Báo cáo khoa học: The calpain 1–a-actinin interaction Resting complex between the calcium-dependant protease and its target in cytoskeleton doc

The calpain 1–a-actinin interaction
Resting complex between the calcium-dependant protease and its target
in cytoskeleton
Fabrice Raynaud
1
, Chantal Bonnal
1
, Eric Fernandez
2
, Laure Bremaud
3
, Martine Cerutti
4
,
Marie-Christine Lebart
1
, Claude Roustan
1
, Ahmed Ouali
2
and Yves Benyamin
1
1
UMR 5539 – CNRS, laboratoire de Motilite
´
Cellulaire – EPHE, cc107, USTL, Montpellier, France;
2
Station de Recherches sur la
Viande, INRA-Theix, St-Gene
`
s-Champanelle, France;

3
Institut ‘Sciences de la Vie et de la Sante
´
’, Genetique Moleculaire Animale,
UR 1061, INRA-Universite
´
de Limoges, Faculte
´
des Sciences, Limoges, France;
4
Laboratoire de Pathologie Compare
´
e,
INRA-CNRS URA5087, Saint Christol Le
`
s Ale
`
s, France
Calpain 1 behaviour toward cytoskeletal targets was inves-
tigated using two a-actinin isoforms from smooth and skel-
etal muscles. These two isoforms which are, respectively,
sensitiveand resistant to calpain cleavage, interactwith the
protease when using in vitro binding assays. The stability
of the complexes in EGTA [K
d(–Ca2+)
¼ 0.5 ± 0.1 l
M
]
was improved in the presence of 1 m
M

calcium ions
[K
d(+Ca2+)
¼ 0.05 ± 0.01 l
M
]. Location of the binding
structures shows that the C-terminal domain of a-actinin
and each calpain subunit, 28 and 80 kDa, participates in
the interaction. In particular, the autolysed calpain form
(76/18) affords a similar binding compared to the 80/28
intact enzyme, with an identified binding site in the cata-
lytic subunit, located in the C-terminal region of the chain
(domain III–IV). The in vivo colocalization of calpain 1
and a-actinin was shown to be likely in the presence of
calcium, when permeabilized muscle fibres were supple-
mented by exogenous calpain 1 and the presence of cal-
pain 1 in Z-line cores was shown by gold-labelled
antibodies. The demonstration of such a colocalization
was brought by coimmunoprecipitation experiments of
calpain 1 and a-actinin from C2.7 myogenic cells. We
propose that calpain 1 interacts in a resting state with
cytoskeletal targets, and that this binding is strengthened
in pathological conditions, such as ischaemia and dystro-
phies, associated with high calcium concentrations.
Keywords: calpain; cytoskeleton; alpha-actinin; muscle;
calcium.
Calpain 1 (Calp1) and calpain 2 (Calp2) are intracellular
Ca
2+
-dependent thiol endoproteases [1], expressed through-

out the animal kingdom, and recently reported in the plant
kingdom [2]. These two proteases are particularly implicated
[1] in the selective proteolysis of factors involved in the cell
cycle, in myocyte fusion, during apoptosis in association
with caspases or in the cleavage of membrane-cytoskeleton
complexes during cell motility phases [3]. Many of the
substrates are transcription and signalling factors with
intracellular presence of less than 2 h [4] or cytoskeletal
proteins with long half-lives, generally specialized in the
cross-link or the membrane anchorage of fibrillar
components [5]. The hypothesis according to which calpains
would be released from complexes with calpastatin (its
natural inhibitor) to join membrane phospholipids where
protease activation is achieved was proposed [6], but the
origin of recognition of specific substrates by calpains [7,8]
remains unclear.
A statistical analysis of the presence of PEST sequences
in the target, critical for calpain recognition [9,10], gives
valuable scores with short half-life proteins, but is not
appropriate in the case of several cytoskeletal actin-binding
proteins [1]. For example, filamin, dystrophin and talin are
known to be cleaved in vivo by calpains. It should be noted
that the accessibility of the calmodulin (CaM)-binding
domain in PEST sequences is an important factor to
consider [11], as demonstrated for IjBa,aCaMand
calpain-binding protein [12]. Moreover, we have shown
recently in muscle fibres [13] the presence of a stable
Ca
2+
-regulated complex between E64-treated Calp1 and

