Ojima, K. et al.

p94 and connectin-mediated signaling pathways.

Possible functions of p94
in connectin-mediated signaling pathways in skeletal muscle cells

KOICHI OJIMA 1, 2, YASUKO ONO 1, SHOJI HATA 1, SUGURU KOYAMA 1, 3
NAOKO DOI1,2 and HIROYUKI SORIMACHI *, 1, 2

1

Department of Enzymatic Regulation for Cell Function, The Metropolitan Institute ofMedical Science (Rinshoken), 3-18-22 Honkomagome, Bunkyo-ku, Tokyo 113-8613,
Japan; 2CREST, Japan Science and Technology Agency (JST), Kawaguchi 332-0012, Japan;
3

Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi,

Bunkyo-ku, Tokyo 113-8657, Japan

Short title: p94 signaling pathways* To whom correspondence should be addressed:
Tel.: +81-3-3823-2181; Fax: +81-3-3823-2181; E-mail: sorimach@rinshoken.or.jp

Key words: p94, connectin/titin, skeletal muscle, muscular dystrophy1Ojima, K. et al.

p94 and connectin-mediated signaling pathways.

Abstract
Calpains are intracellular Ca2+-requiring “modulator proteases”, which
modulate cellular functions by limited and specific proteolysis.

p94/calpain3, askeletal-muscle specific calpain, has been one of the representative calpain species
which indicates physiological importance of calpain proteolytic system; a defect ofproteolytic activity of p94 causes limb girdle muscular dystrophy type2A (LGMD2A,
also called “calpainopathy”).

Immunohistochemical studies on myofibrils showed thatp94 localizes at the Z- and N2-line regions of sarcomeres.

It was also identified by the

yeast two hybrid studies that p94 binds to the N2A and M-line regions of connectin.
Furthermore, genetic studies indicate that p94 is indispensable for skeletal muscles,
although its precise functions are still unclear.

Interestingly, connectin provides

sarcomere not only with elasticity but also with binding sites to various multi-functional
proteins such as muscle ankyrin repeat proteins (MARPs), muscle RING finger proteins(MURFs), titin-capping protein (T-cap/telethonin), sarcomeric-α-actinin, p94 etc.
Binding sites for these proteins are not randomly placed along connectin but rather
accumulated in the Z-, N2-, and/or M-line regions, indicating the existence of “signal
complexes” unique to each regions. The concept of these complexes are strongly
supported by the facts that mutations of connectin or its binding proteins in theseregions severely perturb muscle functions, as in the case of LGMD2A caused bymutations in the p94 gene.

Therefore, it is hypothesized that the “signal complexes” inthe Z-, N2-, and M-lines modulate muscle cell homeostasis by transducing signals of
external stimulations/stresses to trigger appropriate response at various different cellular2

Ojima, K. et al.

p94 and connectin-mediated signaling pathways.

events such as protein modification and gene expressions.

In this article, we

performed detailed immunohistochemical analyses of p94 on isolated single myofibers.
Together with recent findings about p94, it is suggested that sarcomeric localization of
p94, especially its M-line localization, is affected by the combination of cellular
contexts such as contractile status of myofibrils, fiber type compositions, sarcomericmaturation, and the composition of the “signal complexes” in each region.

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p94 and connectin-mediated signaling pathways.

Introduction
Calpains comprise a large family of intracellular Ca2+-requiring cysteine
proteases, which cleave their substrates at limited and specific sites to modulate theirfunctions, thus called “modulator proteases”. In mammals, 14 genes for calpains exist,
which can be classified into two groups. One group is expressed in most of the tissues
ubiquitously (“ubiquitous calpains”).

The conventional calpains, such as µ-calpain

and m-calpain, belong to this group (Suzuki et al. 2004).

The other is predominantly

expressed in a specific tissue, such as p94 in skeletal muscle and nCL-2/-2’ in stomach(Sorimachi et al. 1993; Sorimachi et al. 1995; Hata et al. 2001).
Conventional calpain forms a heterodimer composed of the larger catalytic
subunit (“µCL (µ-calpain large subunit)” and “mCL” for µ- and m-calpains,respectively) and the smaller regulatory subunit (common for both, “30K” named after
its approximate molecular mass).

The large subunits are divided into four functional

domains (I-IV) (Figure 1, for review, see Goll et al. 2003; Suzuki et al. 2004).
Domain I is autolyzed upon activation, involved in regulation of activity and subunit
dissociation.

