Hannah A. DeBerg, Benjamin H. Blehm, Janet Sheung, Andrew R. Thompson, Carol S. Bookwalter, Seyed F. Torabi, Trina A. Shroer, Christopher L. Berger, Yi Lu, Kathleen M. Trybus and Paul R. Selvin
Disruptions in microtubule motor-transport are associated with a variety of neurodegenerative diseases. Post-translational modification of the cargo binding domain of the light- and heavy-chains of kinesin has been shown to regulate transport, but less is known about how modifications of the motor domain affect transport. Here we report on the effects of phosphorylation of a mammalian kinesin motor domain by the kinase JNK3 at a conserved serine residue (S175 in the B isoform and S176 in the A and C isoforms). Phosphorylation of this residue has been implicated in Huntington's disease, but the mechanism by which S175 phosphorylation affects transport is unclear. The ATPase, microtubule binding affinity, and processivity, are unchanged between a phosphomimetic S175D and a non-phosphorylatable S175A construct. However, we find that application of force differentiates between the two. Placement of negative charge at S175, through phosphorylation or mutation, leads to a lower stall force and decreased velocity under a load of 1 pN or greater. Sedimentation velocity experiments also show that addition of a negative charge at S175 favors the autoinhibited conformation of kinesin. These observations imply that when cargo is transported by both dynein and phosphorylated kinesin, a common occurrence in the cell, there may be a bias that favors motion toward the minus-end of microtubules. Such bias could be used to tune transport in healthy cells when properly regulated, but contribute to a disease state when misregulated.
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