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The Journal of Neuroscience, May 1, 2001, 21(9):3151-3160
Src, Fyn, and Yes Are Not Required for Neuromuscular Synapse
Formation But Are Necessary for Stabilization of Agrin-Induced Clusters
of Acetylcholine Receptors
Cynthia L.
Smith1,
Peggy
Mittaud2,
Elizabeth
D.
Prescott1,
Christian
Fuhrer2, and
Steven J.
Burden1
1 Molecular Neurobiology Program, Skirball Institute
for Biomolecular Medicine, New York University Medical School, New
York, New York 10016, and 2 Brain Research Institute,
University of Zürich-Irchel, CH-8057 Zürich, Switzerland
 |
ABSTRACT |
Mice deficient in src and fyn or
src and yes move and breathe poorly and
die perinatally, consistent with defects in neuromuscular function. Src
and Fyn are associated with acetylcholine receptors (AChRs) in
muscle cells, and Src and Yes can act downstream of ErbB2,
suggesting roles for Src family kinases in signaling pathways regulating neuromuscular synapse formation. We studied neuromuscular synapses in src
/;
fyn/ and
src/; yes/
mutant mice and found that muscle development, motor axon pathfinding, clustering of postsynaptic proteins, and synapse-specific transcription are normal in these double mutants, showing that these pairs of kinases
are not required for early steps in synapse formation. We generated
muscle cell lines lacking src and fyn and
found that neural agrin and laminin-1 induced normal clustering of
AChRs and that agrin induced normal tyrosine phosphorylation of the AChR 
subunit in the absence of Src and Fyn. Another Src family member, most likely Yes, was associated with AChRs and phosphorylated by agrin in myotubes lacking Src and Fyn, indicating that Yes may
compensate for the loss of Src and Fyn. Nevertheless, PP1 and
PP2, inhibitors of Src-class kinases, did not inhibit agrin signaling,
suggesting that Src class kinase activity is dispensable for
agrin-induced clustering and tyrosine phosphorylation of AChRs. AChR
clusters, however, were less stable in myotubes lacking Src and Fyn but
not in PP1- or PP2-treated wild-type cells. These data show that the
stabilization of agrin-induced AChR clusters requires Src and Fyn and
suggest that the adaptor activities, rather than the kinase activities,
of these kinases are essential for this stabilization.
Key words:
AChR; Src; Yes; cytoskeleton; agrin; neuromuscular
synapse
| |
INTRODUCTION |
Two distinct signaling pathways,
each activated by receptor tyrosine kinases (RTKs), are thought to have
an important role in neuromuscular synapse formation. Agrin, an ~200
kDa protein expressed in motor neurons, stimulates MuSK, a skeletal
muscle-specific RTK, and activates a signaling pathway that leads to a
redistribution of several proteins, including acetylcholine receptors
(AChRs), to newly formed synapses (Fallon and Gelfman, 1989
; McMahan,
1990; Glass and Yancopoulos, 1997; Sanes and Lichtman, 1999). In
addition to this post-translational pathway for postsynaptic
differentiation, locally provided synaptic signals activate
transcription of several genes, including AChR
subunit genes, in the synaptic nuclei of developing and adult muscle,
leading to enhanced AChR synthesis at synaptic sites (Merlie and Sanes,
1985; Klarsfeld et al., 1991; Simon et al., 1992). Neuregulin-1
(NRG-1), which is expressed by motor neurons and skeletal muscle and is
concentrated at neuromuscular synapses, is currently the best candidate
for the signal that activates synapse-specific transcription (Fischbach
and Rosen, 1997; Burden, 1998). NRG-1 activates ErbBs, members
of the epidermal growth factor receptor family of RTKs, and ErbB
stimulation is thought to activate a signaling pathway in muscle cells
that culminates in enhanced transcription of certain genes, including
AChR subunit genes, in nuclei near the activated ErbB
receptor (Carraway and Burden, 1995; Lemke, 1996). The NRG-1 and agrin
signaling pathways, which contribute to the high density of AChRs in
the postsynaptic membrane, may be linked, because agrin can induce
AChR gene expression in cultured myotubes (Jones et al.,
1996), and this induction is dependent on signaling through ErbB2
(Meier et al., 1998).
Despite the reported roles for calcium, Rac, and Cdc42 in
agrin-induced clustering of AChRs (Megeath and Fallon, 1998; Weston et
al., 2000), little is known about signaling downstream from MuSK and
how agrin stimulates clustering and tyrosine phosphorylation of AChRs
(Wallace et al., 1991; Ferns et al., 1996). Because the kinase
inhibitor staurosporine inhibits agrin-stimulated clustering and
phosphorylation of AChRs, without blocking tyrosine phosphorylation of
MuSK (Wallace, 1994; Ferns et al., 1996; Fuhrer et al., 1997), at least
one kinase is downstream of MuSK and necessary for agrin signaling. The
downstream kinase(s) linking MuSK activation to clustering and
phosphorylation of AChRs may include an Src-like kinase, because Src
and Fyn are associated with AChRs and Src can phosphorylate the AChR
subunit in vitro (Fuhrer and Hall, 1996; Fuhrer et al.,
1997; Mohamed and Swope, 1999). Furthermore, agrin causes activation
and tyrosine phosphorylation of AChR-associated Src family kinases, a
process that requires rapsyn and correlates with AChR clustering
(Mittaud et al., 2001). Signaling downstream from ErbBs has been
studied more extensively, and these studies have shown that several
signaling molecules, including Shc, Ras, mitogen-activated protein
kinases (MAPKs), and SHP2, act downstream from ErbBs in
muscle (Si et al., 1996; Tansey et al., 1996; Altiok et al., 1997;
Tanowitz et al., 1999; Won et al., 1999). Src and Yes can act
downstream of ErbB2 in mouse mammary tumor cells (Muthuswamy et al.,
1994; Muthuswamy and Muller, 1995) and might therefore participate in
synapse-specific transcription in muscle.
