Hostname: page-component-586b7cd67f-g8jcs Total loading time: 0 Render date: 2024-11-22T05:33:34.610Z Has data issue: false hasContentIssue false
  • English
  • Français

Purification of the NF2 Tumor Suppressor Protein from Human Erythrocytes

Published online by Cambridge University Press:  02 December 2014

Hitesh K. Jindal ,
Kazumi Yoshinaga ,
Pil-Soo Seo ,
Mohini Lutchman ,
Patrick A. Dion ,
Guy A. Rouleau ,
Toshihiko Hanada  and
Athar H. Chishti
Hitesh K. Jindal
Affiliation:
Departments of Medicine, Anatomy, and Cellular Biology, St. Elizabeth's Medical Center, Tufts University School of Medicine, Boston, MA, U.S.A
Kazumi Yoshinaga
Affiliation:
Department of Pharmacology, UIC Cancer Center, University of Illinois College of Medicine, Chicago, IL, U.S.A.
Pil-Soo Seo
Affiliation:
Department of Pharmacology, UIC Cancer Center, University of Illinois College of Medicine, Chicago, IL, U.S.A.
Mohini Lutchman
Affiliation:
Departments of Medicine, Anatomy, and Cellular Biology, St. Elizabeth's Medical Center, Tufts University School of Medicine, Boston, MA, U.S.A
Patrick A. Dion
Affiliation:
Center for the Study of Brain Diseases, CHUM Research Center- Notre Dame Hospital, Montreal, QC, Canada
Guy A. Rouleau
Affiliation:
Center for the Study of Brain Diseases, CHUM Research Center- Notre Dame Hospital, Montreal, QC, Canada
Toshihiko Hanada
Affiliation:
Department of Pharmacology, UIC Cancer Center, University of Illinois College of Medicine, Chicago, IL, U.S.A.
Athar H. Chishti*
Affiliation:
Departments of Medicine, Anatomy, and Cellular Biology, St. Elizabeth's Medical Center, Tufts University School of Medicine, Boston, MA, U.S.A Department of Pharmacology, UIC Cancer Center, University of Illinois College of Medicine, Chicago, IL, U.S.A.
*
Department of Pharmacology, UIC Cancer Center, University of Illinois College of Medicine, Chicago, IL, 60612, U.S.A.
Rights & Permissions [Opens in a new window]

Abstract

Core share and HTML view are not available for this content. However, as you have access to this content, a full PDF is available via the ‘Save PDF’ action button.
Background:

Neurofibromatosis type 2 (NF2) is an autosomal dominant disease predisposing individuals to the risk of developing tumors of cranial and spinal nerves. The NF2 tumor suppressor protein, known as Merlin/Schwanomin, is a member of the protein 4.1 superfamily that function as links between the cytoskeleton and the plasma membrane.

Methods:

Upon selective extraction of membrane-associated proteins from erythrocyte plasma membrane (ghosts) using low ionic strength solution, the bulk of NF2 protein remains associated with the spectrin-actin depleted inside-out-vesicles. Western blot analysis showed a ~70 kDa polypeptide in the erythrocyte plasma membrane. Furthermore, quantitative removal of NF2 protein from the inside-out-vesicles was achieved using 1.0 M potassium iodide, a treatment known to remove tightly-bound peripheral membrane proteins.

Results:

These results suggest a novel mode of NF2 protein association with the erythrocyte membrane that is distinct from the known membrane interactions of protein 4.1. Based on these biochemical properties, several purification strategies were devised to isolate native NF2 protein from human erythrocyte ghosts. Using purified and recombinant NF2 protein as internal standards, we quantified approximately ~41-65,000 molecules of NF2 protein per erythrocyte.

Conclusion:

We provide evidence for the presence of NF2 protein in the human erythrocyte membrane. The identification of NF2 protein in the human erythrocyte membrane will make it feasible to discover novel interactions of NF2 protein utilizing powerful techniques of erythrocyte biochemistry and genetics in mammalian cells.