the N-terminal region of the titin located between the Z-band
and the N2-line in the I-band of myofibrils. This titin region,
rich in PEST sequences, was reported to show a marked
calcium binding ability related to acidic sequences [14]. In
the absence of Ca
2+
ions, a weak interaction between the
Ca
2+
-binding titin fragments and Calp1 was observed.
On the other hand, several other calpain substrates
deprived of PEST sequences but containing calmodulin-
binding domains [15] or EF-hand sequences [16] were
Correspondence to Y. Benyamin, UMR 5539, laboratoire de Motilite
´
Cellulaire – EPHE, Bt. 24, cc107, USTL, place E. Bataillon F-34095
Montpellier cedex 5, France.
Fax: + 33 4 67144927, Tel.: + 33 4 67143813,
E-mail:
Abbreviations: ask, skeletal muscle a-actinin; asm, smooth muscle
a-actinin;Calp1,calpain1(l-calpain); Calp2, calpain 2 (m-calpain);
CaM, calmodulin; ELISA, enzyme-linked immunosorbant assay;
FITC, fluorescein 5-isothiocyanate; Seph-ask, Sepharose-insolubilized
skeletal muscle a-actinin.
Note: web pages are available at />umr5539/, />(Received 29 July 2003, accepted 30 September 2003)
Eur. J. Biochem. 270, 4662–4670 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03859.x
reported. It was thus suggested that the calpain domains IV
and VI, which have a CaM-like structure and are members
of the penta-EF-hand family of proteins [1], could be the
binding structures of interfaces with cytoskeleton compo-

nents [17] and contribute to link the two subunits in calpains
[18]. Indeed, the two subunits of 80 kDa (domains I–IV)
and 30 kDa (domains V–VI), contain altogether 10–11
putative EF-hands motifs in domains IV, VI and II
[1,19,20], from which five to six are estimated to be
functional. A negatively charged loop of domain III also
offers Ca
2+
binding capacity [21,22], which maximizes
binding to eight equivalents of Ca
2+
in agreement with a
previous in vitro evaluation [23]. Domain III, which includes
binding sites for calpastatin and phospholipids [1,18],
appears as the regulation centre between the CaM-like
domains IV and VI and the catalytic domain II, which is in
interactionwithdomainIIIthroughCa
2+
-regulated salt
bridges [20]. Thus, according to a previous hypothesis, the
interaction between a PEST sequence, a CaM-binding
domain, or a Ca
2+
-binding motif and a CaM-like domain,
would place the catalytic site of calpain in close proximity to
the substrate.
To test this hypothesis, we have investigated Calp1
interaction with two a-actinin isoforms, either resistant or
sensitive to calpain 1 proteolysis, purified from chicken
skeletal and smooth muscles, respectively. The a-actinin

family displays two EF-hand motifs in the C-terminal
domain [24], presents low PEST scores after analysis, and
several isoforms are calpain substrates [16,25–27]. Our study
of calpain 1–a-actinin interaction suggests the importance
of calmodulin-like domains and EF-hand motifs. Finally, it
allows dissociation of two aspects in the protease behaviour
toward its target, binding and cleavage, in relation to the
presence of Ca
2+
ions.
Materials and methods
Proteins
Bovine Calp1 (80/30) was isolated [28] from the bovine
skeletal Rectus abdominus muscle, excised within 1 h post-
stunning (INRA slaughterhouse, Clermont-Fd, France).
Smooth (asm) and skeletal (ask) muscle a-actinins were
purified from chicken gizzard and breast muscles, respect-
ively, obtained immediately after killing (Avigar slaughter-
house, Gard, France). Purification procedures were
described previously [29,30].
Human (887 b) cDNA (calpain 28 kDa regulatory
subunit) was expressed as C-terminal His-tagged protein
in the pET16b vector (Roche Diagnostics). The construct
was transformed into competent BL21(DE3) Escherichia
coli. Expression was induced by adding 1 m
M
isopropyl
thio-b-
D
-galactoside for 3 h at 37 °C. Slurry Ni/NTA were

added to supernatant after bacteria lysis and gently mixed
for 30 min. Solid phase was packed in a column before
washing twice with the lysis buffer, adjusted to 20 m
M
imidazole, pH 8, and the elution performed with the same
buffer adjusted to 250 m
M
imidazole. Eluted fractions were
analysed by SDS/PAGE and Coomassie blue staining.
Human 80 kDa catalytic subunit (microcalpain) was
expressed [31] in Spodoptra frugiperda (SF9) cells using a
recombinant 80 kDa subunit baculovirus. Sf9 cell pellets
were lysed in 100 m
M
NaCl, 2 m
M
EGTA, 0.1% (v/v)
Triton X-100, 20 m
M
Tris/HCl, pH 7.5 buffer, supplemen-
ted with antiproteases cocktail (Roche) and cleared at
23 000 g for 15 min. Supernatant was incubated (4 °C,
60 min) with 800 lg of anti-Calp1 Ig (a purified low affinity
antibody subfraction [32]), then supplemented with Seph-
arose-protein A (Pharmacia, Uppsala, Sweden) and gently
mixed for 60 min. Solid phase was washed four times with
the lysis buffer, before a batch elution with 0.6
M
KI, 2 m
M

