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The actin-cytoskeleton linker protein ezrin is regulated during osteosarcoma metastasis by PKC

Oncogene volume 28, pages 792–802 (2009)Cite this article

Abstract

Ezrin is a member of the ERM (ezrin, radixin, moesin) protein family and links F-actin to the cell membrane following phosphorylation. Ezrin has been associated with tumor progression and metastasis in several cancers including the pediatric solid tumors, osteosarcoma and rhabdomyosarcoma. In this study, we were surprised to find that ezrin was not constitutively phosphorylated but rather was dynamically regulated during metastatic progression in osteosarcoma. Metastatic osteosarcoma cells expressed phosphorylated ERM early after their arrival in the lung, and then late in progression, only at the invasive front of larger metastatic lesions. To pursue mechanisms for this regulation, we found that inhibitors of PKC (protein kinase C) blocked phosphorylation of ezrin, and that ezrin coimmunoprecipitated in cells with PKCα, PKCι and PKCγ. Furthermore, phosphorylated forms of ezrin and PKC had identical expression patterns at the invasive front of pulmonary metastatic lesions in murine and human patient samples. Finally, we showed that the promigratory effects of PKC were linked to ezrin phosphorylation. These data are the first to suggest a dynamic regulation of ezrin phosphorylation during metastasis and to connect the PKC family members with this regulation.

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References

  • Berryman M, Franck Z, Bretscher A . (1993). Ezrin is concentrated in the apical microvilli of a wide variety of epithelial cells whereas moesin is found primarily in endothelial cells. J Cell Sci 105 (Part 4): 1025–1043.

    CAS  PubMed  Google Scholar 

  • Blobe GC, Obeid LM, Hannun YA . (1994). Regulation of protein kinase C and role in cancer biology. Cancer Metastasis Rev 13: 411–431.

    Article  CAS  PubMed  Google Scholar 

  • Bornancin F, Parker PJ . (1997). Phosphorylation of protein kinase C-alpha on serine 657 controls the accumulation of active enzyme and contributes to its phosphatase-resistant state. J Biol Chem 272: 3544–3549.

    Article  CAS  PubMed  Google Scholar 

  • Bretscher A, Chambers D, Nguyen R, Reczek D . (2000). ERM-Merlin and EBP50 protein families in plasma membrane organization and function. Annu Rev Cell Dev Biol 16: 113–143.

    Article  CAS  PubMed  Google Scholar 

  • Bretscher A, Edwards K, Fehon RG . (2002). ERM proteins and merlin: integrators at the cell cortex. Nat Rev Mol Cell Biol 3: 586–599.

    Article  CAS  PubMed  Google Scholar 

  • Bretscher A, Reczek D, Berryman M . (1997). Ezrin: a protein requiring conformational activation to link microfilaments to the plasma membrane in the assembly of cell surface structures. J Cell Sci 110 (Part 24): 3011–3018.

    CAS  PubMed  Google Scholar 

  • Brown MJ, Nijhara R, Hallam JA, Gignac M, Yamada KM, Erlandsen SL et al. (2003). Chemokine stimulation of human peripheral blood T lymphocytes induces rapid dephosphorylation of ERM proteins, which facilitates loss of microvilli and polarization. Blood 102: 3890–3899.

    Article  CAS  PubMed  Google Scholar 

  • Bruce B, Khanna G, Ren L, Landberg G, Jirstrom K, Powell C et al. (2007). Expression of the cytoskeleton linker protein ezrin in human cancers. Clin Exp Metastasis 24: 69–78.

    Article  CAS  PubMed  Google Scholar 

  • Cant SH, Pitcher JA . (2005). G protein-coupled receptor kinase 2-mediated phosphorylation of ezrin is required for G protein-coupled receptor-dependent reorganization of the actin cytoskeleton. Mol Biol Cell 16: 3088–3099.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Chuan YC, Pang ST, Cedazo-Minguez A, Norstedt G, Pousette A, Flores-Morales A . (2006). Androgen induction of prostate cancer cell invasion is mediated by ezrin. J Biol Chem 281: 29938–29948.

