MULTIPLE MOLECULAR INTERACTIONS IMPLICATE CONNECTIN/TITIN N2A REGION
AS A MODULATING SCAFFOLD FOR P94/CALPAIN 3 ACTIVITY IN SKELETAL MUSCLE*
Chikako Hayashi§‡1, Yasuko Ono§1,2, Naoko Doi§¶, Fujiko Kitamura§, Mai Tagami§, Reiko Mineki**,║Takao Arai‡, Hayao Taguchi‡, Mitsuaki Yanagida†, Stephanie Hirner , Dietmar Labeit║, Siegfried
Labeit║, and Hiroyuki Sorimachi§¶2
From the §Department of Enzymatic Regulation for Cell Functions (Calpain Project), The Tokyo Metropolitan
Institute of Medical Science (Rinshoken), Tokyo, Japan, ‡Department of Applied Biological Science, Facultyof Science and Technology, Tokyo University of Science, Chiba, Japan, ¶CREST, Japan Science and Technology (JST), Saitama, Japan, **Biomedical Research Center, Juntendo University Graduate School of Medicine,║
Institute für Anästhesiologie und Operative Intensivmedizin, Universitätsklinikum Mannheim, 68167 Mannheim, Germany, †Institute for Environmental and Gender Specific Medicine, Graduate School of Medicine,
Juntendo University, Chiba, Japan
Running head: Calpain/connectin/MARP N2A-complex modulated by proteolysis
Address correspondence to: Yasuko Ono or Hiroyuki Sorimachi, Department of Enzymatic Regulation for Cell
Functions (Calpain Project), The Tokyo Metropolitan Institute of Medical Science (Rinshoken), 3-18-22 Honkomagome, Bunkyo-ku, Tokyo 113-8613, Japan. Fax: 81-3-3823-2359; E-mail: yakoono@rinshoken.or.jp orsorimach@rinshoken.or.jp

Intriguingly, a connectin N2A fragment withthe mdm deletion possessed enhanced resistanceto proteases including p94, and its interaction
with MARPs was weakened. Our data support
a model that MARP2–p94 signaling converges
within the N2A connectin segment, and that the
mdm deletion disrupts their coordination. These
results also implicate the dynamic nature ofconnectin molecule as a regulatory scaffold of
p94 functions.
Calpain (EC 3.4.22.18, clan CA, family C2) isa family of Ca2+-requiring and papain-like proteases comprising the products of 15 different genes
in humans (1-3). Calpain is considered a modulator protease, as it modulates functions of substrates.
More than 10 calpain molecules are expressed inskeletal muscle, including p94/calpain 3, a skeletal
muscle-specific isoform, as well as the conventional μ- and m-calpains, whose catalytic subunits
are called calpain 1 and 2, respectively. The functions of calpain in skeletal muscle have been investigated regarding pathogenic conditions such as
atrophy and muscular dystrophy (4-6), and the molecular mechanisms involved in myogenesis (7-9).
The physiological relevance of p94 to skeletal
muscle integrity is an urgently pursued issue. Defective p94 protease activity by gene mutations
causes a muscular dystrophy categorized as cal-

p94/calpain 3 is a skeletal muscle-specificCa2+-regulated cysteine protease (calpain), andgenetic loss of p94 protease activity causes muscular dystrophy (calpainopathy). In addition, asmall in-frame deletion in the N2A region ofconnectin/titin that impairs p94/connectin interaction causes a severe muscular dystrophy(mdm) in mice. Since p94 via its interaction with
the N2A and M-line regions of connectin becomes part of the connectin filament systemthat serves as a molecular scaffold for the myofibril, it has been proposed that structural andfunctional integrity of the p94-connectin complex is essential for myocytes health and maintenance.
In this study, we have surveyed the interactions made by p94 and connectin N2A inside
COS7 cells. This revealed that p94 binds toconnectin at multiple sites including newly identified loci in the N2A and PEVK regions of connectin. Functionally, p94–N2A interactions suppress p94 autolysis and protected connectinfrom proteolysis. The connectin N2A region
also contains a binding site for the muscle ankyrin repeat proteins (MARPs), a protein family involved in the cellular stress responses.
MARP2/Ankrd2 competed with p94 for binding
to connectin and was also proteolyzed by p94.

1

MARP2 (Ankrd2 or Arpp) expressed primarily inskeletal muscle, and MARP3 (DARP) expressed inboth muscle tissues. MARPs become strongly
upregulated in myocytes under a variety of acutestresses; cardiac injury and hypertrophy (MARP1)(31-33), stretch or denervation of skeletal muscle(MARP2) (34,35), and metabolic challenge(MARP3) (36). MARP1 and 2 are upregulated inskeletal muscle in chronic pathogenic conditions
such as Duchenne muscular dystrophy (37-39).
MARPs are localized both in the nucleus and thesarcomere N2A region, and MARP1 and 2 alsointeract with nuclear factors involved in transcriptional regulation (40-42). Therefore, MARPs arethought to link nuclear transcriptional activity andsarcomere functions.
Another example of the physiological significance of the connectin N2A region is that the homozygosity of the in-frame deletion in the connectin N2A region, Ttnmdm/mdm, causes muscular
dystrophy, mdm, in mice (43-45). The mdm deletion overlaps partially with the C-terminus of the
p94 binding region and compromises the interaction between connectin I81-I83 (Fig. 1A) and p94(23,44). Intriguingly, in mdm skeletal muscle,
MARP1 and 2 are upregulated significantly, respectively, and their specific targeting to the sarcomeric I-band region were observed in microscopic analysis of myofibrils from Ttnmdm/mdm mice
(44).
Most previous studies focused on single molecule such as calpain, MARP, or connectin. Here,
we have focused on the simultaneous relationships
among these three molecules. We examined theeffect of the mdm deletion on properties of connectin fragments containing binding sites for both
MARPs and p94. As a result, the sensitivity ofN2A connectin to proteases including p94 wasshown to be suppressed by the mdm deletion. Identification of multiple binding sites for p94 in N2A
connectin, and N2A connectin and MARP2 as
substrates for p94 protease activity provided a new
aspect of the relationships among these molecules.
We propose that p94-connectin interaction regulates and facilitates proteolytic modulation by p94
on the signaling cascade through N2A connectinand MARP2.

painopathy (10). The calpainopathy-type muscular
dystrophy contrasts with other muscular dystrophies with regard to the mode of calpain involvement. Unlike the conventional calpains that are
hyperactivated as a consequence of advanced dystrophic phenotype (i.e., an aberrant increase in intracellular [Ca2+]), and, in turn, aggravate thesymptoms (11), the dystrophic phenotypes of calpainopathy is caused by loss of p94 protease activity from skeletal muscle (12). Phenotypes of transgenic mice in which the p94 protease activity is
manipulated in various ways showed that the regulated proteolytic action of p94 on target proteins is
critical for the maintenance of skeletal muscle
functions (13-16). Thus, identification of p94 substrates is a key to clarify underlying mechanisms.
One of the interesting properties of p94 is itsvery rapid and exhaustive autolysis in protein expression systems examined so far, whereas native
p94 protein can be detected at significant quantities in skeletal muscle where it constitutes a complex with other myofibril components, especiallyconnectin/titin (17-21). Therefore, connectin is acandidate that regulates p94 stability and activity.
Two distinct regions in connectin, the N2A and Cterminus regions, have been identified as p94binding sites by yeast two-hybrid (YTH)3 screening (19,22). These interactions are thought to impede autolytic disassembly of p94, which has been
demonstrated for the N2A fragment in a proteasetrapping assay, a phenomenon we call “p94trapping” (23,24). Interestingly, in primary cultures of skeletal muscle cells, p94 translocatesfrom the M-line to the N2A region as myofibrillogenesis proceeds or as the sarcomere lengthens(25). Together, these observations suggest that p94
and connectin function as a complex in skeletal
muscle cells.
The importance of connectin as a scaffold for
multiple molecular interactions for both the structural and force-generating elements has been recognized (26-29). In fact, the N2A region of connectin appears to be a major site involved in signaltransduction in striated muscle tissues. N-terminal
to the binding site for p94 in the N2A region ofconnectin is a specific insertion sequence, “is”,
which binds to muscle ankyrin repeat proteins(MARPs) (30) (Fig. 1A). There are three MARP
paralogues: MARP1 expressed primarily in cardiac
muscle (also referred to as CARP or Ankrd1),

