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. 1997 Apr 1;323(Pt 1):233–237. doi: 10.1042/bj3230233

Actin is cleaved during constitutive apoptosis.

S B Brown 1, K Bailey 1, J Savill 1
PMCID: PMC1218300  PMID: 9173887

Abstract

Proteases play an important role in the programme of cell death by apoptosis but little is known of the substrates cleaved, particularly in constitutive models of this type of cell death. Neutrophils spontaneously undergo apoptosis in culture without requiring external stimuli. During this process we found biochemical and immunochemical evidence for the cleavage of membrane-associated actin, a component of the cytoskeleton that links polymerized actin to the plasma membrane. Cleavage occurred at a single site at the N-terminus, between residues Val43-Met44, a site devoid of a consensus motif for cleavage by cysteine proteases of the interleukin-1beta-converting enzyme (ICE)-family. Whereas actin cleavage and nuclear/cell surface markers of apoptosis were co-ordinately diminished by zVAD-fmk, an inhibitor of the ICE-like family of proteases, only acetyl-leucyl-leucylnormethional, an inhibitor of calpains, was capable of completely inhibiting actin cleavage. Our results suggest that actin is not a direct substrate for the ICE-like family of proteases. By disabling the cytoskeleton, actin cleavage may be an important component in the capacity of apoptosis to reduce the injurious potential of neutrophils.