The Src family kinases, Src, Fyn, and Yes, are expressed in numerous
cell types, including neurons and muscle, and their activities are
implicated in multiple signaling pathways (Abram and Courtneidge, 2000). Mice deficient in src, fyn, or
yes, nevertheless, show a limited phenotype (Soriano et al.,
1991; Appleby et al., 1992; Grant et al., 1992; Stein et al., 1992,
1994; Umemori et al., 1992; Osterhout et al., 1999). The overlapping
expression patterns and activities of Src family kinases suggest that
the restricted phenotype of these single mutant mice may be
attributable to compensation by other Src family members.
Indeed, mice lacking multiple members of the Src family exhibit a more
severe and complex phenotype than mice lacking a single family member
(Stein et al., 1994). For example,
src/;
fyn/ and
src/;
yes/ double mutant mice move and
breathe poorly at birth and die perinatally, and
src/;
fyn/;
yes/ triple mutant mice die at
embryonic day 9.5 (E9.5) (Klinghoffer et al., 1999).
Because Src family kinases have been implicated in signaling pathways
at the neuromuscular synapse and mice deficient in combinations of
these kinases have a phenotype consistent with defects in neuromuscular function, we studied the formation of neuromuscular synapses in src/;
fyn/ and
src/;
yes/ double mutant mice. We
found that muscle development, motor axon pathfinding, clustering of
postsynaptic proteins, and synapse-specific transcription are normal in
these double mutants. Agrin-induced AChR clusters, however, are less
stable in cultured myotubes lacking Src and Fyn, demonstrating that the
stability of AChR clusters depends on Src and Fyn.
| |
MATERIALS AND METHODS |
Immunohistochemistry. Diaphragm muscles were
dissected from E18.5 embryos (five embryos for each genotype), fixed
for 90 min in 1% formaldehyde, rinsed in PBS, and incubated with 0.1 M glycine in PBS for 15 min. After dissection of
the overlying connective tissue, the muscles were permeabilized in
0.5% Triton X-100 in PBS for 5 min, incubated overnight at 4°C with
rabbit polyclonal antibodies against neurofilament (1:500; Chemicon,
Temecula, CA) and synaptophysin (1:5; Zymed, San Francisco, CA)
in 2% BSA in PBS, washed three times for 20 min in 0.5% Triton X-100
in PBS, and incubated for 3 hr at room temperature with
fluorescein-conjugated goat anti-rabbit IgG (1:200; Jackson
ImmunoResearch) and Texas Red-conjugated 
-bungarotoxin (-BGT)
(Molecular Probes, Eugene, OR). The muscles were washed twice for 20 min in 0.5% Triton X-100 in PBS, twice for 20 min in PBS, post-fixed
in 1% formaldehyde for 10 min, rinsed in PBS, flat-mounted in
Vectashield (Vector Laboratories, Burlingame, CA), and viewed
with optics selective for either fluorescein or Texas Red.
Frozen sections (10 µm) from unfixed E18.5 limbs (two to three
embryos for each genotype) were labeled with antibodies as described
previously (Zhu et al., 1995; DeChiara et al., 1996). The following
primary antibodies were used: affinity-purified rabbit anti-rapsyn
(1:100), mouse anti-utrophin (1:10), rabbit anti-AChE (1:1000; Dr. T. Rosenberry, Mayo Clinic, Jacksonville, FL), rabbit anti-ErbB4
[1:2000 of antibodies #616 (Zhu et al., 1995)], and rabbit anti-MuSK
[1:1000 (Herbst and Burden, 2000)].
In situ hybridization. Ribs and attached intercostal muscles
were dissected from E18.5 src/;
fyn/,
src/;
yes/, and wild-type littermate
control embryos (three to five embryos for each genotype), fixed
overnight in 4% formaldehyde, and embedded in OCT (Tissue Tek, Miles
Inc., Elkhart, IN). Frozen longitudinal sections (10 µm) were
collected on Superfrost slides (Fisher Scientific, Houston, TX) and
processed for in situ hybridization as described previously
(DeChiara et al., 1996) using a RNA probe derived from the
AChR subunit and viewed with dark-field optics.
35S-Radiolabeled sense and antisense
probes were transcribed from T7 or T3 promoters as described previously
(Simon et al., 1992).
Cell culture and production of mutant muscle cell lines.
