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:

PEA-15 potentiates H-Ras-mediated epithelial cell transformation through phospholipase D

Oncogene volume 31, pages 3547–3560 (2012)Cite this article

Subjects

Abstract

The small GTPase H-Ras is a proto-oncogene that activates a variety of different pathways including the extracellular-signal-regulated kinase (ERK)/mitogen-activated protein kinase pathway. H-Ras is mutated in many human malignancies, and these mutations cause the protein to be constitutively active. Phosphoprotein enriched in astrocytes, 15 kDa (PEA-15) blocks ERK-dependent gene transcription and inhibits proliferation by sequestering ERK in the cytoplasm. We therefore investigated whether PEA-15 influences H-Ras-mediated transformation. We found that PEA-15 does not block H-Ras-activated proliferation when H-Ras is constitutively active. We show instead that in H-Ras-transformed mouse kidney epithelial cells, co-expression of PEA-15 resulted in enhanced soft agar colony growth and increased tumor growth in vivo. Overexpression of both H-Ras and PEA-15 resulted in accelerated G1/S cell cycle transition and increased activation of the ERK signaling pathway. PEA-15 mediated these effects through activation of its binding partner phospholipase D1 (PLD1). Inhibition of PLD1 or interference with PEA-15/PLD1 binding blocked PEA-15's ability to increase ERK activation. Our findings reveal a novel mechanism by which PEA-15 positively regulates Ras/ERK signaling and increases the proliferation of H-Ras-transformed epithelial cells through enhanced PLD1 expression and activation. Thus, our work provides a surprising mechanism by which PEA-15 augments H-Ras-driven transformation. These data reveal that PEA-15 not only suppresses ERK signaling and tumorigenesis but also alternatively enhances tumorigenesis in the context of active Ras.

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

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

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

Similar content being viewed by others

References

  • Barrett T, Troup DB, Wilhite SE, Ledoux P, Rudnev D, Evangelista C et al. (2009). NCBI GEO: archive for high-throughput functional genomic data. Nucleic Acids Res 37: D885–D890.

    Article  CAS  PubMed  Google Scholar 

  • Bartholomeusz C, Rosen D, Wei C, Kazansky A, Yamasaki F, Takahashi T et al. (2008). PEA-15 induces autophagy in human ovarian cancer cells and is associated with prolonged overall survival. Cancer Res 68: 9302–9310.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Buchanan FG, McReynolds M, Couvillon A, Kam Y, Holla VR, Dubois RN et al. (2005). Requirement of phospholipase D1 activity in H-RasV12-induced transformation. Proc Natl Acad Sci USA 102: 1638–1642.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Castaneda CA, Cortes-Funes H, Gomez HL, Ciruelos EM . (2010). The phosphatidylinositol 3-kinase/AKT signaling pathway in breast cancer. Cancer Metastasis Rev 29: 751–759.

    Article  CAS  PubMed  Google Scholar 

  • Connell-Crowley L, Harper JW, Goodrich DW . (1997). Cyclin D1/Cdk4 regulates retinoblastoma protein-mediated cell cycle arrest by site-specific phosphorylation. Mol Biol Cell 8: 287–301.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Cordova-Alarcon E, Centeno F, Reyes-Esparza J, Garcia-Carranca A, Garrido E . (2005). Effects of HRAS oncogene on cell cycle progression in a cervical cancer-derived cell line. Arch Med Res 36: 311–316.

    Article  CAS  PubMed  Google Scholar 

  • Degenhardt K, White E . (2006). A mouse model system to genetically dissect the molecular mechanisms regulating tumorigenesis. Clin Cancer Res 12: 5298–5304.

    Article  CAS  PubMed  Google Scholar 

  • Donaldson JG . (2009). Phospholipase D in endocytosis and endosomal recycling pathways. Biochim Biophys Acta 1791: 845–849.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Doti N, Cassese A, Marasco D, Paturzo F, Sabatella M, Viparelli F et al. (2010). Residues 762-801 of PLD1 mediate the interaction with PED/PEA15. Mol Biosyst 6: 2039–2048.

    Article  CAS  PubMed  Google Scholar 

  • Dunn KL, Espino PS, Drobic B, He S, Davie JR . (2005). The Ras-MAPK signal transduction pathway, cancer and chromatin remodeling. Biochem Cell Biol 83: 1–14.

