Apoptosis, or programmed cell death, involves a cascade of regulatory events leading to the activation of specific proteases. However, the key substrates for these proteases remain to be identified. We previously demonstrated that levels of five unidentified polypeptides were specifically increased in neurons from embryonic chicken ciliary ganglia undergoing apoptosis by trophic deprivation. Here we show by microsequencing of two of these polypeptides that they are fragments of actin. One of them represents cleavage of actin at the site of interaction with DNase I. The same actin fragments are also found at early stages of apoptosis in chicken and rat dorsal root ganglion neurons, chicken spinal motoneurons and rat thymocytes. Actin fragmentation may play a role in the apoptotic process, since calpain inhibitors I and II both inhibit neuronal death and suppress actin fragmentation. In contrast, caspase (ICE family) inhibitors, though effective in delaying neuronal death, do not prevent actin cleavage or DNA fragmentation. These results indicate a key role for calpain-like proteases in neuronal programmed cell death and suggest that actin fragmentation in the cell is correlated with subsequent DNA fragmentation.
REFERENCES
Allsopp
T. E.
, Wyatt
S.
, Paterson
H. F.
, Davies
A. M.
(1993
). The proto-oncogene bcl-2 can selectively rescue neurotrophic factor-dependent neurons from apoptosis.
Cell
73
, 295
–307
Alnemri
E. S.
, Livingston
D. J.
, Nicholson
D. W.
, Salvesen
G.
, Thornberry
N. A.
, Wong
W. W.
, Yuan
J.
(1996
). Human ICE/CED-3 protease nomenclature.
Cell
87
, 171
–.Bloch-Gallego
E.
, Huchet
M.
, El M'HamdiS
H.
, Xie
F. K.
, Tanaka
H.
, Henderson
C. E.
(1991
). Survival in vitro of motoneurons identified or purified by novel antibody-based methods is selectively enhanced by muscle-derived factors.
Development
111
, 221
–232
Brancolini
C.
, Benedetti
M.
, Schneider
C.
(1995
). Microfilament reorganization during apoptosis: the role of Gas2, a possible substrate for ICE-like proteases.
EMBO J
14
, 5179
–5190
Brown
S. B.
, Bailey
K.
, Savill
J.
(1997
). Actin is cleaved during constitutive apoptosis.
Biochem. J
323
, 233
–237
Casciola-Rosen
L. A.
, Anhalt
G. J.
, Rosen
A.
(1995
). DNA-dependant protein kinase is one of the subset of autoantigens specifically cleaved early during apoptosis.
J. Exp. Med
182
, 1625
–1634
Cowan
W. M.
, Fawcett
J. W.
, O'Leary
D. D.
, Stanfield
B. B.
(1984
). Regressive events in neurogenesis.
Science
225
, 1258
–1265
Deshmukh
M.
, Vasilakos
J.
, Deckwerth
T. L.
, Lampe
P. A.
, Shivers
B. D.
, Johnson
E. M.
Jr. (1996
). Genetic and metabolic status of NGF-deprived sympathetic neurons saved by an inhibitor of ICE family proteases.
J. Cell Biol
135
, 1341
–1354
Drexler
H. C.
(1997
). Activation of the cell death program by inhibition of proteasome function.
Proc. Nat. Acad. Sci. USA
94
, 855
–860
Ellis
H. M.
, Horvitz
H. R.
(1986
). Genetic control of programmed cell death in the nematode C. elegans.
Cell
44
, 817
–829
Ellis
R. E.
, Yuan
J. Y.
, Horvitz
H. R.
(1991
). Mechanisms and functions of cell death.
Annu. Rev. Cell Biol
7
, 663
–698
Garrels
J. I.
(1979
). Two dimensional gel electrophoresis and computer analysis of proteins synthesized by clonal cell lines.
J. Biol. Chem
254
, 7961
–7977
Gu
Y.
, Sarnecki
C.
, Aldape
R. A.
, Livingston
D. J.
, Su
M. S.
(1995
). Cleavage of poly(ADP-ribose) polymerase by interleukin-1 beta converting enzyme and its homologs TX and Nedd-2.