Experimental Procedures
Mouse experiments– All procedures used for2

HCl, pH 7.5, 150 mM NaCl or CsCl, 1 mM
EDTA-Na or EDTA-K, pH 8.0, and 1% TritonX100) containing protease inhibitors [1 mM
phenylmethylsulfonyl fluoride (PMSF), 0.1 mM
leupeptin, and 1.5 μM aprotinin]. For cells expressing p94, 10 mM iodoacetamide was also included. The cell lysate was centrifuged at
20,630×g at 4°C for 15 min, and the supernatant
was incubated with ANTI-FLAG M2 affinity gel(Sigma) according to the manufacturer’s instructions. For detecting the p94–connectin interaction
and MARP–connectin interaction, the incubation
was carried out for 2 h and 4 h, respectively. Immunoprecipitates were collected by centrifugation
and were rinsed twice with lysis buffer and thentwice with wash buffer (50 mM Tris-HCl, pH 7.5,
150 mM NaCl or CsCl) followed by an incubation
with 3×FLAG peptide (150 ng/μl in wash buffer)for 30 min at 4°C. The supernatant was subjected
to SDS-PAGE and western blot analysis.
Western blot analysis– Proteins were separated
by SDS-PAGE and transferred onto PVDF membranes (Millipore, Billerica, MA). Membranes
were probed with appropriate primary antibodies
and horseradish peroxidase-coupled secondary
antibodies (Nichirei, Tokyo, Japan) followed byvisualization using a POD immunostaining kit(Wako, Osaka, Japan) or ECLTM Western Blotting
Detection Reagent (GE, Buckinghamshire, UK).
Antibodies– Antibodies used in this study include anti-FLAG monoclonal antibody (clone M2,
Stratagene), anti-HA monoclonal antibody (clone
6E2, Cell Signaling Technology, Danvers, MA),
anti-MYC monoclonal antibody (clone 4A6, Invitrogen), anti-slow myosin heavy chain (sMHC)
monoclonal antibody (clone NOQ7.5.4D, Sigma),and anti-developmental MHC (dMHC) monoclonal antibody (NCL-MHCd, Novocastra, Newcastle upon Tyne, UK). Rabbit polyclonal antiMARP1 and MARP2 and goat anti-pIS2 antibodies were described before (23,30). An affinitypurified rabbit anti-TPALKK was generated using
the keyhole limpet hemocyanin-conjugated peptide
TPALKK-C (aa 8558–8563 in mouse connectin,
NP_035782), which corresponds to the N-terminal
region of human connectin N2A proteolyzed by μcalpain. Before use, the antibody was incubated on
ice with peptide RAMLKKTPALKK (aa 8552–
8563 in NP_035782) to absorb the population of
IgG that also recognizes the uncleaved sequence;experimental animals were approved by the Experimental Animal Care and Use Committee of theTokyo Metropolitan Institute of Medical Science.
cDNA constructs– The cDNAs for human andmouse p94/calpain 3 were subcloned into expression vectors pSRD and pAS2-1 or pAS2-1c forprotein expression in mammalian and yeast cells,
respectively, as described previously (12,46). The
yeast expression vectors for the full length and IS2
region of rat p94 were described previously (19).
The mouse cDNAs encoding various regions of
N2A connectin/titin and full-length MARP1, 2,
and 3, were amplified by PCR from mouse skeletal
muscle cDNA using Pfu DNA polymerase (Stratagene, La Jolla, CA). Connectin fragments wereexpressed as the N-terminally FLAG-tagged proteins using the expression vector pSRD (Table I).
MARP1 with an N-terminal hemagglutinin (HA)or MYC epitope was expressed using pSRD.
MARP2 and 3 were expressed using pcDNA3.1 as
the N-terminally HA-tagged proteins (47). An expression vector pACT2 (U29899, Clontech, Mountain View, CA) was used to express cloned connectin cDNAs in a YTH assay. p94:D607A wasconstructed by introducing an Asp607ÆAla mutation into the human p94 cDNA and inserting this
into the XbaI–SacI sites of human p94 cDNA inpFastBac1 (Invitrogen, Carlsbad, CA). This mutant p94 was characterized using “p94-trapping”(23) and used for the biochemical analyses of p94
as described previously (24). cDNA for the FLAGtagged I80-PEVK connectin fragment was subcloned into pFastBac-HTa. Enzymes used for manipulating recombinant DNA were purchased fromTakara Bio (Shiga, Japan) or New England Biolabs (Ipswich, MA). Mutations described here
were introduced by long PCR using Pfu-Turbo
DNA polymerase as described previously (12).
Every nucleotide of all the constructs was verified
by DNA sequencing.
Protein expression in COS7 cells and coimmunoprecipitation assay– COS7 cells were grownin Dulbecco’s modified Eagle’s medium (Sigma,
St. Louis, MO) supplemented with 10% fetal bovine serum that had been heat inactivated before
use at 56°C for 30 min. Electroporation was performed using Gene Pulser (Bio Rad, Hercules,
CA) according to the manufacturer’s instructions.
Cells were harvested 72 h after electroporation andlysed by sonication in lysis buffer (50 mM Tris-3

the final concentration of peptide was 25 mg/ml.
Preparation of an extract from mouse skeletaland cardiac muscle– The skeletal muscle (hamstring) and cardiac muscle tissues dissected frommice were frozen in liquid nitrogen-cooled isopentane. Cryosections from each muscle tissue, 20 μm
thick per section, were lysed in homogenizing buffer [50 mM Tris-HCl, pH 7.5, 5 mM EDTA-K, pH
8.0, and 1 mM dithiothreitol (DTT)] containing
protease inhibitors as follows: 1 mM PMSF, 0.1
mM pepstatin A, and 50 μM calpastatin peptide 42(amino acids 140–181 of human calpastatin,
NP_001035911). The homogenate (total; T) was
fractionated into the soluble fraction (supernatant;S) and insoluble fraction (pellet; P) by centrifugation at 20,630×g for 30 min at 4°C. Equal amountof protein, 5 μg for each sample, was separated by
SDS-PAGE and subjected to western blot analysis.
Histology and immunohistochemistry– The posterior compartments (gastrocnemius (GC) and soleus (Sol)) of the hind limb skeletal muscles weredissected from 10-week-old Ttn+/+ (WT) and
Ttnmdm/mdm mice and frozen in liquid nitrogencooled isopentane. Serial transverse cryosections(7 μm thick) were stained with hematoxylin andeosin followed by the examination using a lightmicroscope (BX 60, Olympus, Tokyo, Japan).
Immunofluorescence on cryosections was performed as previously described (25,30,44) using
antibodies specific for MARP1, MARP2, sMHCand dMHC in combination with appropriate secondary antibodies conjugated with either Alexa
Fluor 488 or 555 (Invitrogen). The nuclei werelabeled with DAPI contained in a mounting medium (VECTORSHIELD Mounting Medium with
DAPI, Vector laboratories, Burlingame, CA). Sections were analyzed on a laser scanning confocal
microscope (LSM510, Carl Zeiss Japan, Tokyo,
Japan) and the images were processed using Photoshop CS2 (Adobe Systems, San Jose, CA).
Expression of recombinant proteins in Sf-9
cells– Recombinant baculovirus was generated
according to the protocol provided by Invitrogen,and the recombinant proteins were expressed as
described previously (48). Briefly, Spodoptera
frugiperda (Sf-9) cells were suspended in infection
medium containing 1/10 vol. of each baculovirus
stock solution at a concentration of 1.0 × 107
cells/ml, left for 1 h with gentle agitation every 15
min, diluted to a concentration of 1.0 × 106

cells/ml, and shaken at 140 rpm at 27°C for 44 to
48 h. Preparation of the cell lysate and immunoprecipitation were performed as described above
for analyzing proteins expressed in COS7 cells.
Bacterial expression and purification of recombinant proteins– cDNA fragments corresponding to N2A region of human connectin, I80–I83 (nt15307–16851 in X90569), and human MARP2 (nt
294–1289 in NM_020349) were amplified by PCRfrom human skeletal muscle cDNA and cloned
into the pET vector. Proteins were expressed in
Escherichia coli BL21(DE3) and purified as described previously (49).
Proteolytic assay for calpains– One μg of recombinant human MARP2 was incubated with
0.25 μg of recombinant human μ-calpain prepared
as described previously (48) in 20 μl of incubationbuffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA-K,
pH 8.0, 1 mM DTT, 1 mM PMSF, 100 μM pepstatin A) with or without 15 mM CaCl2 at 30°C for
60 and 90 min. To measure p94 autolysis and proteolysis of connectin fragment activity, the lysateof Sf-9 cells expressing p94 and/or the connectinfragment was incubated with 5 mM CaCl2 at 37°C.
The protein concentration of cell lysates was adjusted to 0.5 μg/μl. For the proteolytic assay of
MARP2, recombinant His-MARP2 was added to
the lysate of cells expressing p94 at a concentration of 20 ng/μl. The reaction was stopped by theaddition of SDS sample buffer, and the sample wassubjected to SDS-PAGE followed by silver staining or western blot analysis. Where indicated, protease inhibitors were added at following concentrations: 1 mM PMSF, 1 mM leupeptin, 0.1 mME64c, and 7 μM calpastatin domain I (Takara Bio).
Peptide sequencing– After SDS-PAGE, theproteins were blotted onto Pro-Blot membrane(Applied Biosystems, Foster City, CA) and thenvisualized by Coomassie brilliant blue G-250
staining. The target protein bands were excised
and washed three times with an excess amount of
50% (v/v) methanol and then with absolute methanol. The N-terminal sequence was determined using a 491cLC protein sequencer (Applied Biosystems) according to the manufacturer’s instructions.
YTH assay– Saccharomyces cerevisiae strain
AH109 was transformed with a series of combination of expression vectors for various regions ofconnectin and p94 WT and mutants using the
FastTM-Yeast Transformation Kit (G-Biosciences/4

On the other hand, I82-PEVK, in which PpBS
remains intact, did not coimmunoprecipitate either
p94:WT or p94:CS regardless that the FLAG-tag is
N- (Fig. 1B, lane 10, or 1C, lane 5, anti-pIS2) or
C-terminal (data not shown). This suggests that theconnectin local structure affects its ability binding
to p94.
p94 interacts with PEVK-connectin through theproximity of the IS2 region. Because the interaction between PEVK-N and p94:CS was detectablein the YTH assay, we examined further the binding
region for SpBS2 in p94 and compared this withthat for PpBS (Fig. 1D). The interaction was complicated but suggested that the SpBS2 binding region overlaps with that for PpBS in the N-terminalin the proximity of the IS2 region, with a slight
extension toward the C-terminus (Fig. 1D, p94:1–
600 and p94:1–594). Interestingly, the sequence
encoded by exons 15 and 16 of p94 is not necessary for its interaction with SpBS2 if the Cterminal region of p94 is intact (Fig. 1D,
p94:ex15–16–). These data can be explained byassuming that SpBS2 binds to an intact structure in
the proximity of the p94 IS2 region encoded byexon 14, which requires several extra amino acids
at its N- and C-termini.
The N2A-PEVK junction of connectin has a sitethat is susceptible for proteolysis. In the course of
the above experiments, some connectin fragments
were detected as both full-length and a breakdown
products even when these were coexpressed with
p94:CS (Fig. 1C, lanes 2 and 3, anti-FLAG, closed
arrowheads; Table I). In contrast, corresponding
fragments with the mdm-deletion, i.e., I80PEVK(mdm) and I82-PEVK(mdm) lacking 83 aar,appeared to be full-length without significant
breakdown products (Fig. 1B, lane 3, anti-FLAG;
Fig. S1B, lane 2). The same trend was observed
when I80-PEVK and I80-PEVK(mdm) were expressed in Sf-9 cells (see Fig. 2C, lanes 1 and 19,
anti-FLAG).
The proteolytic site in I82-PEVK was determined to be the N-terminus of Ser8934 in the mdm
deletion region (Fig. 1A, arrow [1]; Fig. S1B). Expression of various fragments encompassing
Ser8934 with or without mdm deletion in COS7
cells suggested that fragments without mdm deletion are commonly proteolyzed at the same site
(Fig. S1C). These results indicate that the N2APEVK junction is susceptible to unidentified pro-

