From the Brain Research Institute, University of Zürich, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland
Received for publication, August 3, 2000, and in revised form, January 30, 2001
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ABSTRACT |
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During neuromuscular synaptogenesis, neurally
released agrin induces aggregation and tyrosine phosphorylation of
acetylcholine receptors (AChRs) by acting through both the receptor
tyrosine kinase MuSK (muscle-specific kinase) and the AChR-associated
protein, rapsyn. To elucidate this signaling mechanism, we examined
tyrosine phosphorylation of AChR-associated proteins,
particularly addressing whether agrin activates Src family
kinases bound to the AChR. In C2 myotubes, agrin induced tyrosine
phosphorylation of these kinases, of AChR-bound MuSK, and of the AChR
A common feature of most synapses is the local accumulation of
proteins regulating synaptic responses in the postsynaptic membrane. At the neuromuscular junction, a model system for
synaptogenesis, aggregation of acetylcholine receptors
(AChRs)1 is an early sign of
postsynaptic differentiation during development and occurs at sites of
nerve-muscle contact (1, 2).
Clusters of AChRs and further synaptic components are induced by
neurally released agrin, an essential factor for synaptogenesis (3).
Accordingly, mice deficient for agrin lack differentiated synapses and
nerve-associated clusters of postsynaptic proteins (4). Neurons or
recombinant agrin induce aggregation of AChRs and other muscle
components when added to cultured myotubes (5, 6), a process that
mimics events at developing endplates, as it is blocked by antibodies
specific for nerve-released agrin (7).
Little is known about the signaling pathway of agrin, although a
muscle-specific kinase, MuSK, is part of the agrin receptor (8, 9), and
proteoglycans are thought to play an accessory role in presenting agrin
to its receptor (10). Agrin causes tyrosine phosphorylation of MuSK,
and this kinase is essential for postsynaptic specialization, as shown
in MuSK-deficient mice, in which the phenotype is similar to agrin One protein involved in agrin signaling downstream of MuSK is rapsyn, a
43-kDa protein closely associated with AChRs (16, 17). Rapsyn The individual steps of this postsynaptic assembly, in particular, the
agrin signaling events downstream of MuSK, still remain largely
unknown. Myotubes contain preassembled AChR protein complexes in which,
independently of agrin, AChRs are associated with several muscle
proteins, including rapsyn, MuSK, and Src-related kinases (21). Agrin
selectively increases, in a rapsyn-dependent way, the
association between AChRs and MuSK, suggesting that agrin causes a link
of these preassembled AChR protein complexes with the MuSK primary
synaptic scaffold, thereby driving postsynaptic assembly.
Proteins that bind to MuSK or the AChR are likely to regulate this
mechanism. Activated MuSK contains several phosphorylated tyrosine
residues in its cytoplasmic tail, which may act as docking sites for
signal transducing molecules (22). One of these residues, a
juxtamembrane tyrosine within an NPXY consensus, is
indeed required for agrin-induced AChR phosphorylation and clustering,
suggesting the involvement of a PTB (phosphotyrosine-binding) domain
adaptor protein (23, 24). Another critical step in agrin's signaling pathway are calcium fluxes, as the fast chelator, BAPTA-AM, inhibits agrin-induced AChR clustering (25). However, the nature of this calcium-dependent step is unknown.
Moreover, several observations indicate the existence of a critical
kinase downstream of MuSK. Thus, staurosporine does not affect
agrin-induced activation of MuSK but inhibits AChR In several signaling pathways, the functional roles of Src kinases are
indicated by an increase in their kinase activities following
extracellular stimulation (30). Similarly to muscle, Src family kinases
associate with ionotropic neurotransmittor receptors in the central
nervous system, e.g. Src with NMDA receptors and Lyn with
AMPA receptors (31, 32). The associated kinases regulate receptor
channel properties and form receptor-bound signaling cascades, as shown
for Lyn, which is activated by AMPA stimulation of cerebellar neurons,
leading to activation of the mitogen-activated protein kinase
pathway and possibly synaptic plasticity (31).
To begin to understand the role of the Src family in postsynaptic
differentiation in muscle and in agrin-induced signaling events
downstream of MuSK, we examined the effect of agrin on tyrosine
phosphorylation of proteins associated with the AChR, in particular the
Src family members. We demonstrate that agrin causes activation and
phosphorylation of AChR-bound Src class kinases, which requires rapsyn
and correlates with AChR phosphorylation and aggregation. Src-related
kinases thus cause AChR phosphorylation induced by agrin and may play a
role in AChR clustering, suggesting that the Src family not only
regulates ionotropic receptors channel properties but also contributes
to the synaptic localization of these receptors.
Cell Culture--
Cell culture reagents were purchased from Life
Technologies, Inc. C2 (C2C12) and S27 mouse muscle cells were
maintained at 37 °C in 8% CO2 and propagated in
Dulbecco's modified Eagle's medium with 4.5 g/liter
D-glucose containing 20% fetal bovine serum, 0.5% chick
embryo extract, 2 mM glutamine, and
penicillin/streptomycin. After reaching 90-100% confluence, cells
were shifted to fusion medium containing Dulbecco's modified Eagle's
medium, 5% horse serum, and 2 mM glutamine and fed daily.
C2 myotubes were used for experiments after 2 days in this medium,
while S27 cells were used after 2-3 days, by which time myotubes
similar to C2 had formed. We noticed that the morphology of the S27
myotubes varied between different experiments, in that myotubes were
occasionally wider than in C2 (see Fig. 8, S27 cells treated with 50 nM agrin). Furthermore, the fusion of myoblasts to form
myotubes was less efficient in the case of S27, such that most cultures
had more unfused cells than C2. Nevertheless, these morphological
variations did not affect the outcome of our experiments, because
agrin-induced clustering of AChRs and phosphotyrosine-containing
proteins, as well as phosphorylation of MuSK and AChR-bound Src family
kinases, were consistently absent in S27 cells irrespective of their
morphology. Rapsyn Antibodies--
To detect AChR-associated phosphotyrosine
proteins by immunoblotting, we used a mixture of two commercially
available mouse monoclonal antibodies, 4G10 (Upstate Biotechnology,
Lake Placid, NY) and PY20 (Transduction Laboratories, Lexington, KY).
