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Molecular and Cellular Biochemistry

, Volume 335, Issue 1–2, pp 173–180 | Cite as

Neutral endopeptidase is a myristoylated protein

  • Rong Zheng
  • Akio Horiguchi
  • Katsuyuki Iida
  • Jungoo Lee
  • Ruoqian Shen
  • Oscar B. GoodmanJr.
  • David M. Nanus
Article

Abstract

Neutral endopeptidase (NEP) is a cell-surface peptidase normally expressed by prostate epithelial cells and lost in ~50% of primary prostate cancers. NEP directly associates with multiple proteins at the cell surface including Ezrin/Radixin/Moesin (ERM) proteins and the PTEN tumor suppressor protein. Analysis of the N-terminal sequence of the NEP cytosolic domain (N-terminal MGKSESQMDI TDINTPKPKK KQRWTR) identified a myristoylation consensus site. Mutation of Gly-2 to Arg significantly decreased 3H-myristoylation activity, and correlated with translocation of NEP from the plasma membrane to a perinuclear domain as demonstrated by immunofluorescence staining and Western blotting with an NEP-specific antibody. Removal of this myristoylation residue did not affect NEP enzymatic specific activity. Myristoylated NEP recruited more PTEN protein to the cell membrane fraction than unmyristoylated NEP. These data demonstrate that NEP is myristoylated at Gly-2 and that this modification is an intrinsic signal for membrane targeting.

Keywords

Myristoylation Neutral endopeptidase Prostate cancer 

Notes

Acknowledgments

This work was supported by NIH grants RO1 DK060908-02, RO1 CA80240, R01 HD33000, the Robert H. McCooey Memorial Cancer Research Fund, Ronald and Susan Lynch Professorship in Urologic Oncology and Brady Urology Foundation of the Department of Urology (to Lee, J.). and DOD grant PC061655. The authors also thank Liza Lau and Samrina Kahlon for technical support.