Domain II is the cysteine protease domain, which shows weak similarity

to papain and cysteine cathepsins. In the absence of Ca2+, the domain II is further
divided into two subdomains (IIa and IIb) to retain inactive “open” structure.

Domain

III is a Ca2+-binding domain, the 3-dimensional, but not primary, structure of which issimilar to that of the C2 domain.

The C2 domain was originally identified as aCa2+-binding motif in protein kinase C and later found in many, mainly Ca2+-regulated,
proteins. Domain IV is also a Ca2+-binding domain, which contains 5 EF-hand4Ojima, K. et al.

p94 and connectin-mediated signaling pathways.

Ca2+-binding motifs.

In the course of more calpain homologues being identified, it

was noticed that many of these molecules possess unique primary structures incombination with calpain protease domain, in which the regions surrounding active site
residues, Cys, His, and Asn, are highly conserved (Sorimachi and Suzuki 2001; Suzuki
et al. 2004).
It has been demonstrated that calpain activity is indispensable for a variety ofcellular functions, such as cell differentiation (Cottin et al. 1994; Moyen et al. 2004),
cell cycle (Raynaud et al. 2004), cell migration, and cell adhesion (Glading et al. 2002;
Franco et al. 2004).

Calpains are also implicated in human diseases, for instances,

Alzheimer’s disease (Saito et al. 1993), type 2 diabetes (Horikawa et al. 2000), andmuscular dystrophies (Richard et al. 1995).

These reports indicate that calpains playroles in various tissues in various ways to regulate cellular functions, although theactual physiological roles of each calpain are still unclear.
p94 is one of the most distinct calpain members among calpain family in thefollowing aspects.

First, p94 is predominantly expressed in skeletal muscles, with lessamounts of its several different splicing variants being expressed in skeletal muscle andnon-muscle cells (Ma et al. 1998; Welm et al. 2002; Kawabata et al. 2003; Tullio et al.
2003).

In sarcomeres, p94 binds to connectin (also called “titin”; in this manuscript

we use “connectin” hereafter) at the N2A and M-line regions as originally shown by the
yeast two-hybrid assays (Sorimachi et al. 1995).
Secondly, p94 contains three unique sequences, namely NS (N-terminal
sequence), IS1 (insertion sequence 1), and IS2, which are located at the N-terminus, in5Ojima, K. et al.

p94 and connectin-mediated signaling pathways.

the subdomain IIb, and between domains III and IV, respectively (Figure 1).

The NS

sequence is rich in Pro, has no similarity to the N-termini of other calpains, and serves
as the autolytic sites upon activation (Rey and Davies 2002). The IS1 region isinserted in the N-terminal region of domain IIb (Sorimachi et al., 1993). The IS1
region includes another site of autolysis, and is involved in extremely rapid autolytic
activity of p94 (see below).

A part of the IS2 region and its periphery are the bindingsite for the N2A region of connectin (Sorimachi et al. 1995).

A putative nuclear

localization signal sequence is also included in the IS2 region (Sorimachi et al. 1993;
Sorimachi et al. 1995).
Thirdly, Ca2+ dependence of activation of p94 is different from those forubiquitous conventional calpains, µ- and m-calpains.

Activation of µ- and m-calpains

requires more than 10 and 100 µΜ of Ca2+, respectively, at minimum in vitro.
However, it has been shown that p94 autolyzes in the absence of Ca2+, strongly
suggesting different regulatory mechanisms for proteolytic activity of p94 (Sorimachi etal. 1993).
Fourthly, p94 shows exhaustive rapid autolysis at least in vitro.

The half lifeof p94 is less than 10 minutes in vitro (Sorimachi et al. 1993). Native p94 extracted
from skeletal muscles, however, is mainly in unprocessed form immediately after
extraction (Kinbara et al. 1998).

Overexpression of wild type p94 in transgenic mice

did not show any obvious phenotype (Spencer et al. 2002). These results indicate thatp94 proteolytic activity is under a certain control in vivo unlike in vitro.
Finally, the gene for p94, CAPN3, was identified as a gene responsible for6Ojima, K. et al.

p94 and connectin-mediated signaling pathways.

limb girdle muscular dystrophy type 2A (LGMD2A) (Richard et al. 1995).

Since

LGMD2A is caused by a defect of calpain homologue, it is also called “calpainopathy”.
It should be noted that LGMD2A describes phenotype, whereas calpainopathy
corresponds rather to genotype.

Thus, both are used for human, but for animals otherthan human, “calpainopathy” is preferred to be used.