Mice that were heterozygous for src (C57BL/6J × 129S7/SvEvBrd hybrid), fyn (C57BL/6J × 129S7/SvEvBrd
hybrid), or yes (129S7/SvEvBrd) were purchased from The
Jackson Laboratory (Bar Harbor, ME). These mice were interbred and
maintained on a hybrid C57FL/6J and 129S7/SvEvBrd background. Mice that
were heterozygous for MuSK, generously provided by Regeneron
Pharmaceuticals (Tarrytown, NY), were extensively backcrossed into a
C57BL/6 background. Muscle cell lines from src/,
fyn/,
src/;
fyn/, or wild-type littermate
control embryos, which carried a copy of the H-2Kb-tsA58 transgene,
were derived essentially as described previously (Herbst and Burden,
2000). Embryos were genotyped, limbs were dissected free from bones,
tissue was dissociated in 2% trypsin (Sigma, St. Louis, MO) and 0.01%
DNase (Sigma) in PBS, and cells were resuspended in DMEM containing
glutamine, 10% fetal bovine serum (Gemini Bio-Products, Calabasas,
CA), 10% horse serum, 2% chick embryo extract (Life Technologies,
Gaithersburg, MD), penicillin-streptomycin, and 20 U/ml recombinant
mouse interferon-
(Life Technologies). Cells were preplated on a
Petri dish for 20 min at 33°C to preferentially deplete connective
tissue cells, and the less-adherent cells in the supernatant were
transferred to Matrigel-coated tissue culture dishes. Clones of cells
were isolated and expanded under permissive conditions and checked for
their ability to differentiate into myotubes by culturing in DMEM
containing glutamine, 10% fetal bovine serum (Gemini Bio-Products), 10% horse serum, 2% chick embryo extract (Life Technologies), and
penicillin-streptomycin at 39°C. The genotypes of established mutant
cell lines were reconfirmed by PCR. The mutations in src and
fyn, generated by introducing a PGK-neomycin gene
into the first coding exon, result in protein null mutations (Soriano
et al., 1991; Stein et al., 1992). Because
src/;
fyn/;
yes/ triple mutant embryos die
at E9.5 (Klinghoffer et al., 1999), ~1 d before the appearance of
myoblasts, we could not isolate muscle cell lines from triple mutant embryos.
AChR clustering assay. Myoblasts were induced to
differentiate into myotubes by growing cells in differentiation medium
at 39°C for 3 d. Myotubes were treated overnight with 0.5 nM recombinant neural agrin N4 (Hoch et al.,
1994) or 60 nM laminin-1 (Sigma). Src class
kinase inhibitors, PP1 and PP2 (5 µM),
were added to cultures for 24 hr before the addition of agrin or
laminin-1, and a fresh aliquot of inhibitor was added together with
agrin; this concentration of PP1 and PP2 is 1000-fold greater than the IC50 for Lck and Fyn (Hanke et al., 1996) and 5- to 10-fold greater than reported to fully inhibit Src kinase activity
in a variety of cell types (Hanke et al., 1996; Liu et al., 1999;
Mocsai et al., 1999; Osterhout et al., 1999). Similar results were
obtained with a 1-5 hr preincubation time of PP1 and PP2 and without
further addition of inhibitor together with agrin. We also used
CGP77675, a different and more potent Src family kinase inhibitor
(Novartis, Summit, NJ), at 0.1-60 µM (Missbach
et al., 1999). To analyze the stability of agrin-induced AChR clusters,
myotubes were treated overnight with neural agrin and subsequently
maintained in differentiation medium lacking agrin for 1-5 hr. For
treatment with PP1 and PP2, all media contained the inhibitors,
including a 24 hr pretreatment before addition of agrin. Myotubes were
fixed in 1% formaldehyde and stained with Texas Red-conjugated -BGT
(1:5000) in 2% BSA in PBS. Myotubes were washed in PBS, post-fixed in
1% formaldehyde, and mounted under a coverslip. AChR clusters were
counted from 20 or more random fields in each experiment, and the
mean ± SEM number of clusters per field was determined.
Affinity purification of AChRs and Western blotting. Limbs
from E18.5 embryos were homogenized in a Polytron in ice-cold
buffer (50 mM NaCl, 30 mM
triethanolamine, pH 7.5, 5 mM EGTA, 5 mM EDTA, 50 mM NaF, 2 mM Na orthovanadate, 50 mM
Na pyrophosphate, 10 mM p-nitrophenylphosphate, 1 mM
benzamidine, 1 mM PMSF, and 25 µg/ml each of
aprotinin, leupeptin, and pepstatin). An equal volume of ice-cold
buffer containing 2% NP-40 was added, the lysate was extracted for 30 min at 4°C, and insoluble material was removed by centrifugation for
5 min at 4°C.
Total protein (2.5 mg) from cleared lysates were incubated with
biotin-conjugated -BGT (Molecular Probes) for 30 min at 4°C, followed by the addition of streptavidin-coupled agarose beads (Sigma)
and incubated at 4°C for 1 hr. Bound proteins were eluted from the
beads by heating the samples to 80°C for 5 min in SDS-PAGE sample
buffer. Proteins were resolved in 10% polyacrylamide gels and
transferred to polyvinylidene difluoride membranes.
Tyrosine-phosphorylated proteins were detected by probing membranes
with antibodies to phosphotyrosine (4G10; Upstate Biochemicals, Lake
Placid, NY), followed by incubation with horseradish
peroxidase-conjugated secondary antibodies (Jackson ImmunoResearch,
West Grove, PA). Labeled bands were visualized with enhanced
chemiluminescence (ECL; Amersham Pharmacia Biotech, Arlington Heights,
IL). Blots were stripped in 10% acetic acid for 2 hr at room
temperature and reprobed with antibodies to the AChR subunit
[monoclonal antibody 124 (mAb124)].