dithiothreitol, 20 m
M
Tris/HCl, pH 7.5 and dialysis against
the interaction buffer.
Proteolysis and protein modifications
Calp1 autolysis was conducted [1,20,33] during 10 min at
20 °Cin1m
M
CaCl
2
,20m
M
Tris/HCl buffer, pH 7.5, to
obtain the autolysed form (76/18) or during 120 min in the
same buffer at 20 °C to conduct a more complete degra-
dation. Autolysis kinetics were followed by SDS/PAGE and
stopped (2 m
M
EGTA) after an optimal incubation time.
Skeletal a-actinin cleavage was performed [30] with
thermolysin (1 : 25 enzyme/substrate, w/w). The 30, 55
and 10 kDa domains issued from the cleavage were purified
on a PorosHQ/H (Boehringer, Manheim) FPLC column
using the procedure previously described with fish ask. ask
and asm (1 mgÆmL
)1
) were treated in 1 m
M
CaCl
2

,1m
M
dithiothreitol, 50 m
M
KCl, 20 m
M
Tris/HCl buffer, pH 7.5
by Calp1 during 2 h at 20 °C using a protease/substrate
ratio of 1 : 10 (w/w) [1,25,30]. Proteolysis was stopped by
2m
M
EGTA, and the residual Calp1 discarded by the
FPLC procedure. Sepharose-insolubilized skeletal muscle
a-actinin (Seph-ask) was obtained (1 mg proteinÆmL
)1
gel)
with BrCN-activated Sepharose 4B (Pharmacia).
Protein labelling was performed with biotin succinimide
ester [34] or fluorescein isothiocyanate (FITC) [35]. Biotin-
amidocaproate N-hydroxysuccinimide ester, E64 calpain
inhibitor and fluorescein isothiocyanate were purchased
from Sigma Chemical Co. Thermolysin was from Serva
(Heidelberg, Germany).
Antibody specificities
The anti-Calp1 and anti-Calp1,2 Igs are directed against a
specific sequence (539–553 in domain IV) and a conserved
sequence (330–344 in the subdomain IIb) of the unprocessed
80 kDa subunit (SwissProt, ID number P07384), respect-
ively. Sequences were chosen according to their accessibility
and helicoidal content criteria before synthesis and coupling

to hemocyanin using glutaraldehyde [36]. Rabbit anti-
(a-actinin) Igs cross-reacting with ask and asm [30] were
fractionated with the 30 kDa, 55 kDa and 10 kDa Seph-
arose 4B-insolubilized fragments, issued from ask thermo-
lysin cleavage. Anti-rabbit IgG conjugated with alkaline
phosphatase was obtained from Biosys (Compiegne,
France). Monoclonal (His)
6
antibody was from Qiagen.
Binding analysis
ELISA was performed [29] in microtitration plates (Poly-
sorp, Nalgen Nunc International, Denmark). Incubation
steps were carried out at 20 °Cin150m
M
NaCl, 0.5%
gelatine, 3% gelatine hydrolysate, 0.05% Tween 20, 20 m
M
Ó FEBS 2003 Calpain 1–a-actinin interaction (Eur. J. Biochem. 270) 4663
Tris/HCl buffer, pH 7.4. Each assay monitored at 405 nm
was conducted in triplicate and the mean value was plotted
after subtraction of nonspecific absorption.
In spectrofluorescence experiments, interactions of the
fluorescein labelled Calp1 were performed at 20 °Cin
50 m
M
KCl, 20 m
M
Tris/HCl buffer, pH 7.4, using a
Perkin-Elmer luminescence spectrometer LS 50. The exci-
tation was set at 494 nm and the emission spectrum

recorded between 510 and 550 nm. Fluorescence changes
were deduced from the area of emission spectra [35].
ELISA and fluorescence binding assays were conducted
in the presence of 1 m
M
EGTA or 1 m
M
CaCl
2
supple-
mented with 10 l
M
E64 to saturate all Ca
2+
binding sites,
including the non EF-hand ones [21,22,37] and avoid
proteolytic processes. The parameters K
d
(apparent disso-
ciation constant) and A
max
(maximum effect) were obtained
(
CURVEFIT
software developed by K. Raner Software,
Mount Waverley, Victoria, Australia) by nonlinear least
squares fitting of the experimental data points to the
following equation
A ¼ A
max