A defect of proper proteolytic

functions of p94 is responsible for LGMD2A as revealed by properties of 10
independent missense pathogenic mutants of p94 (Ono et al. 1998).

Some mutations

such as S744G, R769Q, and S606L retained autolytic activity (Ono et al. 1998, Jenne etal., 2005), but S744G and R769Q were shown to be unable to proteolyze p94 substrates,
indicating that proper recognition and cleavage of substrates, including p94 itself, by
p94 are essential for functional skeletal muscle cells.

Since then, several roles of p94

in skeletal muscle cells have been suggested. However, it has yet to be clarified as tohow, when, and where p94 functions in skeletal muscle.
In addition to being a binding partner for p94, connectin is a unique andimportant constituent of sarcomeres. Connectin is the largest protein in mammals(molecular weight is more than 3,000 kDa) and is exclusively expressed in striatedmuscles (Maruyama 1976; Wang et al. 1979; Labeit and Kolmerer 1995).
molecule of connectin extends to a half size of sarcomeres.

A single

The N-terminal end ofconnectin is located in the Z-line and its C-terminal end is located in the M-line.
Several important functions in skeletal muscles are proposed for connectin.
One of the classical roles of connectin in striated muscle is to give elasticity to
sarcomeres by generating passive tension, which is attributable to the molecular

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p94 and connectin-mediated signaling pathways.

structures in the I-band connectin (Granzier and Labeit 2004).

In brief, the I-band

region of connectin is composed of structurally distinct two motifs; tandemly arranged
IG (immunoglobulin-like) motifs and the PEVK domain which is rich in Pro, Glu, Val
and Lys.

Because of its unusual amino acid composition, the PEVK domain has no

specific secondary structure, and, belongs to “intrinsically unstructured protein (IUP)”
family (Tompa 2002). The molecular basis for generating passive tension is explained
as follows; the length of sarcomeres extends, the tandem IG-modules are unfolded
followed by extension of the PEVK domains (Granzier and Labeit 2004).
Recently, another aspect of biological functions of connectin has beenhighlighted, i.e., a platform for various ligands in sarcomeres.

Connectin provides

with many specific protein binding sites at the Z-line, N2A, and M-line regions, notonly for other myofibrillar proteins, such as sarcomeric-α-actinin (s-α-actinin) (Ohtsuka
et al. 1997; Sorimachi et al. 1997), myosin heavy chain (MHC) (Labeit et al. 1992;
Soteriou et al. 1993), myosin-binding protein C (Labeit et al. 1992; Soteriou et al.
1993), etc. Importantly, there are many non-structural, so-called regulatory proteinsthat interact with connectin at the Z-line, N2A, and M-line regions.

These include

muscle ankyrin repeat proteins (MARPs) (Miller et al. 2003), muscle-specific RING
finger protein-1 (MURF-1) (Centner et al. 2001), p94/calpain 3 (Sorimachi et al. 1995),
T-cap/telethonin (Valle et al. 1997; Gregorio et al. 1998; Ojima et al. 1999), etc.

Someof these connectin binding proteins were reported to localize in the nucleus in certain
conditions, suggesting a linkage between functions of connectin and nuclear response
against cellular circumstances.

Furthermore, close to the C-terminus at the M-line

8

Ojima, K. et al.

p94 and connectin-mediated signaling pathways.

region, connectin has a serine/threonine-kinase domain similar to that of the smoothmuscle myosin light chain kinase (MLCK) (Labeit et al. 1992; Labeit and Kolmerer
1995; Gregorio et al. 1999).

Therefore, it is also suggested that connectin exerts its

functions through its own enzymatic activity. Together, these properties of connectin
prompts an idea that skeletal muscle cells have unique signal complexes which include
distinct regions of connectin and proteins binding to these regions as criticalcomponents.

In fact, mutations of connectin or its binding proteins in these regions

severely perturb muscle functions, as in the case of LGMD2A caused by mutations in
the p94 gene (Richard et al. 1995), tibial muscular dystrophy (TMD) and muscular
dystrophy with myositis (MDM) caused by mutations in the M- and N2A-line regionsof connectin, respectively (Udd et al. 1993; Garvey et al. 2002), and LGMD2G causedby T-cap mutations (Moreira et al. 2000). The impact of those findings is that thosemutations are not readily explained as to their effect on sarcolemmal and/or myofibril
structures. In other words, it is suggested that connectin is responsible for skeletal
muscle integrity not only as a structural and elastic component but also as a link
between sarcolemma and intracellular signal transduction systems.

Here, weperformed immunohistochemical analyses of p94 using single myofibers.

