Regulation of SLIT-Robo Signaling by Scaffolding Proteins

Thesis (Ph.D.)--University of Washington, 2015Axon guidance receptors in the growth cone respond to secreted molecular cues, directing axons towards their appropriate targets of innervation. Many of these receptors complex with scaffolding proteins which recruit protein kinases and protein phosphatases to control the efficacy, context, and duration of neuronal phosphorylation events. The A-Kinase Anchoring Protein AKAP79/150 interacts with protein kinase A (PKA), protein kinase C (PKC), and protein phosphatase 2B (PP2B, calcineurin) to modulate second messenger signaling events. In a mass spectrometry based screen for additional AKAP79/150 binding partners, we have identified the Roundabout axonal guidance receptor Robo2 and its ligands Slit2 and Slit3. Biochemical and cellular approaches confirm that a linear sequence located in the cytoplasmic tail of Robo2 (residues 991-1070) interfaces directly with sites on the anchoring protein. Additional studies show that AKAP79/150 interacts with the Robo3 receptor in a similar manner. Immunofluorescent staining detects overlapping expression patterns for murine AKAP150, Robo2, and Robo3 in a variety of brain regions including hippocampal region CA1 and the islands of calleja. In vitro kinase assays, peptide spot array mapping, and proximity ligation assay staining approaches establish that human AKAP79-anchored PKC selectively phosphorylates the Robo3.1 receptor subtype on serine 1330. In a parallel set of experiments, we also identified an interaction between Robo3 and the 14-3-3 family of adaptor proteins. Binding between these proteins can be disrupted using the R18 14-3-3 competitor peptide, confirming the specificity of this interaction. In addition, PKC activation decreases the binding of 14-3-3 to the Robo3 receptor. These finding suggest that scaffolding proteins interact with specific Robo receptor subcomplexes, providing enhanced regulation of this family of axon guidance molecules