J. Biol. Chem
270
, 18715
–18718
Henderson
C. E.
(1996
). Programmed cell death in the developing nervous system.
Neuron
17
, 579
–585
Hengartner
M. O.
, Ellis
R. E.
, Horvitz
H. R.
(1992
). Caenorhabditis elegans gene ced-9 protects cells from programmed cell death.
Nature
356
, 494
–499
Humblet
C.
, Deman
J.
, Franzen
R.
, Greimers
R.
, Boniver
J.
, Defresne
M. P.
(1995
). Spontaneous and induced apoptosis after whole body radiation exposure: experimental approaches. Observations in radio-induced thymic lymphomagenesis.
Stem Cells
1
, 129
–135
Kabsch
W.
, Mannherz
H. G.
, Suck
D.
, Pai
E. F.
, Holmes
K. C.
(1990
). Atomic structure of the actin:DNase I complex [see comments].
Nature
347
, 37
–44
Kayalar
C.
, Ord
T.
, Testa
M. P.
, Zhong
L. T.
, Bredesen
D. E.
(1996
). Cleavage of actin by interleukin 1 beta-converting enzyme to reverse DNase I inhibition.
Proc. Nat. Acad. Sci. USA
93
, 2234
–2238
Kuida
K.
, Lippke
J. A.
, Ku
G.
, Harding
M. W.
, Livingston
D. J.
, Su
M.
, Flavell
R. A.
(1995
). Altered cytokine export and apoptosis in mice deficient in interleukin-1-beta converting-enzyme.
Science
267
, 2000
–2003
Kuida
K.
, Zheng
T. S.
, Na
S.
, Kuan
C.
, Yang
D.
, Karasuyama
H.
, Rakic
P.
, Flavell
R. A.
(1996
). Decreased apoptosis in the brain and premature lethality in CPP32-deficient mice.
Nature
384
, 368
–372
Lazarides
E.
, Lindberg
U.
(1974
). Actin is the naturally occurring inhibitor of deoxyribonuclease I.
Proc. Nat. Acad. Sci. USA
71
, 4742
–4746
Lazebnik
Y. A.
, Kaufmann
S. H.
, Desnoyers
S.
, Poirier
G. G.
, Earnshaw
W. C.
(1994
). Cleavage of poly(ADP-ribose) polymerase by a proteinase with properties like ICE.
Nature
371
, 346
–347
Lazebnik
Y. A.
, Takahashi
A.
, Moir
R. D.
, Goldman
R. D.
, Poirier
G. G.
, Kaufmann
S. H.
, Earnshaw
W. C.
(1995
). Studies of the lamin proteinase reveal multiple parallel biochemical pathways during apoptotic execution.
Proc. Nat. Acad. Sci. USA
92
, 9042
–906
Levi-Montalcini
R.
(1964
). Events in the developing nervous system.
Prog. Brain Res
4
, 1
–29
Li
P.
, Allen
H.
, Banerjee
S.
, Franklin
S.
, Herzog
L.
, Johnston
C.
, Mcdowell
J.
, Paskind
M.
, Rodman
L.
, Salfeld
J.
, Towne
E.
, Tracey
D.
, Wardwell
S.
, Wei
F. Y.
, Wong
W.
, Kamen
R.
, Seshadri
T.
(1995
). Mice deficient in IL-1-beta-converting enzyme are defective in production of mature IL-1-beta and resistant to endotoxic-shock.
Cell
80
, 401
–411
Loret
C.
, Sensenbrenner
M.
, Labourdette
G.
(1988
). Maturation-related gene expression of rat astroblasts in vitro studied by two-dimensional polyacrylamide gel electrophoresis.
Cell Differ Dev
25
, 37
–45
MacCarthy
N. J.
, Whyte
M. K. B.
, Gilbert
C. S.
, Evan
G.
(1997
). Inhibition of Ced-3/ICE proteases does not prevent cell death induced by oncogenes, DNA damage, or the Bcl-2 homologue Bak.
J. Cell Biol
136
, 215
–227
Manthorpe
M.