Togetherwith recent findings about p94 from other studies, the nature of p94 localization insarcomeres was suggested to be dynamic, reflecting local structural change in connectin
as well as p94 itself.

In this article, we discuss functions of p94 and connectin

regarding signal transduction pathways in skeletal muscle, based on our
immunohistochemical analyses of p94 and other recent findings about p94.

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p94 and connectin-mediated signaling pathways.

Material and methods
Single myofibers were prepared as described previously (Bischoff 1986; Ojima
et al. 2004). In short, dissected extensor digitorum longus (EDL) muscles from
C57BL/6 mice were digested with 0.5% type I collagenase (Worthington Biochemical,
Lakewood, NJ) at 37oC for 120 min. Separated single myofibers were fixed with 4%paraformaldehyde in PBS for 20 minutes. After washing with 0.5% triton X-100 in
PBS, specimens were blocked with 5% goat serum (Vector Laboratory, Burlingame,CA) in PBS for 15 minutes, and then incubated with anti-s-α-actinin antibody (1:1000;
EA-53, Sigma Chemical, St. Louis, MO) and antiserum against NS domain of p94(1:300) (Sorimachi et al. 1995) for 16 hours at 4oC.

The reactions were detected withsecondary antibodies conjugated with Alexa 488 or Alexa 555 (Molecular Probes Inc.,
Eugene, OR).

Double-stained myofibers were analysed on a laser scanning confocal

microscopic system (LSM 510; Carl Zeiss, Inc., Germany), which employed a Zeiss
Axiovert inverted microscope.

Images were recorded and processed with LSM510

imaging software.

10Ojima, K. et al.

p94 and connectin-mediated signaling pathways.

Results
After binding of p94 to connectin was originally reported, localization of p94
in the myofibrils have been studied (Sorimachi et al. 1995; Baghdiguian et al. 1999;
Keira et al. 2003).

Although histochemical analysis using skeletal muscle sections has

been successful in showing that p94 is localized in the N2-line region of sarcomeres andin the nuclei, we examined the localization of p94 in single myofibers to obtain further
insight into p94 localization.

Single fiber staining can potentially give us information

about more precise 3-dimensional localization of the protein of interest within
individual cells.
Clear staining at the Z-lines where sarcomeric-α-actinin (s-α-actinin) locates(arrows in Figure 2) was observed using anti-pNS antiserum, being consistent with the
immuno-staining results shown previously (Sorimachi et al. 1995).

In addition,

anti-pNS antiserum gave weak signals in the region surrounding the Z-lines(arrowheads in Figure 2), which is likely to correspond to p94 binding to the N2A
regions of connectin. Since the myofibers used here were not treated with relaxationbuffer, sarcomeres observed in our experiments were relatively shortened (generally lessthan 1.9 µm).

Therefore, it was anticipated that the N2A regions of connectin was

tangled, i.e., tandem IG motifs were individually folded and packed together not being
regularly exposed for antiserum access.

This may be one of the reasons why p94

staining in the N2A regions was detected not as clear distinctive lines but as hazystructures.
Yeast two-hybrid studies showed that p94 interacts with the M-line region of11

Ojima, K. et al.

p94 and connectin-mediated signaling pathways.

connectin as well as the N2A regions of connectin (Sorimachi et al. 1995; Kinbara et al.
1997).

However, we did not observe p94 at the M-line region in sarcomeres of single

fibers prepared in this study.
sarcomeres?

Why p94 was not detected at the M-line regions in

One of the possibilities is that the used muscle fibers might lack the p94

binding site in the M-line connectin, i.e., “is7” region between the last two IG motifs,
M9 and M10 (Kinbara et al. 1997). The M-line region of connectin is subjected tovarious alternative splicing and several C-terminal domains, including is7 region, in the
M-line region of connectin, were shown to be excluded in fast myofibers (Labeit andKolmerer 1995).

Single myofibers used in this study were prepared from EDL

muscles, which are mostly composed of fast myofibers, and, thus, contained little
number of is7 region. This could explain the absence of p94 detection in the M-lines.
Second possibility is that the contractile status of sarcomeres might account for thechange of p94 localization.

When skeletal muscles exhibit spontaneous contractions,extension and contraction of sarcomeres might alter the local structure of p94 bindingsites in connectin affecting the localization of p94.

Therefore, when the sarcomeresare in its contracted status as was discussed above, it might be difficult that p94 binds to
the M-line region of connectin, leading to the loss of detection of p94 on the M-lines.
It is also possible that the epitope of anti-pNS antiserum, i.e., the N-terminal portion of
p94, is buried upon its binding to the M-line region of connectin.