Genotech, St. Louis, MO), according to the manufacturer’s instructions. Cotransformants were selected on plates with SD medium that lacked Leu
and Trp (SD-LW) and the expression of reporter
genes by growth on plates that also lacked His andAde (SD-LWHA) were measured according to the
manufacturer’s instructions (Clontech).
RESULTS
I80-PEVK connectin/titin preferentially coimmunoprecipitates with full-length p94 rather thanautolyzed fragments. We surveyed for potential
p94-connectin/titin interactions by a coimmunoprecipitation thus extending our previous YTH
studies (19,22,44) by a complementary approach.
p94:WT expressed in COS7 cells autolyzes
very rapidly at IS1 and only a 55 kDa fragment isdominantly detectable (Fig. 1B, lane 1, anti-pIS2,
closed arrowhead; Fig. 1D, arrow [1]) (20). However, when I80-PEVK, I80-PEVK(mdm), or I81I83 (previously called mCN48 (23)) (Fig. 1A) was
expressed with p94:WT, the full-length p94, as
well as the autolyzed fragment, was detected incoimmunoprecipitates (Fig. 1B, lane 8, 9, or 12,
open arrowhead). This suggests that the full-length
p94 binds to the connectin fragment more efficiently than the autolyzed 55 kDa fragment does.
In apparent contrast to our previous conclusion(23), the mdm deletion in a connectin fragment
I80-PEVK did not abolish the interaction with p94(also confirmed when using protease inactive mutant p94:C129S(CS); data not shown). However,subsequent analysis indicated that the I80-PEVK
fragment contains in addition to the previously
identified p94 binding site in I82-I83 (see Fig. 1A,
PpBS: primary p94 binding site) at least one additional binding site.
Identification of novel p94 binding sites in the
N2A-PEVK region of connectin. Coimmunoprecipitation studies using p94:CS located additionalbinding sites for p94 within connectin’s I80-is and
PEVK-N regions, respectively (Fig. 1C, lane 12
and 13), which were designated as secondary p94
binding site 1 and 2 (SpBS1 and 2), as shown in
Fig. 1A. Since interaction between p94:WT and
I80-I81 was not detectable (Fig. 1B, lane 11),
autolyzed fragments appear to be insufficient formediating interaction with SpBS1 only. It is also
possible that SpBS1 only can interact with p94
very weakly and cannot prevent p94 from autolysis.

5tease(s) in COS7 and Sf-9 cells in the context offragment structures used in this study, and that the
mdm deletion confers resistance to this proteolytic
attack.
I80-PEVK connectin is proteolyzed by μcalpain in the “is” region. Proteolysis of connectin by calpains including p94 was reported under several different experimental conditions(16,50). Thus, we co-incubated purified recombinant proteins of I80–I83 and μ-calpain (Fig. 2A),
because p94:WT is not available as a purified protein. Two proteolytic sites in the “is” region, flanking the MARP-binding region of N2A connectin,were revealed (Fig. 2B, arrows (b)/[2] and (c)/[3]).
A specific antibody for the N-terminus of theproteolyzed fragment (b) (Fig. 2B), anti-TPALKK,
was generated using the corresponding mouseconnectin sequence. This antibody reacted withfragment (b), but not with (a), (c), or (d) (data notshown, see Experimental Procedures), and wasused for further analysis of connectin proteolysis.
Connectin is proteolyzed by p94 and an unknown endogenous protease at the same site as μcalpain. The detected amount of I80-PEVK decreased in the presence of coexpressed p94:WT,
but not p94:CS, both in Sf-9 (data not shown) and,to a lesser extent, COS7 cells (see Fig. 5A, lane 9
vs 10, Input, anti-FLAG), suggesting that I80PEVK is a substrate for p94. Therefore, we further
examined proteolytic processes of I80-PEVK using Sf-9 and p94:D607A(DA) missense mutant(24).
Incubation of the lysate of Sf-9 cells expressing either p94:DA or p94:CS demonstrated that
p94:DA has moderate Ca2+-dependent activity,
which was inhibited by leupeptin+E64c but not by
calpastatin, representing a unique property of p94(Fig. S2A) (24). Moreover, as in the case of
p94:WT, the full-length p94:DA was predominantly coimmunoprecipitated with the connectinfragment (Fig. S2B, lane 3, open arrowhead).
Surprisingly, when the lysate of Sf-9 cells expressing I80-PEVK alone was incubated with Ca2+,
I80-PEVK was proteolyzed generating the 43 kDa
breakdown product detectable by anti-TPALKK(Fig. 2C, lanes 1–6). This indicates that an unknown endogenous Sf-9 protease proteolyzes I80PEVK at the same site as μ-calpain. The proteolysis was inhibited by leupeptin+E64c+calpastatin(Fig. 2C, lane 6, anti-FLAG), but not by calpas-

tatin alone (data not shown). Considering thatsome calpain species are insensitive to calpastatin(51), it is possible that endogenous calpain homologue(s) in Sf-9 cells proteolyze I80-PEVK.
When p94:DA was coexpressed, proteolysis of
I80-PEVK was accelerated (Fig. 2C, lanes 7–12,
anti-FLAG). The peak of the 43 kDa band amountshifted to 40 min from 60 min of that without
p94:DA (lane 5 vs 10, anti-TPALKK). These datasuggested the involvement of p94:DA in the proteolysis of I80-PEVK directly and/or indirectly;
p94:DA proteolyzes I80-PEVK in the same manner as μ-calpain and/or activates unknown Sf-9
protease(s). In contrast, coexpression of p94:CS
did not accelerate, but slightly slowed, the proteolysis of I80-PEVK (Fig. 2C, lanes 13-18). It is,thus, inferred that interaction with p94:CS protects
I80-PEVK from the endogenous protease(s), andaccordingly, it is likely that p94:DA binds to I80PEVK and directly proteolyses it when activatedby Ca2+.
Differential proteolytic susceptibility of I80PEVK and I80-PEVK(mdm). Judging from patterns of breakdown products, proteolysis of I80PEVK (Figs. 2B & C, (e)) probably occurs first at
the N-terminus of Ser8934 generating the 62 kDa
fragment (Fig. 2B, arrow [1] and fragment (f)),
followed by that of Ser8558 ([2] and (g)), and thenthat of Gln8646/Thr8647 ([3] and (h), ca. 10 kDa too small to detect).
On the other hand, I80-PEVK(mdm) was notproteolyzed as intensively as I80-PEVK was (Fig.
2C, anti-FLAG, lanes 19–23). The presence of coexpressed p94:DA did not significantly enhance
the proteolysis, but autolysis of p94:DA with I80PEVK(mdm) proceeded more slowly than with
I80-PEVK (Fig. 2C, lanes 25–29 vs 7–11). AntiTPALKK antibody faintly detected a 67 kDa bandprobably corresponding to the fragment (i) (Fig.
2B), generation of which was slightly enhanced
and suppressed by coexpression with p94:DA andp94:CS, respectively (Fig. 2C, (i)).
Our results suggest that the “is” region of I80PEVK becomes further sensitized to proteases including p94 after a rate-limiting proteolytic step
occurred in the junction of I83-PEVK. This model
would explain why I80-PEVK(mdm) is resistant toproteolysis. Additionally, our data suggested that
p94:CS protects connectin from proteolysis at sites
in the “is” region as well as in the junction of I83-

6

their cellular distribution in a manner consistent
with our in vitro results, i.e., connectin-binding
activity is MARP1 >> MARP2. It should be notedthat these molecular properties are essentially thesame both in WT and Ttnmdm/mdm (Table II), whichraises a question as to a relationship between
MARP1 and MARP2 under mdm condition.
Characteristics of MARP1 and 2 in the modeof induction in Ttnmdm/mdm skeletal muscle. To characterize the expression of MARP1 and 2 in relation to the integrity and the fiber type in Ttnmdm/mdm
skeletal muscle, expression of MARP1, 2, dMHC,and sMHC were simultaneously analyzed in situ.
As previously reported, dMHC-positive fibers
were absent in WT (data not shown), whereas degeneration/regeneration of muscle fibers is apparent in 10-wk-old Ttnmdm/mdm skeletal muscle (Figs.
4B & D, dMHC), and abnormal morphology suchas clusters of small-sized regenerating fibers and alarge percentage of fibers with irregular outlines
and central nuclei was apparent (Fig. 4C, HE) (45).
In WT, essentially no MARP1-positive fiber was
detectable (data not shown), and MARP2 was detectable only at low frequency and signal intensity(Fig. 4A), consistent with the above western blot
data (Fig. 3D). Some MARP2-positive fibers werealso positive for sMHC (Fig. 4A, *).
In Ttnmdm/mdm muscle, MARP1-positive fibers
were sparsely identified, and significantly more
fibers were positive for MARP2, sMHC and/or
dMHC in mdm than in WT (Figs. 4B–E, Table III).
MARP1 was not detectable in dMHC-positive fibers (Fig. 4B), suggesting that induction of
MARP1 is not significant in regenerating fibers.
There was a trend that MARP1-positive fibers
were also MARP2-positive and had central nuclei(Fig. 4C, arrow). Expression of MARP2 seemed
independent from fiber-types specified by MHC(Figs. 4C & D). Fibers with relatively intense
MARP2 signal tended to have central nuclei regardless of their fiber sizes (Fig. 4C, arrowhead).
Although frequency was low, MARP1 and 2 weredetected in or at the periphery of central nuclei in
Ttnmdm/mdm (Fig. 4E). Because of low frequency,
correlation between nuclear localization of
MARP1/2 and the fiber type or size was not clear.
These observations demonstrate that MARP1
and 2 are not mutually exclusive as to their
upregulation in Ttnmdm/mdm muscle. The presence ofmyofibers in which only MARP2 increased (Fig.