Résumé:

RÉSUMÉ: Contexte:

La neurofibromatose de type 2 (NF2) est une maladie dominante autosomique qui prédispose au développement de tumeurs au niveau des nerfs crâniens et des nerfs spinaux. La protéine codée par le gène suppresseur de tumeurs NF2, connue sous le nom de Merlin/Schwannomin, fait partie de la superfamille des protéines 4,1 impliquées dans l’interface entre le cytosquelette et la membrane plasmatique.

Méthodes:

Lors de l’extraction sélective des protéines associées à la membrane cellulaire d’érythrocytes plasmatiques (fantômes d’hématies) au moyen d’une solution dont la force ionique est faible, la majeure partie de la protéine NF2 demeure associée aux vésicules inversées dépourvues de spectrine-actine. L’analyse par buvardage Western a démontré la présence d’un polypeptide de ˜70 kDa dans la membrane plasmatique erythrocytaire. L’élimination quantitative de la protéine NF2 des vésicules inversées a été effectuée au moyen d’iodure de potassium 1,0 M, un traitement qui extrait les protéines membranaires périphériques fortement liées.

Résultats:

Ces résultats sont compatibles avec un nouveau mode d’association de la protéine NF2 à la membrane érythrocytaire qui est distinct des interactions membranaires connues au sujet des protéines 4,1. Plusieurs stratégies de purification fondées sur ces propriétés biochimiques ont été élaborées pour isoler la protéine NF2 native des fantômes d’hématies humaines. Nous avons quantifié approximativement ˜41-65 000 molécules de protéine NF2 par érythrocyte en utilisant la protéine NF2 purifiée et la protéine NF2 recombinante comme standards internes.

Conclusion:

Ces données sont compatibles avec la présence de la protéine NF2 dans la membrane des érythrocytes humains. L’identification de la protéine NF2 dans la membrane des érythrocytes humains permettra de découvrir de nouveaux modes d’interactions de la protéine NF2 dans les cellules de mammifères au moyen de techniques puissantes de biochimie et de génétique érythrocytaire.

Type
Original Articles
Information
Canadian Journal of Neurological Sciences , Volume 33 , Issue 4 , November 2006 , pp. 394 - 402
Copyright
Copyright © The Canadian Journal of Neurological 2006

