Skip to main content

Advertisement

SpringerLink
Log in

Identification of the primary caspase 3 cleavage site in alpha II-spectrin during apoptosis

  • Published:
Apoptosis Aims and scope Submit manuscript

Abstract

Alpha II-spectrin is one of the major proteins responsible for maintaining the cytoskeletal integrity of the cell. The caspase 3-mediated cleavage of alpha II-spectrin during apoptotic cell death may play an important role in altering membrane stability and the formation of apoptotic bodies. In this study, we identified the primary caspase 3 cleavage site in alpha II-spectrin. We found that the transcriptional inhibitor, actinomycin D, induced caspase 3 activation and that caspase 3 activation is coincident with the cleavage of alpha II-spectrin protein at a primary cleavage site. Deletion analysis and site directed mutagenesis identified the primary cleavage site in alpha II spectrin at amino acid 1185 (DETD). The primary caspase 3 cleavage site in alpha II spectrin is conserved in immature and mature B cells. Our results indicate that alpha II-spectrin is initally cleaved at a caspase 3 consensus site and this primary event likely alters the structural conformation of the protein exposing subsequent cleavage sites and altering cytoskeletal integrity. Identification of the primary cleavage site for caspase 3 may help to elucidate the role of alpha II-spectrin in membrane stability and apoptosis as well as provide new insights into alpha II-spectrin autoantibody formation associated with the autoimmune disease, Sjögren's syndrome.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  1. Deng H, Lee JK, Goldstein LS, Branton D. Drosophila development requires spectrin network formation. J Cell Biol 1995; 128: 71-79.

    Google Scholar 

  2. Morrow JS, Rimm LD, Kennedy PS, Cianci DC, Sinard HJ, Weed AS. Membrane stability and mosaics: The spectrin cytoskeleton. Handbook of Physiology 1997; 485-540.

  3. Haneji N, Nakamura T, Takio K, et al. Identification of alphafodrin as a candidate autoantigen in primary Sjögren's syndrome. Science 1997; 276: 604-607.

    Google Scholar 

  4. Inoue H, Tsubota K, Ono M, et al. Possible involvement of EBV-mediated alpha-fodrin cleavage for organ-specific autoantigen in Sjögren's syndrome. J Immunol 2001; 166: 5801-5809.

    Google Scholar 

  5. Manthrope R, Jacobsson LT, Kirtava Z, Theander E. Epidemiology of Sjögren's syndrome, especially its primary form. Ann Med Interne (Paris) 1998; 149: 7-11.

    Google Scholar 

  6. Kerr JF, Wyllie AH, Currie AR. Apoptosis: A basic biological phenomenon with wide-ranging implications in tissue kinetics. Br J Cancer 1972; 26: 239-257.

    Google Scholar 

  7. King LB, Monroe JG. Immunobiology of the immature B cell: Plasticity in the B-cell antigen receptor-induced response fine tunes negative selection. Immunol Rev 2000; 176: 86-104.

    Google Scholar 

  8. Gottschalk RA, Quintans J. Apoptosis in B lymphocytes: The WEHI-231 perspective. Immunology and Cell Biology 1995; 73: 8-16.

    Google Scholar 

  9. Plas DR, Thompson CB. Cell metabolism in the regulation of programmed cell death. Trends Endocrinol Metab 2002; 13: 75-78.

    Google Scholar 

  10. Earnshaw WC, Martins LM, Kaufmann SH. Mammalian caspases: Structure, activation, substrates, and functions during apoptosis. Annu Rev Biochem 1999; 68: 383-424.

    Google Scholar 

  11. Salvesen GS, Dixit VM. Caspases: Intracellular signaling by proteolysis. Cell 1997; 91: 443-446.

    Google Scholar 

  12. Cohen GM. Caspases: The executioners of apoptosis. Biochem J 1997; 326: 1-16.

    Google Scholar 

  13. Faleiro L, Kobayashi R, Fearnhead H, Lazebnik Y. Multiple species of CPP32 and Mch2 are the major active caspases present in apoptotic cells. EMBO J 1997; 16: 2271-2281.

    Google Scholar 

  14. Brown TL, Patil S, Cianci CD, Morrow JS, Howe PH. TGF beta induces caspase 3 independent cleavage of alpha II-spectrin (alpha-fodrin) coincident with apoptosis. J Biol Chem 1999; 274: 23256-23262.

    Google Scholar 

  15. Wang KK, Posmantur R, Nath R, et al. Simultaneous degradation of alpha II-and beta II-spectrin by caspase 3 (CPP32) in apoptotic cells. J Biol Chem 1998; 273: 22490-22497.