The yeast

two-hybrid studies indicated that binding of p94 to the M-line connectin requires whole
molecule of p94 (Sorimachi et al. 1995), suggesting that the structure at the whole
molecule level is important for the interaction between p94 and the M-line connectin.

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p94 and connectin-mediated signaling pathways.

Discussion
Functions of p94 at the N2A region of connectin
Skeletal muscles always undergo physical stresses.

In particular, sarcomeric

as well as sarcolemmal components receive the mechanical stresses during spontaneouscontractions.

How can skeletal muscles sense mechanical stresses and prepare

themselves to dynamic change in extracellular and intracellular conditions?

Recently,

it has been proposed that the connectin and its associated protein complexes insarcomeres are involved in mechanical sensing and response (Granzier and Labeit 2004).
In particular, three regions of connectin, the Z-, N2A-, and M-line regions, are found tobind several signaling molecules as well as structural proteins to form “signal
complexes” (Figure 3). These signal complexes are considered to be important not
only for muscle functions but also for mechanisms of muscle diseases. For, a defect of
one of the molecules included in these complexes such as T-cap/telethonin, myotilin,
p94, and connectin itself causes various types of muscular dystrophies (Richard et al.
1995; Salmikangas et al. 1999; Hauser et al. 2000; Moreira et al. 2000; Hackman et al.
2002), and others such as MARPs and MURF1 are upregulated upon muscle atrophies(Bodine et al. 2001; Lecker et al. 2004; Nikawa et al. 2004; Witt et al. 2004).
In the N2-line region, the N2A element is one of the splicing variants ofconnectin, and consists of several immunoglobulin-like (IG) motifs as well as uniquesequences in between (Figure 3).
the N2A region of connectin.

p94 is the first molecule that was shown to bind to

p94 interacts with two IG motifs, I82 and I83, in the

N2A region, which is proximate to the N-terminus of the PEVK domain (Figure 3B)

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p94 and connectin-mediated signaling pathways.

(Sorimachi et al. 1995).
Muscle ankyrin repeat proteins (MARPs) also participate in the N2A protein
complex on connectin. Members of MARPs family include cardiac ankyrin repeat
protein (CARP) (Baumeister et al. 1997; Kuo et al. 1999), diabetes related ankyrin
repeat protein (DARP) (Ikeda et al. 2003), and ankyrin-repeat domain protein 2(Ankrd2/Arpp) (Pallavicini et al. 2001).

Although expression patterns of three

MARPS, CARP, DARP, and Ankrd2, are slightly different in striated muscles, they all
have an ability to interact with a tyrosine-rich binding motif between two IG motifs, I80
and I81, of connectin, where the N-terminus of the p94 binding site in the N2A region is
adjacent (Miller et al. 2003; Witt et al. 2004).
MARPs contain the nuclear localization signal, and possibly function astranscription regulators. For example, CARP regulates Nkx2.5-dependent muscle gene
expression both in cardiac and skeletal muscles during development (Baumeister et al.
1997).

CARP also forms complex with the transcription factor, Y-box protein 1a(YB-1a), to suppress expression of muscle-specific genes such as myosin heavy chain(MHC) genes (Zou et al. 1997).

It should be noted that a defect of MHC isoform

change was observed in transgenic mice over-expressing a splicing variant of p94
lacking IS1 region (Spencer et al. 2002). As p94 also contains potential nuclear
localization signal, p94 and MARPs can potentially shuttle between myofibrils andmyonuclei. Thus, using the N2A region of connectin as a scaffold, MARPs and p94
may interact with each other to form signal complex, which may transduce signals frommyofibril to the nuclei.

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p94 and connectin-mediated signaling pathways.

Skeletal muscle phenotypes of MDM (muscular dystrophy with myositis)
mice represent another indication for the importance of p94 and MARPs in the N2A
region.

The mdm allele contains a retro-transposon insertion that spontaneously

occurred within the connectin gene (Garvey et al. 2002).

This allele causes theexpression of a connectin lacking 83 amino acid residues in the N2A region, which
includes the p94 binding region.

Mouse homozygotes of mdm, Ttnmdm/mdm, show

severe muscular dystrophy phenotypes, and die within 3 months from birth (Garvey etal. 2002; Witt et al. 2004).