    Article  CAS  PubMed  Google Scholar 

  • Ferro E, Trabalzini L . (2010). RalGDS family members couple Ras to Ral signalling and that′s not all. Cell Signal 22: 1804–1810.

    Article  CAS  PubMed  Google Scholar 

  • Formisano P, Perruolo G, Libertini S, Santopietro S, Troncone G, Raciti GA et al. (2005). Raised expression of the antiapoptotic protein ped/pea-15 increases susceptibility to chemically induced skin tumor development. Oncogene 24: 7012–7021.

    Article  CAS  PubMed  Google Scholar 

  • Formstecher E, Ramos JW, Fauquet M, Calderwood DA, Hsieh JC, Canton B et al. (2001). PEA-15 mediates cytoplasmic sequestration of ERK MAP kinase. Dev Cell 1: 239–250.

    Article  CAS  PubMed  Google Scholar 

  • Frankel P, Ramos M, Flom J, Bychenok S, Joseph T, Kerkhoff E et al. (1999). Ral and Rho-dependent activation of phospholipase D in v-Raf-transformed cells. Biochem Biophys Res Commun 255: 502–507.

    Article  CAS  PubMed  Google Scholar 

  • Haling JR, Wang F, Ginsberg MH . (2010). Phosphoprotein enriched in astrocytes 15 kDa (PEA-15) reprograms growth factor signaling by inhibiting threonine phosphorylation of fibroblast receptor substrate 2alpha. Mol Biol Cell 21: 664–673.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Hancock JF . (2007). PA promoted to manager. Nat Cell Biol 9: 615–617.

    Article  CAS  PubMed  Google Scholar 

  • Joseph T, Bryant A, Frankel P, Wooden R, Kerkhoff E, Rapp UR et al. (2002). Phospholipase D overcomes cell cycle arrest induced by high-intensity Raf signaling. Oncogene 21: 3651–3658.

    Article  CAS  PubMed  Google Scholar 

  • Joseph T, Wooden R, Bryant A, Zhong M, Lu Z, Foster DA . (2001). Transformation of cells overexpressing a tyrosine kinase by phospholipase D1 and D2. Biochem Biophys Res Commun 289: 1019–1024.

    Article  CAS  PubMed  Google Scholar 

  • Lavoie JN, L′Allemain G, Brunet A, Muller R, Pouyssegur J . (1996). Cyclin D1 expression is regulated positively by the p42/p44MAPK and negatively by the p38/HOGMAPK pathway. J Biol Chem 271: 20608–20616.

    Article  CAS  PubMed  Google Scholar 

  • Lazarov M, Kubo Y, Cai T, Dajee M, Tarutani M, Lin Q et al. (2002). CDK4 coexpression with Ras generates malignant human epidermal tumorigenesis. Nat Med 8: 1105–1114.

    Article  CAS  PubMed  Google Scholar 

  • Lin HJ, Eviner V, Prendergast GC, White E . (1995). Activated H-ras rescues E1A-induced apoptosis and cooperates with E1A to overcome p53-dependent growth arrest. Mol Cell Biol 15: 4536–4544.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Lovec H, Sewing A, Lucibello FC, Muller R, Moroy T . (1994). Oncogenic activity of cyclin D1 revealed through cooperation with Ha-ras: link between cell cycle control and malignant transformation. Oncogene 9: 323–326.

    CAS  PubMed  Google Scholar 

  • Lu Z, Hornia A, Joseph T, Sukezane T, Frankel P, Zhong M et al. (2000). Phospholipase D and RalA cooperate with the epidermal growth factor receptor to transform 3Y1 rat fibroblasts. Mol Cell Biol 20: 462–467.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Macaluso M, Russo G, Cinti C, Bazan V, Gebbia N, Russo A . (2002). Ras family genes: an interesting link between cell cycle and cancer. J Cell Physiol 192: 125–130.

    Article  CAS  PubMed  Google Scholar 

  • Nandan MO, Yoon HS, Zhao W, Ouko LA, Chanchevalap S, Yang VW . (2004). Kruppel-like factor 5 mediates the transforming activity of oncogenic H-Ras. Oncogene 23: 3404–3413.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Noh DY, Ahn SJ, Lee RA, Park IA, Kim JH, Suh PG et al. (2000). Overexpression of phospholipase D1 in human breast cancer tissues. Cancer Lett 161: 207–214.