AChRs were similarly isolated from cultured myotubes. Briefly, myotubes
were stimulated for 30-40 min with 0.5 nM neural agrin. AChRs were precipitated from cellular lysates by biotinylated -BGT
and streptavidin-agarose or by -BGT covalently coupled to Sepharose
beads (Fuhrer et al., 1997). Phosphorylation of AChR subunits and
of AChR-bound Src family kinases was analyzed by phosphotyrosine
immunoblotting as described above. AChR-associated Src family kinases
were detected by blotting with Src-CT, an antiserum reactive
with Src, Fyn, and Yes in muscle; kinase-specific antibodies were used
to detect individual kinases in total cellular extracts of myotubes
(Fuhrer and Hall, 1996). For quantitation of Yes in immunoblots of cell
extracts, films were scanned using a computerized densitometer
(Scantouch 210; Nikon, Tokyo, Japan) and NIH Image J 1.04b software.
Signals were normalized for the AChR subunit detected in parallel
samples using mAb124.
| |
RESULTS |
Motor axons innervate skeletal muscle in
src/; fyn/
and src/;
yes/ embryos
src/;
fyn/ and
src/;
yes/ mice fail to move or
breathe regularly and die shortly after birth, suggesting a potential
deficit in neuromuscular function (Stein et al., 1994). We studied
synapse formation in diaphragm muscles because its thin structure
allows synaptic sites to be readily visualized in whole-mount
preparations. The main intramuscular nerve, visualized by staining with
antibodies to neurofilament, is oriented perpendicular to the long axis
of the muscle fibers and extends through the central region of the muscle (Fig. 1A).
Branches of the main intramuscular nerve terminate adjacent to the main
nerve, and these nerve terminals can be visualized by staining with
antibodies to synaptophysin, a synaptic vesicle protein (Fig.
1A, insets). AChRs, visualized by
staining with Texas Red-conjugated -BGT, are clustered in the muscle
membrane at these synaptic sites (Fig. 1B).
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Figure 1.
Motor axons innervate skeletal muscle in
src/;
fyn/ and
src/;
yes/ embryos. Whole mounts of
E18.5 diaphragm muscle were stained with antibodies to neurofilaments
(NF) to label axons, with antibodies to
synaptophysin (SYN, insets) to label
nerve terminals and with Texas Red-conjugated -BGT to label AChRs.
Synaptic sites in wild-type muscle (wt) are located
adjacent to the main intramuscular nerve and are characterized by
arborized nerve terminals (insets) and clustered AChRs
(-BGT). The position of intramuscular axons,
the branching of nerve terminals, and the pattern of AChRs is similar
in src/;
fyn/,
src/;
yes/, and wild-type
embryos.
|
|
Innervation of the diaphragm muscle in
src/;
fyn/ E18.5 embryos is normal.
The main intramuscular nerve is positioned properly in the central
region of the diaphragm muscle, the arrangement and structure of the
muscle fibers appears normal, and the size and shape of nerve terminals
are similar to those in wild-type mice (Fig.
1C,D). Innervation is likewise unaffected in
src/;
yes/ mutant mice (Fig.
1E,F). Although the width of
the endplate zone is slightly narrower in
src/;
yes/ double mutants, the length
of the entire muscle, like that of the embryo, is reduced ~25%
(Stein et al., 1994), and the decreased width of the endplate zone is
proportional to the decreased size of the muscle. Furthermore, the size
and shape of nerve terminals and AChR clusters are normal (Fig.
1E,F, insets).
Therefore, expression of Src and Fyn or Src and Yes are not essential
for the generation, proliferation, and fusion of myoblasts, the growth
of spinal motor axons to muscle, or the differentiation of nerve terminals.
Postsynaptic proteins are clustered normally in skeletal muscle of
src/;
fyn/ and
src/;
yes/ embryos
Neural agrin, released from motor nerve terminals, activates MuSK
and induces clustering of several proteins, including AChRs, at
postsynaptic sites in skeletal muscle (Burden, 1998; Sanes and
Lichtman, 1999). Because Src and Fyn have been proposed to act
downstream from MuSK (Fuhrer et al., 1997), we examined whether synaptic proteins, which are normally clustered by agrin, are clustered
at synaptic sites in src/;
fyn/ and
src/;
yes/ mutant embryos.
We stained frozen sections of E18.5 hindlimb muscle with Texas
Red-conjugated -BGT to mark AChR clusters at postsynaptic sites and
with antibodies to rapsyn, utrophin, acetylcholinesterase, ErbB4, or
MuSK. We found that each of these proteins, which are clustered at the
postsynaptic membrane in normal mice, is coclustered with AChRs in
hindlimb muscle from
src/;fyn/
and src/;
yes/ mutant embryos (Fig.
2). Thus, postsynaptic proteins, which
are normally clustered by agrin, are concentrated at neuromuscular synapses in src/;
fyn/ and
src/;
yes/ mutant embryos.
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Figure 2.
Postsynaptic proteins are concentrated at synaptic
sites in src/;
fyn/ and
src/;
yes/ embryos. Frozen sections of
muscle from E18.5 hindlimbs were stained with Texas Red-labeled -BGT
and antibodies to rapsyn (RAP), utrophin
(UTR), acetylcholinesterase (AChE),
ErbB4, and MuSK. Rapsyn, utrophin, acetylcholinesterase, ErbB4, and
MuSK are colocalized with AChRs in muscle from
src/;
fyn/ (B)
and src/;
yes/ (C)
embryos, as in muscle from wild-type embryos
(A).
