Phosphatidylinositol 3-kinase, Cdc42, and Rac1 act downstream of Ras in integrin-dependent neurite outgrowth in N1E-115 neuroblastoma cells

Pas and Rho family GTPases have been ascribed important roles in signalling pathways determining cellular morphology and growth. Here we investigated the roles of the GTPases Pas, Cdc42, Rad, and Rho and that of phosphatidyli nositol 3-kinase (PI 3-kinase) in the pathway leading from serum starvation to neurite outgrowth in N1E-115 neuroblastoma cells. Serum-starved cells g rown on a laminin matrix exhibited integrin-dependent neurite outgrowth. Ex pression of dominant negative mutants of Pas, PI 3-kinase, Cdc42, or Rad al l blocked this neurite outgrowth, while constitutively activated mutants of Pas, PI 3-kinase, or Cdc42 were each sufficient to promote outgrowth even in the presence of serum. A Ras(H40C:G12V) double mutant which binds prefer entially to PI 3-kinase also promoted neurite formation. Activated Ras(G12V )-induced outgrowth required PI 3-kinase activity, but activated PI 3-kinas e-induced outgrowth did not require Pas activity. Although activated Rad by itself did not induce neurites, neurite outgrowth induced by activated CdC 42(G12V) was Rac1 dependent. Cdc42(G12V)-induced neurites appeared to lose their normal polarization, almost doubling the average number of neurites p roduced by a single cell. Outgrowth induced by activated Ras or PI 3-kinase required both Cdc42 and Rad activity, but Cdc42(G12V)-induced outgrowth di d not need Ras or PI 3-kinase activity. Active Rho(G14V) reduced outgrowth promoted by Ras(G12V). Finally, expression of dominant negative Jun N-termi nal kinase or extracellular signal-regulated kinase did not inhibit outgrow th, suggesting these pathways are not essential for this process. Our resul ts suggest a hierarchy of signalling where Ras signals through PI 3-kinase to Cdc42 and Rad activation (and Rho inactivation), culminating in neurite outgrowth, Thus, in the absence of serum factors, Pas may initiate cell cyc le arrest and terminal differentiation in N1E-115 neuroblastoma cells.