Lamina-associated polypeptide 2 alpha (LAP2 alpha) is a non-membrane-bound isoform of the LAP2 family involved in nuclear structure organization. Using various cell systems, including Jurkat, HL-60, and HeLa cells, and different death-inducing agents, such as anti-Fas antibody, topoisomerase inhibitors, and staurosporine, we found that LAP2 alpha was cleaved during apoptosis as rapidly as lamin B in a caspase-dependent manner yielding stable N- and C-terminal fragments of approximately 50 and 28 kDa, respectively. Based on fragment size and localization of immunoreactive epitopes, four potential cleavage sites were mapped between amino acids 403–485. These sites were located within a domain that has previously been described to be essential and sufficient for association of LAP2 alpha with chromosomes, suggesting that LAP2 alpha cleavage impairs its chromatin-binding properties. Immunofluorescence microscopy demonstrated that, unlike full length protein, apoptotic fragments did not colocalize with condensed chromatin, but remained in the nuclear compartment as long as a single nucleus was visible. Subfractionation analyses showed that the N-terminal LAP2 alpha fragment was extracted from intranuclear structures in detergent/salt buffers, whereas the C-terminal fragment remained associated with a residual framework devoid of chromatin. Our data suggest that early cleavage of LAP2 alpha) is important for chromatin reorganization during apoptosis.

REFERENCES

Alsheimer
M.
,
Fecher
E.
,
Benavente
R.
(
1998
).
Nuclear envelope remodelling during rat spermiogenesis: distribution and expression pattern of LAP2/thymopoietins.
J. Cell Sci
111
,
2227
2234
Buendia
B.
,
Santa-Maria
A.
,
Courvalin
J. C.
(
1999
).
Caspase-dependent proteolysis of integral and peripheral proteins of nuclear membranes and nuclear pore complex proteins during apoptosis.
J. Cell Sci
112
,
1743
1753
Cohen
G. M.
(
1997
).
Caspases: the executioners of apoptosis.
Biochem. J
326
,
1
16
Cross
T.
,
Griffiths
G.
,
Deacon
E.
,
Sallis
R.
,
Gough
M.
,
Watters
D.
,
Lord
J. M.
(
2000
).
PKC-delta is an apoptotic lamin kinase.
Oncogene
19
,
2331
2337
Dechat
T.
,
Gotzmann
J.
,
Stockinger
A.
,
Harris
C. A.
,
Talle
M. A.
,
Siekierka
J. J.
,
Foisner
R.
(
1998
).
Detergent-salt resistance of LAP2alpha in interphase nuclei and phosphorylation-dependent association with chromosomes early in nuclear assembly implies functions in nuclear structure dynamics.
EMBO J
17
,
4887
4902
Dechat
T.
,
Korbei
B.
,
Vaughan
O. A.
,
Vlcek
S.
,
Hutchison
C. J.
,
Foisner
R.
(
2000
).
Lamina-associated polypeptide 2binds intranuclear A-type lamins.
J. Cell Sci
113
,
3473
3484
Dechat
T.
,
Vlcek
S.
,
Foisner
R.
(
2000
).
Review: Lamina-associated polypeptide 2 isoforms and related proteins in cell cycle-dependent nuclear structure dynamics.
J. Struct. Biol
129
,
335
345
Duband-Goulet
I.
,
Courvalin
J. C.
,
Buendia
B.
(
1998
).
LBR, a chromatin and lamin binding protein from the inner nuclear membrane, is proteolyzed at late stages of apoptosis.
J. Cell Sci
111
,
1441
1451
Foisner
R.
(
1997
).
Dynamic organisation of intermediate filaments and associated proteins during the cell cycle.
BioEssays
19
,
297
305
Foisner
R.
,
Gerace
L.
(
1993
).
Integral membrane proteins of the nuclear envelope interact with lamins and chromosomes, and binding is modulated by mitotic phosphorylation.
Cell
73
,
1267
1279
Furukawa
K.
,
Pante
N.
,
Aebi
U.
,
Gerace
L.
(
1995
).
Cloning of a cDNA for lamina-associated polypeptide 2 (LAP2) and identification of regions that specify targeting to the nuclear envelope.
EMBO J
14
,
1626
1636
Furukawa
K.
,
Glass
C.
,
Kondo
T.
(
1997
).
Characterization of thechromatin binding activity of lamina-associated polypeptide (LAP) 2.
Biochem. Biophys. Res. Commun
238
,
240
246
Furukawa
K.
,
Fritze
C. E.
,
Gerace
L.
(
1998
).
The major nuclear envelope targeting domain of LAP2 coincides with its lamin binding region but is distinct from its chromatin interaction domain.
J. Biol. Chem
273
,
4213
4219
Furukawa
K.
(
1999
).
LAP2 binding protein 1 (L2BP1/BAF) is a candidate mediator of LAP2-chromatin interaction.
