Role of the p70S6K pathway in regulating the actin cytoskeleton and cell migration

Abstract

We have examined the role of endogenous 70-kDa S6 kinase (p70 S6K) in actin cytoskeletal organization and cell migration in Swiss 3T3 fibroblasts. Association of p70S6K with the actin cytoskeleton was demonstrated by cosedimentation of p70S6K with F-actin and by subcellular fractionation in which p70S6K activity was measured in the F-actin cytoskeletal fraction. Immunocytochemical studies showed that p70S6K, Akt1, PDK1, and p85 phosphoinositide 3-kinase (PI 3-kinase) were localized to the actin arc, a caveolin-enriched cytoskeletal structure located at the leading edge of migrating cells. Using a phospho-specific antibody to mammalian target of rapamycin (mTOR), we find that activated mTOR is enriched at the actin arc, suggesting that activation of the p70S6K signaling pathway is important to cell migration. Using the actin arc to assess migration, epidermal growth factor (EGF) stimulation was found to induce actin arc formation, an effect that was blocked by rapamycin treatment. We show further that actin stress fibers may function to down-regulate p70 S6K. Fibronectin stimulated stress fiber formation in the absence of growth factors and caused an inactivation of p70S6K. Conversely, cytochalasin D and the Rho kinase inhibitor Y-27632, both of which cause stress fiber disruption, increased p70S6K activity. These studies provide evidence that the p70S6K pathway is important for signaling at two F-actin microdomains in cells and regulates cell migration. Show more