ýL=ðK
d
þ½LÞ
where A is the measured effect and [L] is the ligand
concentration.
Cell culture
C2.7 myoblasts derived from the C2 mouse myogenic cell
line [38] were cultured in DMEM (Gibco-BRL/Life Tech-
nology) supplemented with 2 m
M
glutamax (Gibco-BRL),
100 lgÆmL
)1
penicillin, 100 lgÆmL
)1
streptomycin (Gibco-
BRL), and 20% fetal bovine serum (Gibco-BRL). Cells in
proliferation (confluence stage) were lysed in 0.1% Triton
X-100, 150 m
M
NaCl, 2 m
M
EGTA, antiprotease cocktail
(Roche), 20 m
M
Tris/HCl buffer (lysis buffer), then centri-
fuged.
Protein cosedimentation and immunoprecipitation
Cosedimentations were performed at 20 °Cusing2.5lgof
Calp1 (80 kDa/28 kDa) mixed with 2.5 lgofthe10min

autolysed form (76 kDa/18 kDa), 10 lgofthe120min
autolysed form or 10 lg of the calpain 28 kDa subunit,
incubated with 50 lg of insolubilized Seph-ask in 200 lLof
50 m
M
Tris/HCl pH 7.4 buffer supplemented with 150 m
M
NaCl, 1 m
M
EGTA, 0.1% NP-40, and 0.25% gelatine.
After 60-min incubation, the solid phase was washed four
times with 50 m
M
Tris/HCl, pH 7.4, 1 m
M
EGTA, 0.1%
NP-40 and resuspended in 60 lL of Laemmli loading
buffer. Thirty microlitres of the suspension were analysed by
SDS/PAGE and Western blotting.
The C2.7 line cell lysate supernatant obtained from 10
7
cells was incubated for 1 h at room temperature with 50 lg
of anti-Calp1 Ig, then with insolubilized Sepharose-protein
A (Sigma) in lysis buffer supplemented by 1% bovine serum
albumin. After extensive washing in lysis buffer, the solid
phase was treated at 100 °C in Laemmli buffer. A control
assay in the same conditions, but without anti-Calp1 Ig, was
performed.
Electrophoresis (SDS/PAGE) was made [39] using 7.5%
resolving gels and stained with Coomassie blue or silver.

Molecular mass standards were from Bio-Rad and Phar-
macia. Western blotting [40] was performed using the
appropriate antibody.
Calp1 enrichment of permeabilized muscle fibres
Glycerinated fibres were obtained as previously reported
[25]. Briefly, small fibre bundles (1 · 5 mm) taken from
freshly excised bovine longissiumus muscle, were stretched
between two pins and immersed in 30 m
M
Tris/HCl,
pH 7.5, containing 50% glycerol, 5 m
M
EDTA and anti-
proteases cocktail during 5 h, diced into small pieces
(0.8 · 0.2 mm), and maintained in the same solution for
18 h, before extensive washing in 200 m
M
KCl, antiprotease
cocktail (Roche), 30 m
M
Tris/HCl pH 7.5, to flush out
endogenous calpains and calpastatin complexes (Western
blotting controls). Samples were then incubated under
continuous mild stirring, with Calp1 (0.5 mgÆmL
)1
)in
30 m
M
Tris/HCl pH 7.5 containing 5 m
M

dithiothreitol,
2m
M
EGTA or in 30 m
M
Tris/HCl, pH 7.5, containing
5m
M
dithiothreitol, 1 m
M
CaCl
2
,and10l
M
E64 for 1 h at
room temperature. Exogenous Calp1 added to permeabi-
lized fibres was located using the postembedding procedure
[41,42] performed with a gold (10 nm)-labelled secondary
antibody (Sigma) diluted to 1 : 50.
Results
Interaction of Calp1 with skeletal and smooth muscle
a-actinins
Specificity of the antibodies directed against the specific
sequence 539–553 in subdomain IV (anti-Calp1) and the
conserved (Calp1 and Calp2) sequence 330–344 in subdo-
main II (anti-Calp1,2) was assessed (Fig. 1A) by Western
blotting using bovine skeletal muscle crude extract and the
purified Calp1. Calp1 in muscle fibre extract appears as a
unique band at the 80 kDa level (Fig. 1Ab,e). In particular,
no cross-reactivity of anti-Calp1 was detected toward the

