Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Original Article
  • Published:

Rapamycin inhibits F-actin reorganization and phosphorylation of focal adhesion proteins

Abstract

An early event of cell migration is characterized as the rapid reorganization of the actin cytoskeleton. Recently, we have demonstrated that rapamycin inhibits tumor cell motility. To understand the underlying mechanism, this study was set to determine whether rapamycin inhibition of cell motility is related to its prevention of F-actin reorganization. We found that rapamycin prevented type I insulin-like growth factor (IGF-I)-stimulated F-actin reorganization in human rhabdomyosarcoma (Rh30), Ewing sarcoma (Rh1), glioblastoma (U-373) and prostate carcinoma (PC-3) cells, and concurrently inhibited phosphorylation of focal adhesion proteins, including focal adhesion kinase (FAK), paxillin and p130Cas in the cells. The effect of rapamycin was blocked by expression of a rapamycin-resistant mutant of mTOR (mTORrr), but not a kinase-dead mTORrr. Downregulation of raptor mimicked the effect of rapamycin. Cells infected with a recombinant adenovirus expressing constitutively active and rapamycin-resistant mutant of p70 S6 kinase 1 (S6K1) conferred to resistance to rapamycin. Further, IGF-I failed to stimulate F-actin reorganization and phosphorylation of the focal adhesion proteins in the S6K1-downregulated cells. Expression of constitutively hypophosphorylated eukaryotic initiation factor 4E (eIF4E)-binding protein 1 (4E-BP1-5A) inhibited IGF-I-stimulated F-actin reorganization, but did not alter the cellular protein or phosphorylation levels of the focal adhesion proteins. The results suggest that rapamycin inhibits IGF-I-induced F-actin reorganization and phosphorylation of the focal adhesion proteins by disruption of mTOR–raptor complex. Both S6K1 and 4E-BP1 pathways, mediated by the mTOR–raptor complex, are involved in the regulation of IGF-I-stimulated F-actin reorganization, but only the former controls IGF-I-stimulated phosphorylation of the focal adhesion proteins.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
Figure 8
Figure 9

Similar content being viewed by others

References

  • Berven LA, Willard FS, Crouch MF . (2004). Role of the p70S6K pathway in regulating the actin cytoskeleton and cell migration. Exp Cell Res 296: 183–195.

    Article  CAS  Google Scholar 

  • Boffa DJ, Luan F, Thomas D, Yang H, Sharma VK, Lagman M et al. (2004). Rapamycin inhibits the growth and metastatic progression of non-small cell lung cancer. Clin Cancer Res 10: 293–300.

    Article  CAS  Google Scholar 

  • Casamassima A, Rozengurt E . (1998). Insulin-like growth factor I stimulates tyrosine phosphorylation of p130Cas, focal adhesion kinase, and paxillin. Role of phosphatidylinositol 3′-kinase and formation of a p130Cas Crk complex. J Biol Chem 273: 26149–26156.

    Article  CAS  Google Scholar 

  • Erbay E, Chen J . (2001). The mammalian target of rapamycin regulates C2C12 myogenesis via a kinase-independent mechanism. J Biol Chem 276: 36079–36082.

    Article  CAS  Google Scholar 

  • Fonseca BD, Smith EM, Lee VH, Mackintosh C, Proud CG . (2007). PRAS40 is a target for mammalian target of rapamycin complex 1 and is required for signaling downstream of this complex. J Biol Chem 282: 24514–24524.

    Article  CAS  Google Scholar 

  • Frias MA, Thoreen CC, Jaffe JD, Schroder W, Sculley T, Carr SA et al. (2006). mSin1 is necessary for Akt/PKB phosphorylation, and its isoforms define three distinct mTORC2s. Curr Biol 16: 1865–1870.

    Article  CAS  Google Scholar 

  • Gan B, Yoo Y, Guan JL . (2006). Association of focal adhesion kinase with tuberous sclerosis complex 2 in the regulation of s6 kinase activation and cell growth. J Biol Chem 281: 37321–37329.