The mouse monoclonal antiserum 88B, reactive with the AChR Expression of Agrin Constructs--
To express soluble
C-terminal agrin forms, COS cells were grown at 37 °C in 8%
CO2 using Dulbecco's modified Eagle's medium, 10% fetal
calf serum, 2 mM glutamine, and penicillin/streptomycin. Cells were transfected with neural (4,8) or muscle (0,0) agrin expression vectors (C-Ag12,4,8 and C-Ag12,0,0,
respectively), and the medium, containing soluble agrin, was collected
for 3 days as described previously (26).
Isolation of AChR Protein Complexes and
Immunoprecipitation--
Myotubes were lysed in a mild extraction
buffer containing 1% Nonidet P-40 and an abundance of protease and
phosphatase inhibitors, and AChRs were precipitated with
In Vitro Phosphorylation and Reprecipitation of Phosphotyrosine
Proteins and Src-related Kinases--
To identify the 60-kDa
AChR-associated phosphoprotein, AChR protein complexes were
precipitated as described above. Complexes were dissociated by an
incubation at 80 °C for 10 min in dissociation buffer containing 50 mM NaCl, 30 mM triethanolamine, pH 7.5, 5 mM EDTA, 5 mM EGTA, 1% Triton X-100, 0.5%
SDS, 0.5% deoxycholate, 50 mM NaF, 3 mM sodium
orthovanadate, 50 µM phenylarsine oxide, 10 mM
p-nitrophenylphosphate and 1 mM
phenylmethylsulfonyl fluoride. After harsh vortexing and
centrifugation, the proteins released into the supernatant were
reprecipitated with anti-phosphotyrosine antibodies (4G10) covalently
coupled to agarose beads (Upstate Biotechnology). These precipitates
were washed three times with wash buffer (0.4% Triton X-100, 120 mM NaCl, 30 mM triethanolamine, pH 7.5, 5 mM EGTA, 5 mM EDTA, 2 mM sodium
orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 10 mM p-nitrophenylphosphate, 50 µM
phenylarsine oxide, 50 mM NaF) and resuspended in SDS
sample buffer for SDS-PAGE and src-CT immunoblotting.
The kinase activity of AChR-associated Src family kinases was assayed
by incubating
For both methods, the quantitation of signals on films was performed by
scanning with a computerized densitometer (Nikon Scantouch 210) and
using the NIH Image J 1.04b software (National Institutes of Health,
Bethesda, MD). Background signals, originating from control
precipitations including excess of free Tyrosine Kinase Inhibitors--
To examine the effects of kinase
inhibitors on tyrosine phosphorylation of proteins bound to the AChR,
C2 myotubes were incubated for 5 to 6 h with 1-20 nM
staurosporine (Sigma), 1 µM herbimycin A (Life
Technologies, Inc.), or carrier controls. After stimulation with agrin,
cells were extracted and analyzed by Immunoblotting--
To detect proteins in AChR, src-CT, or MuSK
precipitations, proteins were eluted into SDS sample buffer, separated
by SDS-PAGE, and transferred to nitrocellulose. Membranes were probed
with the appropriate antibodies, and immunoreactive bands were
visualized using horseradish peroxidase-conjugated secondary antibodies
and enhanced chemiluminescence (ECL, Amersham Pharmacia Biotech). For
reprobing, blots were stripped by incubating them for 20 min in 200 mM glycine, 0.1% Tween 20, pH 2.5.
Immunocytochemical Staining and Fluorescence Microscopy--
To
visualize the distribution of AChRs and phosphotyrosine proteins in C2
and S27 myotubes, cells were grown and fused on chamber slides (Nunc,
Life Technologies, Inc.) and treated for 15 h with agrin
concentrations ranging from 0.5 to 100 nM. Cells were
incubated with 100 nM tetramethylrhodamine-conjugated
Agrin Induces Tyrosine Phosphorylation of Several Proteins
Associated with the AChR--
In our initial experiments, we analyzed
agrin-induced tyrosine phosphorylation of proteins associated with the
AChR by preparing extracts from agrin-treated and untreated C2
myotubes; AChRs were precipitated with
We stimulated C2 myotubes for increasing times with either recombinant
neural agrin (agrin 4,8) or the predominant agrin isoform of muscle
(agrin 0,0), which is much less effective in aggregating AChRs (6, 33).
To determine whether this AChR-associated 60-kDa phosphotyrosine
protein represents one or several members of the Src family, immunoblots were stripped and reprobed with pan-Src antibodies (src-CT)
that recognize Src, Fyn, and Yes in muscle cells (28). These assays
revealed that the protein indeed comigrates precisely with AChR-bound
Src members and that the protein amounts of these associated kinases
are not affected by agrin (Fig.
2A). We have previously
reported that two members of the Src family, Src and Fyn, are
associated with AChRs and that they migrate with identical mobility in
gel electrophoresis and immunoblotting experiments (28). Further
reprobing studies using Fyn-specific antibodies confirmed that the
observed 60-kDa phosphotyrosine band indeed comigrates with AChR-bound
Fyn (Fig. 2B). The amounts of AChR-bound Src and Fyn are
unchanged by agrin (26), a finding similar to the results using src-CT
antibodies (Fig. 2A). Therefore, both kinases are associated
constitutively with the AChR independently of agrin, and either one or
both of them may become phosphorylated strictly in response to
agrin.