PEVK.
Effect of connectin structure on the MARP–
connectin interaction. We next examined the
MARP-binding activity of the connectin N2A
fragment as another important property of connectin that could be affected by the mdm deletion.
First, each MARP protein had a different I80PEVK binding activity: MARP1 ≈ MARP3>
MARP2 (Fig. 3A, lanes 2–4, IP, anti-HA). Theinteraction between connectin and MARP2, a predominant paralogue in skeletal muscle, was further
weakened by deleting the region I82-PEVK (Fig.
3B, IP, lane 4 vs 5), or by introducing the mdm
deletion (Fig. 3C, IP, anti-HA, lane 3 vs 7). The
same trend was observed for the MARP1 (data notshown, and Fig. 3C, IP, anti-MYC, lane 2 vs 5).
Coexpressing MARP1 with MARP2 and I80PEVK(mdm), an expected condition in Ttnmdm/mdm
skeletal muscle, decreased the amount of MARP2(Fig. 3C, IP, anti-HA, lane 6 vs 7), but not MARP1(anti-MYC, lane 6 vs 5), coimmunoprecipitatedwith the connectin fragment.
These results indicate that the region Cterminal to “is” (including those connectin sequences deleted in mdm) affects MARP-connectininteraction, possibly by modulating the structure of“is” surrounded by immunoglobulin domains. Additionally, MARP1 interferes with MARP2 inbinding to connectin, both in WT and in mdm.
Distinct subcellular distribution of MARP1
and 2 in striated muscle. To examine whether thein vitro MARP-connectin interaction observed
above corresponds to in vivo situation, expressionof MARP1 and 2 in muscles from WT and
Ttnmdm/mdm mice (N=3 for each) were compared(Fig. 3D, WT-1–3 and mdm-1–3, respectively).
In both WT and Ttnmdm/mdm, MARP1 was enriched in cardiac muscle in the insoluble myofibrillar bound fraction, consistent with its strong binding to connectin. In skeletal muscle, MARP1 wasobserved in insoluble fraction only in Ttnmdm/mdm,
where MARP1 is upregulated as previously reported (44). In contrast, MARP2 was detected predominantly in the soluble fraction of skeletal muscle, and undetectable in cardiac muscle, suggesting
very weak, if not zero, interaction of MARP2 withmyofibrils. In Ttnmdm/mdm skeletal muscle, where it
was robustly upregulated, MARP2 was detectedslightly in the insoluble fractions as well.
These data show that MARP1 and 2 differ in7

MARP2 is proteolyzed by μ-calpain at its Nterminal region. The proteolytic site in MARP2
cleaved by μ-calpain corresponded to Arg77 (Fig.
6A, closed arrow, lanes 7 and 8, Fig. 6B). Previously, two connectin-binding sites in MARP2 were
determined: 24–42 and 188–205 (Fig. 6B, bidirectional arrows) (30). MARP2Δ1–79, however, interacted with I80-PEVK as efficiently as the fulllength MARP2 (Fig. 6C, lanes 2 and 3, IP, antiFLAG), while the N-terminal MARP2 fragment,
MARP2:1–76–EGFP, did not show detectableconnectin-binding (data not shown). This suggeststhat the second ankyrin motif is sufficient for
MARP2-connectin interaction under the conditions
used, and that properties of MARP2 other thanconnectin-binding are modified by proteolysis.

4C, arrowhead) implies different time points ofaction for MARP1 and 2 during regeneration ofmuscle, and could explain a small population of
MARP2 recovered in the insoluble fraction (Fig.
3D).
Relationships among p94, MARP2 and connectin. Because both p94 and MARP2 show predominant expression in skeletal muscle and interaction with N2A connectin, the relationship between them may be deteriorated by the mdm deletion.
Less p94:CS and MARP2 coimmunoprecipitated with I80-PEVK when both were coexpressed(Fig. 5A, IP, anti-pIS2, lane 8 vs 10; anti-HA, lane
8 vs 6), indicating that p94 and MARP2 affects
their interaction to connectin with each other. Further less MARP2 coimmunoprecipitated with connectin when p94:WT was used (Fig. 5A, IP, antiHA, lanes 6–8). Considering that the amounts ofimmunoprecipitated I80-PEVK were similar regardless of coexpression of p94:WT or CS (Fig.
5A, IP, anti-FLAG, lanes 6–8), and that p94:WT
protein existed much less than p94:CS, the observed decrease in coprecipitated MARP2 probably results from the proteolysis of MARP2 by
p94:WT during their interactions with connectin.
Thus, proteolysis of MARP2 by p94 and the effect of connectin on it were further examined. First,
purified recombinant MARP2 was proteolyzed
Ca2+-dependently in the Sf-9 lysate expressing
p94:DA, which was inhibited by leupeptin+E64c,
but not by calpastatin (Fig. 5B, lanes 11–15, antiMARP2). The proteolysis was not observed in thepresence of p94:CS or absence of p94 (Fig. S2C).
Therefore, in addition to I80-PEVK, our studies
also identified MARP2 as a substrate for p94, atleast in vitro.
When the Sf-9 lysate coexpressing p94:DA
with I80-PEVK was used, MARP2 was proteolyzed faster than without I80-PEVK (Fig. 5B,
lanes 1–3 vs 11–13, anti-MARP2). Since the initialamount of p94:DA decreased when coexpressedwith I80-PEVK (Fig. 5B, anti-pIS2, lane 1 vs 11),it was predicted that I80-PEVK, as a scaffold, expedites proteolysis of MARP2 by p94:DA. Results
obtained with I80-PEVK(mdm) or I80-PEVK did
not differ (Fig. 5B, lanes 6–8), indicating that I80PEVK(mdm), although resistant to proteolysis itself, does not perturb proteolysis of MARP2 by
p94.

DISCUSSION
In this study, we characterized the relationships
among p94, MARPs, and N2A connectin in the
WT and mdm contexts. We found that (1) novel
binding sites for p94 exist in the N-terminal(SpBS1) and C-terminal (SpBS2) regions adjacentto the previously identified site (PpBS); (2) preautolytic full-length p94 preferentially interacts
with N2A connectin; (3) proteases including p94
and the conventional calpains proteolyze the N2A
region of connectin at several sites (Fig. 7A,
closed arrowheads), which is compromised in the
fragments with the mdm deletion (Fig. 7B, open
arrowheads); (4) the efficiency of binding between
the connectin “is” region and MARPs is also affected by the mdm deletion.
Considering the huge molecular size of connectin, characteristics of subfragment might provide only limited functional insights into wholeconnectin molecules in vivo. However, our in vitro
results demonstrate for the N2A region of connectin a propensity to undergo dynamic changes
imposed intramolecularly by the local molecular
structures. Identification of N2A connectin and
MARP2 as possible p94 substrates implicates that
the N2A region of connectin serves as a versatilescaffold for p94, which stabilizes p94 and facilitates proteolysis of MARP2 by p94 to modify its
functions (Fig. 7A).
Localization of p94 activity and targets. Identification of sarcomeric proteins such as α-actinin
and connectin as p94 binding partners emphasizes
the importance of these interactions to the regula-

8

Structure of connectin fragments affecting its
susceptibility to proteolysis. The physiologicalrelevance of connectin proteolysis previously reported (53) remains unknown to date. In cardiac
myocytes, doxorubicin treatment causes degradation of connectin by m-calpain to produce a proteolyzed fragment designated T2 (54). The major
proteolytic sites of connectin were suggested to bein the elastic I-band region, where N2A and its
alternative splicing isoform, N2B, are located, andthe adjacent PEVK region (55). μ-calpain is also
reported to interact with connectin and generate
proteolyzed fragments encompassing N1 (the Cterminal to Z-line) and N2A regions (56).
Proteolysis by p94 of connectin fragments corresponding to the Z-line, PEVK, and M-line regions has also been reported (16,50). In this study,
p94 was shown to target the same proteolytic sitein N2A connectin as μ-calpain. Because this proteolytic site resides in “is”, the binding site for
MARP2 and for p94 itself, our results provide a
decisive basis for further investigation on the effect of p94 on the MARP2–connectin interaction.
We have shown that the mdm deletion caused
protection from proteolysis in the region of the
mdm deletion and, rather unexpectedly, within “is”.
These results indicate that proteolysis of N2A connectin proceeds step-by-step changing its structure
accordingly, i.e., proteolytic sites within “is” region are exposed only after the proteolysis between I83 and PEVK. One should, thus, consider
that structures of connectin fragments used in experiments may affect the results as to identificationof proteolytic sites.
The protease(s) responsible for spontaneous
proteolysis of N2A connectin in COS7 cells has(have) not been identified; no inhibitory effect wasevident for several different protease inhibitors,
including leupeptin, ALLNal, E-64d, NH4Cl, AcYVAD-CMK, or, Z-D-CH2-DCB (data not shown).
Relationships between MARPs and connectinwith or without the mdm deletion. Genetic studies
using mice lacking MARPs suggested that MARP
paralogues have functional redundancy as both
structural components and signaling molecules(57). Our study, however, showed that MARPs aredifferent in their connectin-binding activity, and,accordingly, in cellular distribution. MARP1 and 2
were shown to be differently induced under the
mdm condition. This is consistent with previous

tion of p94, especially its stability (19,21,23,25),
and that p94 is also a component for signal transduction inherent in skeletal muscle structure. So
far, the physiological relevance of p94 localization
to the sarcomere has been unclear. Our results present three possibilities, which may be interrelated.
First, more efficient binding of the full-length
p94 than its autolyzed fragment to N2A connectincan be inferred as a molecular mechanism for condensing protease activity in the proximity of p94
substrates (i.e., connectin itself and MARP2) andfor releasing a remnant of enzyme after completionof p94 functions in a given context (Fig. 7A).
Second, protection effect of p94:CS against
proteolysis of N2A connectin may result from alteration of connectin local structures by p94 binding so that connectin is not proteolyzed spontaneously and/or randomly by yet unknown proteases,
one of candidates of which is conventional calpain.
Third, the negative effect of p94:CS on the
MARP2–connectin interaction could alternatively
control the extent of proteolysis of MARP2 by p94
as well as its functions. One of the novel p94 binding sites of connectin, e.g., SpBS1, may directlycompete with MARP binding to “is”. Furthermore,
the presence of several p94–connectin interactionsin I80-PEVK may trigger structural changes that
affect the efficiency of MARP binding.
Activity of p94-connectin complex in Ttnmdm/mdm
skeletal muscle. The effect of the mdm deletion on
p94 stabilities was apparently in conflict with previous results. Decrease of p94 in Ttnmdm/mdm skeletal muscle (43) corroborates the idea that the mdm
deletion abrogates p94–connectin interaction required for p94 regulation. In contrast, I80PEVK(mdm) was able to coprecipitate p94:WT(Fig. 1B), and autolysis of p94:DA coexpressedwith I80-PEVK(mdm) was retarded (Fig. 2C). One
interpretation is that I80-PEVK(mdm) can bind
p94 at SpBSs, but is distinct from WT not only in
its resistance to proteolysis, but also in that it hinders certain aspects of p94 protease activity.
In this context, we hypothesize that p94connectin N2A complex represents one of functional p94 protease units; p94 as a catalytic subunit
and connectin as a regulatory subunit. The observation that gait deficits found in Ttnmdm/+ mice
were restored by p94 overexpression indicates thatthe mdm deletion does not produce dominant phenotypes but causes a decrease in p94 activity (52).

