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

Abstract

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 GSK3β plays a role in motor function. Here, we report that a particular site on kinesin 1 motor domain (KHC), S314, is phosphorylated by GSK3β in vivo. The GSK3β-phosphomimetic-KHCS314D stalled kinesin 1 motility without dissociating from microtubules, indicating that constitutive GSK3β phosphorylation of the motor domain acts as a STOP. In contrast, uncoordinated mitochondrial motility was observed in CRISPR/Cas9-GSK3β 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 GSK3β 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.

Disruption of axonal transport in neurodegeneration

“…Phosphotransferase activity regulates motor protein function ( Figure 2 ). For example, the kinase glycogen synthase kinase-3β (GSK3β) phosphorylates KHC to inhibit axonal transport and also phosphorylates KLCs to release cargoes ( 15 , 16 ). Studies using perfusion of kinases and their inhibitors into squid axoplasm showed that the stress-activated protein kinases c-Jun N-terminal kinase 3 (JNK3) and p38 mitogen-activated protein kinase (p38 MAPK) directly phosphorylate KHCs to inhibit anterograde transport ( 17 , 18 ).…”

Phosphoregulation of Kinesins Involved in Long-Range Intracellular Transport

“…A recent study further suggested that Glycogen Synthase Kinase 3β (GKS3β) could phosphorylate Drosophila KHC at the S314 residue in the α6 helix interfacing the head and the neck-linker domain (Banerjee et al, 2021). The phosphomimetic S314D and the phosphodeficient S314A substitutions severely affected the motor function.…”

“…Although it did not detach the motor from the microtubule, the ATPase activity and microtubule gliding in vitro were significantly reduced. The mutations also affected the axonal transport of mitochondria in Drosophila (Banerjee et al, 2021). The neck-linker domain moves to a significant extent during the ATPase cycle to generate the force along microtubule during the ATPase cycle (Rice et al, 1999;Vale and Milligan, 2000), which is likely to transiently increase tension on the α6 segment during each stepping cycle (Qin et al, 2020).…”

Inhibition of glycogen synthase kinase 3 by lithium, a mechanism in search of specificity

“…Phosphorylation of kinesins subunits on different sites by GSK3 can lead to different outcomes. For example, GSK3β phosphorylation of KIF1A at S402 impairs transport but does not regulate mobility, whereas phosphorylation of kinesin-1 motor domain at S314 halts motility without detaching from microtubules ( Gan et al, 2020 ; Banerjee et al, 2021 ). In SH-SY5Y cells and neuronal hippocampal cultures, GSK3 phosphorylation of KLC1 and KLC2 results in decreased association of cargoes from the kinesin cargo system, indicating failed delivery of cargos to their target locations ( Du et al, 2010 ; Morel et al, 2012 ).…”