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|
Synapse-specific transcription is normal
in src/;
fyn/ and
src/;
yes/ mutant embryos
Synapse-specific transcription of AChR subunit genes
leads to localization of AChR mRNAs at synaptic sites and
contributes to accumulation of AChR protein in the postsynaptic
membrane (Burden, 1998; Sanes and Lichtman, 1999). To determine whether
expression of Src and Fyn or Src and Yes are required for
synapse-specific transcription, we examined whether AChR
mRNAs are restricted to the endplate zone of muscle from
double mutant mice. We found that AChR subunit
transcripts are concentrated normally in the endplate zone of muscle
from src/;
fyn/ and
src/;
yes/ mutant embryos (Fig.
3). AChR 
subunit mRNA is
likewise patterned normally in muscle from these mutant embryos (data
not shown). Thus, Src and Fyn are not necessary for synapse-specific
transcription.
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Figure 3.
AChR subunit gene expression is
restricted to synaptic sites in
src/;
fyn/ and
src/;
yes/ mutant embryos. Frozen
longitudinal sections of E18.5 intercostal muscles from wild-type,
src/;
fyn/, and
src/;
yes/ embryos were hybridized to
an AChR subunit antisense probe. The positions of ribs
(R) and attached intercostal muscles
(M) are indicated. AChR subunit transcripts are localized to the endplate zone
(arrows) in muscles from wild-type,
src/;
fyn/, and
src/;
yes/ embryos. Labeling with a
sense subunit probe is uniform and not greater than the background
from the emulsion (data not shown).
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Src and Fyn are not required for tyrosine phosphorylation of the
AChR subunit
Because Src can phosphorylate the AChR subunit in
vitro and because Src and Fyn are associated with AChRs in
cultured muscle cells (Fuhrer and Hall, 1996; Fuhrer et al., 1997), we
asked whether the AChR subunit is phosphorylated in
src/;
fyn/ and
src/;
yes/ mutant embryos. We prepared
muscle lysates from wild-type and mutant embryos, affinity-purified
AChRs with biotin-conjugated -BGT, fractionated AChR subunits by
SDS-PAGE, and probed Western blots with antibodies to the AChR subunit or to phosphotyrosine. We found that the AChR subunit is
phosphorylated normally in src/,
fyn/,
yes/,
src/;
fyn/, and
src/;
yes/ mutant embryos (Fig.
4). Because MuSK is necessary for
agrin-induced AChR phosphorylation in cultured myotubes, we analyzed,
as a control, AChR phosphorylation in MuSK mutant
embryos and found it to be strongly reduced (Fig. 4).
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Figure 4.
The AChR subunit is
tyrosine-phosphorylated
in src/;
fyn/ and
src/;
yes/ embryos. AChRs were
affinity-purified, using biotin-conjugated -BGT, from limbs of
wild-type, MuSK/,
src/,
fyn/,
yes/,
src/;
fyn/, and
src/;
yes/ embryos, and the subunits
were fractionated by SDS-PAGE. Western blots were probed with
antibodies to phosphotyrosine (4G10; anti-pTyr),
stripped, and reprobed with antibodies to the AChR subunit
(mAb124). The AChR subunit is tyrosine-phosphorylated in wild-type,
src/,
fyn/,
yes/,
src/;
fyn/, and
src/;
yes/ embryos but not in
MuSK/ embryos.
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Although these results suggest that Src, Fyn, and Yes are dispensable
for agrin-induced clustering and phosphorylation of the AChR subunit, it is possible that ligands other than agrin stimulate
clustering of AChRs and tyrosine phosphorylation of the subunit
in vivo. Therefore, we measured AChR clustering and subunit phosphorylation in src/,
fyn/, or
src/;fyn/
mutant muscle cells that were grown in vitro and treated
with agrin.
We derived muscle cell lines from wild-type and mutant embryos by
crossing the H2KTts transgene into mice that were heterozygous for
either src or fyn and intercrossing double
heterozygotes that carried the transgene (Jat et al., 1991; Morgan et
al., 1994). Single or double mutant muscle cells grew and fused to form
differentiated myotubes, although adhesion to the substrate was weaker
in the case of double mutant cells. Thus, early steps of muscle
development in cell culture are primarily normal in the absence of Src
and Fyn, consistent with our findings that these kinases are
dispensable for muscle development in vivo. Importantly, we
found that agrin stimulates normal clustering of AChRs in muscle cells
lacking Src, Fyn, or Src and Fyn (Fig.
5A,B).
Thus, the ability of myotubes to cluster AChRs in response to agrin is
not dependent on expression of Src and Fyn.
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Figure 5.
Muscle cells lacking Src and/or Fyn respond
normally to agrin. A, Myotubes, derived from wild-type,
src/,
fyn/, or
src/;
fyn/ mice, were treated with
neural agrin for 18 hr and stained with Texas Red-conjugated -BGT to
label AChR clusters. Agrin induces clustering of AChRs in wild-type,
src/,
fyn/, and
src/;
fyn/ myotubes, and the size and
shape of AChR clusters is similar in all cases. B, The
number of agrin-induced AChR clusters in wild-type myotubes was
assigned as 100%, and all other values are expressed relative to wild
type. Agrin stimulates a similar increase in the number of AChR
clusters in wild-type, src/,
fyn/, and
src/;
fyn/ myotubes. Myotubes not
treated with agrin had few, if any, AChR clusters (data not shown; see
Fig. 6). C, Agrin stimulates tyrosine phosphorylation of
the AChR subunit in wild-type,
src/,
fyn/, and
src/;
fyn/ myotubes. Wild-type and
mutant myotubes were treated with neural agrin, and AChRs were
affinity-purified and fractionated by SDS-PAGE. Western blots were
probed with antibodies to phosphotyrosine (4G10), stripped, and
reprobed with antibodies to the AChR subunit (mAb124). Agrin
stimulates normal tyrosine phosphorylation of the AChR subunit in
src/,
fyn/, and
src/;
fyn/ myotubes. We examined two
wild-type, two src/, three
fyn/, and two
src/;
fyn/ cell lines and found that
each cell line responded normally to agrin.