References

  1. 1.
    Farazi TA, Waksman G, Gordon JI (2001) The biology and enzymology of protein N-myristoylation. J Biol Chem 276:39501–39504CrossRefPubMedGoogle Scholar
  2. 2.
    Gordon JI (1990) Protein N-myristoylation: simple questions, unexpected answers. Clin Res 38:517–528PubMedGoogle Scholar
  3. 3.
    Han KK, Martinage A (1992) Post-translational chemical modification(s) of proteins. Int J Biochem 24:19–28CrossRefPubMedGoogle Scholar
  4. 4.
    Johnson DR, Bhatnagar RS, Knoll LJ, Gordon JI (1994) Genetic and biochemical studies of protein N-myristoylation. Annu Rev Biochem 63:869–914CrossRefPubMedGoogle Scholar
  5. 5.
    Boutin JA (1997) Myristoylation. Cell Signal 9:15–35CrossRefPubMedGoogle Scholar
  6. 6.
    Mende I, Malstrom S, Tsichlis PN, Vogt PK, Aoki M (2001) Oncogenic transformation induced by membrane-targeted Akt2 and Akt3. Oncogene 20:4419–4423CrossRefPubMedGoogle Scholar
  7. 7.
    Selvakumar P, Lakshmikuttyamma A, Shrivastav A, Das SB, Dimmock JR, Sharma RK (2007) Potential role of N-myristoyltransferase in cancer. Prog Lipid Res 46:1–36CrossRefPubMedGoogle Scholar
  8. 8.
    Podell S, Gribskov M (2004) Predicting N-terminal myristoylation sites in plant proteins. BMC Genomics 5:37CrossRefPubMedGoogle Scholar
  9. 9.
    Kahn RA, Randazzo P, Serafini T, Weiss O, Rulka C, Clark J, Amherdt M, Roller P, Orci L, Rothman JE (1992) The amino terminus of ADP-ribosylation factor (ARF) is a critical determinant of ARF activities and is a potent and specific inhibitor of protein transport. J Biol Chem 267:13039–13046PubMedGoogle Scholar
  10. 10.
    Preininger AM, Parello J, Meier SM, Liao G, Hamm HE (2008) Receptor-mediated changes at the myristoylated amino terminus of Galpha(il) proteins. Biochemistry 47:10281–10293CrossRefPubMedGoogle Scholar
  11. 11.
    Shen R, Sumitomo M, Dai J, Hardy DO, Navarro D, Usmani B, Papandreou CN, Hersh LB, Shipp MA, Freedman LP, Nanus DM (2000) Identification and characterization of two androgen response regions in the human neutral endopeptidase gene. Mol Cell Endocrinol 170:131–142CrossRefPubMedGoogle Scholar
  12. 12.
    Shipp MA, Look AT (1993) Hematopoietic differentiation antigens that are membrane-associated enzymes: cutting is the key!. Blood 82:1052–1070PubMedGoogle Scholar
  13. 13.
    Ino K, Suzuki T, Uehara C, Nagasaka T, Okamoto T, Kikkawa F, Mizutani S (2000) The expression and localization of neutral endopeptidase 24.11/CD10 in human gestational trophoblastic diseases. Lab Invest 80:1729–1738CrossRefPubMedGoogle Scholar
  14. 14.
    Saito T, Iwata N, Tsubuki S, Takaki Y, Takano J, Huang SM, Suemoto T, Higuchi M, Saido TC (2005) Somatostatin regulates brain amyloid beta peptide Abeta42 through modulation of proteolytic degradation. Nat Med 11:434–439CrossRefPubMedGoogle Scholar
  15. 15.
    Papandreou CN, Usmani B, Geng Y, Bogenrieder T, Freeman R, Wilk S, Finstad CL, Reuter VE, Powell CT, Scheinberg D, Magill C, Scher HI, Albino AP, Nanus DM (1998) Neutral endopeptidase 24.11 loss in metastatic human prostate cancer contributes to androgen-independent progression. Nat Med 4:50–57CrossRefPubMedGoogle Scholar
  16. 16.
    Turner AJ, Tanzawa K (1997) Mammalian membrane metallopeptidases: NEP, ECE, KELL, and PEX. Faseb J 11:355–364PubMedGoogle Scholar
  17. 17.
    Iwase A, Shen R, Navarro D, Nanus DM (2004) Direct binding of neutral endopeptidase 24.11 to ezrin/radixin/moesin (ERM) proteins competes with the interaction of CD44 with ERM proteins. J Biol Chem 279:11898–11905CrossRefPubMedGoogle Scholar
  18. 18.
    Sumitomo M, Iwase A, Zheng R, Navarro D, Kaminetzky D, Shen R, Georgescu MM, Nanus DM (2004) Synergy in tumor suppression by direct interaction of neutral endopeptidase with PTEN. Cancer Cell 5:67–78CrossRefPubMedGoogle Scholar
  19. 19.
    D’Adamio L, Shipp MA, Masteller EL, Reinherz EL (1989) Organization of the gene encoding common acute lymphoblastic leukemia antigen (neutral endopeptidase 24.11): multiple miniexons and separate 5′ untranslated regions. Proc Natl Acad Sci USA 86:7103–7107CrossRefPubMedGoogle Scholar
  20. 20.
    King MJ, Sharma RK (1991) N-myristoyl transferase assay using phosphocellulose paper binding. Anal Biochem 199:149–153CrossRefPubMedGoogle Scholar
  21. 21.
    Horiguchi A, Zheng R, Goodman OB Jr, Shen R, Guan H, Hersh LB, Nanus DM (2007) Lentiviral vector neutral endopeptidase gene transfer suppresses prostate cancer tumor growth. Cancer Gene Ther 14:583–589CrossRefPubMedGoogle Scholar
  22. 22.
    Zambrano N, Minopoli G, de Candia P, Russo T (1998) The Fe65 adaptor protein interacts through its PID1 domain with the transcription factor CP2/LSF/LBP1. J Biol Chem 273:20128–20133CrossRefPubMedGoogle Scholar
  23. 23.
    Katagiri YU, Ohmi K, Tang W, Takenouchi H, Taguchi T, Kiyokawa N, Fujimoto J (2002) Raft.1, a monoclonal antibody raised against the raft microdomain, recognizes G-protein beta1 and 2, which assemble near nucleus after shiga toxin binding to human renal cell line. Lab Invest 82:1735–1745PubMedGoogle Scholar