, Fagnani
R.
, Skaper
S. D.
, Varon
S.
(1986
). An automated colorimetric microassay for neuronotrophic factors.
Brain Res
390
, 191
–198
Martin
S. J.
, Green
D. R.
(1995
). Protease activation during apoptosis: death by a thousand cuts?.
Cell
82
, 349
–352
Martin
S. J.
, O'Brien
G. A.
, Nishioka
W. K.
, McGahon
A. J.
, Mahboubi
A.
, Saido
T. C.
, Green
D. R.
(1995
). Proteolysis of fodrin (non-erythroid spectrin) during apoptosis.
J. Biol. Chem
270
, 6425
–6428
Marwitt
R.
, Pilar
G.
, Weakly
J. N.
(1971
). Characterization of two ganglion cell populations in avian ciliary ganglia.
Brain Res
25
, 317
–334
Mashima
T.
, Naito
M.
, Fujita
N.
, Noguchi
K.
, Tsuruo
T.
(1995
). 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
217
, 1185
–1192
Mashima
T.
, Naito
M.
, Noguchi
K.
, Miller
D. K.
, Nicholson
D. W.
, Tsuruo
T.
(1997
). Actin cleavage by CPP-32/apopain during the development of apoptosis.
Oncogene
14
, 1007
–1012
Merril
C. R.
, Goldman
D.
, Sedman
S. A.
, Ebert
M. H.
(1981
). Ultrasensitive stain for proteins in polyacrylamide gels shows regional variation in cerebrospinal fluid proteins.
Science
211
, 1437
–1438
Milankov
K.
, De
B. U.
(1993
). Cytochemical localization of actin and myosin aggregates in interphase nuclei in situ.
Exp. Cell Res
209
, 189
–199
Milligan
C. E.
, Prevette
D.
, Yaginuma
H.
, Homma
S.
, Cardwell
C.
, Fritz
L. C.
, Tomaselli
K. J.
, Oppenheim
R. W.
, Schwartz
L. M.
(1995
). Peptide inhibitors of the ICE protease family arrest programmed cell death of motoneurons in vivo and in vitro.
Neuron
15
, 385
–393
Miura
M.
, Zhu
H.
, Rotello
R.
, Hartwieg
E. A.
, Yuan
J.
(1993
). Induction of apoptosis in fibroblasts by IL-1 beta-converting enzyme, a mammalian homolog of the C. elegans cell death gene ced-3.
Cell
75
, 653
–660
O'Farrell
P. H.
(1975
). High resolution two-dimensional electrophoresis of proteins.
J. Biol. Chem
250
, 4007
–4021
Oppenheim
R. W.
(1985
). Naturally occurring cell death during neural development.
Trends Neurosci
8
, 487
–493
Oppenheim
R. W.
(1991
). Cell death during development of the nervous system.
Annu. Rev. Neurosci
14
, 453
–501
Peitsch
M. C.
, Polzar
B.
, Stephan
H.
, Crompton
T.
, MacDonald
H. R.
, Mannherz
H. G.
, Tschopp
J.
(1993
). Characterization of the endogenous deoxyribonuclease involved in nuclear DNA degradation during apoptosis (programmed cell death).
EMBO J
12
, 371
–377
Polverino
A. J.
, Patterson
S. D.
(1997
). Selective activation of caspases during apoptotic induction in HL-60 cells.
J. Biol. Chem
272
, 7013
–7021
Polzar
B.
, Nowak
E.
, Goody
R. S.
, Mannherz
H. G.
(1989
). The complex of actin and deoxyribonuclease I as a model system to study the interactions of nucleotides, cations and cytochalasin D with monomeric actin.
Eur. J. Biochem
182
, 267
–275
Roberts-Lewis
J. M.
, Savage
M. J.
, Marcy
V. R.
, Pinsker
L. R.
, Siman
R.
(1994
). Immunolocalization of calpain I-mediated spectrin degradation to vulnerable neurons in the ischemic gerbil brain.
J. Neurosci
14
, 3934
–3944
Sadoul
R.