purified Calp2 (not shown) or Calp3 (p94) in extract
(Fig. 1A,b). As expected, anti-Calp1,2 was able to detect
both Calp1 (Fig. 1A,f) and Calp2 (not shown).
Chicken a-actinins extracted from skeletal muscle (ask)
and gizzard (asm) were assayed as substrates of Calp1. As
shown in Fig. 1B, upon Calp1 treatment in the presence of
1m
M
CaCl
2
, asm is deprived of a segment of about 5 kDa
in contrast to the ask isoform which resists to proteolysis.
The asm 95 kDa truncated protein did not react with the
antibody directed against the C-terminal 10 kDa fragment
of a-actinin [25,30], indicating that the deleted segment is
located at the C-terminal extremity (not shown). Similar
calpain proteolysis was previously reported for fish a-actinin
[25,30,43] and for nonmuscle isoforms [16] in contrast to
porcine, bovine or rabbit skeletal muscles isoforms [44,45]
which resist.
Using two independent methods, interaction of a-actinins
with Calp1 was investigated. In solid phase assay (ELISA),
we observed that ask binds to coated Calp1 in the absence
of Ca
2+
ions with a significant affinity (Fig. 2A). Apparent
dissociation constant [K
d(–Ca2+)
], calculated from five
experiments performed in the presence of 1 m

M
EGTA,
corresponds to 0.5 ± 0.1 l
M
. A similar affinity (0.3 ±
0.1 l
M
) was observed when ask was immobilized instead of
4664 F. Raynaud et al. (Eur. J. Biochem. 270) Ó FEBS 2003
Calp1 (Fig. 2A, inset). An equivalent result was obtained
with asm [K
d(– Ca2+)
] ¼ 1.0 ± 0.4 l
M
), except that the
95 kDa fragment generated after Calp1 cleavage did not
bind calpain (Fig. 2B). These experiments were confirmed
using fluorescent assays in which increasing amounts of ask
were added to FITC-labelled Calp1 (Fig. 2C). When the
interaction was conducted in the presence of 1 m
M
calcium
(and E64 as calpain inhibitor), a tenfold increase in affinity
[K
d(+Ca2+)
¼ 0.05 ± 0.01 l
M
] was observed (Fig. 2C,
Fig. 1. Protein patterns. (A) Specificity of the anticalpain 1 antibodies.
Bovine skeletal muscle extract (a,b), purified bovine Calp1 (c,e), and

10-min autolysed Calp1 (d) were stained by Coomassie blue (a), by
silver (c,d) or tested by Western blotting (b,e,f) using anti-Calp1 (b,e)
and anti-Calp1,2 (f) Igs. (B) Proteolysis of smooth muscle a-actinin (a)
cleaved by Calp1 and revealed by anti-(a-actinin) after 30 min (b) and
120 min (c). The arrowhead points to the 95 kDa proteolysis product,
and the arrow indicates the position of the rabbit muscle phosphory-
lase B (97 kDa). (C), Western blotting of skeletal a-actinin (a) cleaved
by thermolysin (b) and the FPLC purified C-terminal 10 kDa frag-
ment (c) using anti-(a-actinin).
Fig. 2. a-Actinin–Calp1 interactions. (A) Solid phase immunoassay
(ELISA) between coated Calp1 (j) or coated 10 min-autolysed Calp1
(h) and increasing ask concentrations or between coated ask and
increasing Calp1 concentrations (inset). Binding was monitored at
405 nm using biotin-labelled proteins (ask or Calp1) and streptavidin–
alkaline phosphatase-labelled (1 : 2000 diluted). (B) Solid phase
immunoassay (ELISA) between coated Calp1 and increasing amounts
of intact asm (s) or the 95 kDa cleaved asm (d). The experimental
conditions were those described in (A). (C) Decrease in the fluores-
cence (DF) of FITC–Calp1 (1 lgÆmL
)1
) in interaction with increasing
concentrations of ask in the presence of 1 m
M
EGTA or 1 m
M
CaCl
2
(inset).
Ó FEBS 2003 Calpain 1–a-actinin interaction (Eur. J. Biochem. 270) 4665
inset) in comparison to the binding [K

d(–Ca2+)
¼
0.5 ± 0.1 l
M
] obtained in the absence of calcium. A similar
calcium effect [K
d(+Ca2+)
¼ 0.2 ± 0.04 l
M
; K
d(–Ca2+)
¼
1±0.2l
M
] was observed with asm (not shown).
In conclusion, ask and ask isoforms are able to bind
Calp1 in the absence and in the presence of calcium, with an
important increase in the affinity in the presence of calcium.
Furthermore, the cleavage of asm by calpain 1 generates a
fragment of 95 kDa which is unable to interact with Calp1.
This indicates the presence of a calpain binding site in the
C-terminal domain of smooth muscle a-actinin.
Involvement of the C-terminal domain of ask
in Calp1 binding
In order to locate the region responsible for the binding of
Calp1 on ask isoform, which is not sensitive to calpain
cleavage, we tested the three fragments issued from the
proteolysis of ask by thermolysin (Fig. 1Ca,b): the 30
(N-terminal actin binding domain), the 60 (spectrin-like
repeats, central domain), and the 10 kDa (C-terminal

