expressing the dominant negative Rac, N17Rac.…”

“…In rac1, mtl double mutants, axons are misrouted across the midline (14). Biochemical and genetic studies have revealed an important role of Cdc42 and Rac in Robo repulsion (15,16). In contrast to the Slit-dependent negative regulation of Cdc42 in vertebrates, where the Slit͞Robo GAPs (srGAPs) link the Robo cytoplasmic domain directly to Cdc42 (16), how Slit stimulation leads to Rac activation in both vertebrates and Drosophila is unknown.…”

Inhibition of medulloblastoma cell invasion by Slit

“…Interaction of srGAP's with the Rho-GTPase, Cdc42 is essential for the repulsive activity of this guidance cue (Wong et al, 2001). Other studies support a role for Phosphatidyl inositol 3 kinase, Dock and Pak, and the Abelson (Abl) kinase and Enabled (Ena) proteins further implicating actin and microtubule reorganization in Slit-Robo signaling (Bashaw et al, 2000;Fan et al, 2003;Wang et al, 2003). Our time-lapse studies showed a change in cell morphology, as the presence of Slit2 aggregates increased the number of noninvading ruffling cells.…”

“…Slit stimulation leads to recruitment of a Dock and Pak complex to the Robo receptor and increases Rac activation (Fan et al, 2003). In contrast, Vilse, a conserved family of RhoGAPs, regulate the repellent response to Slit by binding to the intracellular domain of Robo receptors and promoting Rac inactivation (Lundstrom et al, 2004).…”

“…Cdc42 and Rac1 play roles in Slit-mediated repulsion (Wong et al, 2001;Fan et al, 2003;Lundstrom et al, 2004). To assess Rho-GTPase activation by Slit2, medulloblastoma and glioma cells were cultured in medium conditioned by HEK or Slit2 cells and pulldown assays of activated Cdc42, Rac1 and RhoA were conducted.…”