In neurons, amyloid β-protein precursor (APP) is transported by binding to kinesin-1, mediated by JNK-interacting protein 1b (JIP1b), which generates the enhanced fast velocity (EFV) and efficient high frequency (EHF) of APP anterograde transport. Previously, we showed that EFV requires conventional interaction between the JIP1b C-terminal region and the kinesin light chain 1 (KLC1) tetratricopeptide repeat, whereas EHF requires a novel interaction between the central region of JIP1b and the coiled-coil domain of KLC1. We found that phosphorylatable Thr466 of KLC1 regulates the conventional interaction with JIP1b. Substitution of Glu for Thr466 abolished this interaction and EFV, but did not impair the novel interaction responsible for EHF. Phosphorylation of KLC1 at Thr466 increased in aged brains, and JIP1 binding to kinesin-1 decreased, suggesting that APP transport is impaired by aging. We conclude that phosphorylation of KLC1 at Thr466 regulates the velocity of transport of APP by kinesin-1 by modulating its interaction with JIP1b. INTRODUCTIONAmyloid β-protein precursor (APP), a type I membrane protein that is processed to form amyloid β-protein (Aβ), is deeply implicated in Alzheimer's disease pathogenesis. The APP gene generates three main isoforms: APP695, APP750, and APP770. Although APP is ubiquitously expressed in many tissues, APP695 is expressed exclusively and at high levels in neurons (reviewed in Suzuki and Nakaya, 2008;Huang and Mucke, 2012).One of the most important functions of APP in neurons is as a cargo receptor for kinesin-1, a conventional kinesin, which was first identified as an anterograde molecular motor in squid giant axon (Vale et al., 1985). Functional kinesin-1 forms a tetramer comprising two kinesin heavy chains (KHCs) and two kinesin light chains (KLCs), and plays an essential role in anterograde transport of many cargoes, including membrane vesicles, organelles, and RNA. This transport function is especially critical in neurons, which form huge numbers of connections with each other via extended neurites (reviewed in Hirokawa et al., 2010;Bentley and Banker, 2016). Kinesin-1 is the most extensively analyzed anterograde motor of the kinesin superfamily of proteins (KIFs); accordingly, many of its biophysical properties, including generation of mechanical force driven by ATP hydrolysis, are well understood (reviewed in Lu and Gelfand, 2017