UNC-104 is the <i>C. elegans</i> homolog of kinesin-3 KIF1A known for its fast shuffling of STVs (synaptic vesicle protein transport vesicles) in axons. SYD-2 is the homolog of liprin- in <i>C. elegans</i> known to activate UNC-104, however, signals that trigger SYD-2 binding to the motor remain unknown. Because SYD-2 is a substrate of PTP-3/LAR PTPR, we speculate a role of this phosphatase in SYD-2-mediated motor activation. Indeed, co-immunoprecipitation assays revealed increased interaction between UNC-104 and SYD-2 in <i>
ptp-3</i> knockout worms. Intramolecular FRET analysis in living nematodes demonstrates that SYD-2 largely exists in an open conformation state in <i>
ptp-3</i> mutants. These assays also revealed that non-phosphorylatable SYD-2 (Y741F) exists predominately in folded conformations while phosphomimicking SYD-2 (Y741E) primarily exists in open conformations. Increased UNC-104 motor clustering was observed along axons likely as a result of elevated SYD-2 scaffolding function <i>in
ptp-3</i> mutants. Also, both motor velocities as well as cargo transport speeds were visibly increased in neurons of <i>
ptp-3</i> mutants. Lastly, epistatic analysis revealed that PTP-3 is upstream of SYD-2 to regulate its intramolecular folding.