A stop or go switch: glycogen synthase kinase 3 beta phosphorylation of the kinesin 1 motor domain at Ser314 halts motility without detaching from microtubules

It is more than 25 years since the discovery that kinesin 1 is phosphorylated by several protein kinases. However, fundamental questions still remain as to how specific protein kinase(s) contribute to particular motor functions under physiological conditions. Because, within an whole organism, kinase cascades display considerable crosstalk and play multiple roles in cell homeostasis, deciphering which kinase(s) is/are involved in a particular process has been challenging. Previously, we found that GSK3 beta plays a role in motor function. Here, we report that a particular site on kinesin 1 motor domain (KHC), S314, is phosphorylated by GSK3 beta in vivo. The GSK3 beta-phosphomimetic-KHCS314D stalled kinesin 1 motility without dissociating from microtubules, indicating that constitutive GSK3 beta phosphorylation of the motor domain acts as a STOP. In contrast, uncoordinated mitochondrial motility was observed in CRISPR/Cas9-GSK3 beta non-phosphorylatable-KHCS314A Drosophila larval axons, owing to decreased kinesin 1 attachment to microtubules and/or membranes, and reduced ATPase activity. Together, we propose that GSK3 beta phosphorylation fine-tunes kinesin 1 movement in vivo via differential phosphorylation, unraveling the complex in vivo regulatory mechanisms that exist during axonal motility of cargos attached to multiple kinesin 1 and dynein motors.