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Previous Article | Next Article 
A MAP Kinase-Signaling Pathway Mediates Neurite Outgrowth on L1
and Requires Src-Dependent Endocytosis
Ralf-Steffen
Schmid,
Wendy M.
Pruitt, and
Patricia F.
Maness
Department of Biochemistry, School of Medicine, University of North
Carolina, Chapel Hill, North Carolina 27599-7260
The neural cell adhesion molecule L1 mediates the axon
outgrowth, adhesion, and fasciculation necessary for proper development of synaptic connections. Mutations of human L1 cause an X-linked mental
retardation syndrome termed CRASH (corpus callosum hypoplasia, retardation, aphasia, spastic paraplegia, and hydrocephalus), and L1
knock-out mice display defects in neuronal process extension resembling
the CRASH phenotype. Little is known about the biochemical or
cellular mechanism by which L1 performs neuronal functions. Here it is
demonstrated that clustering of L1 with antibodies or L1 protein in
rodent B35 neuroblastoma and cerebellar neuron cultures induced the
phosphorylation/activation of the mitogen-activated protein kinases
(MAPKs) and extracellular signal-regulated kinases 1 and 2. MAPK activation was essential for L1-dependent neurite outgrowth,
because chemical inhibitors
[2-(2'-amino-3'-methoxyphenyl)-oxanaphthalen-4-one and
1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)butadiene] of
the MAPK kinase MEK strongly suppressed neurite outgrowth by cerebellar neurons on L1. The nonreceptor tyrosine kinase
pp60c-src was required for L1-triggered MAPK phosphorylation,
as shown in src-minus cerebellar neurons and by
expression of the kinase-inactive mutant Src(K295M) in B35
neuroblastoma cells. Phosphatidylinositol 3-kinase (PI3-kinase)
and the small GTPase p21rac were identified as
signaling intermediates to MAPK by phosphoinositide and Rac-GTP assays
and expression of inhibitory mutants. Antibody-induced endocytosis of
L1, visualized by immunofluorescence staining and confocal microscopy
of B35 cells, was blocked by expression of kinase-inactive Src(K295M)
and dominant-negative dynamin(K44A) but not by inhibitors of MEK or
PI3-kinase. Dynamin(K44A) also inhibited L1 antibody-triggered MAPK
phosphorylation. This study supports a model in which pp60c-src
regulates dynamin-mediated endocytosis of L1 as an essential step in
MAPK-dependent neurite outgrowth on an L1 substrate.
Key words:
neurite outgrowth; endocytosis; neural cell adhesion
molecule; signal transduction; Src; MAP kinase; PI3-kinase
Copyright © 2000 Society for Neuroscience 0270-6474/00/20114177-12$05.00/0
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