    Article  CAS  PubMed  Google Scholar 

  • Dard N, Louvet-Vallee S, Santa-Maria A, Maro B . (2004). Phosphorylation of ezrin on threonine T567 plays a crucial role during compaction in the mouse early embryo. Dev Biol 271: 87–97.

    Article  CAS  PubMed  Google Scholar 

  • Elliott BE, Meens JA, SenGupta SK, Louvard D, Arpin M . (2005). The membrane cytoskeletal crosslinker ezrin is required for metastasis of breast carcinoma cells. Breast Cancer Res 7: R365–R373.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Fievet BT, Gautreau A, Roy C, Del Maestro L, Mangeat P, Louvard D et al. (2004). Phosphoinositide binding and phosphorylation act sequentially in the activation mechanism of ezrin. J Cell Biol 164: 653–659.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Forte E, Orsatti L, Talamo F, Barbato G, De Francesco R, Tomei L . (2008). Ezrin is a specific and direct target of protein tyrosine phosphatase PRL-3. Biochim Biophys Acta 1783: 334–344.

    Article  CAS  PubMed  Google Scholar 

  • Gautreau A, Louvard D, Arpin M . (2000). Morphogenic effects of ezrin require a phosphorylation-induced transition from oligomers to monomers at the plasma membrane. J Cell Biol 150: 193–203.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Herbert JM . (1993). Protein kinase C: a key factor in the regulation of tumor cell adhesion to the endothelium. Biochem Pharmacol 45: 527–537.

    Article  CAS  PubMed  Google Scholar 

  • Hishiya A, Ohnishi M, Tamura S, Nakamura F . (1999). Protein phosphatase 2C inactivates F-actin binding of human platelet moesin. J Biol Chem 274: 26705–26712.

    Article  CAS  PubMed  Google Scholar 

  • Ilmonen S, Vaheri A, Asko-Seljavaara S, Carpen O . (2005). Ezrin in primary cutaneous melanoma. Mod Pathol 18: 503–510.

    Article  CAS  PubMed  Google Scholar 

  • Khanna C, Khan J, Nguyen P, Prehn J, Caylor J, Yeung C et al. (2001). Metastasis-associated differences in gene expression in a murine model of osteosarcoma. Cancer Res 61: 3750–3759.

    CAS  PubMed  Google Scholar 

  • Khanna C, Prehn J, Yeung C, Caylor J, Tsokos M, Helman L . (2000). An orthotopic model of murine osteosarcoma with clonally related variants differing in pulmonary metastatic potential. Clin Exp Metastasis 18: 261–271.

    Article  CAS  PubMed  Google Scholar 

  • Khanna C, Wan X, Bose S, Cassaday R, Olomu O, Mendoza A et al. (2004). The membrane-cytoskeleton linker ezrin is necessary for osteosarcoma metastasis. Nat Med 10: 182–186.

    Article  CAS  PubMed  Google Scholar 

  • Kivela T, Jaaskelainen J, Vaheri A, Carpen O . (2000). Ezrin, a membrane-organizing protein, as a polarization marker of the retinal pigment epithelium in vertebrates. Cell Tissue Res 301: 217–223.

    Article  CAS  PubMed  Google Scholar 

  • Kobel M, Gradhand E, Zeng K, Schmitt WD, Kriese K, Lantzsch T et al. (2006). Ezrin promotes ovarian carcinoma cell invasion and its retained expression predicts poor prognosis in ovarian carcinoma. Int J Gynecol Pathol 25: 121–130.

    Article  PubMed  Google Scholar 

  • Koivunen J, Aaltonen V, Koskela S, Lehenkari P, Laato M, Peltonen J . (2004). Protein kinase C alpha/beta inhibitor Go6976 promotes formation of cell junctions and inhibits invasion of urinary bladder carcinoma cells. Cancer Res 64: 5693–5701.

    Article  CAS  PubMed  Google Scholar 

  • Krieg J, Hunter T . (1992). Identification of the two major epidermal growth factor-induced tyrosine phosphorylation sites in the microvillar core protein ezrin. J Biol Chem 267: 19258–19265.