References

1. Lutchman, M, Rouleau, GA. Neurofibromatosis type 2: a new mechanism of tumor suppression. Trends Neurosci. 1996; 19(9): 373–7.CrossRefGoogle ScholarPubMed
2. Parry, DM, MacCollin, MM, Kaiser-Kupfer, MI, Pulaski, K, Nicholson, HS, Bolesta, M, et al. Germ-line mutations in the neurofibromatosis 2 gene: correlations with disease severity and retinal abnormalities. Am J Hum Genet. 1996; 59(3):529–39.Google ScholarPubMed
3. Bretscher, A, Edwards, K, Fehon, RG. ERM proteins and merlin: integrators at the cell cortex. Nat Rev Mol Cell Biol. 2002; 3(8): 586–99.Google Scholar
4. Gautreau, A, Louvard, D, Arpin, M. ERM proteins and NF2 tumor suppressor: the Yin and Yang of cortical actin organization and cell growth signaling. Curr Opin Cell Biol. 2002; 14(1): 104–9.Google Scholar
5. Conboy, J, Kan, YW, Shohet, SB, Mohandas, N. Molecular cloning of protein 4.1, a major structural element of the human erythrocyte membrane skeleton. Proc Natl Acad Sci USA. 1986; 83(24):9512–6.Google Scholar
6. Lim, DJ, Rubenstein, AE, Evans, DG, Jacks, T, Seizinger, BG, Baser, ME, et al. Advances in neurofibromatosis 2 (NF2): a workshop report. J Neurogenet. 2000; 14(2):63–106.CrossRefGoogle ScholarPubMed
7. McClatchey, AI, Saotome, I, Mercer, K, Crowley, D, Gusella, JF, Bronson, RT, et al. Mice heterozygous for a mutation at the Nf2 tumor suppressor locus develop a range of highly metastatic tumors. Genes Dev. 1998; 12(8):1121–33.Google Scholar
8. Kissil, JL, Wilker, EW, Johnson, KC, Eckman, MS, Yaffe, MB, Jacks, T. Merlin, the product of the Nf2 tumor suppressor gene, is an inhibitor of the p21-activated kinase, Pak1. Mol Cell. 2003; 12(4):841–9.CrossRefGoogle ScholarPubMed
9. Shaw, RJ, Paez, JG, Curto, M, Yaktine, A, Pruitt, WM, Saotome, I, et al. The Nf2 tumor suppressor, Merlin, functions in rac-dependent signaling. Develop Cell. 2001; 1:63–72.CrossRefGoogle ScholarPubMed
10. Shaw, RJ, McClatchey, AI, Jacks, T. Regulation of the neurofibromatosis type 2 tumor suppressor protein, Merlin, by adhesion and growth arrest stimuli. J Biol Chem. 1998; 273(13): 7757–64.CrossRefGoogle ScholarPubMed
11. Rong, R, Tang, X, Gutmann, DH, Ye, K. Neurofibromatosis 2 (NF2) tumor suppressor merlin inhibits phosphatidylinositol 3-kinase through binding to PIKE-L. Proc Natl Acad Sci U.S.A. 2004; 101(52):18200–5.Google Scholar
12. Manchanda, N, Lyubimova, A, Ho, HY, James, MF, Gusella, JF, Ramesh, N, et al. The NF2 tumor suppressor Merlin and the ERM proteins interact with N-WASP and regulate its actin polymerization function. J Biol Chem. 2005; 280(13):12517–22.CrossRefGoogle ScholarPubMed
13. Chene, P. The role of tetramerization in p53 function. Oncogene. 2001; 20(21):2611–7.CrossRefGoogle ScholarPubMed
14. Resh, MD. Membrane targeting of lipid modified signal transduction proteins. Subcell Biochem. 2004; 37:217–32.CrossRefGoogle ScholarPubMed
15. Johnson, KC, Kissil, JL, Fry, JL, Jacks, T. Cellular transformation by a FERM domain mutant of the Nf2 tumor suppressor gene. Oncogene. 2002; 21(39):5990–7.CrossRefGoogle ScholarPubMed
16. Jannatipour, M, Dion, P, Khan, S, Jindal, H, Fan, X, Laganiere, J, et al. Schwannomin isoform-1 interacts with syntenin via PDZ domains. J Biol Chem. 2001; 276(35):33093–100.Google Scholar
17. Pearson, MA, Reczek, D, Bretscher, A, Karplus, PA. Structure of the ERM protein moesin reveals the FERM domain fold masked by an extended actin binding tail domain. Cell. 2000; 101(3): 259–70.Google Scholar
18. Nguyen, R, Reczek, D, Bretscher, A. Hierarchy of merlin and ezrin N-and C-terminal domain interactions in homo- and heterotypic associations and their relationship to binding of scaffolding proteins EBP50 and E3KARP. J Biol Chem. 2001; 276(10): 7621–9.CrossRefGoogle ScholarPubMed
19. Xiao, GH, Gallagher, R, Shetler, J, Skele, K, Altomare, DA, Pestell, RG, et al. The NF2 tumor suppressor gene product, merlin, inhibits cell proliferation and cell cycle progression by repressing cyclin D1 expression. Mol Cell Biol. 2005; 25(6):2384–94.Google Scholar