    Article  CAS  PubMed  Google Scholar 

  • Pastorino S, Renganathan H, Caliva MJ, Filbert EL, Opoku-Ansah J, Sulzmaier FJ et al. (2010). The death effector domain protein PEA-15 negatively regulates T-cell receptor signaling. FASEB J 24: 2818–2828.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Radig K, Schneider-Stock R, Rose I, Mittler U, Oda Y, Roessner A . (1998). p53 and ras mutations in Ewing′s sarcoma. Pathol Res Pract 194: 157–162.

    Article  CAS  PubMed  Google Scholar 

  • Ramos JW . (2008). The regulation of extracellular signal-regulated kinase (ERK) in mammalian cells. Int J Biochem Cell Biol 40: 2707–2719.

    Article  CAS  PubMed  Google Scholar 

  • Ramos JW, Hughes PE, Renshaw MW, Schwartz MA, Formstecher E, Chneiweiss H et al. (2000). Death effector domain protein PEA-15 potentiates Ras activation of extracellular signal receptor-activated kinase by an adhesion-independent mechanism. Mol Biol Cell 11: 2863–2872.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Renganathan H, Vaidyanathan H, Knapinska A, Ramos JW . (2005). Phosphorylation of PEA-15 switches its binding specificity from ERK/MAPK to FADD. Biochem J 390: 729–735.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Revet I, Huizenga G, Chan A, Koster J, Volckmann R, van Sluis P et al. (2008). The MSX1 homeobox transcription factor is a downstream target of PHOX2B and activates the Delta-Notch pathway in neuroblastoma. Exp Cell Res 314: 707–719.

    Article  CAS  PubMed  Google Scholar 

  • Rizzo MA, Shome K, Watkins SC, Romero G . (2000). The recruitment of Raf-1 to membranes is mediated by direct interaction with phosphatidic acid and is independent of association with Ras. J Biol Chem 275: 23911–23918.

    Article  CAS  PubMed  Google Scholar 

  • Sathyan KM, Nalinakumari KR, Kannan S . (2007). H-Ras mutation modulates the expression of major cell cycle regulatory proteins and disease prognosis in oral carcinoma. Mod Pathol 20: 1141–1148.

    Article  CAS  PubMed  Google Scholar 

  • Scott SA, Selvy PE, Buck JR, Cho HP, Criswell TL, Thomas AL et al. (2009). Design of isoform-selective phospholipase D inhibitors that modulate cancer cell invasiveness. Nat Chem Biol 5: 108–117.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Shi M, Zheng Y, Garcia A, Xu L, Foster DA . (2007). Phospholipase D provides a survival signal in human cancer cells with activated H-Ras or K-Ras. Cancer Lett 258: 268–275.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Streit M, Velasco P, Brown LF, Skobe M, Richard L, Riccardi L et al. (1999). Overexpression of thrombospondin-1 decreases angiogenesis and inhibits the growth of human cutaneous squamous cell carcinomas. Am J Pathol 155: 441–452.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Tan TT, Degenhardt K, Nelson DA, Beaudoin B, Nieves-Neira W, Bouillet P et al. (2005). Key roles of BIM-driven apoptosis in epithelial tumors and rational chemotherapy. Cancer Cell 7: 227–238.

    Article  CAS  PubMed  Google Scholar 

  • Uchida N, Okamura S, Kuwano H . (1999). Phospholipase D activity in human gastric carcinoma. Anticancer Res 19: 671–675.

    CAS  PubMed  Google Scholar 

  • Uchida N, Okamura S, Nagamachi Y, Yamashita S . (1997). Increased phospholipase D activity in human breast cancer. J Cancer Res Clin Oncol 123: 280–285.

    Article  CAS  PubMed  Google Scholar 

  • Vaidyanathan H, Opoku-Ansah J, Pastorino S, Renganathan H, Matter ML, Ramos JW . (2007). ERK MAP kinase is targeted to RSK2 by the phosphoprotein PEA-15. Proc Natl Acad Sci USA 104: 19837–19842.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Viparelli F, Cassese A, Doti N, Paturzo F, Marasco D, Dathan NA et al. (2008). Targeting of PED/PEA-15 molecular interaction with phospholipase D1 enhances insulin sensitivity in skeletal muscle cells. J Biol Chem 283: 21769–21778.