    Article  CAS  Google Scholar 

  • Govindarajan G, Eble DM, Lucchesi PA, Samarel AM . (2000). Focal adhesion kinase is involved in angiotensin II-mediated protein synthesis in cultured vascular smooth muscle cells. Circ Res 87: 710–716.

    Article  CAS  Google Scholar 

  • Guba M, von Breitenbuch P, Steinbauer M, Koehl G, Flegel S, Hornung M et al. (2002). Rapamycin inhibits primary and metastatic tumor growth by antiangiogenesis: involvement of vascular endothelial growth factor. Nat Med 8: 128–135.

    Article  CAS  Google Scholar 

  • Guertin DA, Sabatini DM . (2007). Defining the role of mTOR in cancer. Cancer Cell 12: 9–22.

    Article  CAS  Google Scholar 

  • Guvakova MA, Surmacz E . (1999). The activated insulin-like growth factor I receptor induces depolarization in breast epithelial cells characterized by actin filament disassembly and tyrosine dephosphorylation of FAK, Cas, and paxillin. Exp Cell Res 251: 244–255.

    Article  CAS  Google Scholar 

  • Hara K, Maruki Y, Long X, Yoshino K, Oshiro N, Hidayat S et al. (2002). Raptor, a binding partner of target of rapamycin (TOR), mediates TOR action. Cell 110: 177–189.

    Article  CAS  Google Scholar 

  • Hosoi H, Dilling MB, Shikata T, Liu LN, Shu L, Ashmun RA et al. (1999). Rapamycin causes poorly reversible inhibition of mTOR and induces p53-independent apoptosis in human rhabdomyosarcoma cells. Cancer Res 59: 886–894.

    CAS  Google Scholar 

  • Huang S, Shu L, Dilling MB, Easton J, Harwood FC, Ichijo H et al. (2003). Sustained activation of the JNK cascade and rapamycin-induced apoptosis are suppressed by p53/p21Cip1. Mol Cell 11: 1491–1501.

    Article  CAS  Google Scholar 

  • Jacinto E, Facchinetti V, Liu D, Soto N, Wei S, Jung SY et al. (2006). SIN1/MIP1 maintains rictor-mTOR complex integrity and regulates Akt phosphorylation and substrate specificity. Cell 127: 125–137.

    Article  CAS  Google Scholar 

  • Jacinto E, Loewith R, Schmidt A, Lin S, Ruegg MA, Hall A et al. (2004). Mammalian TOR complex 2 controls the actin cytoskeleton and is rapamycin insensitive. Nat Cell Biol 6: 1122–1128.

    Article  CAS  Google Scholar 

  • Kadowaki T, Koyasu S, Nishida E, Sakai H, Takaku F, Yahara I et al. (1986). Insulin-like growth factors, insulin, and epidermal growth factor cause rapid cytoskeletal reorganization in KB cells. Clarification of the roles of type I insulin-like growth factor receptors and insulin receptors. J Biol Chem 261: 16141–16147.

    CAS  Google Scholar 

  • Kanellopoulou C, Muljo SA, Kung AL, Ganesan S, Drapkin R, Jenuwein T et al. (2005). Dicer-deficient mouse embryonic stem cells are defective in differentiation and centromeric silencing. Genes Dev 19: 489–501.

    Article  CAS  Google Scholar 

  • Kim DH, Sarbassov DD, Ali SM, King JE, Latek RR, Erdjument-Bromage H et al. (2002). mTOR interacts with raptor to form a nutrient-sensitive complex that signals to the cell growth machinery. Cell 110: 163–175.

    Article  CAS  Google Scholar 

  • Kim DH, Sarbassov DD, Ali SM, Latek RR, Guntur KV, Erdjument-Bromage H et al. (2003). GbetaL, a positive regulator of the rapamycin-sensitive pathway required for the nutrient-sensitive interaction between raptor and mTOR. Mol Cell 11: 895–904.

