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;

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-

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: [email protected]
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 http://www.dbs.univ-montp2.fr/
umr5539/, http://www.ephe.univ-montp2.fr/
(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

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-

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

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

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

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

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+)

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).

(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

(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

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

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],

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

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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