Motor domain phosphorylation modulates kinesin-1 transport.
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 (Ser-175 in the B isoform and Ser-176 in the A and C isoforms). Phosphorylation of this residue has been implicated in Huntington disease, but the mechanism by which Ser-175 phosphorylation affects transport is unclear. The ATPase, microtubule-binding affinity, and processivity are unchanged between a phosphomimetic S175D and a nonphosphorylatable S175A construct. However, we find that application of force differentiates between the two. Placement of negative charge at Ser-175, through phosphorylation or mutation, leads to a lower stall force and decreased velocity under a load of 1 piconewton or greater. Sedimentation velocity experiments also show that addition of a negative charge at Ser-175 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.
From the Physics Department and Center for the Physics of Living Cells.
Hannah A
HA
DeBerg
From the Physics Department and Center for the Physics of Living Cells.
Benjamin H
BH
Blehm
From the Physics Department and Center for the Physics of Living Cells.
Janet
J
Sheung
the Department of Molecular Physiology and Biophysics, University of Vermont, Burlington, Vermont 05405.
Andrew R
AR
Thompson
the Department of Molecular Physiology and Biophysics, University of Vermont, Burlington, Vermont 05405.
Carol S
CS
Bookwalter
the Departments of Biochemistry.
Seyed F
SF
Torabi
the Department of Biology, The Johns Hopkins University, Baltimore, Maryland 21218.
Trina A
TA
Schroer
the Department of Molecular Physiology and Biophysics, University of Vermont, Burlington, Vermont 05405.
Christopher L
CL
Berger
the Departments of Biochemistry; Chemistry, University of Illinois, Urbana, Illinois 61801.
Yi
Y
Lu
the Department of Molecular Physiology and Biophysics, University of Vermont, Burlington, Vermont 05405.
Kathleen M
KM
Trybus
From the Physics Department and Center for the Physics of Living Cells. Electronic address: selvin@illinois.edu.
Paul R
PR
Selvin
D019943:Amino Acid Substitution; D000818:Animals; D002417:Cattle; D004398:Dyneins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006801:Humans; D006816:Huntington Disease / Q000235:genetics / Q000378:metabolism; D016547:Kinesins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D048057:Mitogen-Activated Protein Kinase 10 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020125:Mutation, Missense; D010766:Phosphorylation / Q000235:genetics; D017434:Protein Structure, Tertiary; D021381:Protein Transport / Q000235:genetics; D061987:Sf9 Cells; D018411:Spodoptera
C488101:KIF5B protein, human; D048057:Mitogen-Activated Protein Kinase 10; D004398:Dyneins; D016547:Kinesins
Jun N-terminal Kinase (JNK); Kinesin; Molecular Motors; Phosphorylation; Single-molecule Biophysics