Intriguingly, CARP is more abundantly expressed inskeletal muscles of mdm mice than in those of wild type mice, whereas yeast two-hybrid
assay showed that the N2A region of mdm-type connectin could not bind p94 (Witt etal., 2004). These results show a possibility that a loss of the interaction between p94
and the N2A region causes up-regulation of CARP and severe muscle degeneration.
Considering the importance of CARP in transcriptional regulation of several muscle
genes, it is interesting to speculate that expression of several skeletal muscle genes
could be regulated by a balance between p94 and CARP, which may compete for the
N2A binding site because of the close proximity of both sites, and/or by proteolytic
regulation of CARP by p94. At least, mdm mice studies clearly indicate that the N2A
region of connectin plays critical roles in maintaining skeletal muscle homeostasis.

Functions of p94 at the M-line region of connectin
The M-line region of connectin is another attractive region when one
considers connectin-mediated signal transduction pathways. For, the M-line region of15

Ojima, K. et al.

p94 and connectin-mediated signaling pathways.

connectin contains (1) the binding site for MURF-1 (Centner et al. 2001), (2) aserine/threonine kinase domain (Labeit and Kolmerer 1995), and (3) another bindingsite for p94 (Sorimachi et al. 1995; Kinbara et al. 1997).
MURF-1 interacts with two IG-motifs, A168 and A169, of connectin, which islocated at periphery of the M-line (Figure 3C) (Centner et al. 2001).

Several lines ofevidence support that MURF-1 is a multi functional protein involved in skeletal muscle
homeostasis.

First, exogenously expressed MURF-1 disrupted the assembly of

myofibrillar proteins around the M-lines, suggesting that MURF-1 has a critical role inconstruction of connectin-based M-line structure (McElhinny et al. 2002). Secondly,
MURF-1 binds to glucocorticoid modulatory element binding protein-1 (GMEB-1),
which controls transcription in response to cellular glucocorticoid levels (McElhinny et
al. 2002), suggesting a role in regulating gene expression. Thirdly, MURF-1 has beenshown to function as ubiquitin ligase (Bodine et al. 2001), and one of splicing variantsof MURF-1 interacts with small ubiquitin related modifier-3 (SUMO-3) (Dai and Liew2001), suggesting that MURF-1 regulates protein turnover via ubiquitin and/or SUMO
systems.
Disruption of a part of the connectin gene corresponding to both the bindingsite for MURF-1 and the kinase domain resulted in embryonic lethality, suggesting that
MURF-1 binding and/or connectin kinase activity is essential for muscle development(Gotthardt et al. 2003). It is possible that the kinase activity is sterically modulated byassociation of MURF-1 with connectin. For, the MURF-1 binding site on connectin is
IG-motifs, A168 and A169, whereas the kinase domain is located between the next two

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p94 and connectin-mediated signaling pathways.

IG-motifs, A170 and M1.

The p94 binding site in the M-line region of connectin is

located between the two IG-motifs, M8 and M10, at the C-terminus of connectin (M9and a unique sequence called “is7”, see Figure 3C) (Sorimachi et al. 1995; Kinbara et al.
1997).

Since connectin molecules extending from the opposite side of sarcomere

overlap with each other in the M-line region, p94 bound to the M-line connectin cancontact with MURF-1 binding to another connectin extending in the reverse direction(Figure 3C) (Gregorio et al. 1999). Thus, there might be functional interaction
between p94 and MURF-1 such as proteolysis of MURF-1 by p94 at least in response tocertain signal(s).
In tibial muscular dystrophy (TMD), skeletal muscles of anterior lower legs are
preferentially affected and show atrophy (Udd et al. 1993). TMD is caused bymutations in the extremely C-terminal IG-motif of connectin, namely, M10, which islocated next to the p94 binding site in the M-line connectin. In TMD muscles, theexpression of myomesin, which binds to IG-motif M4 of the M-line connectin, wasshown to be intact, suggesting that the sarcomeric localization of M4 epitope of
connectin is not altered (Hackman et al., 2002). It is, however, found that in TMD
skeletal muscles, M8 and M9 regions of M-line connectin, which is N-terminal to theaffected M10, could not be immunohistologically detected. Therefore, the mutation in
M10 might disrupt the structure of its proximity, and as Hackman and colleagues
discussed, binding of p94 to the adjacent M9+is7 region may be compromised.

On theother hand, yeast two-hybrid study showed that only M9+is7 region is enough forinteracting with p94 (Kinbara et al., 1997).

At present, it is unclear whether or not p94

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p94 and connectin-mediated signaling pathways.

is dissociated from the C-terminal region of connectin in TMD skeletal muscles.