AChR-associated Kinases of the Src Family Are Activated by Neural
Agrin--
To confirm that the 60-kDa AChR-associated phosphoprotein
indeed represents one or several members of the Src family bound to the
AChR, we isolated large amounts of AChR protein complexes from
agrin-treated and untreated C2 myotubes using
Interestingly, when Src members were isolated from agrin-treated
myotubes by a direct immunoprecipitation with src-CT antibodies, their
phosphotyrosine content was unaffected in comparison with untreated
cells (Fig. 3C). Thus, agrin specifically activates those
Src-related kinases that are associated with AChRs without affecting
the total cellular kinase pool.
Agrin-induced Phosphorylation of AChR-bound Src Members Correlates
with AChR Phosphorylation--
We asked how activation of
AChR-associated Src members correlates with phosphorylation of the AChR
subunits and MuSK. For this purpose, C2 myotubes were treated for
various times with a wide range of agrin concentrations (20 pM-5 nM). A 5-min incubation with 0.5 nM neural agrin was sufficient to induce significant phosphorylation of AChR-bound Src family kinases and the receptor Staurosporine Selectively Blocks Agrin-induced Phosphorylation of
AChR-associated Src Family Kinases and the AChR--
To analyze the
role of Src family activation in agrin-induced AChR phosphorylation and
clustering, we used the kinase inhibitors herbimycin and staurosporine.
Both of these inhibitors block AChR
Herbimycin, in contrast, abolished phosphorylation of all proteins seen
in In the Absence of Rapsyn, AChR-bound Src Members Are Not Activated
by Agrin--
To further correlate Src family autophosphorylation with
AChR clustering, we analyzed the effect of neural agrin on tyrosine phosphorylation of AChR-bound proteins in rapsyn-deficient myotubes. These cells were derived from rapsyn knockout mice and do not aggregate
AChRs in response to agrin (21). Agrin induces normal phosphorylation
of total cellular as well as AChR-associated MuSK in these cells,
whereas AChR
Therefore, we examined phosphorylation of AChR-bound Src members by
agrin in rapsyn In S27 Myotubes, Agrin Fails to Cause Phosphorylation of AChR-bound
Src Family Kinases--
As a final correlation between agrin-induced
Src member activation and AChR clustering, we analyzed Src class
phosphorylation in S27 myotubes, genetic variants of C2 defective in
heparan and chondroitin sulfate proteoglycan biosynthesis (34, 35). In these cells, even high concentrations of agrin up to 100 nM
do not induce AChR clustering and
To determine which step in the agrin signaling pathway is affected in
S27 cells, we examined agrin-induced phosphorylation and clustering
events in these cells. In C2, 0.5 nM neural agrin was
sufficient to induce abundant co-clustering of AChRs and
phosphotyrosine proteins, as observed by double-label
immunofluorescence microscopy using rhodamine-conjugated
Based on the absence of agrin-activated AChR-associated MuSK in S27, we
examined phosphorylation of the total cellular pool of MuSK by neural
agrin in these cells, using MuSK immunoprecipitation followed by
phosphotyrosine immunoblotting. 100 nM neural agrin did not
cause prominent MuSK activation in S27, whereas in C2 cells, 0.5 nM caused a strong phosphorylation (Fig. 7B). We
noticed that the majority of MuSK isolated from S27 cells migrated with a lower molecular weight than in C2, consistent with defective glycosylation of MuSK in the mutant cells. Our phosphotyrosine blotting
assays, however, sometimes revealed weak agrin-induced phosphorylation
of a MuSK population with the same apparent molecular weight as in C2
(Fig. 7B). It is therefore possible that MuSK exists in
several forms in S27 cells, because of differential glycosylation, and
that some of these subpopulations react slightly differently to
treatment with high doses of agrin. Together, these experiments show
that even high concentrations of agrin fail to cause clustering of
AChRs and phosphorylation of AChR-bound Src members in S27. Although we
cannot exclude the possibility that further, unknown genetic
defects in these cells prevent AChR clustering, our results are
consistent with the assumption that, similar to herbimycin-treated C2
cells, MuSK is not activated by agrin in S27 cells, which causes
defects in the activation of AChR-bound Src family kinases, in AChR
phosphorylation, and in clustering of AChRs and
phosphotyrosine-containing proteins.
In this study, we demonstrate that agrin activates Src family
kinases that are associated with the AChR. The activation occurs downstream of MuSK, requires rapsyn, and correlates closely with agrin-induced AChR clustering. These data indicate that Src family kinases are part of a local AChR-bound signaling cascade and that they
may play a role in the assembly of AChR protein complexes into a
postsynaptic apparatus.
Activation of Src Family Kinases by Agrin--
Several of our
observations indicate that AChR-associated kinases of the Src family
are activated by agrin. In agrin-treated myotubes, the phosphotyrosine
content of these kinases is increased, as shown by both kinase
reprobing and phosphotyrosine reprecipitation experiments. Furthermore,
in kinase assays in vitro, incorporation of phosphate into
the kinases is augmented by agrin. Such in vitro phosphorylation assays are widely used to analyze the activation of Src
class kinases in response to various stimuli, e.g. growth factors, and an increase in phosphate incorporation indicates kinase
autophosphorylation and thus activation (30). We can exclude the
possibility that AChR-bound Src family kinases are phosphorylated by MuSK, because staurosporine blocks Src
phosphorylation without affecting MuSK autophosphorylation (see also
below). Thus, our kinase assays most likely reflect Src family
autophosphorylation. In our experiments, we used a high affinity Src
family antibody against a conserved epitope of Src, Fyn, and Yes. As
both Src and Fyn have been shown to be associated with AChRs (28), we therefore assume that either one or both of these kinases are activated
by agrin. The affinities of specific anti-Src or anti-Fyn antibodies in
immunoprecipitations were too low to show an activation of the
respective individual
kinase.2
Kinases of the Src family are regulated in complex ways, both by
tyrosine phosphorylation and by intramolecular protein interactions mediated by their SH2 and SH3 domains (30, 36). Activation can be
achieved by binding of proline-rich sequences of other proteins to the
SH3 domain, by interaction of external phosphotyrosine peptides
with the SH2 domain, or by C-terminal dephosphorylation, all of which
lead to the active open Src conformation (36, 37). In all cases,
activation requires autophosphorylation of a tyrosine in the activation
loop of the kinase domain.