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Selected References

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  1. An B., Dou Q. P. Cleavage of retinoblastoma protein during apoptosis: an interleukin 1 beta-converting enzyme-like protease as candidate. Cancer Res. 1996 Feb 1;56(3):438–442. [PubMed] [Google Scholar]
  2. Brancolini C., Benedetti M., Schneider C. Microfilament reorganization during apoptosis: the role of Gas2, a possible substrate for ICE-like proteases. EMBO J. 1995 Nov 1;14(21):5179–5190. doi: 10.1002/j.1460-2075.1995.tb00202.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bruno S., Lassota P., Giaretti W., Darzynkiewicz Z. Apoptosis of rat thymocytes triggered by prednisolone, camptothecin, or teniposide is selective to G0 cells and is prevented by inhibitors of proteases. Oncol Res. 1992;4(1):29–35. [PubMed] [Google Scholar]
  4. Carson D. A., Ribeiro J. M. Apoptosis and disease. Lancet. 1993 May 15;341(8855):1251–1254. doi: 10.1016/0140-6736(93)91154-e. [DOI] [PubMed] [Google Scholar]
  5. Casciola-Rosen L. A., Anhalt G. J., Rosen A. DNA-dependent protein kinase is one of a subset of autoantigens specifically cleaved early during apoptosis. J Exp Med. 1995 Dec 1;182(6):1625–1634. doi: 10.1084/jem.182.6.1625. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Casciola-Rosen L. A., Miller D. K., Anhalt G. J., Rosen A. Specific cleavage of the 70-kDa protein component of the U1 small nuclear ribonucleoprotein is a characteristic biochemical feature of apoptotic cell death. J Biol Chem. 1994 Dec 9;269(49):30757–30760. [PubMed] [Google Scholar]
  7. Cotter T. G., Lennon S. V., Glynn J. M., Green D. R. Microfilament-disrupting agents prevent the formation of apoptotic bodies in tumor cells undergoing apoptosis. Cancer Res. 1992 Feb 15;52(4):997–1005. [PubMed] [Google Scholar]
  8. Darmon A. J., Nicholson D. W., Bleackley R. C. Activation of the apoptotic protease CPP32 by cytotoxic T-cell-derived granzyme B. Nature. 1995 Oct 5;377(6548):446–448. doi: 10.1038/377446a0. [DOI] [PubMed] [Google Scholar]
  9. Dransfield I., Buckle A. M., Savill J. S., McDowall A., Haslett C., Hogg N. Neutrophil apoptosis is associated with a reduction in CD16 (Fc gamma RIII) expression. J Immunol. 1994 Aug 1;153(3):1254–1263. [PubMed] [Google Scholar]
  10. Earnshaw W. C. Nuclear changes in apoptosis. Curr Opin Cell Biol. 1995 Jun;7(3):337–343. doi: 10.1016/0955-0674(95)80088-3. [DOI] [PubMed] [Google Scholar]
  11. Emoto Y., Manome Y., Meinhardt G., Kisaki H., Kharbanda S., Robertson M., Ghayur T., Wong W. W., Kamen R., Weichselbaum R. Proteolytic activation of protein kinase C delta by an ICE-like protease in apoptotic cells. EMBO J. 1995 Dec 15;14(24):6148–6156. doi: 10.1002/j.1460-2075.1995.tb00305.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Fawthrop D. J., Boobis A. R., Davies D. S. Mechanisms of cell death. Arch Toxicol. 1991;65(6):437–444. doi: 10.1007/BF01977355. [DOI] [PubMed] [Google Scholar]
  13. Fernandes-Alnemri T., Litwack G., Alnemri E. S. CPP32, a novel human apoptotic protein with homology to Caenorhabditis elegans cell death protein Ced-3 and mammalian interleukin-1 beta-converting enzyme. J Biol Chem. 1994 Dec 9;269(49):30761–30764. [PubMed] [Google Scholar]
  14. Haslett C., Guthrie L. A., Kopaniak M. M., Johnston R. B., Jr, Henson P. M. Modulation of multiple neutrophil functions by preparative methods or trace concentrations of bacterial lipopolysaccharide. Am J Pathol. 1985 Apr;119(1):101–110. [PMC free article] [PubMed] [Google Scholar]
  15. Homburg C. H., de Haas M., von dem Borne A. E., Verhoeven A. J., Reutelingsperger C. P., Roos D. Human neutrophils lose their surface Fc gamma RIII and acquire Annexin V binding sites during apoptosis in vitro. Blood. 1995 Jan 15;85(2):532–540. [PubMed] [Google Scholar]
  16. Kaufmann S. H., Desnoyers S., Ottaviano Y., Davidson N. E., Poirier G. G. Specific proteolytic cleavage of poly(ADP-ribose) polymerase: an early marker of chemotherapy-induced apoptosis. Cancer Res. 1993 Sep 1;53(17):3976–3985. [PubMed] [Google Scholar]