    Article  CAS  Google Scholar 

  • Konstantopoulos N, Clark S . (1996). Insulin and insulin-like growth factor-1 stimulate dephosphorylation of paxillin in parallel with focal adhesion kinase. Biochem J 314: 387–390.

    Article  CAS  Google Scholar 

  • Leopoldt D, Yee Jr HF, Rozengurt E . (2001). Calyculin-A induces focal adhesion assembly and tyrosine phosphorylation of p125Fak, p130Cas, and paxillin in Swiss 3T3 cells. J Cell Physiol 188: 106–119.

    Article  CAS  Google Scholar 

  • LeRoith D, Roberts Jr CT . (2003). The insulin-like growth factor system and cancer. Cancer Lett 195: 127–137.

    Article  CAS  Google Scholar 

  • Leventhal PS, Shelden EA, Kim B, Feldman EL . (1997). Tyrosine phosphorylation of paxillin and focal adhesion kinase during insulin-like growth factor-I-stimulated lamellipodial advance. J Biol Chem 272: 5214–5218.

    Article  CAS  Google Scholar 

  • Liu L, Li F, Cardelli JA, Martin KA, Blenis J, Huang S . (2006). Rapamycin inhibits cell motility by suppression of mTOR-mediated S6K1 and 4E-BP1 pathways. Oncogene 25: 7029–7040.

    Article  CAS  Google Scholar 

  • Loewith R, Jacinto E, Wullschleger S, Lorberg A, Crespo JL, Bonenfant D et al. (2002). Two TOR complexes, only one of which is rapamycin sensitive, have distinct roles in cell growth control. Mol Cell 10: 457–468.

    Article  CAS  Google Scholar 

  • Luan FL, Ding R, Sharma VK, Chon WJ, Lagman M, Suthanthiran M . (2003). Rapamycin is an effective inhibitor of human renal cancer metastasis. Kidney Int 63: 917–926.

    Article  CAS  Google Scholar 

  • Malik RK, Parsons JT . (1996). Integrin-dependent activation of the p70 ribosomal S6 kinase signaling pathway. J Biol Chem 271: 29785–29791.

    Article  CAS  Google Scholar 

  • Mothe-Satney I, Yang D, Fadden P, Haystead TA, Lawrence Jr JC . (2000). Multiple mechanisms control phosphorylation of PHAS-I in five (S/T)P sites that govern translational repression. Mol Cell Biol 20: 3558–3567.

    Article  CAS  Google Scholar 

  • Nave BT, Ouwens M, Withers DJ, Alessi DR, Shepherd PR . (1999). Mammalian target of rapamycin is a direct target for protein kinase B: identification of a convergence point for opposing effects of insulin and amino-acid deficiency on protein translation. Biochem J 344: 427–431.

    Article  CAS  Google Scholar 

  • O’Reilly KE, Rojo F, She QB, Solit D, Mills GB, Smith D et al. (2006). mTOR inhibition induces upstream receptor tyrosine kinase signaling and activates Akt. Cancer Res 66: 1500–1508.

    Article  Google Scholar 

  • Pearce LR, Huang X, Boudeau J, Pawlowski R, Wullschleger S, Deak M et al. (2007). Identification of Protor as a novel Rictor-binding component of mTOR complex-2. Biochem J 405: 513–522.

    Article  CAS  Google Scholar 

  • Ridley AJ, Schwartz MA, Burridge K, Firtel RA, Ginsberg MH, Borisy G et al. (2003). Cell migration: integrating signals from front to back. Science 302: 1704–1709.

    Article  CAS  Google Scholar 

  • Sancak Y, Thoreen CC, Peterson TR, Lindquist RA, Kang SA, Spooner E et al. (2007). PRAS40 is an insulin-regulated inhibitor of the mTORC1 protein kinase. Mol Cell 25: 903–915.

    Article  CAS  Google Scholar 

  • Sarbassov DD, Ali SM, Kim DH, Guertin DA, Latek RR, Erdjument-Bromage H et al. (2004). Rictor, a novel binding partner of mTOR, defines a rapamycin-insensitive and raptor-independent pathway that regulates the cytoskeleton. Curr Biol 14: 1296–1302.