Based on these mechanisms, our observed Src family activation does not
appear to be mainly mediated by C-terminal dephosphorylation but rather
by interacting proteins that bind to the Src family SH2 and/or SH3
domains, thereby leading to the increase in overall phosphotyrosine
content. A good candidate for such an interacting protein is MuSK, a
portion of which is associated with the AChR and becomes phosphorylated
in response to agrin (26). Interestingly, the cytoplasmic domain of
MuSK contains two proline-rich regions, one of which corresponds to the
consensus requirements for Src SH3 binding, as it contains the typical
PXXP motif preceded by a leucine (38, 39). These MuSK
peptide motifs and/or the autophosphorylated cytoplasmic tyrosine
residues of activated MuSK (22) could thus bind to Src class kinases
causing kinase opening, autophosphorylation, and activation. Such a
mechanism would work most efficiently and rapidly if the proteins
involved (AChRs, MuSK, and Src family kinases) were to form a
preassembled complex. Indeed, we have previously demonstrated that
these proteins interact in myotubes, before the addition of agrin (21),
and data in this study indicate that Src family activation occurs
rapidly, as early as 5 min after addition of agrin, and is highly
correlated with the activation of MuSK.
Src family activation was observed only for that fraction of kinases
associated with AChRs but not on the level of the total cellular kinase
pool. In contrast, the activation of Src members in other signaling
systems, such as mitogenic responses to growth factors or adhesion on
fibronectin, involves and is detectable on the whole cellular kinase
pool (40, 41). This illustrates an important difference between Src
family signaling in cell proliferation or adhesion and in postsynaptic
assembly. The latter process requires correct protein interactions,
presumably driven by local signaling events, and would be hard to
reconcile with signaling occurring throughout myotubes as a whole. The
need for local signaling, combining kinase activation with protein
interactions, is illustrated by MuSK, which, in addition to signaling,
also mediates protein interactions involving its extracellular domain,
and where, unlike in the case of growth factor receptors, the activated
kinase domain is not sufficient to reproduce all aspects of signaling
(20, 42).
Src Family Activation and AChR Phosphorylation--
Our AChR
precipitation procedure combined with phosphotyrosine immunoblotting
also revealed agrin-induced phosphorylation of AChR
Which tyrosine kinase phosphorylates the AChR in response to agrin?
Several of our experiments suggest that both the AChR
Interestingly, as in staurosporine-treated C2 myotubes, agrin-induced
Src member activation and AChR phosphorylation are also inhibited in
rapsyn-deficient cells, showing that rapsyn is necessary for Src family
activation by agrin. In fibroblasts, rapsyn interacts with and
activates both MuSK and Src members, causing AChR Agrin-induced Src Family Activation and Postsynaptic
Differentiation--
Studies in several signaling systems show that an
activation of Src family kinases by extracellular stimuli is an
indication of a functional role of these kinases in the respective
pathways (30). In our case, the activation of AChR-bound Src family
kinases by agrin correlates closely with agrin-induced AChR clustering as judged by several criteria, including the differential effects of
neural and muscle agrin, the inhibition by staurosporine and herbimycin, and the absence of agrin-induced Src member activation and
AChR clustering in rapsyn-deficient myotubes. Furthermore, also in S27
myotubes treated with high concentrations of agrin, AChR clustering and
phosphorylation of AChR-bound Src members are not observed (Table
I). As staurosporine blocks Src
family but not MuSK autophosphorylation, AChR-associated Src class
kinases indeed appear to be the previously proposed kinase(s)
downstream of MuSK that are important for AChR clustering (26).
Furthermore, by inference, Src family activation shows the same dose
dependence on neural agrin as AChR clustering, because it correlates
strictly with AChR
Agrin-activated Src class kinases could act at different steps in the
pathway leading from muscle innervation to formation of a fully
differentiated postsynaptic apparatus. We have previously suggested
that preassembled protein complexes containing AChRs in association
with postsynaptic elements may be the building blocks for agrin-induced
formation of the postsynaptic membrane (21) and that this mechanism
involves a rapsyn-dependent link of such complexes to the
MuSK-based primary synaptic scaffold (20). Preassembled AChR complexes
contain rapsyn, Src members, and MuSK, and signaling by these kinases
may render the complexes competent for such a link by the
phosphorylation of parts of the complexes and/or further proteins. This
mechanism could involve tyrosine phosphorylation of rapsyn, as rapsyn
is a substrate for Src family kinases when coexpressed in fibroblasts
(29). However, in preliminary experiments we did not observe tyrosine
phosphorylation of AChR-bound rapsyn due to
agrin,3 although small
amounts of phosphorylation may have remained undetected.
Alternatively, as AChR-associated Src class kinases, in contrast to
MuSK, are still activated after prolongated incubation with agrin
in vitro, they may play a role in growth and stabilization of postsynaptic protein clusters. Interestingly, besides rapsyn, several cytoskeletal proteins concentrated at the neuromuscular junction or in Torpedo synaptic membranes, including talin,
paxillin, and
Finally, maturation of the postsynaptic apparatus involves a series of
cytoskeletal rearrangements, which ultimately lead to formation of
postsynaptic folds, including segregation of several postsynaptic
proteins between crests and troughs (51). At the mammalian
neuromuscular junction, as well as in Torpedo electrocytes, tyrosine phosphorylation in the postsynaptic membrane increases during
this late stage of synaptic maturation (52, 53). This activity could
originate from AChR-bound tyrosine kinases, e.g. Src
members, and may cause correct colocalization of proteins like utrophin
or syntrophin isoforms with AChRs at the crests of the folds, whereas
other proteins, such as sodium channels or dystrophin, are enriched in
the troughs (51, 54). Indeed, some of these proteins as well as
dystrobrevin isoforms, which may also be differentially distributed,
are tyrosine-phosphorylated in Torpedo or have tyrosine
phosphorylation consensus sites (51, 55). Thus, Src family-mediated
phosphorylation of such proteins may determine their precise
postsynaptic localization with respect to AChRs and contribute to later
aspects of postsynaptic maturation.