J. Cell Sci
112
,
2485
2492
Gant
T. M.
,
Harris
C. A.
,
Wilson
K. L.
(
1999
).
Roles of LAP2 proteins in nuclear assembly and DNA replication: truncated LAP2beta proteins alter lamina assembly, envelope formation, nuclear size, and DNA replication efficiency in Xenopus laevis extracts.
J. Cell Biol
144
,
1083
1096
Gant
T. M.
,
Wilson
K. L.
(
1997
).
Nuclear assembly. Annu. Rev. Cell.
Dev. Biol
13
,
669
695
Gerace
L.
,
Burke
B.
(
1988
).
Functional organization of the nuclear envelope.
Annu. Rev. Cell Biol
4
,
335
374
Gerner
C.
,
Holzmann
K.
,
Grimm
R.
,
Sauermann
G.
(
1998
).
Similarity between nuclear matrix proteins of various cells revealed by an improved isolation method.
J. Cell. Biochem
71
,
363
374
Gotzmann
J.
,
Foisner
R.
(
1999
).
Lamins and lamin-binding proteins in functional chromatin organization.
Crit. Rev. Eukaryot. Gene Expr
9
,
257
265
Gruenbaum
Y.
,
Wilson
K. L.
,
Harel
A.
,
Goldberg
M.
,
Cohen
M.
(
2000
).
Review: Nuclear lamins—structural proteins with fundamental functions.
J. Struct. Biol
129
,
313
323
Kipp
M.
,
Schwab
B. L.
,
Przybylski
M.
,
Nicotera
P.
,
Fackelmayer
F. O.
(
2000
).
Apoptotic cleavage of scaffold attachment factor A (SAF-A) by caspase-3 occurs at a noncanonical cleavage site.
J. Biol. Chem
275
,
5031
5036
Laemmli
U. K.
(
1970
).
Cleavage of structural proteins during the assembly of the head of bacteriophage T4.
Nature
227
,
680
685
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
9046
Leist
M.
,
Single
B.
,
Castoldi
A. F.
,
Kuhnle
S.
,
Nicotera
P.
(
1997
).
Intracellular adenosine triphosphate (ATP) concentration: a switch in the decision between apoptosis and necrosis.
J. Exp. Med
185
,
1481
1486
Lin
F.
,
Blake
D. L.
,
Callebaut
I.
,
Skerjanc
I. S.
,
Holmer
L.
,
McBurney
M. W.
,
Paulin-Levasseur
M.
,
Worman
H. J.
(
2000
).
MAN1, an inner nuclear membrane protein that shares the LEM domain with lamina-associated polypeptide 2 and emerin.
J. Biol. Chem
275
,
4840
4847
Loewinger
L.
,
McKeon
F.
(
1988
).
Mutations in the nuclear lamin proteins resulting in their aberrant assembly in the cytoplasm.
EMBO J
7
,
2301
2309
Mandal
M.
,
Maggirwar
S. B.
,
Sharma
N.
,
Kaufmann
S. H.
,
Sun
S. C.
,
Kumar
R.
(
1996
).
Bcl-2 prevents CD95 (Fas/APO-1)-induced degradation of lamin B and poly(ADP-ribose) polymerase and restores the NF-kappaB signaling pathway.
J. Biol. Chem
271
,
30354
30359
Manilal
S.
,
thi Man
N.
,
Sewry
C. A.
,
Morris
G. E.
(
1996
).
The Emery-Dreifuss muscular dystrophy protein, emerin, is a nuclear membrane protein.
Hum. Mol. Genet
5
,
801
808
Morris
G. E.
,
Manilal
S.
(
1999
).
Heart to heart: from nuclear proteins to Emery-Dreifuss muscular dystrophy.
Hum. Mol. Genet
8
,
1847
1851
Neamati
N.
,
Fernandez
A.
,
Wright
S.
,
Kiefer
J.
,
McConkey
D. J.
(
1995
).
Degradation of lamin B1 precedes oligonucleosomal DNA fragmentation in apoptotic thymocytes and isolated thymocyte nuclei.
J. Immunol
154
,
3788
3795
Oberhammer
F. A.
,
Hochegger
K.
,
Froschl
G.
,
Tiefenbacher
R.
,
Pavelka
M.
(
1994
).
Chromatin condensation during apoptosis is accompanied by degradation of lamin A+B, without enhanced activation of cdc2 kinase.
J. Cell Biol
126
,
827
837
Pyrpasopoulou
A.
,
Meier
J.
,
Maison
C.
,
Simos
G.
,
Georgatos
S. D.
(
1996
).
The lamin B receptor (LBR) provides essential chromatin docking sites at the nuclear envelope.
EMBO J
15
,
7108
7119
Rao
L.
,
Perez
D.
,
White
E.
(
1996
).
Lamin proteolysis facilitates nuclear events during apoptosis.
J. Cell Biol
135
,
1441
1455
Ricci
J. E.
,
Maulon
L.
