Neurofilament heavy chain side arm phosphorylation regulates axonal transport of neurofilaments.
Neurofilaments possess side arms that comprise the carboxy-terminal domains of neurofilament middle and heavy chains (NFM and NFH); that of NFH is heavily phosphorylated in axons. Here, we demonstrate that phosphorylation of NFH side arms is a mechanism for regulating transport of neurofilaments through axons. Mutants in which known NFH phosphorylation sites were mutated to preclude phosphorylation or mimic permanent phosphorylation display altered rates of transport in a bulk transport assay. Similarly, application of roscovitine, an inhibitor of the NFH side arm kinase Cdk5/p35, accelerates neurofilament transport. Analyses of neurofilament movement in transfected living neurons demonstrated that a mutant mimicking permanent phosphorylation spent a higher proportion of time pausing than one that could not be phosphorylated. Thus, phosphorylation of NFH slows neurofilament transport, and this is due to increased pausing in neurofilament movement.
Department of Neuroscience, The Institute of Psychiatry, Denmark Hill, London SE5 8AF, UK.
Steven
S
Ackerley
Paul
P
Thornhill
Andrew J
AJ
Grierson
Janet
J
Brownlees
Brian H
BH
Anderton
P Nigel
PN
Leigh
Christopher E
CE
Shaw
Christopher C J
CC
Miller
D000595:Amino Acid Sequence / Q000235:genetics; D000818:Animals; D001370:Axonal Transport / Q000187:drug effects / Q000235:genetics*; D001369:Axons / Q000187:drug effects / Q000378:metabolism*; D001665:Binding Sites / Q000187:drug effects / Q000235:genetics; D019556:COS Cells; D051360:Cyclin-Dependent Kinase 5; D018844:Cyclin-Dependent Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins; D009154:Mutation / Q000235:genetics; D009420:Nervous System / Q000378:metabolism*; D016900:Neurofilament Proteins / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D017434:Protein Structure, Tertiary / Q000187:drug effects / Q000502:physiology; D011687:Purines / Q000494:pharmacology; D011993:Recombinant Fusion Proteins; D000077546:Roscovitine; D012694:Serine / Q000378:metabolism
D004791:Enzyme Inhibitors; D008164:Luminescent Proteins; D016900:Neurofilament Proteins; D011687:Purines; D011993:Recombinant Fusion Proteins; D000077546:Roscovitine; C051774:neurofilament protein H; D049452:Green Fluorescent Proteins; D012694:Serine; D051360:Cyclin-Dependent Kinase 5; C495905:CDK5 protein, human; D018844:Cyclin-Dependent Kinases