DOI: 10.1126/science.278.5336.294. Caspase-3-Generated Fragment of Gelsolin: Effector of Morphological Change in Apoptosis

Caspase-3-Generated Fragment of Gelsolin: Effector of Morphological Change in Apoptosis

10.1126/science.278.5336.294

Crossref journal-article

American Association for the Advancement of Science (AAAS)

Science (221)

Abstract

The caspase-3 (CPP32, apopain, YAMA) family of cysteinyl proteases has been implicated as key mediators of apoptosis in mammalian cells. Gelsolin was identified as a substrate for caspase-3 by screening the translation products of small complementary DNA pools for sensitivity to cleavage by caspase-3. Gelsolin was cleaved in vivo in a caspase-dependent manner in cells stimulated by Fas. Caspase-cleaved gelsolin severed actin filaments in vitro in a Ca 2+ -independent manner. Expression of the gelsolin cleavage product in multiple cell types caused the cells to round up, detach from the plate, and undergo nuclear fragmentation. Neutrophils isolated from mice lacking gelsolin had delayed onset of both blebbing and DNA fragmentation, following apoptosis induction, compared with wild-type neutrophils. Thus, cleaved gelsolin may be one physiological effector of morphologic change during apoptosis.

Bibliography

Kothakota, S., Azuma, T., Reinhard, C., Klippel, A., Tang, J., Chu, K., McGarry, T. J., Kirschner, M. W., Koths, K., Kwiatkowski, D. J., & Williams, L. T. (1997). Caspase-3-Generated Fragment of Gelsolin: Effector of Morphological Change in Apoptosis. Science, 278(5336), 294â298.

Authors 11

Srinivas Kothakota (first)
Toshifumi Azuma (additional)
Christoph Reinhard (additional)
Anke Klippel (additional)
Jay Tang (additional)
Keting Chu (additional)
Thomas J. McGarry (additional)
Marc W. Kirschner (additional)
Kirston Koths (additional)
David J. Kwiatkowski (additional)
Lewis T. Williams (additional)

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Dates

Type	When
Created	23 years, 1 month ago (July 27, 2002, 5:50 a.m.)
Deposited	1 year, 7 months ago (Jan. 12, 2024, 11:48 p.m.)
Indexed	6 days, 21 hours ago (Aug. 23, 2025, 12:59 a.m.)
Issued	27 years, 10 months ago (Oct. 10, 1997)
Published	27 years, 10 months ago (Oct. 10, 1997)
Published Print	27 years, 10 months ago (Oct. 10, 1997)

Funders 0

None

BibTeX

@article{Kothakota_1997, title={Caspase-3-Generated Fragment of Gelsolin: Effector of Morphological Change in Apoptosis}, volume={278}, ISSN={1095-9203}, url={http://dx.doi.org/10.1126/science.278.5336.294}, DOI={10.1126/science.278.5336.294}, number={5336}, journal={Science}, publisher={American Association for the Advancement of Science (AAAS)}, author={Kothakota, Srinivas and Azuma, Toshifumi and Reinhard, Christoph and Klippel, Anke and Tang, Jay and Chu, Keting and McGarry, Thomas J. and Kirschner, Marc W. and Koths, Kirston and Kwiatkowski, David J. and Williams, Lewis T.}, year={1997}, month=oct, pages={294–298} }

