The phosphorylation of kinesin regulates its binding to synaptic vesicles.
Membrane organella are transported bidirectionally in cells, and the axonal transport system has provided an ideal model system for studying this bidirectional transport. Kinesin and cytoplasmic dynein were identified as candidates for the motor molecules of fast axonal transport, which transport organella along microtubules anterogradely and retrogradely. However, the mechanism that controls this bidirectional transport is unknown. Our previous work revealed that kinesin in axons was associated abundantly with anterogradely transported membranous organella, most of which are believed to be precursors of synaptic vesicles and axonal plasma membranes, while the fractions bound to retrogradely transported ones were very small (Hirokawa, N., Sato-Yoshitake, R., Kobayashi, N., Pfister, K. K., Bloom, G. S., and Brady, S. T. (1991) J. Cell Biol. 114, 295-302). Here we demonstrated in vitro that the binding of kinesin to synaptic vesicles was concentration-dependent and saturable and could be released by high salt concentration. When kinesin was phosphorylated by cAMP-dependent protein kinase, its binding to symaptic vesicles was significantly reduced. By motility assay and by statistical analysis using electron microscopy, we further revealed that synaptic vesicles preincubated with phosphorylated kinesin associated less frequently with microtubules than synaptic vesicles preincubated with unphosphorylated kinesin. The phosphorylation of kinesin should therefore play an essential role in regulating the direction of fast axonal transport by inhibiting its binding to membrane organella, thus releasing it from membrane organella at nerve terminals.
Sato-Yoshitake
R
R
Department of Anatomy and Cell Biology, School of Medicine, University of Tokyo, Japan.
Yorifuji
H
H
Inagaki
M
M
Hirokawa
N
N
D000818:Animals; D001921:Brain / Q000378:metabolism*; D002540:Cerebral Cortex / Q000378:metabolism*; D016547:Kinesins / Q000302:isolation & purification / Q000378:metabolism* / Q000648:ultrastructure; D007700:Kinetics; D008854:Microscopy, Electron; D008870:Microtubules / Q000378:metabolism / Q000648:ultrastructure; D008961:Models, Structural; D010766:Phosphorylation; D011485:Protein Binding; D051381:Rats; D013572:Synaptic Vesicles / Q000378:metabolism* / Q000648:ultrastructure
D016547:Kinesins