, Fernandez
P. A.
, Quiquerez
A. L.
, Martinou
I.
, Malki
M.
, Schroter
M.
, Becherer
J. D.
, Irmler
M.
, Tschopp
J.
, Martinou
J. C.
(1996
). Involvement of the proteasome in the programmed cell death of NGF-deprived sympathetic neurons.
EMBO J
15
, 3845
–3852
Shiokawa
D.
, Ohyama
H.
, Yamada
T.
, Tanuma
Si
(1997
). Purification and properties of DNase gamma from apoptotic rat thymocytes.
Biochem. J
326
, 675
–681
Sleath
P. R.
, Hendrickson
R. C.
, Kronheim
S. R.
, March
C. J.
, Black
R. A.
(1990
). Substrate specificity of the protease that processes human interleukin-1 beta.
J. Biol. Chem
265
, 14526
–14528
Song
Q.
, Lees-Miller
S. P.
, Kumar
S.
, Zhang
Z.
, Chan
D. W.
, Smith
G. C.
, Jackson
S. P.
, Alnemri
E. S.
, Litwack
G.
, Khanna
K. K.
, Lavin
M. F.
(1996
). DNA-dependent protein kinase catalytic subunit: a target for an ICE-like protease in apoptosis.
EMBO J
15
, 3238
–3246
Song
Q.
, Wei
T.
, Lees-Miler
S.
, Alnemri
E.
, Watters
D.
, Lavin
M. F.
(1997
). Resistance of actin to cleavage during apoptosis.
Proc. Nat. Acad. Sci. USA
94
, 157
–162
Squier
M. K.
, Miller
A. C.
, Malkinson
A. M.
, Cohen
J. J.
(1994
). Calpain activation in apoptosis.
J. Cell. Physiol
159
, 229
–237
Stabach
P. R.
, Cianci
C. D.
, Glantz
S. B.
, Zhang
Z.
, Morrow
J. S.
(1997
). Site-directed mutagenesis of alpha II spectrin at codon 1175 modulates its mu-calpain susceptibility.
Biochemistry
36
, 57
–65
Takahashi
A.
, Earnshaw
W. C.
(1996
). ICE-related proteases in apoptosis.
Curr. Opin. Genet. Dev
6
, 50
–55
Torriglia
A.
, Chaudun
E.
, Chanyfournier
F.
, Jeanny
J. C.
, Courtois
Y.
, Counis
M. F.
(1995
). Involvement of DNase II in nuclear degeneration during lens cell differentiation.
J. Biol. Chem
270
, 28579
–28585
Troy
C. M.
, Derossi
D.
, Prochiantz
A.
, Greene
L. A.
, Shelanski
M. L.
(1996
). Downregulation of Cu/Zn superoxide dismutase leads to cell death via the nitric oxide-peroxynitrite pathway.
J. Neurosci
16
, 253
–261
Troy
C. M.
, Stefanis
L.
, Prochiantz
A.
, Greene
L. A.
, Shelanski
M. L.
(1996
). The contrasting roles of ICE family proteases and interleukin-1beta in apoptosis induced by trophic factor withdrawal and by copper/zinc superoxide dismutase down-regulation.
Proc. Nat. Acad. Sci. USA
93
, 5635
–5640
Vandekerckhove
J.
, Weber
K.
(1979
). The complete amino-acid sequence of actins from bovine aorta, bovine heart, bovine fast skeletal muscle and rabbit slow skeletal muscle.
Differentiation
14
, 123
–133
Villa
P.
, Miehe
M.
, Sensenbrenner
M.
, Pettmann
B.
(1994
). Synthesis of specific proteins in trophic factor-deprived neurons undergoing apoptosis.
J. Neurochem
62
, 1468
–1475
Villa
P.
, Kaufmann
S. H.
, Earnshaw
W. C.
(1997
). Caspases and caspase inhibitors.
Trends Biochem. Sci
22
, 388
–393
Zimmer
D. B.
, Goldstein
M. A.
(1987
). DNase I interactions with filaments of skeletal muscles.
J. Muscle Res. Cell Motil
8
, 30
–38
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