As

described above, a peripheral region of the M-line connectin contains the kinase domain
and the binding sites for MURF-1 and p94, forming a possible signal complex.
mutation must perturb functions of this signal complex.

TMD

Given the facts that the heart

muscle is not affected in TMD patients, it is predicted that molecular mechanisms of
TMD pathogenesis involves the components of skeletal muscle specific signal complex
at the M-line region of connectin. Since p94 is not expressed in mature heart, it ispossible that p94 in the M-line region would play a critical role(s).

Possible functions of p94 in the Z-lines
Our immunohistochemical studies showed clear staining of p94 in the Z-lines(Figure1) (Sorimachi et al. 1995). The Z-line region of connectin, however, is not
responsible for p94 binding (data not shown), or an alternate binding partner for p94 in
Z-lines has not yet been identified. Besides p94, the Z-lines contains various protein
components including the N-terminal region of connectin, s-α-actinin (Sorimachi et al.
1997), T-cap/telethonin (Gregorio et al. 1998; Mues et al. 1998), myotilin (Salmikangas
et al. 1999), subunit of K+-channel (minK) (Furukawa et al. 2001), and barbed end ofthin filament protein complex, etc. The importance of the Z-line structure has been
discussed, e.g., as to that it links myofibrils to sarcolemma through costameres (Evarsti2003).

Mutations in the genes for T-cap/telethonin and myotilin cause limb-girdle

muscular dystrophies, more directly suggesting that complex in the Z-line is alsoessential for proper functions of muscles. Thus, again, it is tempting to imagine a18

Ojima, K. et al.

p94 and connectin-mediated signaling pathways.

signal complex specifically formed at the sarcomere Z-line regions where p94 also plays
a crucial role in signal transduction pathway linking sarcolemma and the nuclei.
p94 is the unique mutual factor identified in the Z-, N2-, and M-line region.

So far,It might

be, therefore, the case that p94 facilitates coordinated signal transduction by adjusting
distinct signal complex in each region.

19

Ojima, K. et al.

p94 and connectin-mediated signaling pathways.

Conclusion
The concept of “connectin- and p94-mediated stress response pathways” has
now become more convincing based on immunohistological data, biochemical analyses
as well as genetic studies related to skeletal muscle pathology.

Under the normal

conditions, in skeletal muscles, p94 binds to connectin where p94 activity may besuppressed to keep a pool of inactive form of p94 in sarcomeres. Detailed evaluationof immunostained single myofibrils suggested that the localization of p94 in thesarcomere Z-, N2A, and M-line regions might be regulated under the muscle conditions
such as contractility.
The connectin-bound, quiescent p94 molecules are in close proximity to otherconnectin binding proteins including MARPs, MURF-1, etc.

When skeletal muscles

receive mechanical stresses/signals, p94 could dissociate from connectin, transform into
an active protease in the myonuclei and other intracellular regions of muscle cells.
These activities might prompt appropriate cellular responses, such as gene expressions,in coordinate with other connectin binding proteins.

Although much has yet to beelucidated, the impact of recent studies on p94 including this study is that a greatchange of viewpoints on physiological properties not only of p94 but also of connectinhave been introduced. In other words, p94 is not just a protease binding to asarcomeric protein, and connectin is not just a platform for various protein ligands, butrather, together, these molecules work as unique devices crucially important for muscle
homeostasis.

p94 has brought about a great change of viewpoints on physiological

functions of connectin, and, vice versa, so has connectin on those of p94.

20

Ojima, K. et al.

p94 and connectin-mediated signaling pathways.

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p94 and connectin-mediated signaling pathways.

Acknowledgement
We would like to thank Dr Siegfried Labeit and Dr Christian Witt in
Universitätsklinikum Mannheim, Dr Carol C. Gregorio in University of Arizona, and Dr
Sumiko Kimura in University of Chiba for valuable discussions.

This work was

supported in part by a Grant-in-Aid for Scientific Research on Priority Areas (Cell
Cycle) from the Ministry of Education, Science, Sports and Culture, a Grant-in-Aid for
Scientific Research and Research Fellowships for Young Scientists from JSPS (theJapan Society for the Promotion of Science), a Research Grant (14B-4) for Nervous andMental Disorders from the Ministry of Health, Labour and Welfare, and “Ground-based
Research Announcement for Space Utilization” promoted by the Japan Space Forum.
This paper is dedicated to the memory of Prof. Koscak Maruyama.

We all miss him.

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p94 and connectin-mediated signaling pathways.

