A MAP kinase-signaling pathway mediates neurite outgrowth on L1 and requires Src-dependent endocytosis

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 terme d CRASH (corpus callosum hypoplasia, retardation, aphasia, spastic parapleg ia, and hydrocephalus), and L1 knock-out mice display defects in neuronal p rocess 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 pho sphorylation/activation of the mitogen-activated protein kinases (MAPKs) an d extracellular signal-regulated kinases 1 and 2. MAPK activation was essen tial 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 suppress ed neurite outgrowth by cerebellar neurons on L1. The nonreceptor tyrosine kinase pp60(c-src) was required for L1-triggered MAPK phosphorylation, as s hown in src-minus cerebellar neurons and by expression of the kinase-inacti ve mutant Src(K295M) in B35 neuroblastoma cells. Phosphatidylinositol 3-kin ase (PI3-kinase) and the small GTPase p21(rac) were identified as signaling intermediates to MAPK by phosphoinositide and Rac-GTP assays and expressio n of inhibitory mutants. Antibody-induced endocytosis of L1, visualized by immunofluorescence staining and confocal microscopy of B35 cells, was block ed by expression of kinase-inactive Src( K295M) and dominant-negative dynam in(K44A) but not by inhibitors of MEK or PI3-kinase. Dynamin(K44A) also inh ibited L1 antibody-triggered MAPK phosphorylation. This study supports a mo del in which pp60(c-src) regulates dynamin-mediated endocytosis of L1 as an essential step in MAPK-dependent neurite outgrowth on an L1 substrate.