EF-hand domain). In solid phase assay, we observed
(Fig. 3A) that the purified 10 kDa fragment (Fig. 1Cc)
was the only one to bind Calp1 with a detectable affinity.
We confirmed this result using fluorescent assays (Fig. 3B)
and found a higher affinity in the presence of calcium
[K
d(+Ca2+)
¼ 1.0 ±0.1 l
M
] in comparison with its absence
[K
d(–Ca2+)
¼ 2.5 ±0.3 l
M
]. Thus, a Calp1 binding site is
included in the C-terminal domain of the ask molecule,
and this binding is independent of the susceptibility of the
a-actinin isoform to calpain proteolysis.
Identification of the calpain 1 subunit implicated
in a-actinin interaction
The regulatory (28 kDa) and the catalytic (80 kDa) sub-
units, expressed as recombinant proteins, were assayed for
binding activity toward skeletal muscle a-actinin. We
observed (Fig. 4) that in the presence of 1 m
M
Ca
2+
the
two subunits interact with ask, the catalytic 80 kDa subunit
having a better affinity [K

d(+Ca2+)
¼ 0.5 ± 0.1 l
M
]than
the regulatory 28 kDa chain [K
d(+Ca2+)
¼ 1.4 ± 0.2 l
M
].
In the absence of calcium (1 m
M
EGTA), the 28 kDa
subunit interaction is very weak [K
d(–Ca2+)
>10l
M
]in
contrast to that of the 80 kDa chain. Binding between the
28 kDa subunit and ask in the presence of calcium, was
confirmed [K
d(+Ca2+)
¼ 2.5 ± 0.5 l
M
) using fluorescence
assays (not shown) and cosedimentation experiments using
Seph-ask (Fig. 5A).
Similarly, we have shown that the intact (80 kDa/
28 kDa) calpain 1 (Fig. 1A) and the 10-min autolysed
(76 kDa/18 kDa) form (Fig. 1Ad) have the same binding
ability toward ask in the absence of calcium (Figs 2A and

5B, 10 min) as in its presence (not shown). Furthermore, the
76, 50 and 30 kDa fragments issued from the 120 min
autolysis of Calp1, and recognized by anti-Calp1 (Fig. 5Ba,
120 min), cosedimented with ask (Fig. 5Bb, 120 min). It
can be observed (Fig. 5Bb,c, 120 min) that only the 76 kDa
fragment is recognized by anti-Calp1 (domain IV) and anti-
Calp1,2 (domain II), which locates the 50 kDa and the
30 kDa fragments in the C-terminal region (domains
III–IV) of the catalytic subunit.
Thus, the 28 kDa subunit (probably its C-terminal
18 kDa region) in a calcium-dependent fashion, and the
C-terminal part (domains III–IV) of the 80 kDa subunit,
are implicated in the interface linking calpain 1 to skeletal
muscle a-actinin.
Colocalization of microcalpain and a-actinin
in myogenic cells
Calpains and a-actinin were previously located in Z-disks
[44], and adhesion structures [5], without evidence of strong
molecular proximity. To confirm that the a-actinin–Calp1
interaction was physiologically relevant, we first performed
coimmunoprecipitation studies. As shown in Fig. 5C,
a-actinin was coprecipitated with calpain 1 from a cell
Fig. 3. Interactions of ask domains with Calp1. (A) Solid phase
immunoassay (ELISA) between coated 30 (u), 60 (j)and10 kDa(s)
ask fragments and increasing amounts of biotin-labelled Calp1.
Binding was determined at 405 nm using streptavidin–alkaline
phosphatase labelling (1 : 2000). (B) Fluorescence decrease (DF) of
FITC-labelled Calp1 (1 lgÆmL
)1
) in interaction with increasing