9connectin molecule, which may desensitize theconnectin-MARP interaction to respond to a
stretch signal transmitted as structural changes inconnectin. Since a release of MARP2 N-terminal
part upon proteolysis by μ-calpain retained bindingof MARP2 to connectin, the interaction of MARP2
with molecules other than connectin could be affected. In this regard, previously reported interactions of MARP2 with YB-1 or myopalladin(42,59), which is mediated by the N-terminal region of MARP2, represent good candidates.
As a condition where the proteolysis of sarcomeric components above described bears biological significance, the maintenance and remodeling of once established muscle tissues should beconsidered. This process must require coordinatedevents including proteolytic dismissal of damaged
proteins and reorganization of newly synthesized
proteins (61-63). Together with reported functionsof muscle proteins, our data suggest that the N2Acomplex comprising connectin, MARP2 and p94 iscapable of playing a key role in such a scenario.
This protease machinery is allowed to be dynamic and sensitive accordingly to cellular context,
because of unique properties of connectin including its huge molecular size, intramolecular effects
on its interaction with MARPs and on susceptibility to proteolysis, and the selective recruitment of
p94 activity. Moreover, the proposed functions of
MARPs in accommodating muscles to stress byregulating gene expression indicates a link between the N2A complex and the nucleus. Recent
proteomic approaches also indicate that p94 is involved in the regulation of metabolism and protein
synthesis (64,65). The interlocking behavior ofconnectin with multiple activities of skeletal muscle would assure a balanced projection of p94 activity to molecules of diverging functions. Perturbing this by, e.g., the mdm deletion, should leadto serious dysfunction of muscles.

studies that MARP1 and 2 respond differentiallydepending on the quantity and/or quality of“stress” (58).
Multiple cellular events should
control MARP–connectin interaction in muscles(59), and whether MARPs interact with connectin
as a monomer, dimer, or complex with other proteins is one of critical future issues to be investigated (49).
Studies on fiber type specificity of MARPs
showed that high level expression of MARP1 isoften associated with small regenerating fibers,
which are labeled by embryonic MHC (corresponding to dMHC in this study), in Duchenne
muscular dystrophy, but not in other muscular dystrophies (60). In Ttnmdm/mdm skeletal muscle, theexpression of MARP1 was not detected in dMHCpositive fibers, whereas MARP2 was detected infibers positive for sMHC or dMHC or negative forboth. The same trend of MARP2 was reported in
Amyotrophic lateral sclerosis (38).
Altogether, these observations suggest that themolecular mechanisms underlying the induction of
MARP1 and 2 in Ttnmdm/mdm are distinct, and thatthese mechanisms are different from those in othermuscular dystrophy conditions. In other words,
different primary causes (e.g., deletion in dystrophin or connectin) of the symptom collectively
described as “dystrophy” distinguish the molecularmechanism of each symptom.
Consistent with a short life span (10-12 wk) of
Ttnmdm/mdm mice, skeletal muscle from 10-wk-old
mice examined in this study presented advanced
dystrophic symptoms. The expression trend of
MARP1 and 2 observed here may be related to astage of disease progression. One of directions forthe future study is comparative analysis of the timecourse for expression of MARP1 and 2 in relation
to muscle fiber types and sizes.
Regulated proteolysis as a modulator for muscle functions. Proteolysis of connectin in “is” region by calpains including p94 is predicted to release connectin-MARP complex from the entire

10

REFERENCES
1.

Goll, D. E., Thompson, V. F., Li, H., Wei, W., and Cong, J. (2003) Physiol. Rev. 83, 731-801

2.

Suzuki, K., Hata, S., Kawabata, Y., and Sorimachi, H. (2004) Diabetes 53 Suppl 1, S12-18

3.

Sorimachi, H., Ishiura, S., and Suzuki, K. (1997) Biochem. J. 328, 721-732

4.

Jackman, R. W., and Kandarian, S. C. (2004) Am. J. Physiol. Cell Physiol. 287, C834-843

5.

Spencer, M. J., and Mellgren, R. L. (2002) Hum. Mol. Genet. 11, 2645-2655

6.

Sugita, H., Ishiura, S., Suzuki, K., and Imahori, K. (1980) Muscle Nerve 3, 335-339

7.

Dedieu, S., Poussard, S., Mazeres, G., Grise, F., Dargelos, E., Cottin, P., and Brustis, J. J. (2004) Exp. Cell
Res. 292, 187-200

8.

Raynaud, F., Carnac, G., Marcilhac, A., and Benyamin, Y. (2004) Exp. Cell Res. 298, 48-57

9.

Dedieu, S., Mazeres, G., Dourdin, N., Cottin, P., and Brustis, J. J. (2003) J. Mol. Biol. 326, 453-465

10.

Richard, I., Broux, O., Allamand, V., Fougerousse, F., Chiannilkulchai, N., Bourg, N., Brenguier, L.,
Devaud, C., Pasturaud, P., Roudaut, C., Hillaire, D., Passos-Bueno, M., Zatz, M., Tischfield, J. A., Fardeau,
M., Jackson, C. E., Cohen, D., and Beckmann, J. S. (1995) Cell 81, 27-40

11.

Alderton, J. M., and Steinhardt, R. A. (2000) Trends Cardiovasc. Med. 10, 268-272

12.

Ono, Y., Shimada, H., Sorimachi, H., Richard, I., Saido, T. C., Beckmann, J. S., Ishiura, S., and Suzuki, K.
(1998) J. Biol. Chem. 273, 17073-17078

13.

Tagawa, K., Taya, C., Hayashi, Y., Nakagawa, M., Ono, Y., Fukuda, R., Karasuyama, H., ToyamaSorimachi, N., Katsui, Y., Hata, S., Ishiura, S., Nonaka, I., Seyama, Y., Arahata, K., Yonekawa, H., Sorimachi, H., and Suzuki, K. (2000) Hum. Mol. Genet. 9, 1393-1402

14.

Spencer, M. J., Guyon, J. R., Sorimachi, H., Potts, A., Richard, I., Herasse, M., Chamberlain, J., Dalkilic, I.,
Kunkel, L. M., and Beckmann, J. S. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 8874-8879

15.

Richard, I., Roudaut, C., Marchand, S., Baghdiguian, S., Herasse, M., Stockholm, D., Ono, Y., Suel, L.,
Bourg, N., Sorimachi, H., Lefranc, G., Fardeau, M., Sebille, A., and Beckmann, J. S. (2000) J. Cell Biol.
151, 1583-1590

16.

Kramerova, I., Kudryashova, E., Tidball, J. G., and Spencer, M. J. (2004) Hum. Mol. Genet. 13, 1373-1388

17.

Maruyama, K. (1976) J. Biochem. 80, 405-407

18.

Tokuyasu, K. T., and Maher, P. A. (1987) J. Cell Biol. 105, 2795-2801

19.

Sorimachi, H., Kinbara, K., Kimura, S., Takahashi, M., Ishiura, S., Sasagawa, N., Sorimachi, N., Shimada,H., Tagawa, K., Maruyama, K., and et al. (1995) J. Biol. Chem. 270, 31158-31162

20.

Sorimachi, H., Toyama-Sorimachi, N., Saido, T. C., Kawasaki, H., Sugita, H., Miyasaka, M., Arahata, K.,
Ishiura, S., and Suzuki, K. (1993) J. Biol. Chem. 268, 10593-10605

11

21.

Kinbara, K., Ishiura, S., Tomioka, S., Sorimachi, H., Jeong, S. Y., Amano, S., Kawasaki, H., Kolmerer, B.,
Kimura, S., Labeit, S., and Suzuki, K. (1998) Biochem. J. 335, 589-596

22.

Kinbara, K., Sorimachi, H., Ishiura, S., and Suzuki, K. (1997) Arch. Biochem. Biophys. 342, 99-107

23.

Ono, Y., Torii, F., Ojima, K., Doi, N., Yoshioka, K., Kawabata, Y., Labeit, D., Labeit, S., Suzuki, K., Abe,K., Maeda, T., and Sorimachi, H. (2006) J. Biol. Chem. 281, 18519-18531

24.

Ono, Y., Hayahi, C., Doi, N., Tagami, M., and Sorimachi, H. (2008) FEBS Lett. in press

25.

Ojima, K., Ono, Y., Doi, N., Yoshioka, K., Kawabata, Y., Labeit, S., and Sorimachi, H. (2007) J. Biol.
Chem. 282, 14493-14504

26.

Maruyama, K. (1997) FASEB J. 11, 341-345

27.

Centner, T., Fougerousse, F., Freiburg, A., Witt, C., Beckmann, J. S., Granzier, H., Trombitas, K., Gregorio,
C. C., and Labeit, S. (2000) Adv. Exp. Med. Biol. 481, 35-49, 50-52

28.

Tskhovrebova, L., and Trinick, J. (2003) Nat. Rev. Mol. Cell Biol. 4, 679-689

29.

Miller, M. K., Granzier, H., Ehler, E., and Gregorio, C. C. (2004) Trends Cell Biol. 14, 119-126

30.

Miller, M. K., Bang, M. L., Witt, C. C., Labeit, D., Trombitas, C., Watanabe, K., Granzier, H., McElhinny,
A. S., Gregorio, C. C., and Labeit, S. (2003) J. Mol. Biol. 333, 951-964

31.

Aihara, Y., Kurabayashi, M., Saito, Y., Ohyama, Y., Tanaka, T., Takeda, S., Tomaru, K., Sekiguchi, K.,
Arai, M., Nakamura, T., and Nagai, R. (2000) Hypertension 36, 48-53

32.

Carson, J. A., Nettleton, D., and Reecy, J. M. (2002) FASEB J. 16, 207-209

33.

Chen, Y. W., Nader, G. A., Baar, K. R., Fedele, M. J., Hoffman, E. P., and Esser, K. A. (2002) J. Physiol.
545, 27-41

34.