  17. Kayalar C., Ord T., Testa M. P., Zhong L. T., Bredesen D. E. Cleavage of actin by interleukin 1 beta-converting enzyme to reverse DNase I inhibition. Proc Natl Acad Sci U S A. 1996 Mar 5;93(5):2234–2238. doi: 10.1073/pnas.93.5.2234. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Kerr J. F., Wyllie A. H., Currie A. R. Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br J Cancer. 1972 Aug;26(4):239–257. doi: 10.1038/bjc.1972.33. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Kosower N. S., Glaser T., Kosower E. M. Membrane-mobility agent-promoted fusion of erythrocytes: fusibility is correlated with attack by calcium-activated cytoplasmic proteases on membrane proteins. Proc Natl Acad Sci U S A. 1983 Dec;80(24):7542–7546. doi: 10.1073/pnas.80.24.7542. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  21. Lazebnik Y. A., Kaufmann S. H., Desnoyers S., Poirier G. G., Earnshaw W. C. Cleavage of poly(ADP-ribose) polymerase by a proteinase with properties like ICE. Nature. 1994 Sep 22;371(6495):346–347. doi: 10.1038/371346a0. [DOI] [PubMed] [Google Scholar]
  22. Lazebnik Y. A., Takahashi A., Moir R. D., Goldman R. D., Poirier G. G., Kaufmann S. H., Earnshaw W. C. Studies of the lamin proteinase reveal multiple parallel biochemical pathways during apoptotic execution. Proc Natl Acad Sci U S A. 1995 Sep 26;92(20):9042–9046. doi: 10.1073/pnas.92.20.9042. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Lee A., Whyte M. K., Haslett C. Inhibition of apoptosis and prolongation of neutrophil functional longevity by inflammatory mediators. J Leukoc Biol. 1993 Oct;54(4):283–288. [PubMed] [Google Scholar]
  24. Martin S. J., Green D. R. Protease activation during apoptosis: death by a thousand cuts? Cell. 1995 Aug 11;82(3):349–352. doi: 10.1016/0092-8674(95)90422-0. [DOI] [PubMed] [Google Scholar]
  25. Martin S. J., O'Brien G. A., Nishioka W. K., McGahon A. J., Mahboubi A., Saido T. C., Green D. R. Proteolysis of fodrin (non-erythroid spectrin) during apoptosis. J Biol Chem. 1995 Mar 24;270(12):6425–6428. doi: 10.1074/jbc.270.12.6425. [DOI] [PubMed] [Google Scholar]
  26. Martin S. J., Reutelingsperger C. P., McGahon A. J., Rader J. A., van Schie R. C., LaFace D. M., Green D. R. Early redistribution of plasma membrane phosphatidylserine is a general feature of apoptosis regardless of the initiating stimulus: inhibition by overexpression of Bcl-2 and Abl. J Exp Med. 1995 Nov 1;182(5):1545–1556. doi: 10.1084/jem.182.5.1545. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Mashima T., Naito M., Fujita N., Noguchi K., Tsuruo T. Identification of actin as a substrate of ICE and an ICE-like protease and involvement of an ICE-like protease but not ICE in VP-16-induced U937 apoptosis. Biochem Biophys Res Commun. 1995 Dec 26;217(3):1185–1192. doi: 10.1006/bbrc.1995.2894. [DOI] [PubMed] [Google Scholar]
  28. Mirabelli F., Salis A., Vairetti M., Bellomo G., Thor H., Orrenius S. Cytoskeletal alterations in human platelets exposed to oxidative stress are mediated by oxidative and Ca2+-dependent mechanisms. Arch Biochem Biophys. 1989 May 1;270(2):478–488. doi: 10.1016/0003-9861(89)90529-8. [DOI] [PubMed] [Google Scholar]
  29. Miura M., Zhu H., Rotello R., Hartwieg E. A., Yuan J. Induction of apoptosis in fibroblasts by IL-1 beta-converting enzyme, a mammalian homolog of the C. elegans cell death gene ced-3. Cell. 1993 Nov 19;75(4):653–660. doi: 10.1016/0092-8674(93)90486-a. [DOI] [PubMed] [Google Scholar]
  30. Neamati N., Fernandez A., Wright S., Kiefer J., McConkey D. J. Degradation of lamin B1 precedes oligonucleosomal DNA fragmentation in apoptotic thymocytes and isolated thymocyte nuclei. J Immunol. 1995 Apr 15;154(8):3788–3795. [PubMed] [Google Scholar]
  31. Nicholson D. W., Ali A., Thornberry N. A., Vaillancourt J. P., Ding C. K., Gallant M., Gareau Y., Griffin P. R., Labelle M., Lazebnik Y. A. Identification and inhibition of the ICE/CED-3 protease necessary for mammalian apoptosis. Nature. 1995 Jul 6;376(6535):37–43. doi: 10.1038/376037a0. [DOI] [PubMed] [Google Scholar]