    CAS  PubMed  Google Scholar 

  • Krishnan K, Bruce B, Hewitt S, Thomas D, Khanna C, Helman LJ . (2006). Ezrin mediates growth and survival in Ewing's sarcoma through the AKT/mTOR, but not the MAPK, signaling pathway. Clin Exp Metastasis 23: 227–236.

    Article  CAS  PubMed  Google Scholar 

  • Makitie T, Carpen O, Vaheri A, Kivela T . (2001). Ezrin as a prognostic indicator and its relationship to tumor characteristics in uveal malignant melanoma. Invest Ophthalmol Vis Sci 42: 2442–2449.

    CAS  PubMed  Google Scholar 

  • Matsui T, Maeda M, Doi Y, Yonemura S, Amano M, Kaibuchi K et al. (1998). Rho-kinase phosphorylates COOH-terminal threonines of ezrin/radixin/moesin (ERM) proteins and regulates their head-to-tail association. J Cell Biol 140: 647–657.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Matsui T, Yonemura S, Tsukita S . (1999). Activation of ERM proteins in vivo by Rho involves phosphatidyl-inositol 4-phosphate 5-kinase and not ROCK kinases. Curr Biol 9: 1259–1262.

    Article  CAS  PubMed  Google Scholar 

  • Musashi M, Ota S, Shiroshita N . (2000). The role of protein kinase C isoforms in cell proliferation and apoptosis. Int J Hematol 72: 12–19.

    CAS  PubMed  Google Scholar 

  • Nakamura N, Oshiro N, Fukata Y, Amano M, Fukata M, Kuroda S et al. (2000). Phosphorylation of ERM proteins at filopodia induced by Cdc42. Genes Cells 5: 571–581.

    Article  CAS  PubMed  Google Scholar 

  • Ng T, Parsons M, Hughes WE, Monypenny J, Zicha D, Gautreau A et al. (2001). Ezrin is a downstream effector of trafficking PKC-integrin complexes involved in the control of cell motility. EMBO J 20: 2723–2741.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Ohtani K, Sakamoto H, Rutherford T, Chen Z, Satoh K, Naftolin F . (1999). Ezrin, a membrane-cytoskeletal linking protein, is involved in the process of invasion of endometrial cancer cells. Cancer Lett 147: 31–38.

    Article  CAS  PubMed  Google Scholar 

  • Oshiro N, Fukata Y, Kaibuchi K . (1998). Phosphorylation of moesin by rho-associated kinase (Rho-kinase) plays a crucial role in the formation of microvilli-like structures. J Biol Chem 273: 34663–34666.

    Article  CAS  PubMed  Google Scholar 

  • Pang ST, Fang X, Valdman A, Norstedt G, Pousette A, Egevad L et al. (2004). Expression of ezrin in prostatic intraepithelial neoplasia. Urology 63: 609–612.

    Article  PubMed  Google Scholar 

  • Pearson MA, Reczek D, Bretscher A, Karplus PA . (2000). Structure of the ERM protein moesin reveals the FERM domain fold masked by an extended actin binding tail domain. Cell 101: 259–270.

    Article  CAS  PubMed  Google Scholar 

  • Pietromonaco SF, Simons PC, Altman A, Elias L . (1998). Protein kinase C-theta phosphorylation of moesin in the actin-binding sequence. J Biol Chem 273: 7594–7603.

    Article  CAS  PubMed  Google Scholar 

  • Polesello C, Payre F . (2004). Small is beautiful: what flies tell us about ERM protein function in development. Trends Cell Biol 14: 294–302.

    Article  CAS  PubMed  Google Scholar 

  • Reczek D, Berryman M, Bretscher A . (1997). Identification of EBP50: A PDZ-containing phosphoprotein that associates with members of the ezrin-radixin-moesin family. J Cell Biol 139: 169–179.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Saotome I, Curto M, McClatchey AI . (2004). Ezrin is essential for epithelial organization and villus morphogenesis in the developing intestine. Dev Cell 6: 855–864.

    Article  CAS  PubMed  Google Scholar 

  • Serrador JM, Nieto M, Sanchez-Madrid F . (1999). Cytoskeletal rearrangement during migration and activation of T lymphocytes. Trends Cell Biol 9: 228–233.

