The calpain 1–a-actinin interactionResting complex between the calcium-dependant protease and its target in cytoskeleton Fabrice Raynaud1, Chantal Bonnal1, Eric Fernandez2, Laure Bremaud
Trang 1The calpain 1–a-actinin interaction
Resting complex between the calcium-dependant protease and its target
in cytoskeleton
Fabrice Raynaud1, Chantal Bonnal1, Eric Fernandez2, Laure Bremaud3, Martine Cerutti4,
Marie-Christine Lebart1, Claude Roustan1, Ahmed Ouali2and Yves Benyamin1
1 UMR 5539 – CNRS, laboratoire de Motilite´ Cellulaire – EPHE, cc107, USTL, Montpellier, France; 2 Stationde 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,
sensitive and resistant to calpain cleavage, interact with the
protease when using invitro binding assays The stability
of the complexes in EGTA [Kd(–Ca2+)¼ 0.5 ± 0.1 lM]
was improved in the presence of 1 mM calcium ions
[Kd(+Ca2+)¼ 0.05 ± 0.01 lM] 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 invivo 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
Ca2+-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 invivo 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, a CaM and calpain-binding protein [12] Moreover, we have shown recently in muscle fibres [13] the presence of a stable
Ca2+-regulated complexbetween 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 Ca2+ions, a weak interaction between the
Ca2+-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 cedex5, France.
Fax: + 33 4 67144927, Tel.: + 33 4 67143813,
E-mail: benyamin@univ-montp2.fr
Abbreviations: ask, skeletal muscle a-actinin; asm, smooth muscle
a-actinin; Calp1, calpain 1 (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)
Trang 2reported 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 sixare estimated to be
functional A negatively charged loop of domain III also
offers Ca2+ binding capacity [21,22], which maximizes
binding to eight equivalents of Ca2+in agreement with a
previous invitro 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
interaction with domain III through Ca2+-regulated salt
bridges [20] Thus, according to a previous hypothesis, the
interaction between a PEST sequence, a CaM-binding
domain, or a Ca2+-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 Ca2+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 mM isopropyl
thio-b-D-galactoside for 3 h at 37C 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 mM
imidazole, pH 8, and the elution performed with the same
buffer adjusted to 250 mMimidazole 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 mM NaCl, 2 mM EGTA, 0.1% (v/v) Triton X-100, 20 mMTris/HCl, pH 7.5 buffer, supplemen-ted with antiproteases cocktail (Roche) and cleared at
23 000 g for 15 min Supernatant was incubated (4C,
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 0.6MKI, 2 mM dithiothreitol, 20 mMTris/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
20C in 1 mMCaCl2, 20 mMTris/HCl buffer, pH 7.5, to obtain the autolysed form (76/18) or during 120 min in the same buffer at 20C to conduct a more complete degra-dation Autolysis kinetics were followed by SDS/PAGE and stopped (2 mMEGTA) 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 mMCaCl2, 1 mM dithiothreitol, 50 mMKCl, 20 mMTris/HCl buffer, pH 7.5
by Calp1 during 2 h at 20C using a protease/substrate ratio of 1 : 10 (w/w) [1,25,30] Proteolysis was stopped by
2 mM EGTA, and the residual Calp1 discarded by the FPLC procedure Sepharose-insolubilized skeletal muscle a-actinin (Seph-ask) was obtained (1 mg proteinÆmL)1gel) 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)6antibody was from Qiagen Binding analysis
ELISA was performed [29] in microtitration plates (Poly-sorp, Nalgen Nunc International, Denmark) Incubation steps were carried out at 20C in 150 mM NaCl, 0.5% gelatine, 3% gelatine hydrolysate, 0.05% Tween 20, 20 m
Trang 3Tris/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 20C in
50 mM KCl, 20 mM 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 mM EGTA or 1 mM CaCl2
supple-mented with 10 lME64 to saturate all Ca2+binding sites,
including the non EF-hand ones [21,22,37] and avoid
proteolytic processes The parameters Kd(apparent
disso-ciation constant) and Amax(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
A¼ Amax ½L=ðKdþ ½LÞ
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 mMglutamax(Gibco-BRL),
100 lgÆmL)1penicillin, 100 lgÆmL)1streptomycin
(Gibco-BRL), and 20% fetal bovine serum (Gibco-BRL) Cells in
proliferation (confluence stage) were lysed in 0.1% Triton
X-100, 150 mMNaCl, 2 mMEGTA, antiprotease cocktail
(Roche), 20 mMTris/HCl buffer (lysis buffer), then
centri-fuged