Kemp, T. J., Sadusky, T. J., Saltisi, F., Carey, N., Moss, J., Yang, S. Y., Sassoon, D. A., Goldspink, G., andCoulton, G. R. (2000) Genomics 66, 229-241

35.

Tsukamoto, Y., Senda, T., Nakano, T., Nakada, C., Hida, T., Ishiguro, N., Kondo, G., Baba, T., Sato, K.,
Osaki, M., Mori, S., Ito, H., and Moriyama, M. (2002) Lab. Invest. 82, 645-655

36.

Ikeda, K., Emoto, N., Matsuo, M., and Yokoyama, M. (2003) J. Biol. Chem. 278, 3514-3520

37.

Bakay, M., Zhao, P., Chen, J., and Hoffman, E. P. (2002) Neuromuscul. Disord. 12 Suppl 1, S125-141

38.

Nakamura, K., Nakada, C., Takeuchi, K., Osaki, M., Shomori, K., Kato, S., Ohama, E., Sato, K., Fukayama,M., Mori, S., Ito, H., and Moriyama, M. (2002) Pathobiology 70, 197-203

39.

Nakada, C., Oka, A., Nonaka, I., Sato, K., Mori, S., Ito, H., and Moriyama, M. (2003) Pathol. Int. 53, 653658

40.

Jeyaseelan, R., Poizat, C., Baker, R. K., Abdishoo, S., Isterabadi, L. B., Lyons, G. E., and Kedes, L. (1997)
J. Biol. Chem. 272, 22800-22808

41.

Zou, Y., Evans, S., Chen, J., Kuo, H. C., Harvey, R. P., and Chien, K. R. (1997) Development 124, 793-804

12

42.

Kojic, S., Medeot, E., Guccione, E., Krmac, H., Zara, I., Martinelli, V., Valle, G., and Faulkner, G. (2004) J.
Mol. Biol. 339, 313-325

43.

Garvey, S. M., Rajan, C., Lerner, A. P., Frankel, W. N., and Cox, G. A. (2002) Genomics 79, 146-149

44.

Witt, C. C., Ono, Y., Puschmann, E., McNabb, M., Wu, Y., Gotthardt, M., Witt, S. H., Haak, M., Labeit, D.,
Gregorio, C. C., Sorimachi, H., Granzier, H., and Labeit, S. (2004) J. Mol. Biol. 336, 145-154

45.

Heimann, P., Menke, A., Rothkegel, B., and Jockusch, H. (1996) Cell Tissue Res. 283, 435-441

46.

Herasse, M., Ono, Y., Fougerousse, F., Kimura, E., Stockholm, D., Beley, C., Montarras, D., Pinset, C.,
Sorimachi, H., Suzuki, K., Beckmann, J. S., and Richard, I. (1999) Mol. Cell Biol. 19, 4047-4055

47.

Sato, N., Kawahara, H., Toh-e, A., and Maeda, T. (2003) Mol. Cell Biol. 23, 6662-6671

48.

Ono, Y., Kakinuma, K., Torii, F., Irie, A., Nakagawa, K., Labeit, S., Abe, K., Suzuki, K., and Sorimachi, H.
(2004) J. Biol. Chem. 279, 2761-2771

49.

Witt, S. H., Labeit, D., Granzier, H., Labeit, S., and Witt, C. C. (2005) J. Muscle Res. Cell Motil. 26, 401408

50.

Taveau, M., Bourg, N., Sillon, G., Roudaut, C., Bartoli, M., and Richard, I. (2003) Mol. Cell Biol. 23, 91279135

51.

Laval, M., and Pascal, M. (2002) Biochim. Biophys. Acta. 1570, 121-128

52.

Huebsch, K. A., Kudryashova, E., Wooley, C. M., Sher, R. B., Seburn, K. L., Spencer, M. J., and Cox, G. A.
(2005) Hum. Mol. Genet. 14, 2801-2811

53.

Suzuki, A., Kim, K., and Ikeuchi, Y. (1996) Adv. Biophys. 33, 53-64

54.

Lim, C. C., Zuppinger, C., Guo, X., Kuster, G. M., Helmes, M., Eppenberger, H. M., Suter, T. M., Liao, R.,
and Sawyer, D. B. (2004) J. Biol. Chem. 279, 8290-8299

55.

Helmes, M., Trombitas, K., and Granzier, H. (1996) Circ. Res. 79, 619-626

56.

Raynaud, F., Fernandez, E., Coulis, G., Aubry, L., Vignon, X., Bleimling, N., Gautel, M., Benyamin, Y.,
and Ouali, A. (2005) FEBS J. 272, 2578-2590

57.

Barash, I. A., Bang, M. L., Mathew, L., Greaser, M. L., Chen, J., and Lieber, R. L. (2007) Am. J. Physiol.
Cell Physiol. 293, C218-C227

58.

Hentzen, E. R., Lahey, M., Peters, D., Mathew, L., Barash, I. A., Friden, J., and Lieber, R. L. (2006) J.
Physiol. 570, 157-167

59.

Bang, M. L., Mudry, R. E., McElhinny, A. S., Trombitas, K., Geach, A. J., Yamasaki, R., Sorimachi, H.,
Granzier, H., Gregorio, C. C., and Labeit, S. (2001) J. Cell Biol. 153, 413-427

60.

Nakada, C., Tsukamoto, Y., Oka, A., Nonaka, I., Sato, K., Mori, S., Ito, H., and Moriyama, M. (2004)
Pathobiology 71, 43-51

61.

Zak, R., Martin, A. F., Prior, G., and Rabinowitz, M. (1977) J. Biol. Chem. 252, 3430-5343

62.

Taylor, R. G., Papa, I., Astier, C., Ventre, F., Benyamin, Y., and Ouali, A. (1997) J. Muscle Res. Cell Motil.
18, 285-294

13

63.

Bassaglia, Y., Cebrian, J., Covan, S., Garcia, M., and Foucrier, J. (2005) Exp. Cell Res. 302, 221-232

64.

Cohen, N., Kudryashova, E., Kramerova, I., Anderson, L. V., Beckmann, J. S., Bushby, K., and Spencer, M.
J. (2006) Proteomics 6, 6075-6084

65.

Ono, Y., Hayashi, C., Doi, N., Kitamura, F., Shindo, M., Kudo, K., Tsubata, T., Yanagida, M., and Sorimachi, H. (2007) Biotechnol. J. 2, 565-576

FOOTNOTES*We are grateful to Dr Kimie Murayama in Juntendo University, and to all “Calpain Project” members forexperimental support and valuable discussion. This work was supported in part by research grants
MEXT.KAKENHI 17028055 and 18076007 (to H.S.), JSPS.KAKENHI 18770124 (to Y.O.), 18380085(to H.S.), Research Grant 17A-10 for Nervous and Mental Disorders from the Ministry of Health, Labourand Welfare, and the Takeda Science Foundation research grant (to H.S.), the SFB753 and the DFG(La668/9-1+11-1 to S.L. and La1619/1-1 to D.L.).
1

These authors contributed equally to this work.

2

To whom correspondence should be addressed: Department of Enzymatic Regulation for Cell Functions(Calpain Project), The Tokyo Metropolitan Institute of Medical Science (Rinshoken), 3-18-22 Honkomagome, Bunkyo-ku, Tokyo 113-8613, Japan. Tel.: 81-3-3823-2181; Fax: 81-3-3823-2359; E-mail: yakoono@rinshoken.or.jp or sorimach@rinshoken.or.jp

3

The abbreviations used are: E64c, [L-3-trans-carboxyoxirane-2-carbonyl] L-leucine(3-methylbutyl) amide; mdm, muscular dystrophy with myositis; HA, hemagglutinin; I8x, immunoglobulin motif No. 8x in
the I-band region of connectin/titin; “is”, intervening sequence region in N2A connectin/titin; MARP,
muscle ankyrin-repeat protein; p94, muscle-specific calpain (calpain 3); dMHC and sMHC, developmental and slow-type isoforms of myosin heavy chain; PEVK, Pro, Glu, Val, and Lys-rich region; PMSF,
phenylmethylsulfonyl fluoride; PpBS and SpBS, primary and secondary p94 biding site, respectively; WT,
wild type; CS, C129S protease deficient mutant; DA, D607A missense mutant; YTH, yeast two-hybrid.

FIGURE LEGENDS
Fig. 1. Interaction between p94 and the connectin N2A-PEVK region. A, Schematic structure of the
N2A-PEVK connectin/titin region and constructs used in this study. Numbers correspond to aar in themouse connectin, NP_035782. Horizontal bars above connectin indicate the binding regions for MARP(=”is”) and p94 (=PpBS, primary p94 binding site) identified previously by YTH. The 83-aa span deleted
in mdm is indicated by a bracket. ”I80”–“I83”, “is”, and “PEVK” indicate the 80th to 83rd immunoglobulin(IG) motifs, a specific insertion sequence, and a region rich in Pro, Glu, Val, and Lys, respectively, in theconnectin I-band region. Vertical arrows [1]–[3] and bidirectional arrows indicate the proteolytic sites
and p94 binding sites (SpBS, secondary p94 binding site), respectively, identified in this study. The abilityof each fragment to coimmunoprecipitate p94:WT or p94:CS and its susceptibility to endogenous proteolysis at the junction of I83 and PEVK are summarized on the right. NA, not applicable; ND, not determined. B, Connectin constructs indicated and p94:WT were coexpressed in COS7 cells (Input) and immunoprecipitated by anti-FLAG (IP). Note that the full-length 94 kDa band for p94:WT (open arrowhead),was hardly detectable in the cell lysate before immunoprecipitation (lanes 1-6), but was efficiently coim-