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To determine whether Src or Fyn are required for agrin-induced tyrosine
phosphorylation of the AChR subunit, we treated wild-type or mutant
myotubes with agrin, isolated AChRs, and probed Western blots with
antibodies to the AChR subunit or with antibodies to
phosphotyrosine. We found that agrin stimulates normal tyrosine phosphorylation of the AChR subunit in
src/,
fyn/, or
src/;
fyn/ double mutant myotubes
(Fig. 5C). Thus, agrin induces AChR phosphorylation in
the absence of Src and Fyn.
AChR clustering by laminin-1 does not require Src and Fyn
Integrins are expressed abundantly in developing and adult muscle
(Martin et al., 1996), and in vitro studies have shown that laminin-1, a component of the developing extracellular matrix (Patton
et al., 1997), can initiate AChR clustering independent from agrin and
MuSK (Sugiyama et al., 1997; Montanaro et al., 1998). In addition,
laminin-1, as well as merosin (laminin-2/4), can enhance the AChR
clustering activity of agrin in C2 myotubes (Sugiyama et al., 1997;
Burkin et al., 1998, 2000). Laminins are thought to act via integrins,
possibly 71, and dystroglycan to induce clustering of AChRs
(Burkin et al., 1998, 2000; Montanaro et al., 1998). Because Src class
kinases are required for integrin-mediated signaling in fibroblasts
(Klinghoffer et al., 1999), we asked whether the response to laminin-1
might be aberrant in src/;
fyn/ mutant myotubes.
We measured the number of AChR clusters in myotubes treated with
laminin-1, agrin, or both ligands simultaneously. The number of AChR
clusters in wild-type myotubes was increased twofold by laminin-1,
10-fold by agrin, and 20-fold by agrin plus laminin-1. src/;
fyn/ double mutant myotubes
responded similarly to laminin-1, agrin, and agrin plus laminin-1 (Fig.
6). Thus, Src and Fyn are not required for laminin-1 to stimulate AChR clustering.
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Figure 6.
Laminin-1 induces normal clustering of AChRs in
muscle cells lacking Src and Fyn. Myotubes were stimulated with agrin,
laminin-1, or agrin and laminin-1 overnight, and the number of AChR
clusters per field was determined. The number of AChR clusters in
wild-type myotubes increases twofold by laminin-1, 10-fold by agrin,
and 20-fold by agrin plus laminin-1.
src/;
fyn/ mutant myotubes respond
similarly to laminin and agrin.
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An Src family kinase(s) remains associated with AChRs in
src/;
fyn/ mutant myotubes
Src, Fyn, and Yes are expressed in skeletal muscle cells, but only
Src and Fyn are associated with AChRs in myotubes (Fuhrer and Hall,
1996). Nevertheless, because Src family kinases have overlapping
activities and are known to compensate for one another (Lowell et al.,
1994), we considered the possibility that Yes, and/or other Src-family
kinases, might associate with AChRs in myotubes lacking Src and Fyn. To
assess this possibility, we first analyzed the protein levels of Src
family kinases in myotube extracts by immunoblotting. We found that Yes
is indeed upregulated in src/;
fyn/ myotubes (Fig.
7A,B).
In addition, we affinity-isolated AChRs from wild-type and
src/;
fyn/ double mutant myotubes and
probed Western blots with pan-Src antibodies that react with the
conserved C terminus of Src, Fyn, and Yes (Fuhrer and Hall, 1996). We
found that an Src family kinase(s) copurifies with AChRs in
src/;
fyn/ double mutant myotubes
(Fig. 7C). This kinase most likely represents Yes, because
Yes is upregulated in these mutant cells and Yes is the only known
kinase in muscle to have a C terminus identical to Src and Fyn (Thomas
and Brugge, 1997). The affinities of antibodies specific to Yes were
too low to directly visualize Yes in AChR precipitates by
immunoblotting.
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Figure 7.
An Src-related kinase(s) is associated with
AChRs and activated by agrin in myotubes lacking Src and Fyn.
A, Myotube lysates were analyzed by immunoblotting
using the indicated antibodies. In
src/;
fyn/ cells, Src and Fyn are not
detected, whereas expression of Yes is increased. B,
Quantitation by densitometric scanning shows that the expression of Yes
is greater (2.4-fold) in src/;
fyn/ than in wild-type myotubes
(mean ± SD; from 5 experiments). C, AChRs were
isolated from myotube lysates using -BGT covalently coupled to
Sepharose beads (BGTS) and Src-like kinases detected
using pan-Src (Src-CT) antibodies. As controls,
an excess of free toxin was added (+T), or
control Sepharose was used (CS), or a portion of the
lysate was analyzed without precipitation (L). In
src/;
fyn/ myotubes, an Src-like
kinase(s) is associated with AChRs, although to a lesser overall degree
than in wild-type myotubes. D, Myotubes were treated
with neural agrin, and phosphorylation of AChR-associated proteins was
examined by precipitation with -BGT beads, followed by
phosphotyrosine immunoblotting. An AChR-bound protein of 60 kDa,
representing and comigrating with Src-family kinases (as shown in
C), becomes tyrosine-phosphorylated in response to agrin
in both wild-type and mutant cells. Wild-type,
src+/, and C2 cells gave identical
results.