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      "doi-asserted-by": "crossref",
      "first-page": "13281",
      "DOI": "10.1016/S0021-9258(18)51626-4",
      "volume": "264",
      "author": "Dieffenbach C. W.",
      "year": "1989",
      "unstructured": "Dieffenbach C. W., Sen Gupta D. N., Krause D., Sawzak D., Silverman R. H., J. Biol. Chem. 264, 13281 (1989).",
      "journal-title": "J. Biol. Chem."
    },
    {
      "unstructured": "Native and cleaved gelsolin preparations were analyzed on a BioSil SEC250 column run in 180 mM NaCl with 10 mM phosphate buffer (pH 7.4) at a flow rate of 1 ml/min and monitored at 280 nm. Column fractions were analyzed by SDS-PAGE and Coomassie staining.",
      "key": "e_1_3_1_9_2"
    },
    {
      "key": "e_1_3_1_10_2",
      "doi-asserted-by": "crossref",
      "first-page": "11894",
      "DOI": "10.1073/pnas.92.25.11894",
      "volume": "92",
      "author": "Chu K.",
      "year": "1995",
      "unstructured": "Chu K., Niu X., Williams L. T., Proc. Natl. Acad. Sci. U.S.A. 92, 11894 (1995).",
      "journal-title": "Proc. Natl. Acad. Sci. U.S.A."
    },
    {
      "key": "e_1_3_1_11_2",
      "doi-asserted-by": "crossref",
      "first-page": "21",
      "DOI": "10.1042/bj3150021",
      "volume": "315",
      "author": "Slee E. A.",
      "year": "1996",
      "unstructured": "Slee E. A., et al., Biochem. J. 315, 21 (1996).",
      "journal-title": "Biochem. J."
    },
    {
      "key": "e_1_3_1_12_2",
      "doi-asserted-by": "crossref",
      "first-page": "429",
      "DOI": "10.1084/jem.184.2.429",
      "volume": "184",
      "author": "Liles W. C.",
      "year": "1996",
      "unstructured": "Liles W. C., Kiener P. A., Ledbetter J. A., Aruffo A., Klebanoff S. J., J. Exp. Med. 184, 429 (1996);",
      "journal-title": "J. Exp. Med."
    },
    {
      "key": "e_1_3_1_12_3",
      "doi-asserted-by": "crossref",
      "first-page": "691",
      "DOI": "10.1093/intimm/5.6.691",
      "volume": "5",
      "author": "Takeda Y.",
      "year": "1993",
      "unstructured": "Takeda Y., et al., Int. Immunol. 5, 691 (1993).",
      "journal-title": "Int. Immunol."
    },
    {
      "key": "e_1_3_1_13_2",
      "doi-asserted-by": "crossref",
      "first-page": "1717",
      "DOI": "10.1083/jcb.108.5.1717",
      "volume": "108",
      "author": "Kwiatkowski D. J.",
      "year": "1989",
      "unstructured": "Kwiatkowski D. J., Janmey P. A., Yin H. L., J. Cell. Biol. 108, 1717 (1989).",
      "journal-title": "J. Cell. Biol."
    },
    {
      "key": "e_1_3_1_14_2",
      "doi-asserted-by": "crossref",
      "first-page": "1016",
      "DOI": "10.1042/bst0191016",
      "volume": "19",
      "author": "Weeds A. G.",
      "year": "1991",
      "unstructured": "Weeds A. G., Gooch J., Hawkins M., Pope B., Way M., Biochem. Soc. Trans. 19, 1016 (1991).",
      "journal-title": "Biochem. Soc. Trans."
    },
    {
      "unstructured": "Cells (10 6 ) were fixed with 4% paraformaldehyde and permeabilized with ethanol/acetone. After washing with phosphate-buffered saline (PBS) cells were incubated with TdT and fluorescein-conjugated dUTP at 37\u00b0C for 1 hour. Cells were washed and free DNA ends were visualized by fluorescent microscopy.",
      "key": "e_1_3_1_15_2"
    },
    {
      "key": "e_1_3_1_16_2",
      "doi-asserted-by": "crossref",
      "first-page": "2026",
      "DOI": "10.1073/pnas.91.6.2026",
      "volume": "91",
      "author": "Lowe S. W.",
      "year": "1994",
      "unstructured": "Lowe S. W., Jacks T., Housman D. E., Ruley H. E., Proc. Natl. Acad. Sci. U.S.A. 91, 2026 (1994).",
      "journal-title": "Proc. Natl. Acad. Sci. U.S.A."
    },
    {
      "key": "e_1_3_1_17_2",
      "doi-asserted-by": "crossref",
      "first-page": "41",
      "DOI": "10.1016/0092-8674(95)90369-0",
      "volume": "81",
      "author": "Witke W.",
      "year": "1995",
      "unstructured": "Witke W., et al., Cell 81, 41 (1995).",