In the central nervous system, the Src family is thought to have
various functions, including regulation of glutamate receptors. At
central synapses, NMDA receptors are regulated by tyrosine phosphorylation and associated with Src (c-Src) in a manner similar to muscle AChRs (56). In CA1 pyramidal cells of rat hippocampus, Src activation induces long-term potentiation, apparently by
potentiation of bound NMDA receptors (32). AMPA receptors of the
cerebellum, on the other hand, interact with the Src family kinase Lyn,
which is activated following receptor stimulation (31). Glutamate receptors thus associate with Src members, which, upon activation, contribute to synaptic plasticity in various ways. Compared with these
studies, our findings are the first indication that activation of Src
class kinases may also regulate the distribution and postsynaptic localization of ionotropic neurotransmittor receptors.
In summary, the present data, together with our previous studies (21),
suggest a model for AChR clustering in which agrin activates AChR-bound
MuSK, which requires rapsyn to activate receptor-associated Src
members, which phosphorylate AChR and
subunits, as observed in phosphotyrosine immunoblotting
experiments. Kinase assays revealed that the activity of
AChR-associated Src kinases was increased by agrin, whereas
phosphorylation of the total cellular kinase pool was unaffected. In
both rapsyn-deficient myotubes and staurosporine-treated C2 myotubes,
where AChRs are not clustered, agrin activated MuSK but did not cause
either Src family or AChR phosphorylation. In S27 mutant myotubes,
which fail to aggregate AChRs, no agrin-induced phosphorylation of
AChR-bound Src kinases, MuSK, or AChRs was observed. These results
demonstrate first that agrin leads to phosphorylation and activation of
AChR-associated Src-related kinases, which requires rapsyn, occurs
downstream of MuSK, and causes AChR phosphorylation. Second, this
activation intimately correlates with AChR clustering, suggesting that
these kinases may play a role in agrin-induced AChR aggregation by
forming an AChR-bound signaling cascade.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
animals (11). Furthermore, myotubes lacking MuSK fail to respond to
agrin (9, 11). Agrin also causes tyrosine phosphorylation of the
subunit of the AChR, which may play a role in AChR clustering because, among other observations, the kinase inhibitors herbimycin and staurosporine block both AChR
phosphorylation and AChR clustering (12, 13). However, mutant AChRs, lacking cytoplasmic tyrosine residues
in their
subunits, can still be recruited into clusters upon
expression in myotubes, which also express wild-type AChRs (14). In
addition, it has been shown that AChR tyrosine phosphorylation is
accompanied by a change in receptor desensitization by cholinergic ligands (15).
/
mice
lack aggregates of AChRs and other postsynaptic proteins, and their
cultured myotubes fail to form agrin-induced AChR clusters,
illustrating the importance of rapsyn for postsynaptic organization
(18). Furthermore, rapsyn causes aggregation of AChRs upon expression
in fibroblasts and is necessary for agrin-induced AChR
phosphorylation in myotubes (19, 20). MuSK, however, is still activated
by agrin and localized at mutant endplates of rapsyn-deficient animals,
suggesting that MuSK forms a primary synaptic scaffold to which rapsyn
recruits AChRs and further components of the postsynaptic
apparatus (20, 21).
phosphorylation and AChR clustering, strongly suggesting that a staurosporine-sensitive intermediate kinase downstream of MuSK causes AChR phosphorylation and
aggregation (26). Such a kinase(s) may be one or several members of the
Src family, because Fyn and Fyk associate with AChRs in the
Torpedo electric organ (27), whereas Src (c-Src) and Fyn are
bound to AChRs in mammalian muscle (28). Furthermore, Src can
phosphorylate AChR
subunit fusion proteins (28), and Src members
are regulated by rapsyn when expressed in fibroblasts, causing AChR
phosphorylation in this heterologous system (29).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
and the corresponding wild-type myoblasts were
grown at 33 °C, 5% CO2 in the same medium as C2 cells
with an additional 4 units/ml
-interferon, as described previously
(21). Confluent cultures were fed with C2 fusion medium every second
day and used for experiments after 3-4 days.
and
subunits, was provided by Dr. S. C. Froehner (University of North
Carolina, Chapel Hill). Rat monoclonal antibodies 124 against
the AChR
subunit were a gift from Dr. J. Lindstrom (University of
Pennsylvania, Philadelphia). To detect Src-related kinases, we used
src-CT (Santa Cruz Biotechnology, Santa Cruz, CA), a rabbit polyclonal
antibody raised against the C terminus of Src (c-Src), which is highly conserved in several Src-related kinases. Src-CT reacts with Src, Fyn,
Yes (28), and possibly further kinases of the Src family. Fyn was
detected by a rabbit polyclonal Fyn-specific antiserum (Santa Cruz
Biotechnology). The antiserum against MuSK was as described previously
(26).
-bungarotoxin (
-BT) coupled to Sepharose beads (
-BT-Sepharose
beads) as described previously (21). Alternatively, AChRs were isolated
using 200 nM soluble biotin-conjugated
-BT followed by
streptavidin-coupled agarose beads (Molecular Probes, Eugene, OR),
which gave identical results. As controls, an excess (10 µM) of free
-BT was added, or unconjugated Sepharose
was used. To immunoprecipitate Src members representing the total
cellular Src family pool, myotube extracts were incubated with 1 µg
of src-CT antibodies followed by protein A-Sepharose. Cellular MuSK was
isolated accordingly using polyclonal anti-MuSK antibodies and protein
A-Sepharose.