14munoprecipitated with I80-PEVK, I80-PEVK(mdm), and I81-I83(mCN48) (lanes 8, 9, and 12). C, Mapping of novel p94 binding sites using protease-inactive mutant p94:CS. p94:CS was coimmunoprecipitatedwith PEVK-N and I80–is (lanes 12 and 13), and these regions were designated as SpBS1 and 2 as in A. D,
Summary of YTH assays for binding of p94 to two different regions of connectin, I81-I83 (PpBS) andPEVK-N (SpBS2). Arrows [1]–[3] refer to p94 autolytic sites. The region containing IS2 is enlarged toshow the sequences encoded by exons 14 to 16.
Fig. 2. Proteolysis of N2A connectin by calpain. A, Recombinant human connectin fragment, HisI80–I83, and μ-calpain were co-incubated in the presence of Ca2+ with amounts indicated. Arrowhead (a)and arrows (b)–(d) indicate the full-length (69 kDa) and proteolyzed fragments as shown in B. B, Proteolytic fragments of I80-I83 are schematically summarized. Bidirectional arrows (a)–(d) correspond to (a)–(d) in A. Proteolytic sites for μ-calpain are indicated according to mouse connectin, NP_035782. The
closed arrowhead indicates another proteolytic site for calpain determined previously (48). The span offull-length and proteolytic fragments of I80-PEVK or I80-PEVK(mdm) are represented by bidirectional
arrows (e)–(i); the antibodies used to detect them in Sf-9 cell lysates (Fig. 2C) are shown on the right. The
anti-TPALKK antibody was generated against the mouse connectin sequence corresponding to the Nterminus of proteolyzed fragment (b) (bracket). Horizontal bars indicate binding sites for MARPs (is) andp94 (SpBS1 and PpBS). C, Expression of I80-PEVK or I80-PEVK(mdm) in Sf-9 cells without (-) or with
p94:DA or p94:CS. The cell lysates were incubated for indicated times in the presence of 1 mM PMSF
and 5 mM CaCl2 at 37oC. Note that I80-PEVK, but not I80-PEVK(mdm), was proteolyzed by endogenous
protease(s) in a Ca2+-dependent manner (anti-FLAG and anti-TPALKK, lanes 1–5), which was accelerated
by coexpressing p94:DA (lanes 7–11) or attenuated by p94:CS (lanes 13–17). Proteolysis of I80-PEVK
was inhibited in the presence of calpastatin, leupeptin, and E64c (lanes 6, 12, and 18) but not by calpastatin alone (not shown). Arrows (e)–(g) and (i) correspond to fragments labeled in Fig. 2B. *, nonspecificsignals.
Fig. 3. Differences in connectin-binding activity and subcellular localization among MARPs. A,
Combination of FLAG-tagged I80-PEVK and HA-tagged MARP1–3 as indicated were coexpressed in
COS7 cells (Input) for a coimmunoprecipitation assay (IP). MARP1 and 3 coimmunoprecipitated more
efficiently with the I80-PEVK fragment than did MARP2. B, C-terminal non-binding region of connectin
affected binding to MARP2. Note that both I80-I81 (lane 4) and I80-PEVK (lane 5) contain MARP2 binding site, “is”, but showed distinct binding ability to MARP2. C, MYC-MARP1 or HA-MARP2, or both,were coexpressed with FLAG-tagged I80-PEVK or I80-PEVK(mdm) in COS7 cells as indicated combinations. We noted that less MARP1 or MARP2 coimmunoprecipitated with I80-PEVK(mdm) than with I80PEVK (lanes 2 and 5, IP, anti-MYC; lanes 3 and 7, IP, anti-HA). The interaction between MARP2 andI80-PEVK or I80-PEVK(mdm) was strongly suppressed by coexpression with MARP1, whereas MARP1
was not affected (lanes 6 and 7, anti-HA; lanes 5 and 6, anti-MYC). D, The amount of MARP1 and 2 inwhole tissue homogenates (T), soluble (S), and insoluble fractions (P) of skeletal and cardiac muscles
from three independent wild type (WT-1 to 3) or Ttnmdm/mdm mice (mdm-1 to 3) was examined using antiMARP1 and MARP2 antibodies. For summary of the results, see Table II. Open and closed arrowheads in
A–C, the full-length and the N-terminal proteolyzed fragments of FLAG-connectin, respectively.
Fig. 4. Expression of MARP1 and MARP2 in WT and Ttnmdm/mdm mouse skeletal muscle. MARP1
or 2 (red) and slow (sMHC) or developmental MHC (dMHC) isoform (green) were detected by doublelabeled immunofluorescence on sections of posterior parts of hind limbs from WT (A) and Ttnmdm/mdm (B–E) mice using specific antibodies. Nuclei were stained with DAPI (blue). Representative images were selected. A, In WT muscle, fibers faintly positive for MARP2 were identified at a low frequency. Some ofthese fibers were sMHC-positive (*). B, In the corresponding area of Ttnmdm/mdm muscle, fibers expressing
MARP1 were sparsely observed, where central nuclei were also identified (arrowhead). Expression of
MARP1 was not detected in fibers expressing dMHC (*). C, Two serial sections were stained with anti-

15

MARP1, 2, and sMHC. Fibers expressing MARP2 and/or sMHC were observed more frequently compared to WT. Expression of MARP1 and 2 is not mutually exclusive, and is independent of the muscle
fiber types. Examples of combination of MARP1, 2, and sMHC expressed in the same fiber were shownby arrows (+, +, -), arrowheads (-, +, -), and asterisks (-, +, +). D, Expression of MARP2 was observed
both in dMHC-positive (*) and -negative fibers (arrowheads). E, Occasionally, MARP1 and MARP2
were localized in or at the periphery of central nuclei in Ttnmdm/mdm myofibers. Bars, 20 μm.
Fig. 5. Effects of p94 protease activity on MARP2. A, p94:WT or p94:CS was coexpressed with
FLAG-I80-PEVK and/or HA-MARP2 in COS7 cells for a coimmunoprecipitation assay as described in
the previous Figs. Note that coexpression of p94:WT decreased MARP2 in both the cell lysate and coimmunoprecipitate (lanes 3 and 7, anti-HA), whereas p94:CS decreased MARP2 only in the coimmunoprecipitate (lanes 6 and 8, anti-HA). B, The lysate of Sf-9 cells expressing p94:DA without (-) or with I80PEVK or I80-PEVK(mdm) was incubated with recombinant His-MARP2 in the presence of 5 mM Ca2+
and 1 mM PMSF at 37oC. Autolysis of p94:DA and proteolysis of MARP2 and/or I80-PEVK were inhibited completely by leupeptin and E64c (lanes 5, 10, and 15), but not by calpastatin (lanes 4, 9, and 14).
Open and closed arrowheads indicate the full-length and autolyzed fragments of p94, respectively.
Fig. 6. Effect of proteolysis by μ-calpain on the MARP2–connectin interaction. A, Recombinant
MARP2 was proteolyzed by recombinant human μ-calpain under indicated conditions. Open and closed
arrowheads indicate μ-calpain and its autolyzed fragment, respectively. Open and closed arrows indicate
the full-length and the proteolyzed fragments of MARP2, respectively. B, The N-terminal sequence of
MARP2 proteolyzed by μ-calpain was determined to be R77 (aar in Q9GZV1) (arrow). NLS, nuclear localization signal; PEST, a region rich in Gln, Ser, and Thr flanked by Pro, which is considered to be susceptible for calpain proteolysis; ANK, ankyrin repeat motif. C, HA-MARP2Δ1–79 coimmunoprecipitated
I80-PEVK as efficiently as the full-length MARP2 did. Open and closed arrows, HA-MARP2 and HAMARP2Δ1–79, respectively. *, nonspecific signals.
Fig. 7. A hypothetical scheme of the molecular complex at the N2A connectin. A, Summary of the
N2A region in connectin and the relationships between p94 and MARPs in WT skeletal muscle. MARP2
binds to the connectin “is” region, which is suppressed by the interaction between p94 and connectin. At
least three regions in N2A connectin are involved as scaffolds in p94-connectin complex. Closed arrowheads indicate the proteolytic sites in N2A connectin and MARP2 determined in this study. Unproteolyzed connectin orchestrates multiple signal transductions (upper). Activation of proteolytic machinery,i.e., p94-connectin complex, results in proteolyzed connectin fragments whose interaction with MARP2 aswell as other functions are modulated (lower). MARP2 is also proteolyzed to generate the free N-terminal
fragment, MARP2-(N), and the C-terminal connectin-associated fragment, MARP2-(C). Dissociationfrom connectin prompts autolysis of p94 to terminate its activity. Coordination of these reactions prevents
aggravation of muscle damage triggered under various physiological conditions. B, Stalled proteolysis
incited by the mdm deletion in connectin. In Ttnmdm/mdm skeletal muscle, expression of MARP1 and 2 isupregulated as a result of aberrant signal transduction by connectin with the mdm deletion. When MARP1
and 2 are coexpressed in the same muscle fiber, “is” is dominantly occupied by MARP1. On the other
hand, several fibers express only MARP2, which binds to “is” without interference by MARP1 and is subjected to proteolysis. The mdm deletion impairs the nature of connectin as a substrate for proteases, anddistorts interaction between p94 and connectin, which compromises functions of p94-connectin complex.
These abnormalities cause deficits in cellular response to muscle damage, i.e., dystrophic symptoms observed in Ttnmdm/mdm skeletal muscle. Open arrowheads indicate proteolytic sites in connectin apparently
invalidated by the mdm deletion. Wavy lines represent distorted interaction between p94 and connectin.

16

Table I. Fragments from mouse connectin N2A-PEVK region used in this study

Construct

Amino acid residue in
NP_035782
I80-PEVK
8463–9137
I80-PEVK(mdm)8463–8894/8978–9137
8463–8895
I80–I83Δ
I80–I82
8463–8826
I80–I818463–8738
I80–is8463–8655
I81–I83(mCN48)
8613–8915
I81-I83/PEVK
8613–8998
I81-I83/PEVK(mdm) 8613–8894/8978–8998
I81–I83:I8931term
8613–8930
I81–I83:I8951term
8613–8950
I82-PEVK
8739–9137
PEVK-N
9089–9526

Calculated molecularweight (kDa)a
80
70
52
43
33
24
37
46
37
38
41
48
52

a

Observed molecularweight (kDa)b97, 60c85
5845, 3533, 2626
39
47, 364239
41
66, 31
99

Additional 1.4 kDa for the N-terminal FLAG-tag is included. b For proteins expressed in COS7 cells with
the N-terminal FLAG-tag. c The lower molecular weight corresponds to a proteolytic fragment.

17

Table II. Cellular distribution of MARPs

SkeletalmuscleCardiacmuscle

WTa
mdmb
WT
mdm

MARP1
Sup
Ppt
c
–
–
–
++
–
+
–
++

MARP2
Sup
Ppt
–
±
+++
+
–
–
–
–

a

Mice homozygous for wild type allele of Ttn, Ttn+/+. b Mice homozygous for mdm allele of Ttn,
Ttnmdm/mdm. c Result of western blot analysis in Fig. 3D was summarized quantitatively.