    Article  CAS  PubMed  Google Scholar 

  • Simons PC, Pietromonaco SF, Reczek D, Bretscher A, Elias L . (1998). C-terminal threonine phosphorylation activates ERM proteins to link the cell's cortical lipid bilayer to the cytoskeleton. Biochem Biophys Res Commun 253: 561–565.

    Article  CAS  PubMed  Google Scholar 

  • Srivastava J, Elliott BE, Louvard D, Arpin M . (2005). Src-dependent ezrin phosphorylation in adhesion-mediated signaling. Mol Biol Cell 16: 1481–1490.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Sullivan RM, Stone M, Marshall JF, Uberall F, Rotenberg SA . (2000). Photo-induced inactivation of protein kinase calpha by dequalinium inhibits motility of murine melanoma cells. Mol Pharmacol 58: 729–737.

    Article  CAS  PubMed  Google Scholar 

  • Tsukita S, Oishi K, Sato N, Sagara J, Kawai A . (1994). ERM family members as molecular linkers between the cell surface glycoprotein CD44 and actin-based cytoskeletons. J Cell Biol 126: 391–401.

    Article  CAS  PubMed  Google Scholar 

  • Wald FA, Oriolo AS, Mashukova A, Fregien NL, Langshaw AH, Salas PJ . (2008). Atypical protein kinase C (iota) activates ezrin in the apical domain of intestinal epithelial cells. J Cell Sci 121 (Part 5): 644–654.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Weng WH, Ahlen J, Astrom K, Lui WO, Larsson C . (2005). Prognostic impact of immunohistochemical expression of ezrin in highly malignant soft tissue sarcomas. Clin Cancer Res 11: 6198–6204.

    Article  CAS  PubMed  Google Scholar 

  • Wu KL, Khan S, Lakhe-Reddy S, Jarad G, Mukherjee A, Obejero-Paz CA et al. (2004). The NHE1 Na+/H+ exchanger recruits ezrin/radixin/moesin proteins to regulate Akt-dependent cell survival. J Biol Chem 279: 26280–26286.

    Article  CAS  PubMed  Google Scholar 

  • Yonemura S, Hirao M, Doi Y, Takahashi N, Kondo T, Tsukita S . (1998). Ezrin/radixin/moesin (ERM) proteins bind to a positively charged amino acid cluster in the juxta-membrane cytoplasmic domain of CD44, CD43, and ICAM-2. J Cell Biol 140: 885–895.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Yu Y, Khan J, Khanna C, Helman L, Meltzer PS, Merlino G . (2004). Expression profiling identifies the cytoskeletal organizer ezrin and the developmental homeoprotein Six-1 as key metastatic regulators. Nat Med 10: 175–181.

    Article  CAS  PubMed  Google Scholar 

  • Zhu L, Zhou R, Mettler S, Wu T, Abbas A, Delaney J et al. (2007). High turnover of ezrin T567 phosphorylation: conformation, activity, and cellular function. Am J Physiol Cell Physiol 293: C874–C884.

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

We would like to thank Drs Joseph Briggs and Luowei Li for experimental advice and their critical review of this manuscript.

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Authors and Affiliations

  1. Tumor and Metastasis Biology Section, Center for Cancer Research, National Cancer Institute, Bethesda, MD, USA

    L Ren, S H Hong, J Cassavaugh, T Osborne, S Y Kim & C Khanna

  2. Department of Pediatrics, Memorial Sloan-Kettering Cancer Center, New York, NY, USA

    A J Chou

  3. Department of Pediatrics, Albert Einstein College of Medicine, The Children's Hospital at Montefiore, Bronx, NY, USA

    R Gorlick

  4. Tissue Array Research Program, Laboratory of Pathology, Center for Cancer Research, National Cancer Institute, Bethesda, MD, USA

    S M Hewitt

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  1. L Ren
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Correspondence to C Khanna.

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Supplementary Information accompanies the paper on the Oncogene website (http://www.nature.com/onc)

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Ren, L., Hong, S., Cassavaugh, J. et al. The actin-cytoskeleton linker protein ezrin is regulated during osteosarcoma metastasis by PKC. Oncogene 28, 792–802 (2009). https://doi.org/10.1038/onc.2008.437

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