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AChR-associated Src and Fyn are activated by agrin in wild-type
myotubes, resulting in an increase in their overall tyrosine phosphorylation (Mittaud et al., 2001). To determine whether agrin stimulates similar overall phosphorylation of the Src family kinase(s) associated with AChRs in src/;
fyn/ mutant myotubes, we probed
Western blots with antibodies to phosphotyrosine. Figure 7D
shows that agrin indeed stimulates tyrosine phosphorylation of the Src
family kinase(s) associated with AChRs in
src/;
fyn/ mutant myotubes, as shown
by the comigration of the phosphotyrosine signal with Src-like
kinase(s). Together, these data indicate that an Src family kinase(s)
remains associated with AChRs in src/;
fyn/ double mutant myotubes and
suggest that this kinase(s), which most likely represents Yes, can
compensate for the absence of Src and/or Fyn.
Agrin signaling does not require Src class kinase activity
Agrin-stimulated tyrosine phosphorylation of the AChR subunit
in src/;
fyn/ double mutant myotubes
might be because of the association of an Src family kinase(s) with
AChRs in muscle cells lacking Src and Fyn. Therefore, we asked whether
any Src-like kinase activity is required for agrin to stimulate
tyrosine phosphorylation of the AChR subunit. We pretreated
wild-type and mutant myotubes with 5 µM of the
Src-family kinase inhibitors PP1 and PP2 (Hanke et al., 1996) and
subsequently stimulated myotubes with agrin in the presence of
inhibitor. AChRs were isolated with biotin--BGT, and Western blots
were probed with antibodies to phosphotyrosine. We found that agrin
stimulates normal tyrosine phosphorylation of the AChR subunit in
myotubes treated with either Src family kinase inhibitor (Fig.
8A). Moreover, even in
mutant myotubes lacking Src and Fyn, PP1 and PP2 fail to inhibit
agrin-induced tyrosine phosphorylation of the AChR subunit (Fig.
8B). To determine whether the Src family kinase
inhibitors were active in these myotubes, we probed Western blots of
cell lysates with antibodies to phosphotyrosine. Both inhibitors caused
a marked reduction in total cellular phosphotyrosine, indicating that
the inhibitors were indeed active in these myotubes (Fig.
8C, D). Furthermore, we used a different Src
family kinase inhibitor, CGP77675 (0.1-60 µM)
(Missbach et al., 1999), and higher concentrations of PP1 or PP2
(20-100 µM) and assayed agrin-induced AChR phosphorylation in C2 myotubes. Agrin-induced AChR phosphorylation
was normal in cells treated with 10 µM CGP77675
(data not shown). After several hours at higher concentrations of
CGP77675, PP1, or PP2, some of the myotubes appeared vacuolated and
began to detach from the culture dishes, making it difficult to
determine whether the partial decrease (~50%) in AChR
phosphorylation (data not shown) observed using these conditions was
attributable to a specific effect on agrin-mediated signaling or
specific inhibition of Src family kinases. Together, these results
suggest that agrin-induced phosphorylation of the AChR subunit
requires little, if any, Src class kinase activity. Nonetheless, we
cannot exclude the possibility that several of the three tyrosines in
the long cytoplasmic loop of the AChR subunit are phosphorylated by
agrin and that phosphorylation of only one of these tyrosines is Src
kinase-dependent.
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Figure 8.
PP1 and PP2 do not inhibit agrin signaling in
wild-type and src/;
fyn/ myotubes.
A-D, Myotubes, pretreated with PP1 or PP2, were
stimulated with agrin in the presence of inhibitor. AChRs were isolated
with biotin--BGT, and Western blots were probed with antibodies to
phosphotyrosine (anti-pTyr), followed by
reprobing with antibodies to the AChR subunit. A,
Agrin stimulates normal tyrosine phosphorylation of the AChR subunit in wild-type myotubes treated with either Src family kinase
inhibitor. B, PP1 and PP2 fail to inhibit agrin-induced
tyrosine phosphorylation of the AChR subunit in mutant myotubes
lacking Src and Fyn. C, D, A portion of
the total cellular lysate was analyzed by phosphotyrosine
immunoblotting without precipitation. Both PP1 and PP2, but not PP3, an
inactive isomer, cause a marked reduction in total cellular
phosphotyrosine in wild-type (C) and
src/; fyn
/ mutant (D)
myotubes. E, F, Wild-type myotubes,
pretreated with PP1 or PP2, were stimulated overnight with agrin in the
presence of inhibitor, and AChRs were stained with Texas Red-conjugated
-BGT. Agrin stimulates normal clustering of AChRs in myotubes
treated with either Src family kinase inhibitor, as illustrated in
E and quantitated in F. PP1 and PP2 also
failed to inhibit agrin-induced AChR cluster formation in
src/; fyn
/ mutant myotubes (data not
shown).
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To determine whether Src class kinase activity is required for agrin to
stimulate clustering of AChRs, we pretreated wild-type and
src/;
fyn/ mutant myotubes with PP1 or
PP2 and subsequently added agrin in the presence of inhibitor. We
measured the number of AChR clusters stained with -BGT and found
that agrin stimulates normal clustering of AChRs in myotubes treated
with either inhibitor (Fig.
