INITIATION AND MAINTENANCE OF NGF-STIMULATED NEURITE OUTGROWTH REQUIRES ACTIVATION OF A PHOSPHOINOSITIDE 3-KINASE

Application of nerve growth factor (NGF) to PC12 cells stimulates a pr ogramme of physiological changes leading to the development of a sympa thetic neuron like phenotype, one aspect of which is the development o f a neuronal morphology characterised by the outgrowth of neuritic pro cesses. We have investigated the role of phosphoinositide 3-kinase in NGF-stimulated morphological differentiation through two approaches: f irstly, preincubation with wortmannin, a reputedly specific inhibitor of phosphoinositide kinases, completely inhibited initial morphologica l responses to NGF, the formation of actin filament rich microspikes a nd subsequent neurite outgrowth. This correlated with wortmannin inhib ition of NGF-stimulated phosphatidylinositol(3,4,5)trisphosphate (PtdI nsP(3)) and phosphatidylinositol(3,4)bisphosphate (PtdIns3,4)P-2) prod uction and with inhibition of NGF-stimulated phosphoinositide 3-kinase activity in anti-phosphotyrosine immunoprecipitates. Secondly, the ov erexpression of a mutant p85 regulatory subunit of the phosphoinositid e 3-kinase, which cannot interact with the catalytic p110 subunit, als o substantially inhibited the initiation of NGF-stimulated neurite out growth. In addition, we found that wortmannin caused a rapid collapse of more mature neurites formed following several days exposure of PC12 cells to NGF. These results indicate that NGF-stimulated neurite outg rowth requires the activity of a tyrosine kinase regulated PI3-kinase and suggest that the primary product of this enzyme, PtdInsP(3), is a necessary second messenger for the cytoskeletal and membrane reorganiz ation events which occur during neuronal differentiation.