    Article  CAS  Google Scholar 

  • Sarbassov DD, Guertin DA, Ali SM, Sabatini DM . (2005). Phosphorylation and regulation of Akt/PKB by the rictor-mTOR complex. Science 307: 1098–1101.

    Article  CAS  Google Scholar 

  • Schalm SS, Tee AR, Blenis J . (2005). Characterization of a conserved C-terminal motif (RSPRR) in ribosomal protein S6 kinase 1 required for its mammalian target of rapamycin-dependent regulation. J Biol Chem 280: 11101–11106.

    Article  CAS  Google Scholar 

  • Sekulic A, Hudson CC, Homme JL, Yin P, Otterness DM, Karnitz LM et al. (2000). A direct linkage between the phosphoinositide 3-kinase-AKT signaling pathway and the mammalian target of rapamycin in mitogen-stimulated and transformed cells. Cancer Res 60: 3504–3513.

    CAS  Google Scholar 

  • Shu L, Zhang X, Houghton PJ . (2002). Myogenic differentiation is dependent on both the kinase function and the N-terminal sequence of mammalian target of rapamycin. J Biol Chem 277: 16726–16732.

    Article  CAS  Google Scholar 

  • Smith MA, Morton CL, Phelps D, Girtman K, Neale G, Houghton PJ . (2006). SK-NEP-1 and Rh1 are Ewing family tumor lines. Pediatr Blood Cancer 50: 703–706.

    Article  Google Scholar 

  • Tremblay F, Marette A . (2001). Amino acid and insulin signaling via the mTOR/p70 S6 kinase pathway. A negative feedback mechanism leading to insulin resistance in skeletal muscle cells. J Biol Chem 276: 38052–38060.

    CAS  Google Scholar 

  • Tzatsos A, Kandror KV . (2006). Nutrients suppress phosphatidylinositol 3-kinase/Akt signaling via raptor-dependent mTOR-mediated insulin receptor substrate 1 phosphorylation. Mol Cell Biol 26: 63–76.

    Article  CAS  Google Scholar 

  • Vander Haar E, Lee SI, Bandhakavi S, Griffin TJ, Kim DH . (2007). Insulin signalling to mTOR mediated by the Akt/PKB substrate PRAS40. Nat Cell Biol 9: 316–323.

    Article  CAS  Google Scholar 

  • Wan X, Harkavy B, Shen N, Grohar P, Helman LJ . (2007). Rapamycin induces feedback activation of Akt signaling through an IGF-1R-dependent mechanism. Oncogene 26: 1932–1940.

    Article  CAS  Google Scholar 

  • Wan X, Mendoza A, Khanna C, Helman LJ . (2005). Rapamycin inhibits ezrin-mediated metastatic behavior in a murine model of osteosarcoma. Cancer Res 65: 2406–2411.

    Article  CAS  Google Scholar 

  • Yang Q, Inoki K, Ikenoue T, Guan KL . (2006). Identification of Sin1 as an essential TORC2 component required for complex formation and kinase activity. Genes Dev 20: 2820–2832.

    Article  CAS  Google Scholar 

  • Wullschleger S, Loewith R, Hall MN . (2006). TOR signaling in growth and metabolism. Cell 124: 471–484.

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank Drs Robert T Abraham, Peter J Houghton, John C Lawrence, John Blenis and David M Sabatini for providing cell lines or constructs. This study was supported in part by NIH Grant R01 CA115414 (SH), Louisiana Board of Regents (LEQSF(2006-09)-RD-A-18) (SH), and the Feist-Weiller Cancer Center Award (SH), Louisiana State University Health Sciences Center, Shreveport, LA, USA.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to S Huang.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Liu, L., Chen, L., Chung, J. et al. Rapamycin inhibits F-actin reorganization and phosphorylation of focal adhesion proteins. Oncogene 27, 4998–5010 (2008). https://doi.org/10.1038/onc.2008.137

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/onc.2008.137

Keywords

This article is cited by

Search

Quick links