,
Luciano
F.
,
Guerin
S.
,
Livolsi
A.
,
Mari
B.
,
Breittmayer
J. P.
,
Peyron
J. F.
,
Auberger
P.
(
1999
).
Cleavage and relocation of the tyrosine kinase P59FYN during Fas-mediated apoptosis in T lymphocytes.
Oncogene
18
,
3963
3969
Samejima
K.
,
Svingen
P. A.
,
Basi
G. S.
,
Kottke
T.
,
Mesner
P. W.
Jr.
,
Stewart
L.
,
Durrieu
F.
,
Poirier
G. G.
,
Alnemri
E. S.
,
Champoux
J. J.
, et al. 
. (
1999
).
Caspase-mediated cleavage of DNA topoisomerase I at unconventional sites during apoptosis.
J. Biol. Chem
274
,
4335
4340
Slee
E. A.
,
Harte
M. T.
,
Kluck
R. M.
,
Wolf
B. B.
,
Casiano
C. A.
,
Newmeyer
D. D.
,
Wang
H. G.
,
Reed
J. C.
,
Nicholson
D. W.
,
Alnemri
E. S.
, et al. 
. (
1999
).
Ordering the cytochrome c-initiated caspase cascade: hierarchical activation of caspases-2,3, 6,7, 8, and10 in a caspase-9-dependent manner.
J. Cell Biol
144
,
281
292
Takahashi
A.
,
Musy
P. Y.
,
Martins
L. M.
,
Poirier
G. G.
,
Moyer
R. W.
,
Earnshaw
W. C.
(
1996
).
CrmA/SPI-2 inhibition of an endogenous ICE-related protease responsible for lamin A cleavage and apoptotic nuclear fragmentation.
J. Biol. Chem
271
,
32487
32490
Talanian
R. V.
,
Quinlan
C.
,
Trautz
S.
,
Hackett
M. C.
,
Mankovich
J. A.
,
Banach
D.
,
Ghayur
T.
,
Brady
K. D.
,
Wong
W. W.
(
1997
).
Substrate specificities of caspase family proteases.
J. Biol. Chem
272
,
9677
9682
Thompson
C. B.
(
1995
).
Apoptosis in the pathogenesis and treatment of disease.
Science
267
,
1456
1462
Vlcek
S.
,
Just
H.
,
Dechat
T.
,
Foisner
R.
(
1999
).
Functional diversity of LAP2alpha and LAP2beta in postmitotic chromosome association is caused by an alpha-specific nuclear targeting domain.
EMBO J
18
,
6370
6384
Walker
P. R.
,
Leblanc
J.
,
Smith
B.
,
Pandey
S.
,
Sikorska
M.
(
1999
).
Detection of DNA fragmentation and endonucleases in apoptosis.
Methods
17
,
329
338
Weaver
V. M.
,
Carson
C. E.
,
Walker
P. R.
,
Chaly
N.
,
Lach
B.
,
Raymond
Y.
,
Brown
D. L.
,
Sikorska
M.
(
1996
).
Degradation of nuclear matrix and DNA cleavage in apoptotic thymocytes.
J. Cell Sci
109
,
45
56
Worman
H. J.
,
Evans
C. D.
,
Blobel
G.
(
1990
).
The lamin B receptor of the nuclear envelope inner membrane: a polytopic protein with eight potential transmembrane domains.
J. Cell Biol
111
,
1535
1542
Wyllie
A. H.
,
Kerr
J. F.
,
Currie
A. R.
(
1980
).
Cell death: the significance of apoptosis.
Int. Rev. Cytol
68
,
251
306
Yang
L.
,
Guan
T.
,
Gerace
L.
(
1997
).
Lamin-binding fragment of LAP2 inhibits increase in nuclear volume during the cell cycle and progression into S phase.
J. Cell Biol
139
,
1077
1087
Ye
Q.
,
Worman
H. J.
(
1994
).
Primary structure analysis and lamin B and DNA binding of human LBR, an integral protein of the nuclear envelope inner membrane.
J. Biol. Chem
269
,
11306
11311
Ye
Q.
,
Worman
H. J.
(
1996
).
Interaction between an integral protein of the nuclear envelope inner membrane and human chromodomain proteins homologous to Drosophila HP1.
J. Biol. Chem
271
,
14653
14656
Ye
Q.
,
Callebaut
I.
,
Pezhman
A.
,
Courvalin
J. C.
,
Worman
H. J.
(
1997
).
Domain-specific interactions of human HP1-type chromodomain proteins and inner nuclear membrane protein LBR.
J. Biol. Chem
272
,
14983
14989
Zhou
Q.
,
Snipas
S.
,
Orth
K.
,
Muzio
M.
,
Dixit
V. M.
,
Salvesen
G. S.
(
1997
).
Target protease specificity of the viral serpin CrmA. Analysis of five caspases.
J. Biol. Chem
272
,
7797
7800
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