Protein cosedimentation and immunoprecipitation
Cosedimentations were performed at 20C using 2.5 lg of
Calp1 (80 kDa/28 kDa) mixed with 2.5 lg of the 10 min
autolysed form (76 kDa/18 kDa), 10 lg of the 120 min
autolysed form or 10 lg of the calpain 28 kDa subunit,
incubated with 50 lg of insolubilized Seph-ask in 200 lL of
50 mMTris/HCl pH 7.4 buffer supplemented with 150 mM
NaCl, 1 mM EGTA, 0.1% NP-40, and 0.25% gelatine
After 60-min incubation, the solid phase was washed four
times with 50 mM Tris/HCl, pH 7.4, 1 mMEGTA, 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 107
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 100C 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 mM Tris/HCl,
pH 7.5, containing 50% glycerol, 5 mM 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 mMKCl, antiprotease cocktail (Roche), 30 mM 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 mM Tris/HCl pH 7.5 containing 5 mM dithiothreitol,
2 mM EGTA or in 30 mM Tris/HCl, pH 7.5, containing
5 mMdithiothreitol, 1 mMCaCl2, and 10 lME64 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
1 mMCaCl2, 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 Ca2+ions with a significant affinity (Fig 2A) Apparent dissociation constant [Kd(–Ca2+)], calculated from five experiments performed in the presence of 1 mM EGTA, corresponds to 0.5 ± 0.1 lM A similar affinity (0.3 ± 0.1 l ) was observed when ask was immobilized instead of
Trang 4Calp1 (Fig 2A, inset) An equivalent result was obtained
with asm [Kd(– Ca2+)]¼ 1.0 ± 0.4 lM), 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 mMcalcium
(and E64 as calpain inhibitor), a tenfold increase in affinity
[K ¼ 0.05 ± 0.01 lM] 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).
Trang 5inset) in comparison to the binding [Kd(–Ca2+)¼
0.5 ± 0.1 lM] obtained in the absence of calcium A similar
calcium effect [Kd(+Ca2+)¼ 0.2 ± 0.04 lM; Kd(–Ca2+)¼
1 ± 0.2 lM] 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
[Kd(+Ca2+)¼ 1.0 ±0.1 lM] in comparison with its absence
[Kd(–Ca2+)¼ 2.5 ±0.3 lM] 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 mM Ca2+the
two subunits interact with ask, the catalytic 80 kDa subunit
having a better affinity [Kd(+Ca2+)¼ 0.5 ± 0.1 lM] than
the regulatory 28 kDa chain [Kd(+Ca2+)¼ 1.4 ± 0.2 lM]
In the absence of calcium (1 mM EGTA), the 28 kDa
subunit interaction is very weak [Kd(–Ca2+)> 10 lM] in
contrast to that of the 80 kDa chain Binding between the
28 kDa subunit and ask in the presence of calcium, was
confirmed [Kd(+Ca2+)¼ 2.5 ± 0.5 lM) 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 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) and 10 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) or 1 m M EGTA (d) Glutathione S-transferase (Sigma) was used
as a negative control (j) of the interaction.
Trang 6lysate 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 mMCa2+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, Ca2+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 Ca2+ions,
which is likely considering the invitro 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 invitro and invivo 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
in the presence of 1 mMCa2+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 mM calcium, Calp1 was used at 1 lgÆmL (coated or FITC-protein) supplemented with 10 lME64 to avoid its auto-proteolysis and its aggregation [37] In fact, the affinity increase in the presence of Ca2+ions, was observed from
50 lM CaCl2, 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) and the 80 kDa (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, lane a) incubated in 1 m 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).
Trang 7induced by the saturation of low affinity Ca2+-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],
could lead to a sticky interaction with a-actinin
Neverthe-less, the fact that efficient calcium concentrations (from
50 lM) 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 Ca2+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
Ca2+-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] as ex pression 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 and an acidic Ca2+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 complexincluding 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 transfecinvestiga-tion 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.
Trang 81 Goll, D.E., Thompson, V.F., Li, H., Wei, W & Cong, J (2003)
The calpain system Physiol Rev 83, 731–801.
2 Margis, R & Margis-Pinheiro, M (2003) Phytocalpains:
ortho-logous calcium-dependent cysteine proteinases Trends Plant Sci.
8, 58–62.