-BT-Sepharose-precipitated AChR complexes for 10 min
at 25 °C under phosphorylating conditions (kinase assay buffer: 20 mM Tris, pH 7.4, 10 mM MgCl2, 2 mM MnCl2, 1 mM phenylmethylsulfonyl fluoride, 1 mM dithiothreitol, 1 mM sodium
orthovanadate, 20 µCi of [
-32P]ATP). Reactions were
stopped by the addition of kinase wash buffer on ice (10 mM
Tris, pH 7.2, 200 mM NaCl, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 1 mM sodium
orthovanadate), and beads were washed twice in this buffer. Complexes
were dissociated by an incubation for 10 min at 80 °C in
phosphate-buffered saline, pH 7.4, containing 1% Triton X-100, 0.5%
SDS, 0.5% deoxycholate, 1 mM phenylmethylsulfonyl
fluoride, and 1 mM sodium orthovanadate. Src class kinases
were precipitated from supernatants using src-CT antibodies and protein
A-Sepharose followed by washing in dissociation buffer, elution into
SDS-PAGE sample buffer, SDS-gel electrophoresis, and autoradiography.
-BT, were subtracted from
-BT AChR precipitations. Values from untreated cells were set to
100%, and signals after agrin treatment were calculated accordingly.
Statistical evaluation was performed by ANOVA followed by pairwise
Bonferroni's t tests.
-BT AChR precipitation followed
by phosphotyrosine immunoblotting. Quantitation of signals was carried
out as described previously (26).
-bungarotoxin (Molecular Probes) in fusion medium for 1 h at
37 °C followed by fixation in methanol for 7 min at
20 °C and
permeabilization (5 min in phosphate-buffered saline containing 1%
Triton X-100 and 1 mM sodium orthovanadate).
Phosphotyrosine proteins were detected by PY20 antibodies (Transduction
Laboratories) and fluorescein isothiocyanate-conjugated goat-anti-mouse
secondary antisera (Jackson ImmunoResearch Laboratories, La Roche,
Switzerland) in the presence of 1 mM sodium orthovanadate.
Cells were mounted in glycerol containing p-phenylenediamine
and examined with a microscope (Axioskop 2, Carl Zeiss) equipped with
the appropriate fluorescence filters. Images were processed with a
computer-supported cooled 3CCD camera (Hamamatsu, Hamamatsu-City, Japan).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-bungarotoxin (
-BT)
covalently coupled to Sepharose beads, or using biotinylated
-BT in
combination with streptavidin-agarose, followed by phosphotyrosine
immunoblotting. To assess nonspecific protein binding to the beads,
free
-BT was added prior to the precipitation, or unconjugated
Sepharose was used. Using this procedure allows the specific
isolation of AChRs in association with several postsynaptic proteins
(21).
-BT precipitation and phosphotyrosine immunoblotting revealed a
consistent pattern of AChR-associated phosphotyrosine bands induced by
neural but not muscle agrin (Fig. 1). In
addition to AChR-bound MuSK and the AChR
subunit (26), we detected a band at about 70 kDa. This protein appears to represent the AChR
subunit, based on the cross-reactivity with
-specific antibodies in
stripping and reprobing experiments (Fig. 1). In addition, an
AChR-associated protein of about 60 kDa, i.e. the molecular
mass of Src family kinases, consistently became
tyrosine-phosphorylated in response to neural agrin. Its increase in
phosphorylation occurred in parallel with that of AChR-bound MuSK and
the AChR
and
subunits, as it was first detected after a 15 min
treatment with 0.1 nM neural agrin and reached a peak after
40 min (Fig. 1). Dephosphorylation of this 60-kDa band strictly
paralleled that of the AChR subunits, as all three proteins were still
phosphorylated to an intermediate degree after a 15-h treatment with
neural agrin. MuSK, however, was no longer phosphorylated after this
time. In these assays, agrin-induced tyrosine phosphorylation events
were observed only for proteins associated with the AChR and not when total cellular lysates were analyzed without precipitation (Fig. 1,
L lanes). This selectivity indicates local, AChR-bound
signaling, which does not involve changes in phosphorylation on the
level of a whole myotube.
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Fig. 1.
Neural agrin induces tyrosine phosphorylation
of a 60-kDa protein associated with AChRs. C2 myotubes were
treated for up to 15 h with 0.1 nM neural (4,8) or
muscle (0,0) agrin. AChRs were isolated from cellular lysates using
-BT-Sepharose beads followed by SDS-PAGE and phosphotyrosine
immunoblotting (Tox-P/PY-blot). Middle panel, the
blot was stripped and reprobed with MuSK-specific antibodies
(Tox-P/MuSK-reprobe), whereas a second reprobing using AChR
- and
-specific antisera (monoclonal antibodies 88b) is shown at
the bottom
(Tox-P/
/
-reprobe). Controls for
AChR precipitation included the addition of 10 µM free
-BT (+T) and the use of unconjugated Sepharose
(C). The solid arrowheads
indicate proteins associated with AChRs isolated by
-BT
precipitation. The open arrowhead designates the AChR-bound
protein(s) of ~ 60 kDa in which tyrosine phosphorylation is
induced by neural agrin. L indicates a fraction (0.3%) of
the initial lysate analyzed without precipitation.
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Fig. 2.
Agrin increases tyrosine phosphorylation but
not the protein amount of AChR-bound Src class kinases. C2
myotubes were treated with 0.1 nM (A) or 0.5 nM (B) neural agrin as indicated, and cell
lysates were subjected to precipitation with -BT-Sepharose beads and
immunoblotting. Controls included the addition of 10 µM
free toxin (+T) and unconjugated Sepharose (C).
A, phosphotyrosine immunoblotting followed by reprobing with
pan-Src antibodies (src-CT) reveals that the 60-kDa
phosphoprotein comigrates with Src family kinases (src's).