  24. 24.
    Abbott DW, Holt JT (1997) Finkel-Biskis-Reilly mouse osteosarcoma virus v-fos inhibits the cellular response to ionizing radiation in a myristoylation-dependent manner. J Biol Chem 272:14005–14008CrossRefPubMedGoogle Scholar
  25. 25.
    Grebe M, Xu J, Mobius W, Ueda T, Nakano A, Geuze HJ, Rook MB, Scheres B (2003) Arabidopsis sterol endocytosis involves actin-mediated trafficking via ARA6-positive early endosomes. Curr Biol 13:1378–1387CrossRefPubMedGoogle Scholar
  26. 26.
    Ishitani M, Liu J, Halfter U, Kim CS, Shi W, Zhu JK (2000) SOS3 function in plant salt tolerance requires N-myristoylation and calcium binding. Plant Cell 12:1667–1678CrossRefPubMedGoogle Scholar
  27. 27.
    Lu SX, Hrabak EM (2002) An Arabidopsis calcium-dependent protein kinase is associated with the endoplasmic reticulum. Plant Physiol 128:1008–1021CrossRefPubMedGoogle Scholar
  28. 28.
    Rutschmann F, Stalder U, Piotrowski M, Oecking C, Schaller A (2002) LeCPK1, a calcium-dependent protein kinase from tomato plasma membrane targeting and biochemical characterization. Plant Physiol 129:156–168CrossRefPubMedGoogle Scholar
  29. 29.
    Resh MD (1999) Fatty acylation of proteins: new insights into membrane targeting of myristoylated and palmitoylated proteins. Biochim Biophys Acta 1451:1–16CrossRefPubMedGoogle Scholar
  30. 30.
    Olsen HB, Kaarsholm NC (2000) Structural effects of protein lipidation as revealed by LysB29-myristoyl, des(B30) insulin. Biochemistry 39:11893–11900CrossRefPubMedGoogle Scholar
  31. 31.
    Goldberg J (1998) Structural basis for activation of ARF GTPase: mechanisms of guanine nucleotide exchange and GTP-myristoyl switching. Cell 95:237–248CrossRefPubMedGoogle Scholar
  32. 32.
    Hermida-Matsumoto L, Resh MD (1999) Human immunodeficiency virus type 1 protease triggers a myristoyl switch that modulates membrane binding of Pr55(gag) and p17MA. J Virol 73:1902–1908PubMedGoogle Scholar
  33. 33.
    Shen R, Sumitomo M, Dai J, Harris A, Kaminetzky D, Gao M, Burnstein KL, Nanus DM (2000) Androgen-induced growth inhibition of androgen receptor expressing androgen-independent prostate cancer cells is mediated by increased levels of neutral endopeptidase. Endocrinology 141:1699–1704CrossRefPubMedGoogle Scholar
  34. 34.
    Sumitomo M, Shen R, Walburg M, Dai J, Geng Y, Navarro D, Boileau G, Papandreou CN, Giancotti FG, Knudsen B, Nanus DM (2000) Neutral endopeptidase inhibits prostate cancer cell migration by blocking focal adhesion kinase signaling. J Clin Invest 106:1399–1407CrossRefPubMedGoogle Scholar
  35. 35.
    Zheng R, Iwase A, Shen R, Goodman OB Jr, Sugimoto N, Takuwa Y, Lerner DJ, Nanus DM (2006) Neuropeptide-stimulated cell migration in prostate cancer cells is mediated by RhoA kinase signaling and inhibited by neutral endopeptidase. Oncogene 25:5942–5952CrossRefPubMedGoogle Scholar
  36. 36.
    Goodman OB Jr, Febbraio M, Simantov R, Zheng R, Shen R, Silverstein RL, Nanus DM (2006) Neprilysin inhibits angiogenesis via proteolysis of fibroblast growth factor-2. J Biol Chem 281:33597–33605CrossRefPubMedGoogle Scholar
  37. 37.
    Albrecht M, Mittler A, Wilhelm B, Lundwall A, Lilja H, Aumuller G, Bjartell A (2003) Expression and immunolocalisation of neutral endopeptidase in prostate cancer. Eur Urol 44:415–422CrossRefPubMedGoogle Scholar
  38. 38.
    Osman I, Yee H, Taneja SS, Levinson B, Zeleniuch-Jacquotte A, Chang C, Nobert C, Nanus DM (2004) Neutral endopeptidase protein expression and prognosis in localized prostate cancer. Clin Cancer Res 10:4096–4100CrossRefPubMedGoogle Scholar
  39. 39.
    Fleischmann A, Schlomm T, Huland H, Kollermann J, Simon P, Mirlacher M, Salomon G, Chun FH, Steuber T, Simon R, Sauter G, Graefen M, Erbersdobler A (2008) Distinct subcellular expression patterns of neutral endopeptidase (CD10) in prostate cancer predict diverging clinical courses in surgically treated patients. Clin Cancer Res 14:7838–7842CrossRefPubMedGoogle Scholar
  40. 40.
    Hersh LB, Rodgers DW (2008) Neprilysin and amyloid beta peptide degradation. Curr Alzheimer Res 5:225–231CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC. 2009

Authors and Affiliations

  • Rong Zheng
    • 1
  • Akio Horiguchi
    • 1
  • Katsuyuki Iida
    • 1
  • Jungoo Lee
    • 2
  • Ruoqian Shen
    • 2
  • Oscar B. GoodmanJr.
    • 3
  • David M. Nanus
    • 1
  1. 1.Genitourinary Oncology Research Laboratory, The Division of Hematology and Medical Oncology, Department of MedicineWeill Cornell Medical College of Cornell UniversityNew YorkUSA
  2. 2.Department of UrologyWeill Cornell Medical College of Cornell UniversityNew YorkUSA
  3. 3.Department of Clinical OncologyNevada Cancer InstituteLas VegasUSA

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