title: Trk activation of the ERK1/2 kinase pathway stimulates intermediate chain phosphorylation and recruits cytoplasmic dynein to signaling endosomes for retrograde axonal transport.
abstract: The retrograde transport of Trk-containing endosomes from the axon to the cell body by cytoplasmic dynein is necessary for axonal and neuronal survival. We investigated the recruitment of dynein to signaling endosomes in rat embryonic neurons and PC12 cells. We identified a novel phosphoserine on the dynein intermediate chains (ICs), and we observed a time-dependent neurotrophin-stimulated increase in intermediate chain phosphorylation on this site in both cell types. Pharmacological studies, overexpression of constitutively active MAP kinase kinase, and an in vitro assay with recombinant proteins demonstrated that the intermediate chains are phosphorylated by the MAP kinase ERK1/2, extracellular signal-regulated kinase, a major downstream effector of Trk. Live cell imaging with fluorescently tagged IC mutants demonstrated that the dephosphomimic mutants had significantly reduced colocalization with Trk and Rab7, but not a mitochondrial marker. The phosphorylated intermediate chains were enriched on immunoaffinity-purified Trk-containing organelles. Inhibition of ERK reduced the amount of phospho-IC and the total amount of dynein that copurified with the signaling endosomes. In addition, inhibition of ERK1/2 reduced the motility of Rab7- and TrkB-containing endosomes and the extent of their colocalization with dynein in axons. NGF-dependent survival of sympathetic neurons was significantly reduced by the overexpression of the dephosphomimic mutant IC-1B-S80A, but not WT IC-1B, further demonstrating the functional significance of phosphorylation on this site. These results demonstrate that neurotrophin binding to Trk initiates the recruitment of cytoplasmic dynein to signaling endosomes through ERK1/2 phosphorylation of intermediate chains for their subsequent retrograde transport in axons.
authors.affiliation: Department of Cell Biology, University of Virginia, Charlottesville, VA 22908, USA.
authors.forename: David J
authors.initials: DJ
authors.lastname: Mitchell
authors.forename: Kiev R
authors.initials: KR
authors.lastname: Blasier
authors.forename: Erin D
authors.initials: ED
authors.lastname: Jeffery
authors.forename: Mitchell W
authors.initials: MW
authors.lastname: Ross
authors.forename: Ashok K
authors.initials: AK
authors.lastname: Pullikuth
authors.forename: Dong
authors.initials: D
authors.lastname: Suo
authors.forename: Juyeon
authors.initials: J
authors.lastname: Park
authors.forename: W Russell
authors.initials: WR
authors.lastname: Smiley
authors.forename: Kevin W-H
authors.initials: KW
authors.lastname: Lo
authors.forename: Jeffrey
authors.initials: J
authors.lastname: Shabanowitz
authors.forename: Christopher D
authors.initials: CD
authors.lastname: Deppmann
authors.forename: Jonathan C
authors.initials: JC
authors.lastname: Trinidad
authors.forename: Donald F
authors.initials: DF
authors.lastname: Hunt
authors.forename: Andrew D
authors.initials: AD
authors.lastname: Catling
authors.forename: K Kevin
authors.initials: KK
authors.lastname: Pfister
mesh_terms: D000818:Animals; D001370:Axonal Transport / Q000502:physiology*; D015153:Blotting, Western; D002462:Cell Membrane / Q000378:metabolism / Q000502:physiology; D002470:Cell Survival / Q000502:physiology; D003593:Cytoplasm / Q000502:physiology*; D004398:Dyneins / Q000502:physiology*; D004591:Electrophoresis, Polyacrylamide Gel; D011992:Endosomes / Q000502:physiology*; D004789:Enzyme Activation; D020935:MAP Kinase Signaling System / Q000235:genetics / Q000502:physiology*; D020932:Nerve Growth Factor / Q000502:physiology; D009414:Nerve Growth Factors / Q000494:pharmacology; D009474:Neurons / Q000502:physiology; D015388:Organelles / Q000502:physiology; D016716:PC12 Cells; D010766:Phosphorylation; D010957:Plasmids / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics; D051381:Rats; D020917:Receptor, trkA / Q000502:physiology*; D015398:Signal Transduction / Q000502:physiology; D014162:Transfection
chemical_list: D009414:Nerve Growth Factors; D034741:RNA, Small Interfering; D020932:Nerve Growth Factor; D020917:Receptor, trkA; D004398:Dyneins