concentrations of the 10 kDa fragment in the presence of 1 m
M
CaCl
2
(s)or1m
M
EGTA (d). Glutathione S-transferase (Sigma) was used
as a negative control (j)oftheinteraction.
4666 F. Raynaud et al. (Eur. J. Biochem. 270) Ó FEBS 2003
lysate issued from C2.7 myoblasts, by using rabbit
anti-Calp1 Ig and insolubilized protein A. Furthermore,
after incubation of permeabilized muscle fibres with
exogenous Calp1 in the presence of 1 m
M
Ca
2+
ions, we
observed that calpain 1 was strikingly concentrated in the
Z-disk core (Fig. 6) in comparison with the A- or M-bands.
When calcium was omitted, we were unable to detect a
preferential location of exogenous Calp1 in myofibrils.
Thus, Ca
2+
ions seem to favour the targeting of Calp1 to
Z-line and the interaction of the protease with the compo-
nents of this anchorage structure. However, in the case of
C2.7 cells, coimmunoprecipitation of a-actinin by the anti-
Calp1 Ig was also observed in the absence of Ca
2+
ions,

which is likely considering the in vitro binding analysis.
Discussion
We have investigated the hypothesis, according to which
calpain 1, as calpain 3 (p94) with titin and glial filaments
[46,47], could bind directly to targets in cytoskeleton
through specific and stable interactions. This hypothesis
involves questions related to the origin of interactions
[9,10,12,27] with the targets, the stability of complexes in the
resting stage [3,48] and the activation state [7,33] of the
binding calpain. The topology of the interface with respect
to the catalytic domain II [1] and the cleavage site in target
are also underlying.
Two muscle a-actinin isoforms (ask and asm) with
different calpain cleavage susceptibilities were chosen as
targets and their binding with calpain 1 analysed by
independent in vitro and in vivo approaches. According to
the presented results, Calp1–a-actinin interaction is inde-
pendent of the cleavage susceptibility of the target and
occurred in the absence of calcium, but is improved in its
presence. In the absence of calcium, the apparent dissociation
constant of Calp1–ask (or asm) complexes is measured in the
micromolar order and decreased to the submicromolar level
inthepresenceof1 m
M
Ca
2+
and E64. The autolysed Calp1
(76/18) form and the intact enzyme (80/28) afforded the
same binding ability toward a-actinin.
In the experiments performed in the presence of 1 m

M
calcium, Calp1 was used at 1 lgÆmL (coated or FITC-
protein) supplemented with 10 l
M
E64 to avoid its auto-
proteolysis and its aggregation [37]. In fact, the affinity
increase in the presence of Ca
2+
ions, was observed from
50 l
M
CaCl
2
, and the effect increased with increasing
calcium concentration. Thus, the conformation changes
Fig. 4. Calp1 subunits binding assays. Solid phase immunoassay
(ELISA) between the 30 (s,d)andthe80kDa(h,j)-coated Calp1
subunits and biotin-labelled ask in the presence of 1 m
M
CaCl
2
(open
symbols) or 1 m
M
EGTA (filled symbols). Binding was determined at
405 nm using streptavidin–alkaline phosphatase labelling (1 : 2000).
Fig. 5. Cosedimentation of calpain–a-actinin complexes. (A) Cosedi-
mentation with Seph-ask of His-tagged 30 kDa subunit (a), in the
presence of 1 m
M

CaCl
2
(b) or 1 m
M
EGTA (c). Suspensions were
centrifuged at 2000 g and the pellet revealed after SDS/PAGE by
Western blotting using anti-His
6
Ig (1 : 1000 diluted). (B), cosedi-
mentation of the 10 min autolysed Calp1 supplemented by the intact
Calp1 (left part, lane a) or the 120 min autolysed Calp1 (right part,
lanea)incubatedin1m
M
EGTA with Seph-ask (see Materials and
methods). Pellets (lanes b) are revealed after SDS/PAGE with Coo-
massie blue (left part) or by Western blotting using anticalpain anti-
bodies (right part, lanes b and c). A negative control (c) using inert
Sepharose was included (left part). Anti-Calp1 (lane a,b) and anti-
Calp1,2 (lane c) were used (right part). (C) Coimmunoprecipitation of
Calp1–ask complexes from C2.7 lysate by anti-Calp1 and Sepharose-
protein A. The presence of ask in the pellet was searched in the assay
(a) and in the control performed without the anti-Calp1 (b), after SDS/
PAGE and Western blotting, using anti-(a-actinin).
Ó FEBS 2003 Calpain 1–a-actinin interaction (Eur. J. Biochem. 270) 4667
induced by the saturation of low affinity Ca
2+
-binding sites,
changes mainly localized at level of the 28 kDa subunit [37],
and the exposure of hydrophobic patches on the surface of
the protease which aggregates calpain 1 molecules [37],