3 Dourdin, N., Bhatt, A.K., Dutt, P., Greer, P.A., Arthur, J.S., Elce,
J.S & Huttenlocher, A (2001) Reduced cell migration and
dis-ruption of the actin cytoskeleton in calpain deficient embryonic
fibroblasts J Biol Chem 276, 48382–48388.
4 Hirai, S., Kawasaki, H., Yaniv, M & Suzuki, K (1991)
Degradation of transcription factors, c-Jun and c-Fos, by calpain.
FEBS Lett 287, 57–61.
5 Bhatt, A., Kaverina, I., Otey, C & Huttenlocher, A (2002)
Reg-ulation of focal complexcomposition and disassembly by the
calcium-dependent protease calpain J Cell Sci 115, 3415–3425.
6 Tullio, R.D., Passalacqua, M., Averna, M., Salamino, F., Melloni,
E & Pontremoli, S (1999) Changes in intracellular localization of
calpastatin during calpain activation Biochem J 343, 467–472.
7 Johnson, G.V & Guttmann, R.P (1997) Calpains: intact and
active? Bioessays 19, 1011–1018.
8 Rutledge, T.W & Whiteheart, S.W (2002) SNAP-23 is a target
for calpain cleavage in activated platelets J Biol Chem 277,
37009–37015.
9 Barnes, J.A & Gomes, A.V (1995) PEST sequences in
calmo-dulin-binding proteins Mol Cell Biochem 149, 17–27.
10 Barnes, J.A & Gomes, A.V (2002) Proteolytic signals in the
primary structure of annexins Mol Cell Biochem 231, 1–7.
11 Molinari, M., Anagli, J & Carafoli, E (1995) PEST sequences do
not influence substrate susceptibility to calpain proteolysis J Biol.
Chem 270, 2032–2035.
12 Shumway, S.D., Maki, M & Miyamoto, S (1999) The PEST
domain of IjBa is necessary and sufficient for invitro degradation
by l-calpain J Biol Chem 274, 30874–30881.
13 Fernandez, E., Aubry, L., Benyamin, Y & Ouali, A (2000)
Co-localization of calpain p94 and calcium ions on N1 and N2
lines of bovine muscle fibers Partial evidence for a similar
locali-zation of calpain 1 In Myologie 2000 conference proceedings,
p.160 AFM, Paris, France.
14 Tatsumi, R., Maeda K., Hattori, A & Takahashi, K (2001)
Calcium binding to an elastic portion of connectin/titin filaments.
J Muscle Res Cell Motil 22, 149–162.
15 Wallace, R.W., Tallant, E.A & McManus, M.C (1987)
Human platelet calmodulin-binding proteins: identification and
Ca 2+ -dependent proteolysis upon platelet activation
Biochemis-try 26, 2766–2773.
16 Selliah, N., Brooks, W.H & Roszman, T.L (1996) Proteolytic
cleavage of alpha-actinin by calpain in T cells stimulated with
anti-CD3 monoclonal antibody J Immunol 156, 3215–3221.
17 Molinari, M., Maki, M & Carafoli, E (1995) Purification of
l-calpain by a novel affinity chromatography approach: new
insights into the mechanism of the interaction of the protease with
targets J Biol Chem 270, 14576–14581.
18 Strobl, S., Fernandez-Catalan, C., Braun, M., Huber, R.,
Masu-moto, H., Nakagawa, K., Irie, A., Sorimachi, H., Bourenkow, G.,
Bartunik, H., Suzuki, K & Bode, W (2000) The crystal structure
of calcium-free human l-calpain suggests an electrostatic switch
mechanism for activation by calcium Proc Natl Acad Sci USA
97, 588–592.
19 Hata, S., Sorimachi, H., Nakagawa, K., Maeda, T., Abe, K &
Suzuki, K (2001) Domain II of m-calpain is a Ca 2+ -dependent
cysteine protease FEBS Lett 501, 111–114.
20 Moldoveanu, T., Hosfield, C.M., Lim, D., Elce, J.S., Jia, Z &
Davies, P.L (2002) A Ca 2+ switch aligns the active site of calpain.
Cell 108, 649–660.
21 Tompa, P., Emori, Y., Sorimachi, H., Suzuki, K & Friedrich, P (2001) Domain III of calpain is a Ca2+-regulated phospholipid-binding domain Biochem Biophys Res Commun 280, 1333– 1339.
22 Hosfield, C.M., Moldoveanu, T., Davies, P.L., Elce, J.S & Jia, Z (2001) Calpain mutants with increased Ca 2+ sensitivity and implications for the role of the C(2)-like domain J Biol Chem.