      "journal-title": "Cell"
    },
    {
      "unstructured": "DNA was prepared from cells by standard digestion and organic extraction. Extracted DNA (10 \u03bcg) was separated by electrophoresis in 1.5% agarose and visualized with ethidium staining.",
      "key": "e_1_3_1_18_2"
    },
    {
      "unstructured": "Neutrophils (10 5 ) were treated with TNF+CHX or antibody to Fas rinsed with PBS and incubated in 50 mM tris-HCl (pH 7.4) 1 mM EDTA 10 mM EGTA and 10 \u03bcM digitonin at 37\u00b0C for 10 min. After centrifugation at 15 000 g for 10 min the lysate was incubated in DEVD-AFC (Enzyme Systems Products) for 30 min. Released AFC was measured by spectrofluorimetry with excitation at a wavelength of 380 nm and emission at 460 nm.",
      "key": "e_1_3_1_19_2"
    },
    {
      "key": "e_1_3_1_20_2",
      "doi-asserted-by": "crossref",
      "first-page": "5245",
      "DOI": "10.1128/MCB.16.10.5245",
      "volume": "16",
      "author": "Janicke R. U.",
      "year": "1996",
      "unstructured": "Janicke R. U., Lin X. Y., Lee F. H. H., Porter A. G., Mol. Cell. Biol. 16, 5245 (1996).",
      "journal-title": "Mol. Cell. Biol."
    },
    {
      "key": "e_1_3_1_21_2",
      "doi-asserted-by": "crossref",
      "first-page": "1185",
      "DOI": "10.1006/bbrc.1995.2894",
      "volume": "217",
      "author": "Mashima T.",
      "year": "1995",
      "unstructured": "Mashima T., Naito M., Fujita N., Noguchi K., Tsuruo T., Biochem. Biophys. Res. Commun. 217, 1185 (1995);",
      "journal-title": "Biochem. Biophys. Res. Commun."
    },
    {
      "key": "e_1_3_1_21_3",
      "doi-asserted-by": "crossref",
      "first-page": "2234",
      "DOI": "10.1073/pnas.93.5.2234",
      "volume": "93",
      "author": "Kayalar C.",
      "year": "1996",
      "unstructured": "Kayalar C., Ord T., Testa M. P., Zhong L. T., Bredesen D. E., Proc. Natl. Acad. Sci. U.S.A. 93, 2234 (1996);",
      "journal-title": "Proc. Natl. Acad. Sci. U.S.A."
    },
    {
      "key": "e_1_3_1_21_4",
      "first-page": "157",
      "volume": "94",
      "author": "Song Q.",
      "year": "1997",
      "unstructured": "Song Q., et al., ibid. 94, 157 (1997);",
      "journal-title": "ibid."
    },
    {
      "key": "e_1_3_1_21_5",
      "doi-asserted-by": "crossref",
      "first-page": "5179",
      "DOI": "10.1002/j.1460-2075.1995.tb00202.x",
      "volume": "14",
      "author": "Brancolini C.",
      "year": "1995",
      "unstructured": "Brancolini C., Benedetti M., Schneider C., EMBO J. 14, 5179 (1995).",
      "journal-title": "EMBO J."
    },
    {
      "key": "e_1_3_1_22_2",
      "doi-asserted-by": "crossref",
      "first-page": "497",
      "DOI": "10.1146/annurev.cb.11.110195.002433",
      "volume": "11",
      "author": "Schafer D. A.",
      "year": "1995",
      "unstructured": "Schafer D. A., Cooper J. A., Annu. Rev. Cell Dev. Biol. 11, 497 (1995);",
      "journal-title": "Annu. Rev. Cell Dev. Biol."
    },
    {
      "key": "e_1_3_1_22_3",
      "doi-asserted-by": "crossref",
      "first-page": "11386",
      "DOI": "10.1073/pnas.90.23.11386",
      "volume": "90",
      "author": "Campbell H. D.",
      "year": "1993",
      "unstructured": "Campbell H. D., et al., Proc. Natl. Acad. Sci. U.S.A. 90, 11386 (1993);",
      "journal-title": "Proc. Natl. Acad. Sci. U.S.A."
    },
    {
      "unstructured": "; D. J. Kwiatkowski et al. unpublished data.",
      "key": "e_1_3_1_22_4"
    },
    {
      "key": "e_1_3_1_23_2",
      "first-page": "3228",
      "volume": "55",
      "author": "Tanaka M.",
      "year": "1995",
      "unstructured": "Tanaka M., et al., Cancer Res. 55, 3228 (1995);",
      "journal-title": "Cancer Res."
    },
    {
      "key": "e_1_3_1_23_3",
      "first-page": "4841",
      "volume": "56",
      "author": "Asch H. L.",
      "year": "1996",
      "unstructured": "Asch H. L., et al., ibid. 56, 4841 (1996);",
      "journal-title": "ibid."
    },
    {
