Rapamycin Inhibits Cytoskeleton Reorganization and Cell Motility by Suppressing RhoA Expression and Activity

Abstract

The mammalian target of rapamycin (mTOR) functions in cells at least as two complexes, mTORC1 and mTORC2. Intensive studies have focused on the roles of mTOR in the regulation of cell proliferation, growth, and survival. Recently we found that rapamycin inhibits type I insulin-like growth factor (IGF-1)-stimulated lamellipodia formation and cell motility, indicating involvement of mTOR in regulating cell motility. This study was set to further elucidate the underlying mechanism. Here we show that rapamycin inhibited protein synthesis and activities of small GTPases (RhoA, Cdc42, and Rac1), crucial regulatory proteins for cell migration. Disruption of mTORC1 or mTORC2 by down-regulation of raptor or rictor, respectively, inhibited the activities of these proteins. However, only disruption of mTORC1 mimicked the effect of rapamycin, inhibiting their protein expression. Ectopic expression of rapamycin-resistant and constitutively active S6K1 partially prevented rapamycin inhibition of RhoA, Rac1, and Cdc42 expression, whereas expression of constitutively hypophosphorylated 4E-BP1 (4EBP1-5A) or down-regulation of S6K1 by RNA interference suppressed expression of the GTPases, suggesting that both mTORC1-mediated S6K1 and 4E-BP1 pathways are involved in protein synthesis of the GTPases. Expression of constitutively active RhoA, but not Cdc42 and Rac1, conferred resistance to rapamycin inhibition of IGF-1-stimulated lamellipodia formation and cell migration. The results suggest that rapamycin inhibits cell motility at least in part by down-regulation of RhoA protein expression and activity through mTORC1-mediated S6K1 and 4E-BP1-signaling pathways.The mammalian target of rapamycin (mTOR), 3 a member of the phosphoinositide 3Ј-kinase-related kinase family, is a central controller of cell proliferation, growth, and survival (1). Rapamycin can form a complex with FK506 binding protein 12 and then bind mTOR, selectively inhibiting its kinase activity and function (1). Recently, two mTOR complexes (mTORC1 and mTORC2) have been identified in mammalian cells (1). mTORC1 is composed of mTOR, mLST8 (also termed G-protein ␤-subunit-like protein, G␤L, a yeast homolog of LST8), PRAS40 (proline-rich Akt substrate 40 kDa), and raptor (regulatory-associated protein of mTOR) and is rapamycin-sensitive (2-8). In response to growth factors and nutrients, mTORC1 regulates cell proliferation and growth by modulating many processes, including protein synthesis and ribosome biogenesis through downstream effectors like 4E-BP1 (eukaryotic initiation factor 4E-binding protein 1) and S6K1 (ribosomal p70 S6 kinase 1) (1, 9). mTORC2 consists of mTOR, mLST8, mSin1 (mammalian stress-activated protein kinase-interacting protein 1), rictor (rapamycin-insensitive companion of mTOR), and protor (protein observed with rictor; also named PRR5 (proline-rich protein 5)), and is rapamycin-insensitive (10 -16). mTORC2 phosphorylates Akt and PKC, signals to small GTPases (RhoA and Rac1), and controls cytoskeleton organization (11,(17)(18)(19)(20). Most recent...

Inhibition of Chemokine (CXC Motif) Ligand 12/Chemokine (CXC Motif) Receptor 4 Axis (CXCL12/CXCR4)-mediated Cell Migration by Targeting Mammalian Target of Rapamycin (mTOR) Pathway in Human Gastric Carcinoma Cells

“…As shown in 4B, PI-103, and rapamycin had little effect on the protein expression of these GTPases, whereas PP-242 slightly inhibited protein expression of RhoA and Rac1. The inconsistency between our results and previous report (19) might reflect different cell types used and time of treatment in the studies. We next examined the active form of RhoA, Rac, and Cdc42 upon inhibition of PI3K and mTOR signaling using a pulldown assay that specifically recognizes the active GTP-bound forms.…”

“…The CXCL12/CXCR4 axis and its downstream signaling could be therapeutic targets for preventing metastasis of gastric cancer. Moreover, it has been reported that both mTORC1 and mTORC2 play critical roles in the regulation of tumor cell motility and cancer metastasis (12,19). We found that the CXCL12/CXCR4 axis was able to activate the PI3K/mTOR pathway and stimulate cell migration in human MKN-45 gastric cancer cells.…”

“…It has been reported recently that rapamycin inhibits cytoskeleton reorganization and cell motility stimulated by insulin-like growth factor 1 via suppressing RhoA expression and activity (19). We thus detected the protein levels of Rac1, Cdc42, and RhoA in MKN-45 cells pretreated with PI-103, rapamycin, or PP242 for 6 h before CXCL12 stimulation.…”

Control of mTORC1 signaling by the Opitz syndrome protein MID1

“…Our results presented here strongly suggest that the defect in mTORC1 signaling resulting from dysregulation of the MID1/PP2A axis is one of the avenues leading to OS pathogenesis. Of note, malformations occurring in OS patients have been attributed to defective migration patterns of neural crest cells needed to mediate ventral midline tissue formation in a very specific time window during development (1), and mTORC1 signaling not only is responsible for cytoskeleton dynamics and intracellular transport-both processes that significantly influence the migration potential of cells-but also has a direct regulatory function in migrating cells (52,53). Dysfunctional mTORC1 signaling in OS patients with MID1 mutations is therefore likely to significantly contribute to the OS phenotype.…”

Neuritic complexity of hippocampal neurons depends on WIP‐mediated mTORC1 and Abl family kinases activities

“…2008) and promote RhoA expression and activity (Liu et al. 2010). RhoA activity is also modulated by Abl family kinases (Bradley and Koleske 2009).…”