276, 7404–7407.
23 Michetti, M., Salamino, F., Minafra, R., Melloni, E & Pon-tremoli, S (1997) Calcium-binding properties of human ery-throcyte calpain Biochem J 325, 721–726.
24 Baron, M.D., Davison, M.D., Jones, P & Critchley, D.R (1987) The sequence of chick alpha-actinin reveals homologies to spectrin and calmodulin J Biol Chem 262, 17623–17629.
25 Taylor, R.G., Papa, I., Astier, C., Ventre, F., Benyamin, Y & Ouali, A (1997) Fish muscle cytoskeleton integrity is not depen-dent on intact thin filaments J Muscle Res Cell Motil 18, 285– 294.
26 Arimura, C., Suzuki, T., Yanagisawa, M., Imamura M., Hamada, Y & Masaki, T (1988) Primary structure of chicken skeletal muscle and fibroblast a-actinins deduced from cDNA sequences Eur J Biochem 177, 649–655.
27 Parr, T., Waites, G.T., Patel, B., Millake, D.B & Critchley, D.R (1992) A chick skeletal-muscle a-actinin gene gives rise to two alternatively spliced isoforms which differ in the EF-hand Ca 2+ -binding domain Eur J Biochem 210, 801–809.
28 Thompson, V.F & Goll, D.E (2000) Purification of l-calpain, m-calpain, and calpastatin from animal tissues Methods Mol Biol 144, 3–16.
29 Lebart, M.C., Mejean, C., Roustan, C & Benyamin, Y (1993) Further characterization of the a-actinin–actin interface and comparison with filamin-binding sites on actin J Biol Chem 268, 5642–5648.
30 Papa, I., Mejean, C., Lebart, M.C., Astier, C., Roustan, C., Benyamin, Y., Alvarez, C., Verrez-Bagnis, V & Fleurence, J (1995) Isolation and properties of white skeletal muscle a-actinin from sea trout (Salmo trutta) and bass (Dicentrarchus labrax) Comp Biochem Physiol 112, 271–282.
31 Masumoto, H., Yoshizawa, T., Sorimachi, H., Nishino, T., Ishi-ura, S & Suzuki, K (1998) Overexpression, purification, and characterization of human m-calpain and its active site mutant, m-C105S-calpain, using a baculovirus expression system J Bio-chem 124, 957–961.
32 Cong, J., Thompson, V.F & Goll, D.E (2002) Immunoaffinity purification of the calpains ProteinExpr Purif 25, 283– 290.
33 Cong, J., Goll, D.E., Peterson, A.M., Kapprell, H & P0 (1989) The role of autolysis in activity of the Ca 2+ -dependent proteinases (l-calpain and m-calpain) J Biol Chem 264, 10096–10103.
34 Papa, I., Astier, C., Kwiatek, O., Raynaud, F., Bonnal, C., Lebart, M.C., Roustan, C & Benyamin, Y (1999) a )Actinin-CapZ, an anchoring complexfor thin filaments in Z-line J Muscle Res Cell Motil 20, 187–197.
35 Renoult, C., Blondin, L., Fattoum, A., Ternent, D., Maciver, S.K., Raynaud, F., Benyamin, Y & Roustan, C (2001) Binding of gelsolin domain 2 to Actin: An Actin interface distinct from that of gelsolin domain 1 and from ADF/Cofilin Eur J Biochem 268, 6165–6175.
36 Benyamin, Y., Roustan, C & Boyer, M (1986) Anti-actin anti-bodies: chemical modification allows the selective production of antibodies to the N-terminal region J Immunol Methods 86, 21–29.
37 Dainese, E., Minafra, R., Sabatucci, A., Vachette, P., Melloni, E.
& Cozzani, I (2002) Conformational changes of calpain from human erythrocytes in the presence of Ca 2+ J Biol Chem 277, 40296–40301.
Trang 938 Pinset, C., Montarras, D., Chenevert, J., Minty, A., Barton, P.,
Laurent, C & Gros, F (1988) Control of myogenesis in the mouse
myogenic C2 cell line by medium composition and by insulin:
characterization of permissive and inducible C2 myoblasts
Dif-ferentiation 38, 28–34.
39 Laemmli, U.K (1970) Cleavage of structural proteins during the
assembly of the head of bacteriophage T4 Nature 227, 680–685.