      "doi-asserted-by": "publisher",
      "key": "e_1_3_1_23_4",
      "DOI": "10.1126/science.276.5316.1268"
    },
    {
      "unstructured": "A cDNA library was prepared from mouse embryo (day 14 postcoitus) RNA. The cDNAs were cloned into the expression vector pCS2+ generating 200 000 independent clones. The library was fractionated into smaller groups of 100 different clones each. Each group of cDNAs was used to express [ 35 S]methionine-labeled proteins using a coupled transcription-translation system (TNT system Promega). We incubated 2 ml of the translation mix with 0.5 ml of active or inactivated caspase-3 (0.15 ng/ml) at 30\u00b0C for 0.5 hour. Caspase-3 was inactivated by incubation with 50 mM DEVD-fmk for 30 min before addition to the translation products. Recombinant active caspase-3 was prepared by expressing the sequence of human caspase-3 (YAMA/CPP32 a gift of V. Dixit) in E. coli (2) . Purified caspase-3 (generously provided by A. L. Fear) was fully active when assayed on DEVD\u20137-amino-4-methylcoumarin as previously described (3). Caspase-3 was >80% pure consisting of 17- and 11-kD subunits as assayed by SDS-PAGE.",
      "key": "e_1_3_1_24_2"
    },
    {
      "key": "e_1_3_1_25_2",
      "doi-asserted-by": "crossref",
      "first-page": "1545",
      "DOI": "10.1084/jem.182.5.1545",
      "volume": "182",
      "author": "Martin S. J.",
      "year": "1995",
      "unstructured": "Martin S. J., et al., J. Exp. Med. 182, 1545 (1995).",
      "journal-title": "J. Exp. Med."
    },
    {
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      "doi-asserted-by": "crossref",
      "first-page": "229",
      "DOI": "10.1002/cm.970160403",
      "volume": "16",
      "author": "Huckriede A.",
      "year": "1990",
      "unstructured": "Huckriede A., et al., Cell Motil. Cytoskeleton 16, 229 (1990).",
      "journal-title": "Cell Motil. Cytoskeleton"
    },
    {
      "unstructured": "Rat embryo fibroblasts (REF 52) [",
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    },
    {
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      "DOI": "10.1126/science.271.5250.810"
    },
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      "unstructured": "] were seeded on acid-treated glass cover slips and 70% density cells were microinjected with plasmid DNA pGG-NT (encoding residues 1 through 352 of gelsolin and tagged with EYMPME epitope at the COOH-terminus) or pGG-CT (encoding residues 353 through 731 of gelsolin and tagged with EYMPME epitope at the COOH-terminus) at 0.1 \u03bcg/ml in 100 mM Hepes (pH 7.4). A plasmid encoding green fluorescent protein (GFP) (pEGFPN1 Promega) at 1 ng/ml was coinjected as a marker. Cells were fixed in 4% paraformaldehyde/PBS permeabilized with 0.1% Triton X-100 and stained for the expression of tagged proteins using a mouse monoclonal antibody to EYMPME (50 \u03bcg/ml) and a secondary goat anti-mouse immunoglobulin G coupled to Texas Red (Amersham). Actin filaments were visualized with tetramethyl rhodamine isothiocyanate (TRITC)\u2013Phalloidin (Sigma).",
      "key": "e_1_3_1_27_4"
    },
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      "DOI": "10.1016/S0091-679X(08)60603-2",
      "volume": "43",
      "author": "Becker T. C.",
      "year": "1994",
      "unstructured": "Becker T. C., et al., Methods Cell Biol. 43, 161 (1994).",
      "journal-title": "Methods Cell Biol."
    },
    {
      "unstructured": "We thank G. Pronk for p35 cDNA; V. Dixit for YAMA cDNA; A. L. Fear for active caspase-3; A. Creasey and T. Gesner for help with neutrophil isolation; T. Brown M. Giedlin and T. Shi for fluorescence-activated cell sorter (FACS) analyses; M. Heinzel and G. Harrowe for microscopy; J. Tucker for DNA sequence analyses; and D. A. Fisher and T. Jacks for p53 \u2013/\u2013 cells. Supported by Chiron Corporation and NIH grants RO1 HL54188 and P01 HL48743 to D.J.K.",
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