Fig. 6. The VAB-1 tyrosine kinase domain physically interacts with the juxtamembrane and CC1 region of SAX-3/Robo. (A) Schematic diagrams of the VAB-1 Eph RTK and SAX-3/Robo receptor. VAB-1 Kinase region (669 aa-985 aa) was fused in frame to the GAL-4 DNA-binding domain and used as bait in yeast two-hybrid assays. The SAX-3 intracellular CC1, CC3 and CC2 regions are shown as blue boxes. SAX-3 does not have a recognizable CC0 consensus and CC3 is N terminal to CC2 when compared with other Robo receptors. (B) Deletion constructs of the intracellular portion of SAX-3 were tested for their ability to bind the VAB-1 tyrosine kinase domain. Liquid ß-Galalctosidase units and yeast X-GAL overlay assays indicate the quantity of activation (interaction). The juxtamembrane and CC1 region is necessary and sufficient for the interaction with the VAB-1 kinase region (dashed rectangle). (C) GST `pull-down' experiments confirm that the SAX-3 juxtamembrane and CC1 region (900 aa-1030 aa) is sufficient for the interaction with the VAB-1 intracellular region. MBP-SAX-3 E. coli lysates were incubated with GST or GST-VAB-1. MBP-SAX-3 input (Load), unbound (UB) and bound (B) fractions were analyzed by SDS-PAGE and western blotting using anti-MBP to visualize SAX-3. The SAX-3 interaction is specific to GST-VAB-1 and not GST alone. GST-VAB-1 is also specific for MBP-SAX-3, as it does not bind the MBP degradation products (asterisks) in the Load.