40 Astier, C., Raynaud, F., Lebart, M.C., Roustan, C & Benyamin,
Y (1998) Binding of a native titin fragment to actin is regulated by
PIP2 FEBS Lett 429, 95–98.
41 Bendayan, M., Nanci, A & Kan, F.W (1987) Effect of tissue
processing on colloidal gold cytochemistry J Histochem
Cyto-chem 35, 983–996.
42 Stirling, J.W (1990) Immuno- and affinity probes for electron
microscopy: a review of labeling and preparation techniques.
J Histochem Cytochem 38, 145–157.
43 Tsuchiya, H & Seki, N (1991) Action of calpain on a-actinin
within and isolated from carp myofibrils NipponSuisanGakkaishi
57, 1133–1139.
44 Goll, D.E., Dayton, W.R., Singh, I & Robson, R.M (1991)
Studies of the a-actinin/actin interaction in the Z-disk by using
calpain J Biol Chem 266, 8501–8510.
45 Taylor, R.G., Geesink, G.H., Thompson, V.F., Koohmaraie, M.
& Goll, D.E (1995) Is Z-disk degradation responsible for
post-mortem tenderization? J Anim Sci 73, 1351–1367.
46 Sorimachi, H., Kinbara, K., Kimura, S., Takahashi, M., Ishiura,
S., Sasagawa, N., Sorimachi, N., Shimada, H., Tagawa, K &
Maruyama, K (1995) Muscle-specific calpain, p94, responsible for
limb girdle muscular dystrophy type 2A, associates with connectin
through IS2, a p94-specific sequence J Biol Chem 270, 31158–
31162.
47 Konig, N., Raynaud, F., Feane, H., Durand, M., Mestre-Frances,
N., Rossel, M., Ouali, A & Benyamin, Y (2003) Calpain 3 is
expressed in astrocytes of rat and Microcebus brain J Chem.
Neuroanat 25, 129–136.
48 Delgado, E.F., Geesink, G.H., Marchello, J.A., Goll, D.E &
Koohmaraie, M (2001) Properties of myofibril-bound calpain
activity in longissimus muscle of callipyge and normal sheep.
J Anim Sci 79, 2097–2107.
49 Rami, A (2003) Ischemic neuronal death in the rat hippocampus: the calpain-calpastatin-caspase hypothesis Neurobiol Dis 13, 75–88.
50 Sandmann, S., Prenzel, F., Shaw, L., Schauer, R & Unger, T (2002) Activity profile of calpains I and II in chronically infarcted rat myocardium – influence of the calpain inhibitor CAL 9961.
Br J Pharmacol 135, 1951–1958.
51 Papa, I., Taylor, R., Astier, C., Ventre, F., Lebart, M.C., Roustan, C., Ouali, A & Benyamin, Y (1997) Dystrophin cleavage and sarcolemme detachment are early post mortem changes on bass (Dicentrarchus labrax) white muscle J Food Sci 62, 917–921.
52 Neumar, R.W., Xu, Y.A., Gada, H., Guttmann, R.P & Siman, R (2003) Cross-talk between calpain and caspase proteolytic systems during neuronal apoptosis J Biol Chem 278, 14162–14167.
53 Kwak, K.B., Kambayashi, J., Kang, M.S., Ha, D.B & Chung, C.H (1993) Cell-penetrating inhibitors of calpain block both membrane fusion and filamin cleavage in chick embryonic myo-blasts FEBS Lett 323, 151–154.
54 Graham-Siegenthaler, K., Gauthier, S., Davies, P.L & Elce, J.S (1994) Active recombinant rat calpain II Bacterially produced large and small subunits associate both invivo and invitro J Biol Chem 269, 30457–30460.
55 Yoshizawa, T., Sorimachi, H., Tomioka, S., Ishiura, S & Suzuki,
K (1995) A catalytic subunit of calpain possesses full proteolytic activity FEBS Lett 358, 101–103.
56 Blanchard, A., Ohanian, V & Critchley, D (1989) The structure and function of a-actinin J Muscle Res Cell Motil 10, 280–289.
57 Ohtsuka, H., Yajima, H., Maruyama, K & Kimura, S (1997) Binding of the N-terminal 63 kDa portion of connectin/titin to a-actinin as revealed by the yeast two-hybrid system FEBS Lett.
401, 65–67.
58 Dewitt, S & Hallett, M.B (2002) Cytosolic free Ca2+changes and calpain activation are required for b integrin-accelerated phago-cytosis by human neutrophils J Cell Biol 159, 181–189.