B, a comparison of phosphotyrosine immunoblotting and
reprobing with Fyn-specific antibodies shows identical electrophoretic
mobilities of the agrin-induced 60-kDa phosphoprotein
(src's) and AChR-bound Fyn.
-BT-Sepharose. Complexes were dissociated by an incubation at 80 °C in a buffer containing deoxycholate and SDS. The proteins released were then reprecipitated with phosphotyrosine antibody beads, immunoblotted, and
probed with src-CT antibodies. This assay specifically monitors the
overall levels of tyrosine phosphorylation of Src class kinases bound
to the AChR. We indeed found a significant increase in the phosphotyrosine content of these kinases in response to neural agrin
(Fig. 3A). As Src members
undergo autophosphorylation when activated, we additionally used kinase
assays to examine the effect of neural agrin on phosphate incorporation
into and thus activation of AChR-associated Src kinases. To this end,
-BT-precipitated AChR complexes, isolated from agrin-treated or
untreated cells, were subjected first to in vitro
phosphorylation, using [
-32P]ATP at 25 °C. The
complexes were then dissociated at 80 °C and released Src members
immunoprecipitated with src-CT antibodies followed by gel
electrophoresis and autoradiography. In these assays, neural agrin
significantly increased the phosphate incorporation into Src-related
kinases by ~3-fold, indicative of their activation (Fig.
3B). Together, these results show that one or several
AChR-associated kinases of the Src family are activated by neural agrin
and that the AChR-bound phosphotyrosine band at 60 kDa, as seen on
immunoblots, indeed originates, at least in part, from Src members.
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Fig. 3.
Neural agrin specifically activates
AChR-associated Src-related kinases without affecting tyrosine
phosphorylation of the total cellular Src family pool. C2 myotubes
were stimulated with 0.5 nM agrin as indicated and lysed
under mild conditions. A, cell lysates were incubated with
-BT-Sepharose beads to isolate AChRs including the addition of 10 µM free toxin as a control (+T). Precipitated
AChR protein complexes were dissociated in a buffer containing 0.5%
SDS and deoxycholate at 80 °C followed by reprecipitation with
anti-phosphotyrosine antibodies coupled to agarose beads (Re-IP
PY-aga). Phosphotyrosine contents of AChR-associated Src class
kinases were visualized by src-CT immunoblotting (left).
Immunoblots from three experiments were quantitated by densitometric
scanning of films. After subtracting the values of control
precipitations (+T) from
-BT-precipitated samples,
signals of untreated cells were set to 100% (control), and signals
after agrin treatment were calculated accordingly. Data represent the
mean ± S.D. (right). B,
-BT-precipitated
AChR complexes were subjected to in vitro phosphorylation
(IVP), using phosphorylating conditions and
[
-32P]ATP at 25 °C, followed by dissociation as
described in A, reprecipitation with src-CT antibodies and
protein A-Sepharose (Re-IP src-CT), SDS-PAGE, and
autoradiography (left). The quantitation (right)
shows the mean ± S.D. from six experiments evaluated by
densitometric scanning as described in A. C, Src-related
kinases were isolated from agrin-treated cells using src-CT antibodies
and protein A-Sepharose precipitation followed by phosphotyrosine
immunoblotting. As controls, either the src-CT antibody
(
Ab) or the cellular lysate (
L) was omitted.
*, differs significantly from control, p < 0.05 (by
ANOVA followed by Bonferroni's t test).
and
subunits, as well as of AChR-associated MuSK (Fig.
4). Maximal phosphorylation of these
proteins, particularly at lower agrin concentrations, required a 40-min
incubation with neural agrin. At 20 pM agrin, a slight
decrease in the extent of Src family phosphorylation (relative to
maximal stimulation at higher concentrations) was paralleled by a
decreased phosphotyrosine signal of the AChR
and
subunits.
Muscle agrin had no effect on any phosphorylation. Thus,
phosphorylation of AChR-bound Src-related kinases intimately correlates
with AChR
and
phosphorylation, raising the possibility that
AChR phosphorylation occurs in direct response to Src member
activation.
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Fig. 4.
Correlation between agrin-induced
phosphorylation of AChR-bound Src kinases and AChR subunits. C2
myotubes were treated with neural (4,8) or muscle (0,0) agrin as
indicated, and AChR protein complexes were precipitated with
-BT-Sepharose followed by phosphotyrosine immunoblotting
(Tox-P/PY-blot). The bottom panel is a
short exposure (Short exp.) of the blot to better visualize
the dose dependence and time course of AChR
phosphorylation.
subunit phosphorylation and
AChR aggregation (12, 13), whereas staurosporine does not affect
agrin-induced activation of MuSK (26). In this present study, we
observed that staurosporine inhibits agrin-induced phosphorylation of
AChR-bound Src family kinases and of AChR
and
subunits (Fig.
5A). Using increasing concentrations of staurosporine, Src member phosphorylation was gradually reduced strictly in parallel with AChR phosphorylation (Fig.
5B). Most dramatically, at 20 nM of
staurosporine, agrin did not induce significant phosphorylation of
AChR-associated Src members and AChR
and
subunits, whereas
phosphorylation of MuSK bound to the AChR occurred normally. Thus, MuSK
activation can be uncoupled from phosphorylation of Src kinases and the
AChR, suggesting that the AChR
and
subunits are direct
substrates for AChR-bound Src members but not for MuSK.
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Fig. 5.
Effects of tyrosine kinase inhibitors on
agrin-induced phosphorylation of AChR-bound MuSK, Src family kinases,
and the AChR and
subunits. A, C2 myotubes were treated for 5 h with 1 µM herbimycin (H), the indicated
concentrations of staurosporine (S), or a Me2SO
carrier control (c) and were then stimulated for 40 min with
1 nM neural agrin. Lysates were processed by
-BT-Sepharose precipitation and subjected to phosphotyrosine
immunoblotting. The lower panel shows a shorter exposure
(Short exp.) of the blot to better illustrate tyrosine
phosphorylation of the AChR
subunit. B, four experiments
as shown in A were quantitated by densitometric scanning.