couldleadtoastickyinteractionwitha-actinin. Neverthe-
less, the fact that efficient calcium concentrations (from
50 l
M
) are higher than the physiological ones, may be
considered in view of several pathological situations such as
brain and muscle ischemia [49,50], or several muscle
dystrophies [1], but also during necrosis [51], apoptosis
[52], or myoblast fusion [53] where Ca
2+
ions concentration
increases in unknown proportions. In these cases, the
accumulation of calpain 1 on cytoskeleton could explain
the rapid intervention of the protease and the quick
Ca
2+
-dependent degradation of several cytoskeletal
proteins [25,51].
Binding interface between Calp 1 and ask was further
located in this study. Calpain binding structures were found
within the 10 kDa C-terminal domain of the a-actinin
molecule. The inability of the 95 kDa chain (issued from the
cleavage of asm by Calp1) to interact with calpain 1, could
restrict the location of the binding elements to the last
5 kDa, although we cannot rule out the possibility of
conformational changes induced to the 95 kDa by the
cleavage. We can thus conclude that calpain 1 displays two
distinct behaviours, one consisting of interaction with its
target and the other being responsible for the proper
cleavage action of the target.

The attempt to locate the binding structures on Calp1
implied disposal of the two 28 and 80 kDa isolated subunits
in the correct conformation, which was effective by using
E. coli [54] and SF9-Bacculo virus[55]asexpression
systems, respectively. We have concluded that both subunits
display binding abilities, although the regulatory subunit
(28 kDa) is strongly controlled by calcium which binds to
the CaM-like domain VI. Concerning the catalytic subunit
(80 kDa), the restriction was brought by cosedimentation
assays to the 50 kDa C-terminal part, bearing domains III
and IV. Thus, the ability of the two isolated subunits or the
autoproteolysed Calp1 products (18/76 and 55 kDa) to
interact with ask indicates that the domains III–IV and
VI participate to the interface with the C-terminal region of
a-actinin. These domains concentrate 10 EF-hand motifs
andanacidicCa
2+
binding sequence [1,20,21].
It is noteworthy that the location of a Calp1 binding site in
the C-terminal region of a-actinin [56] situates the protease in
the vicinity of titin [57] and CapZ [34], two proteins described
as a-actinin partners in the Z-line and known as calpain
substrates [1,25,34]. In this context, according to our
experimentation of enrichment of permeabilized fibres by
exogenous Calp1 on Z-line, one could hypothesize that the
two myofibrillar proteins could also bind Calp1, as a-actinin
does. Note that these three proteins strongly participate in
the Z-disk organization, a compartment rapidly proteolysed
during muscle ischemia [25,45] or after a calpain treatment of
isolated myofibrils [25]. Targeting of Z-disk by calpains was

previously suggested [25,44,48] and a quick ask release from
muscles treated by calpains in the presence of calcium was
observed. Furthermore, a-actinin is also located in cellular
adhesion structures [5], in a colocation with integrin, talin
and vinculin [56,58]. Immunoprecipitation of a-actinin from
C2.7 lysate by anti-Calp1 proves the association of the
protease with a-actinin, either in direct contact or in a
complex including the two proteins.
In conclusion, our study proves the interaction between
a-actinin and calpain 1 and locates binding motifs within
regions where the EF-hand domains of the protease and the
cytoskeletal protein are concentrated. The behaviour of
calpain 1 toward cytoskeletal targets appears dual. In its
first state, the protease would oscillate between cytoskeleton
components, calpastatin and phospholipids in membrane
according to the local calcium concentrations. This would
eventually lead to the cleavage of close substrates in
cytoskeleton. This equilibrium is currently under investiga-
tion by using C2.7 cell line transfection assays with calpain 1
CaM-like subdomains.
Acknowledgements
This work was supported by grants from the Association Franc¸ aise
contre les Myopathies (AFM) and PPF network (EPHE). Authors are
grateful to Professor H. Sorimachi for the calpain 1 constructs gift.
Fig. 6. Muscle fibres treatment by inactivated Calp1. Permeabilized
muscle fibres were enriched with exogenous Calp1 in the presence of
either 1 m
M
Ca
2+

ions (A) or 2 m
M
EGTA (B) during 1 h, stained
with anti-Calp1, then with a gold-labelled secondary anti-rabbit IgG.
Z, Z-band; M, M-band; A, A-band.
4668 F. Raynaud et al. (Eur. J. Biochem. 270) Ó FEBS 2003
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