18

Table III. Expression of MARPs and MHCs

Combinations of expression
at a muscle fiber leveld

Overall expression incross-sectional areac
WTa

mdmb

mdm

MARP1

-

+

+

MARP2

+

+++

slow MHC+++

developmental MHC

-

+

+

+

a

+

+

+
+

+

+

+

+

+

Mice homozygous for wild type allele of Ttn, Ttn+/+. b Mice homozygous for mdm allele of Ttn,
Ttnmdm/mdm. c Expression of each protein examined by immunofluorescent study on cross sectionsof posterior segment of hind limbs was categorized based on the positive rate. +++ = >50%; ++ =10-50%; + = 1-10%; – ≈ 0%. d Trend in coexpression of proteins in the same muscle fiber fromTtnmdm/mdm muscle is summarized.

19

Hayashi et al., Fig. 1
A

SpBS1

SpBS2
p94 binding ∆8895-8977 in mdm(Z-line)N-

G8501 MARPs binding
8529
8651 8739

isI808463 [2]I80-PEVK

I81(PpBS)

I82(M-line)
-C

8915

PEVK

I83

[3]

Proteolysis
in I83-PEVK
at [1]
p94:WT p94:CS
IP:FLAG

9137[1]

I80-PEVK(mdm)

+

+

+

+

+

-

-

+

NA

-

+

NA

+

+

NA

-

-

+

ND

+

NA

8738

I80-I818655

I80-is8613

I81-I83(mCN48)

8914
8739

I82-PEVK

9137
9089

PEVK-N

B

9526

C

m
o
I8 ck
0
I8 -PE
0- V
I8 PE K
2 V
I8 -PE K(
0- V mI8 I8 K dm
1 1
)
m -I8
oc 3(
m
I8 k
CN
0
I8 -PE48
0- V)
I8 PE K
2- V
KP
I8 E (m
0
I8 -I8 VK dm
1- 1
)
I8
3(
m
CN
48
)

+ p94:WT+ p94:CS

1 2 3 4 5 6 7 8 9 10 11 12

97
66

m
o
PE ck
V
I8 K0- N
I8 is1
m -I83oc (mPE k CN4
I8 VK8)
0- N
I8 is
1I8
3(
m
CN
48
)

(kDa)

m
o
I8 ck
2
I8 -PE
0 V
m -PE Ko V
I8 ck K2
I8 -PE
0- V
PE K
VK

FLAGconnectin

1 2 3 4 5 6

7 8 9 10 11 12 1314

FLAGconnectin
(kDa)

(kDa)

94
67

45
30

anti-FLAG

(kDa)

97
66
45

43

97
66

30

30

anti-FLAG

45

94

97

anti-pIS2

Input

anti-pIS2

30
Input

Input

IP:
anti-FLAGIP:
anti-FLAG

IP: anti-FLAG(R592)
[3]

D

570

580

.

590

.

600

.

610

.

620

.

630

.

640

.

.

EFILRVFSEKRNLSEEVENTISVDRPVKKKKTKPIIFVSDRANSNKELGVDQESEEGKGKTSPDKQKQSPQPQPGSSD
ex14

A15/A34[2]

p94

Cys129

NS

IIa

ex15

ex16

G275/S297/E323[1]
His334 Asn358

IS1

IIb

Binding to

IS2

III

IV

anti-pIS2 (586-604)
S821

p94:C129S
570

p94:IS2(570-641)

641

C

p94:1-600
C

p94:1-594C-

p94:ex15 16-

C

p94:1-580
p94:581-821
C

p94:1-572

594

641

I81-I83 PEVK-N(mCN48)[PpBS] [SpBS2]
+

+

+

+

+

+

+

-

+

+

+

-

-

-

-

-

Hayashi et al., Fig. 2A

p94 binding[SpBS1]B

G8501µ-calpain

0 0.06 0.12 µg

is

His-I80-I83

0.5 0.5 0.5 µg(kDa) 1

2

[2]

His-I80-I83

30
20
14

8558

I81

I82

8432

[3]

[1]

8934

MHHH---(a) 69 kDa(b) 50 kDa(c) 38 kDa(d)(d) 17 kDa

(a)

CBB

8646
.
QTEP--8647.
TEPV---

-C

PEVK

I83

8647

His-

3

94
67
43

∆8895-8977 in mdm

MARPs binding

I80
15 mM Ca2+, at 30oC, 90’

p94 binding[PpBS]

8946

(c)
8558

.
TPIL---(b)anti-TPALKK

His-FLAG-I80-PEVK
His-FLAG(e)

8463

9137

(f)
(g)
(h)
(i)

mdm deletion

detection
anti-FLAG anti-TPALKK

+
+
-

+
(+) too small+

C
0’ 10’ 25’ 40’ 60’ 0’ 10’ 25’ 40’ 60’ 0’ 10’ 25’ 40’ 60’
leupeptin+E64c- - - - - + - - - - - + - - - - - ++calpastatin(kDa)

0’ 10’ 25’ 40’ 60’

0’ 10’ 25’40’ 60’

0’ 10’ 25’ 40’

60’- - - - - + - - - - - + - - - - - +19 20 21 2223 24 25 26 2728 29 30 31 32 33 34 35 36

1 2 3 4 5 6 7 8 9 10 1112 13 14 15 16 17 18

94
67
43

anti-pIS2

30
(e)

94
67

(f)

anti-FLAG

*

94
67
43

(g)

-

p94:DA
His-FLAG-I80-PEVK

p94:CS-

p94:DA
His-FLAG-I80-PEVK(mdm)

p94:CS(i)

anti-TPALKKHayashi et al., Fig. 3

A

B

HA-MARP1
HA-MARP2
HA-MARP3(kDa)- + - - - + - - - +1 2 3 4

FLAG-connectin
HA-MARP2

97
66

97
66

anti-FLAG MARP1 (44 kDa)
anti-HA

Input

Input

(kDa)

MARP2 (43 kDa)
MARP3 (40 kDa)

45
30

IP: anti-FLAG

97
66

anti-FLAG

45
30- + + + +1 2 3 4 5

(kDa)

45
30

m
o
m ck
oc
I8 k
2I8 PE
0- V
I8 I8 1 K
0PE
VK

FLAG-connectin I80-PEVK

anti-HA
IP: anti-FLAG

anti-FLAG

anti-HA

anti-HAD

C

FLAG-connectin

I80 - PEVK
I80 - PEVK
(mdm)

MYC-MARP1- + - - + + -HA-MARP2- - + - - + +1 2 3 4 5 6 7

(kDa)

WT-1
WT-2
WT-3 mdm-1 mdm-2 mdm-3
T S P T S P T S P T S P T S P T S P

97
66

Input

45

anti-FLAG
anti-MYC(kDa)

anti-HA

IP: anti-FLAG

97
66
45

anti-FLAG
anti-MYC
anti-HA

skeletalmuscleMARP1cardiacmuscle

MARP1

MARP2

MARP2

Hayashi et al., Fig. 4

MARP2

sMHC

merge

*

*

*

MARP1

dMHC

merge

WT

A

Ttnmdm/mdm

B
*

MARP2

Ttnmdm/mdm

C

sMHC

Ttnmdm/mdm*

Ttnmdm/mdm
Ttnmdm/mdm

*

*

dMHC

merge

*

*

MARP1

merge

HE

MARP2

E

*

*

*

MARP1

D

*

MARP2

*

Hayashi et al., Fig. 5

A

FLAG-I80-PEVK
p94
HA-MARP2-+- WT CS- WT CS WT CS- + + + - + + + - -2 3 4 5 6 7 8 9 10(kDa) 1

97
66

anti-FLAG

(kDa)

Input

97
66
45

anti-pIS2

30

anti-HA(kDa)

IP : anti-FLAG

97
66

anti-FLAG

(kDa)

97
66
45

anti-pIS2

30

anti-HAB

0’ 10’ 30’

60’ 0’ 10’ 30’ 60’ 0’ 10’ 30’ 60’

leupeptin+E64c - - - - + - - - - + - - - - +calpastatin - - - + + - - - + + - - - + +1 2 3 4 5 6 7 8 9 10 1112 13 1415
(kDa) 94
67(kDa) 94anti-FLAG

67
43
anti-pIS2

30

43 kDaHis-FLAG-connectin
p94:DA

I80-PEVK+

I80-PEVK(mdm)++ His-MARP2+

anti-MARP2

Hayashi et al., Fig. 6A
5 mM EDTA

15 mM Ca2+

incubation at 30 oC

90’ 60’ 90’ 90’ 60’ 90’µ-calpain
His-MARP2+ - + + + - + +- + + + - + + +(kDa)

1 2 3 4 5 6 7 8

97
66
45
30

Silver-stain

B

R77

1

NLS PEST ANK ANK ANK ANK

human MARP2
connectin binding sites

C
FLAG-connectin I80-PEVK
HA-MARP2

- + -

- - +
1
2 3
(kDa)

HA-MARP2Δ1-79

Input

97
66

anti-FLAG
43 kDa35 kDaanti-HA(kDa)

IP: anti-HA

97
66

anti-FLAG**

43 kDa35 kDaanti-HA333

Hayashi et al., Fig. 7
Asignal transduction on connectin molecule

MARP-connectin
complexvariousstressesConnectin/titin N2A region in WT

MARP2

is

I80

p94-connectin
complex

I81

I82

I83

PEVK

p94

p94

p94

SpBS1

PpBS

SpBS2

proper proteolytic
functions / ONsegmentation of functions of connectin
MARP2-(N)MARP2-(C)I80
free p94

is

I81

I82

I83

p94

PEVK

p94

p94

autolysis

muscle damage

Baberrant signal transduction by connectin molecule

?
MARP1

upregulation

Nucleus

MARP1
MARP1 MARP1

MARP2

MARP2
with
MARP1

MARP2 MARP2

defective
p94-connectin
complex

Connectin/titin N2A region in mdm

MARP1

MARP2

I80

is

I81

I82

I83

p94

p94

SpBS1

PpBS

or

PEVK

deletion in mdm

p94
SpBS2

MARP2
MARP2

inproper proteolytic
functionsmuscle damage

MARP2

MARP2

I80

only
MARP2

is

I81

I82

p94

p94

SpBS1

PpBS