Figure Legends
Fig. 1. Schematic domain structures of p94 and the conventional calpain (µ-, andm-calpain) large subunits.
I, IIa+IIb, III, and IV represent the four calpain domains.

NS, IS1, and IS2 are

p94-specific insertion sequences. Domains I, and III are α-helical, and C2-like
Ca2+-binding domains, respectively. Domain IV contains 5 EF-hand motifs, also
involved in Ca2+-binding.

Subdomains IIa and IIb compose the protease domain when

activated. The active center of calpains is formed by Cys, His, and Asn indicated in
the domain II.

Fig. 2. Low magnification (A-C) and high magnification (D-E) confocal micrographsdemonstrating the localization of p94 and sarcomeric-α-actinin (s-α-actinin).
Single myofibers were stained with anti-s-α-actinin antibody (red in A, C, D, and F) andanti-pNS antiserum (green in B, C, E, and F).
A-B and D-E, respectively.

C and F are superimposed images ofAnti-pNS antiserum clearly visualized p94 in Z-lines,

where anti-s-α-actinin was positive (arrows).

Further, anti-pNS antiserum detectedfaint patterns around Z-lines (arrowheads). These staining could be p94 in the N2A
region of connectin.

Since the N2A region of connectin was not stretched enough(approximately, the length of sarcomeres were less than 1.9 µm), epitopes of antiserum
may not have aligned in order.

Clear M-line staining of p94 was not detected (see text

for possible reasons). Bars indicate 20 µm and 5 µm in C and F, respectively.

29

Ojima, K. et al.

p94 and connectin-mediated signaling pathways.

Fig. 3. A model of p94- and connectin-mediated signal transduction pathways inskeletal muscles.
(A) p94, connectin and associated proteins in a sarcomere
p94 binds to connectin at the N2A and M-line regions based on yeast two hybrid studies
and is also localized in the Z-lines shown by immunofluorescent studies (see Figure 2).
Muscle ankyrin repeat proteins (MARPs), including cardiac ankyrin repeat protein(CARP), diabetes related repeat protein (DARP), and ankyrin-repeat domain protein 2(Ankrd2), are able to interact with the N2A region of connectin.

MURF-1 is located in

the M-line. T-cap, MARPs, myotilin, and s-α-actinin are involved in Z-line protein
complexes.

Ovals with broken lines indicate each p94- and connectin-mediated signaltransduction complexes. Z, N2A, and M indicate Z-lines, N2A- and M-lines,
respectively.
(B) Protein complex at the N2A region of connectin
p94 binds to I82/I83 of immunoglobulin-like (IG) domains, which are adjacently
located to the N-terminus of the PEVK domain.

MARP family interacts with atyrosine rich binding motif between I80 and I81. Two-headed arrow indicates thedeletion site of connectin in mdm mice.
(C) Protein complex at the M-line of connectin
p94 binds to M9+is7 at the periphery of the M-lines.
A168/A169 IG domains of connectin.

MURF-1 interacts with

Connectin molecules run in an antiparallel

fashion and intersect at the Centner of the M-line. p94 could interact with MURF-1
and the kinase domain of the other side of connectin at the periphery of M-lines.

30

Ojima, K. et al.

p94 and connectin-mediated signaling pathways.

Arrows indicate the mutated site in tibial muscular dystrophy (TMD) patients.

M10 isthe last C-terminal domain of connectin.

31

Ojima, K. et al., Figure 1

Cys

HisAsnp94/Calpain 3

NS

IIa

IS1

µCL, mCL

I

IIa

IIb

Cys

HisAsn

IIb

III

III

IS2

IV

IV

Ojima, K. et al., Figure 3

A
Z

M

Z
thin filament

p94
T-cap
MARPs
α-actinin
myotilinp94
MURF1
MURF-1

MARPs

p94

p94

p94

MURF1
MURF-1

N2A

thick filament

p94

T-cap

MARPs

MARPs

connectin

α-actinin
myotilin

N2A

B
connectin
N-terminal MARPs
I80

connectin
C-terminal

p94
I81

I82

I83

PEVK

deletion in mdm

CM-lineconnectin
N-terminal

is1

A167 A168 A169 A170 kinase

MURF1
MURF-1

M1

is2

M2 M3

is3

M4

is4

M5

is5

M6

is6
M7

is7

M8 M9

p94

p94

M10

M9 M8
is7

M10

is6

M7

M6
is5

M5
is4

M4
is3

M3 M2
is2

MURF1
MURF-1M1 kinase A170A169 A168 A167
is1

connectin
N-terminal