Abstract
The phosphatidylinositol 3-kinase (PI3K)/AKT pathway plays important roles in regulating cell motility. TSC2, a downstream target of AKT, is a central player in negatively controlling cell proliferation and protein translation through suppressing the activity of mTOR (mammalian target of rapamycin). However, the function of TSC2 in regulating cell migration remains unclear. Here, we show that TSC2 plays a critical role in the control of cell spreading, polarity, and migration. TSC2-deficient fibroblast cells were impaired in their ability to spread and alter actin cytoskeleton upon stimulation with insulin-like growth factor-1. Using scratch-induced polarization assay, we demonstrate that TSC2((-/-)) fibroblast cells polarized poorly toward the wound compared with wild-type cells. Similarly, knockdown of TSC2 expression in colon cancer cells resulted in a marked decrease in cell motility. Functionally, the activation of CDC42- and RAC1-GTPase was largely reduced in TSC2 knock-out fibroblast and TSC2 knockdown cancer cells. Furthermore, overexpression of an activating p110alpha mutant or short term rapamycin treatment rescued the cell polarization defect in TSC2((-/-)) fibroblast cells. Concurrently, the activation of CDC42 and RAC1 increased. The defect in cell migration and CDC42 and RAC1 activation was reversed by reintroducing TSC2 back into TSC2((-/-)) fibroblast cells. Taken together, we identified a novel role of TSC2 in controlling cell polarity and migration by regulating CDC42 and RAC1 activation.
Highlights
Protein, whereas TSC2 serves as a GTPase activation protein to promote GTP hydrolysis and inactivation of RHEB, a small G-protein activator of mTOR
Whereas loss of TSC2 function in tuberous sclerosis and other hamartoma syndromes leads to hyperactivation of mTOR and its downstream substrate p70S6 kinase (p70S6K), activation of AKT is suppressed strongly due to p70S6K-directed down-regulation of IRS-1 protein
Loss of TSC2 Expression Inhibits Cell Spreading and Disrupts Actin Cytoskeleton Rearrangement—During the experiments to examine the negative feedback regulation mediated by TSC2 using TSC2(Ϫ/Ϫ) rat fibroblast cells, we were intrigued by the observation that the morphology of WT and TSC2(Ϫ/Ϫ) cells showed striking differences
Summary
Reagents—The retroviral expression plasmid for p110␣H1074R, pBabe-p110␣H1074R, and the pBabe-puro control vector were obtained from Addgene. The colon cancer cells including HCT116 and KM20 were infected with virus containing media, and the stable knockdown cells were obtained through selection with puromycin. Generation of p110␣H1074R- or TSC2-overexpressing Stable Cell Lines—293T cells were co-transfected with pBabe-puro vector, pBabe-p110␣H1074R, or pBabe-TSC2, and a packaging plasmid pCL (Addgene) using FuGENE 6. The cell medium was collected at 72 h post-transfection and used directly to infect wild-type and TSC2(Ϫ/Ϫ) REF cells. The phase-contrast images were taken at 0 and 15 h after the scratch wounds were made (4ϫ objective, Nikon TE2000). The control and TSC2 knockdown colon cancer cells were cultured overnight in serum-free medium. The day, cells were trypsinized, resuspended in serum-free medium containing 0.1% bovine serum albumin, and added to the top well of each migration chamber with an. The numbers of migrated cells were counted using an inverted microscope
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