  32. Sarin A., Adams D. H., Henkart P. A. Protease inhibitors selectively block T cell receptor-triggered programmed cell death in a murine T cell hybridoma and activated peripheral T cells. J Exp Med. 1993 Nov 1;178(5):1693–1700. doi: 10.1084/jem.178.5.1693. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Sarin A., Clerici M., Blatt S. P., Hendrix C. W., Shearer G. M., Henkart P. A. Inhibition of activation-induced programmed cell death and restoration of defective immune responses of HIV+ donors by cysteine protease inhibitors. J Immunol. 1994 Jul 15;153(2):862–872. [PubMed] [Google Scholar]
  34. Savill J. S., Wyllie A. H., Henson J. E., Walport M. J., Henson P. M., Haslett C. Macrophage phagocytosis of aging neutrophils in inflammation. Programmed cell death in the neutrophil leads to its recognition by macrophages. J Clin Invest. 1989 Mar;83(3):865–875. doi: 10.1172/JCI113970. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Savill J. Apoptosis in disease. Eur J Clin Invest. 1994 Nov;24(11):715–723. doi: 10.1111/j.1365-2362.1994.tb01067.x. [DOI] [PubMed] [Google Scholar]
  36. Shi L., Kam C. M., Powers J. C., Aebersold R., Greenberg A. H. Purification of three cytotoxic lymphocyte granule serine proteases that induce apoptosis through distinct substrate and target cell interactions. J Exp Med. 1992 Dec 1;176(6):1521–1529. doi: 10.1084/jem.176.6.1521. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Shi L., Kraut R. P., Aebersold R., Greenberg A. H. A natural killer cell granule protein that induces DNA fragmentation and apoptosis. J Exp Med. 1992 Feb 1;175(2):553–566. doi: 10.1084/jem.175.2.553. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Shuster C. B., Herman I. M. Indirect association of ezrin with F-actin: isoform specificity and calcium sensitivity. J Cell Biol. 1995 Mar;128(5):837–848. doi: 10.1083/jcb.128.5.837. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Spudich J. A., Watt S. The regulation of rabbit skeletal muscle contraction. I. Biochemical studies of the interaction of the tropomyosin-troponin complex with actin and the proteolytic fragments of myosin. J Biol Chem. 1971 Aug 10;246(15):4866–4871. [PubMed] [Google Scholar]
  40. Squìer M. K., Miller A. C., Malkinson A. M., Cohen J. J. Calpain activation in apoptosis. J Cell Physiol. 1994 May;159(2):229–237. doi: 10.1002/jcp.1041590206. [DOI] [PubMed] [Google Scholar]
  41. Tinnemans M. M., Lenders M. H., ten Velde G. P., Ramaekers F. C., Schutte B. Alterations in cytoskeletal and nuclear matrix-associated proteins during apoptosis. Eur J Cell Biol. 1995 Sep;68(1):35–46. [PubMed] [Google Scholar]
  42. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Voelkel-Johnson C., Entingh A. J., Wold W. S., Gooding L. R., Laster S. M. Activation of intracellular proteases is an early event in TNF-induced apoptosis. J Immunol. 1995 Feb 15;154(4):1707–1716. [PubMed] [Google Scholar]
  44. Wang X., Zelenski N. G., Yang J., Sakai J., Brown M. S., Goldstein J. L. Cleavage of sterol regulatory element binding proteins (SREBPs) by CPP32 during apoptosis. EMBO J. 1996 Mar 1;15(5):1012–1020. [PMC free article] [PubMed] [Google Scholar]
  45. Whyte M. K., Meagher L. C., MacDermot J., Haslett C. Impairment of function in aging neutrophils is associated with apoptosis. J Immunol. 1993 Jun 1;150(11):5124–5134. [PubMed] [Google Scholar]
  46. Wray W., Boulikas T., Wray V. P., Hancock R. Silver staining of proteins in polyacrylamide gels. Anal Biochem. 1981 Nov 15;118(1):197–203. doi: 10.1016/0003-2697(81)90179-2. [DOI] [PubMed] [Google Scholar]
  47. Wright S. C., Wei Q. S., Zhong J., Zheng H., Kinder D. H., Larrick J. W. Purification of a 24-kD protease from apoptotic tumor cells that activates DNA fragmentation. J Exp Med. 1994 Dec 1;180(6):2113–2123. doi: 10.1084/jem.180.6.2113. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Wulf E., Deboben A., Bautz F. A., Faulstich H., Wieland T. Fluorescent phallotoxin, a tool for the visualization of cellular actin. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4498–4502. doi: 10.1073/pnas.76.9.4498. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Wyllie A. H., Kerr J. F., Currie A. R. Cell death: the significance of apoptosis. Int Rev Cytol. 1980;68:251–306. doi: 10.1016/s0074-7696(08)62312-8. [DOI] [PubMed] [Google Scholar]

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