INVOLVEMENT OF P90(RSK) IN NEURITE OUTGROWTH MEDIATED BY THE CELL-ADHESION MOLECULE L1

L1 is a neural cell adhesion molecule that has been shown to help guid e nascent axons to their targets, This guidance is based on specific i nteractions of L1 with its binding partners and is likely to involve s ignaling cascades that alter cytoskeletal elements in response to thes e binding events. We have examined the phosphorylation of L1 and the r ole it may have in L1-directed neurite outgrowth. Cytosolic extracts f rom nerve growth factor-stimulated PC12 cells were fractionated by ani on-exchange chromatography, and an activity was found that phosphoryla ted the cytoplasmic domain of L1. This activity was then assayed using a battery of L1-derived synthetic peptides, Based on these peptide as says and sequencing of radiolabeled L1 proteolytic fragments, the phos phorylation site was determined to be Ser(1152). Western blot analysis demonstrated that the L1 kinase activity from PC12 cells that phospho rylated this site was co-eluted with the S6 kinase, p90(rsk), Moreover , S6 kinase activity and p90(rsk) immunoreactivity co-immunoprecipitat e with L1 from brain, and metabolic labeling studies have demonstrated that Ser(1152) is phosphorylated in vivo in the developing rat brain. The phosphorylation site is located in a region of high conservation between mammalian L1 sequences as well as L1-related molecules in vert ebrates from fish to birds, We performed studies to investigate the fu nctional significance of this phosphorylation, Neurons were loaded wit h peptides that encompass the phosphorylation site, as well as the fla nking regions, and their effects on neurite outgrowth were observed. T he peptides, which include Ser(1152), inhibit neurite outgrowth on L1 but not on a control substrate, laminin. A nonphosphorylatable peptide carrying a Ser to Ala mutation did not affect neurite outgrowth on ei ther substrate, These data demonstrate that the membrane-proximal 15 a mino acids of the cytoplasmic domain of L1 are important for neurite o utgrowth on L1, and the interactions it mediates may be regulated by p hosphorylation of Ser(1152).