    Article  CAS  PubMed  Google Scholar 

  • Waber PG, Chen J, Nisen PD . (1993). Infrequency of ras, p53, WT1, or RB gene alterations in Wilms tumors. Cancer 72: 3732–3738.

    Article  CAS  PubMed  Google Scholar 

  • Wang HG, Pathan N, Ethell IM, Krajewski S, Yamaguchi Y, Shibasaki F et al. (1999). Ca2+-induced apoptosis through calcineurin dephosphorylation of BAD. Science 284: 339–343.

    Article  CAS  PubMed  Google Scholar 

  • Whitehurst AW, Robinson FL, Moore MS, Cobb MH . (2004). The death effector domain protein PEA-15 prevents nuclear entry of ERK2 by inhibiting required interactions. J Biol Chem 279: 12840–12847.

    Article  CAS  PubMed  Google Scholar 

  • Yang H, Masters SC, Wang H, Fu H . (2001). The proapoptotic protein Bad binds the amphipathic groove of 14-3-3zeta. Biochim Biophys Acta 1547: 313–319.

    Article  CAS  PubMed  Google Scholar 

  • Zeng X, Shaikh FY, Harrison MK, Adon AM, Trimboli AJ, Carroll KA et al. (2010). The Ras oncogene signals centrosome amplification in mammary epithelial cells through cyclin D1/Cdk4 and Nek2. Oncogene 29: 5103–5112.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Zhang Y, Redina O, Altshuller YM, Yamazaki M, Ramos J, Chneiweiss H et al. (2000). Regulation of expression of phospholipase D1 and D2 by PEA-15, a novel protein that interacts with them. J Biol Chem 275: 35224–35232.

    Article  CAS  PubMed  Google Scholar 

  • Zhao C, Du G, Skowronek K, Frohman MA, Bar-Sagi D . (2007). Phospholipase D2-generated phosphatidic acid couples EGFR stimulation to Ras activation by Sos. Nat Cell Biol 9: 706–712.

    CAS  PubMed  Google Scholar 

  • Zhao Y, Ehara H, Akao Y, Shamoto M, Nakagawa Y, Banno Y et al. (2000). Increased activity and intranuclear expression of phospholipase D2 in human renal cancer. Biochem Biophys Res Commun 278: 140–143.

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank Dr Maarit Tiirikainen and the University of Hawaii Cancer Center Genomics Shared Resource laboratory for their assistance with the mRNA expression analysis. We also thank Anna Knapinska, Shirley Young-Robbins and Marci Takemoto for their excellent technical assistance. This work was supported by the National Institutes of Health National Cancer Institute (R01CA93849 to JWR) and National Institute of General Medicine (R01GM088266 to JWR) and the Victoria S and Bradley Geist Foundation (to JWR). MLM was supported by NIH National Center for Reasearch Resources Grant RR016453. DAN and EPW were supported by a grant from the National Institutes of Health (R37CA53370 to EPW).

Author information

Authors and Affiliations

  1. Cancer Biology Program, University of Hawaii Cancer Center, University of Hawaii at Manoa, Honolulu, HI, USA

    F J Sulzmaier, M K G Valmiki, M J Caliva & J W Ramos

  2. Department of Molecular Biosciences and Bioengineering, University of Hawai’i at Manoa, Honolulu, HI, USA

    F J Sulzmaier, M K G Valmiki & J W Ramos

  3. The Cancer Institute of New Jersey, New Brunswick, NJ, USA

    D A Nelson & E P White

  4. Department of Cell and Molecular Biology, John A Burns School of Medicine, University of Hawaii at Manoa, Honolulu, HI, USA

    M J Caliva & M L Matter

  5. Department of Pediatric Oncology/Hematology, Sophia Children's Hospital, Rotterdam, The Netherlands

    D Geerts

Authors
  1. F J Sulzmaier
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. M K G Valmiki
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. D A Nelson
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. M J Caliva
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. D Geerts
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. M L Matter
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. E P White
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. J W Ramos
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to J W Ramos.

Ethics declarations

Competing interests

The authors declare no conflict of interest.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Sulzmaier, F., Valmiki, M., Nelson, D. et al. PEA-15 potentiates H-Ras-mediated epithelial cell transformation through phospholipase D. Oncogene 31, 3547–3560 (2012). https://doi.org/10.1038/onc.2011.514

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/onc.2011.514

Keywords

This article is cited by

Search

Advanced search

Quick links