    Google Scholar 

  16. Yamazaki Y, Tsuruga M, Zhou D, et al. Cytoskeletal disruption accelerates caspase-3 activation and alters the intracellular membrane reorganization in DNA damage-induced apoptosis. Exp Cell Res 2000; 259: 64-78.

    Google Scholar 

  17. Nath R, Huggins M, Glantz SB, et al. Development and characterization of antibodies specific to caspase-3-produced alpha II-spectrin 120 kDa breakdown product: Marker for neuronal apoptosis. Neurochem Int 2000; 37: 351-361.

    Google Scholar 

  18. Brown TL, Patil S, Howe PH. Analysis of TGF?-inducible apoptosis. In: Howe P, ed. Methods in Molecular Biology-“Transforming Growth Factor-Beta Protocols.” Totowa, NJ: Humana Press, 2000: 142: 149-167.

    Google Scholar 

  19. Patil S, Wildey GM, Brown TL, Choy L, Derynck R, Howe PH. Smad7 is induced by CD40 and protects WEHI 231 Blymphocytes from transforming growth factor-beta-induced growth inhibition and apoptosis. J Biol Chem 2000; 275: 38363-38370.

    Google Scholar 

  20. Fischer G, Kent SC, Joseph L, Green DR, Scott, DW. Lymphoma models for B cell activation and tolerance. X. Antimu-mediated growth arrest and apoptosis of murine B cell lymphomas is prevented by the stabilization of Myc. J Exp Med 1994; 179: 221-228.

    Google Scholar 

  21. Warner GL, Ludlow JW, Nelson DA, Gaur A, Scott DW. Anti-immunoglobulin treatment of murine B-cell lymphomas induces active transforming growth factor beta but pRB hypophosphorylation is transforming growth factor beta independent. Cell Growth Diff 1992; 3: 175-181.

    Google Scholar 

  22. Wu M, Bellas RE, Shen J, Yang W, Sonenshein GE. Increased p27Kip1 cyclin dependent kinase inhibitor gene expression following anti-IgM treatment promotes apoptosis of WEHI 231 B cells. J Immunol 1999; 163: 6530-6535.

    Google Scholar 

  23. Wu M, Arsura M, Bellas RE, et al. Inhibition of c-myc expression induces apoptosis of WEHI 231 murine B cells. Mol Cell Biol 1996; 16: 5015-5025.

    Google Scholar 

  24. Lee JR, Koretzky GA. Extracellular signal-regulated kinase-2, but not c-Jun NH2-terminal kinase, activation correlates with surface IgM-medi-ated apoptosis in the WEHI 231 B cell line. J Immunol 1998; 161: 1637-1644.

    Google Scholar 

  25. Fang W, Rivard JJ, Ganser JA, et al. Bcl-xL rescues WEHI 231 B lymphocytes from oxidant-mediated death following diverse apoptotic stimuli. J Immunol 1995; 155: 66-75.

    Google Scholar 

  26. Brown TL, Supriya P, Basnett K, Howe H. Caspase inhibitor BD-fmk distinguishes transforming growth factor betainduced apoptosis from growth inhibition. Cell Growth and Differentiation 1998; 9: 869-879.

    Google Scholar 

  27. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976; 72: 248-254.

    Google Scholar 

  28. Kamesaki H, Nishizawa K, Michaud GY, Cossman J, Kiyono T. TGF-beta 1 induces the cyclin-dependent kinase inhibitor p27Kip1 mRNA and protein in murine B cells. J Immunol 1998; 160: 770-777.

    Google Scholar 

  29. Takahashi K, Tatsuzawa O, Yanagi K, Hayashi Y, Takahashi H. Alpha-fodrin auto-antibody in Sjogren syndrome and other auto-immune diseases in childhood. Eur J Pediatr 2001; 160: 520-521.

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Physiology & Biophysics, Wright State University School of Medicine, Dayton, OH, USA

    S. T. Williams, A. N. Smith & T. L. Brown

  2. Department of Pathology, Yale University School of Medicine, New Haven, Connecticut, USA

    C. D. Cianci & J. S. Morrow

Authors
  1. S. T. Williams
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. A. N. Smith
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. C. D. Cianci
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. J. S. Morrow
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. T. L. Brown
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Williams, S.T., Smith, A.N., Cianci, C.D. et al. Identification of the primary caspase 3 cleavage site in alpha II-spectrin during apoptosis. Apoptosis 8, 353–361 (2003). https://doi.org/10.1023/A:1024168901003

Download citation

  • Issue Date:

  • DOI: https://doi.org/10.1023/A:1024168901003

Search

Navigation