20. Rangwala, R, Banine, F, Borg, JP, Sherman, LS. Erbin regulates mitogen-activated protein (MAP) kinase activation and MAP kinase-dependent interactions between Merlin and adherens junction protein complexes in Schwann cells. J Biol Chem. 2005; 280(12):11790–7.Google Scholar
21. Rong, R, Surace, EI, Haipek, CA, Gutmann, DH, Ye, K. Serine 518 phosphorylation modulates merlin intramolecular association and binding to critical effectors important for NF2 growth suppression. Oncogene. 2004; 23(52):8447–54.CrossRefGoogle ScholarPubMed
22. Kimura, Y, Koga, H, Araki, N, Mugita, N, Fujita, N, Takeshima, H, et al. The involvement of calpain-dependent proteolysis of the tumor suppressor NF2 (merlin) in schwannomas and meningiomas. Nat Med. 1998; 4(8):915–22.Google Scholar
23. Claudio, JO, Lutchman, M, Rouleau, GA. Widespread but cell-type specific expression of the mouse neurofibromatosis type 2 gene. Neuroreport. 1995, 6(14):1942–6.CrossRefGoogle ScholarPubMed
24. Alloisio, N, Dalla Venezia, N, Rana, A, Andrabi, K, Texier, P, Gilsanz, F, et al. Evidence that red blood cell protein p55 may participate in the skeleton-membrane linkage that involves protein 4.1 and glycophorin C. Blood. 1993; 82(4):1323–7.CrossRefGoogle ScholarPubMed
25. Southgate, CD, Chishti, AH, Mitchell, B, Yi, SJ, Palek, J. Targeted disruption of the murine erythroid band 3 gene results in spherocytosis and severe haemolytic anaemia despite a normal membrane skeleton. Nat Genet. 1996; 14(2):227–30.CrossRefGoogle ScholarPubMed
26. Lutchman, M, Rouleau, GA. The neurofibromatosis type 2 gene product, schwannomin, suppresses growth of NIH 3T3 cells. Cancer Res. 1995; 55(11):2270–4.Google Scholar
27. Chishti, AH. Function of p55 and its nonerythroid homologues. Curr Opin Hema. 1998; 5(2):116–21.CrossRefGoogle ScholarPubMed
28. Risinger, MA, Dotimas, EM, Cohen, CM. Human erythrocyte protein 4.2, a high copy number membrane protein, is N-myristylated. J Biol Chem. 1992; 267(8):5680–5.Google Scholar
29. Marfatia, SM, Lue, RA, Branton, D, Chishti, AH. Identification of the protein 4.1 binding interface on glycophorin C and p55, a homologue of the Drosophila discs-large tumor suppressor protein. J Biol Chem. 1995; 270(2):715–9.Google Scholar
30. Ruff, P, Speicher, DW, Husain-Chishti, A. Molecular identification of a major palmitoylated erythrocyte membrane protein containing the src homology 3 motif. Proc Natl Acad Sci USA. 1991; 88(15):6595–9.Google Scholar
31. Lux, SE, Palek, J. Disorders of the red cell membrane. In: Handin, RI, Lux, SE, Stossel, TP, editors. Blood: principles and practice of hematology. Philadelphia: Lippincott Company; 1995. p. 1701–18.Google Scholar
32. Hassoun, H, Hanada, T, Lutchman, M, Sahr, KE, Palek, J, Hanspal, M, et al. Complete deficiency of glycophorin A in red blood cells from mice with targeted inactivation of the band 3 (AE1) gene. Blood. 1998; 91(6):2146–51.CrossRefGoogle ScholarPubMed
33. Trofatter, JA, MacCollin, MM, Rutter, JL, Murrell, JR, Duyao, MP, Parry, DM, et al. A novel moesin-, ezrin-, radixin-like gene is a candidate for the neurofibromatosis 2 tumor suppressor. Cell. 1993; 75(4):826.Google Scholar
34. Rouleau, GA, Merel, P, Lutchman, M, Sanson, M, Zucman, J, Marineau, C, et al. Alteration in a new gene encoding a putative membrane-organizing protein causes neuro-fibromatosis type 2. Nature. 1993; 363(6429):515–21.Google Scholar
35. Chishti, A, Levin, A, Branton, D. Abolition of actin-bundling by phosphorylation of human erythrocyte protein 4.9. Nature. 1988; 334(6184):718–21.CrossRefGoogle Scholar
36. Chishti, A, Faquin, W, Wu, CC, Branton, D. Purification of erythrocyte dematin (protein 4.9) reveals an endogenous protein kinase that modulates actin-bundling activity. J Biol Chem. 1989; 264(15):8985–91.Google Scholar