8E,F). Thus, the ability of
myotubes to cluster AChRs in response to agrin requires little, if any,
Src family kinase activity.
The stability of AChR clusters is reduced in myotubes lacking Src
and Fyn
Src and Fyn may have a role later in synapse maturation or
stabilization. Because double mutant mice die perinatally, we were unable, however, to determine whether Src and Fyn might be required for
changes in synaptic structure and function that occur after birth
(Sanes and Lichtman, 1999). Therefore, we examined whether the
stability of agrin-induced AChR clusters is altered in
src/;
fyn/ double mutant myotubes. We
stimulated myotubes with agrin, withdrew agrin, and measured the number
of intact AChR clusters that remained. The number of AChR clusters in
wild-type myotubes is relatively stable after agrin withdrawal (Fig.
9B,D)
(t1/2 = 10 hr). In contrast, the
number of agrin-induced AChR clusters decreases rapidly in src/;
fyn/ double mutant myotubes
(t1/2 = 80-120 min) (Fig.
9B,D), resulting in the appearance
of microclusters, which are likely to arise by fragmentation of the
larger AChR clusters found in double mutant cells before agrin
withdrawal (Fig. 5A) and in wild-type cells both before and
after agrin withdrawal (Fig. 9A). Unlike
src/;fyn/
double mutant myotubes, the stability of agrin-induced AChR clusters appears normal in src/ or
fyn/ single mutant myotubes
(Fig. 9A,B). Therefore, the
combination of Src and Fyn is required to maintain AChR clusters after
withdrawal of agrin, and either Src or Fyn alone is sufficient to
stabilize agrin-induced AChR clusters.
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Figure 9.
The stability of AChR clusters is reduced in
muscle cells lacking Src and Fyn but not in wild-type myotubes treated
with PP1 or PP2. A, B, We stimulated
myotubes with agrin, withdrew agrin, and visualized AChR clusters that
remained after 3 hr by staining with Texas Red-conjugated -BGT. The
number of remaining AChR clusters is reduced in
src/;
fyn/ myotubes compared with
single mutants or wild-type cells. D, A time course
analysis reveals that the number of AChR clusters in wild-type myotubes
is relatively stable after agrin withdrawal
(t1/2 = 10 hr) but decreases rapidly in
src/;
fyn/ double mutant myotubes
(t1/2 = 80-120 min). C,
Wild-type myotubes were pretreated with PP1, PP2, or PP3 and stimulated
with agrin in the presence of inhibitor. After agrin withdrawal, in the
presence of inhibitor, the number of AChR clusters remaining after 3 hr
was determined. PP1 and PP2 do not significantly reduce AChR cluster
stability.
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Src family kinases can function as adaptor molecules independent from
their kinase activities (Henkemeyer et al., 1990; Xu and Littman, 1993;
Kaplan et al., 1995; Lee-Fruman et al., 1996; Schwartzberg et al.,
1997). For example, reduced tyrosine phosphorylation of focal adhesion
kinase (FAK) and p130Cas in
src/ fibroblasts can be reverted
by expressing the N-terminal region of Src or a mutant form of Src
lacking kinase activity (Kaplan et al., 1995; Schlaepfer et al., 1997).
Therefore, we asked whether the stability of AChR clusters requires the
kinase or adaptor activities of Src and Fyn. We stimulated wild-type
myotubes, pretreated with PP1 or PP2, with agrin, withdrew agrin, and
measured the number of AChR clusters that remained in the presence of
inhibitor. We found that PP1 and PP2 fail to significantly alter the
stability of agrin-induced AChR clusters (Fig. 9C).
Together, these results strongly suggest that the adaptor, rather than
the kinase activities of Src and Fyn, are required to maintain AChR clusters.
| |
DISCUSSION |
Newborn mice mutant for src and fyn or
src and yes move poorly and die shortly after
birth (Stein et al., 1994). Based on the association of Src and Fyn
with the AChR and because Src can act downstream from ErbB2 (Muthuswamy
et al., 1994; Muthuswamy and Muller, 1995; Fuhrer and Hall, 1996;
Fuhrer et al., 1997), we reasoned that these mice may die because of
defects in agrin- or neuregulin-1-mediated signaling at developing
neuromuscular synapses. We found, however, that clustering of
postsynaptic proteins and synapse-specific transcription are normal in
src/;fyn/
and src/;
yes/ mice. Furthermore, agrin
induced normal clustering and tyrosine phosphorylation of AChRs in
muscle cell lines lacking Src and Fyn. Nonetheless, we find that
stability of agrin-induced AChR clusters depends on Src and Fyn, and
our results suggest that the stability and anchoring of AChRs is
dependent on the adaptor rather than the kinase activities of Src and Fyn.
It remains possible that Src and Fyn normally have important roles at
developing neuromuscular synapses and that other Src family kinases
compensate for the lack of Src and Fyn. Indeed, embryos mutant for all
Src, Fyn, and Yes die at E9.5 from severe developmental defects,
indicative of redundancy between these three kinases (Klinghoffer et
al., 1999). Thus, we cannot exclude the possibility that Src and Fyn
normally have a role in agrin and/or ErbB signaling and that other Src
family kinases compensate for their absence. Indeed, we find that an
Src family kinase(s) is associated with AChRs and becomes
phosphorylated and thus presumably activated by agrin in
src/;
fyn/
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TOP
ABSTRACT
INTRODUCTION