Relative intensities of phosphorylation of AChR-bound MuSK, Src
members, and AChR subunits from cells not treated with agrin were set
to 0%. Agrin-induced signals of cells incubated with carrier and
lacking staurosporine were set to 100% (control), and intensities of
signals from staurosporine-treated cells were calculated accordingly.
The data are shown as the mean ± S.D.
-BT-precipitates, including MuSK, Src kinases, and the AChR (Fig.
5A). Together, these results demonstrate that herbimycin and
staurosporine exert their inhibitory effect on AChR phosphorylation and
clustering by interfering with distinct steps in the agrin signaling
pathway. Whereas herbimycin inhibits MuSK, staurosporine blocks
autophosphorylation of AChR-bound Src kinases, suggesting that Src
activation is downstream of MuSK and involved in AChR clustering.
subunit phosphorylation is strongly reduced,
suggesting that rapsyn is required for a step downstream of MuSK that
leads to AChR phosphorylation and clustering (20, 21).
/
myotubes. In control wild-type cells, agrin
induced phosphorylation of AChR-bound Src family kinases and the AChR
and
subunits as in C2 myotubes (Fig.
6). In contrast, in the rapsyn-deficient
myotubes, these phosphorylations were strongly reduced in both
agrin-treated and untreated cells. Control experiments confirmed that
rapsyn-deficient and wild-type cells express identical levels of MuSK
and Src members and that the amount of
-BT-isolated AChR is
indistinguishable (Fig. 6; Ref. 21). These results concur with
those on staurosporine in C2 myotubes, demonstrating that rapsyn is
required for agrin-induced phosphorylation of AChR-bound Src family
kinases and that this activation occurs downstream of MuSK. The data
further suggest that AChR
and
subunits are direct substrates
for these Src class kinases.
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Fig. 6.
In rapsyn-deficient myotubes, neural agrin
does not cause phosphorylation of AChR-bound Src members and AChR
and
subunits.
Rapsyn
/
and wild-type (W.T.) myotubes were treated with
1 nM neural (4,8) or muscle agrin (0,0) as indicated and
analyzed by
-BT-Sepharose precipitation followed by phosphotyrosine
immunoblotting. A shorter exposure of the film revealed strong
agrin-induced AChR
phosphorylation in the wild type and no signal
at all in mutant cells (not shown). Src family kinases
(src's) were identified by stripping and reprobing the blot
with src-CT antibodies (lower panel). The amounts of
-BT-isolated AChRs were equal between the two cell types (not
shown).
phosphorylation (10, 12).
-BT and
phosphotyrosine antibodies (Fig. 8). The same agrin concentration also
caused phosphorylation of AChR-bound MuSK, Src family kinases and the
AChR
and
subunits in C2 cells (Fig.
7A, see also Fig. 4). In
contrast, in S27 myotubes, 50 and 100 nM agrin did not
induce comparable phosphorylation of any of these proteins, even though
Src family kinases were associated normally with the AChR and AChRs
were present in normal amounts (Fig. 7A). Similarly, 100 nM neural agrin failed to induce the clustering of AChRs
and phosphotyrosine-containing proteins in these cells (Fig.
8). Some aggregates of
phosphotyrosine-containing proteins were occasionally observed in
agrin-treated S27 cells, but these were much smaller and less intense
than the typical clusters in C2 (Fig. 8).
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Fig. 7.
Agrin fails to activate AChR-bound Src family
kinases in S27 myotubes. A, S27 and C2 myotubes were
treated with neural (4,8) or muscle agrin (0,0) as indicated. AChRs
were isolated with biotinylated -BT followed by streptavidin-agarose
precipitation and phosphotyrosine immunoblotting. As controls, an
excess (10 µM) of free toxin was added (+T).
To ensure equal amounts of
-BT-precipitated AChRs and AChR-bound Src
family kinases, parallel cells were subjected to
-BT precipitation
and AChR
subunit or src-CT immunoblotting (middle and
bottom). For comparison, fractions (L) of the
initial lysate were loaded. B, cells treated with agrin as
indicated were lysed and processed by MuSK immunoprecipitation and
phosphotyrosine immunoblotting (top). As a control, the MuSK
antibody was omitted (
Ab). To compare the amounts of MuSK
between both cell types, fractions of cellular lysates (L)
were analyzed by MuSK immunoblotting (bottom). The majority
of MuSK (solid arrowheads) migrates with a lower molecular
weight in S27 than in C2 cells. A fragment of the MuSK antibody that
comigrates with the majority of MuSK in S27 cells is denoted by the
asterisk; the open arrowhead indicates
phosphorylation of a minor population of MuSK in S27 that migrates as
in C2.
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Fig. 8.
Agrin induces co-clustering of AChRs and
phosphotyrosine-containing proteins in C2 but not in S27 myotubes.
C2 and S27 myotubes were treated for 15 h with the indicated
concentrations of neural agrin. Cells were double-labeled for AChRs
with rhodamine-conjugated -BT (left panels) and for
phosphotyrosine proteins using anti-phosphotyrosine antibodies followed
by fluorescein isothiocyanate-coupled secondary antisera (right
panels). Whereas 0.5 nM of agrin induces pronounced
co-clustering of AChRs and phosphotyrosine-containing proteins in C2,
no comparable protein aggregates are observed in S27 myotubes, not even
at 100 nM of agrin. Scale bar, 20 µm.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
subunits, which
has been described previously in the chick (43). Our present data are
the first report of this modification in mammalian myotubes, and they
indicate that AChR
phosphorylation caused by agrin highly
correlates with AChR clustering. Thus, neural but not muscle agrin
induces both phosphorylation and aggregation. Furthermore, herbimycin
and staurosporine inhibit both agrin-induced AChR clustering and
phosphorylation, and phosphorylation of
is not observed in
rapsyn
/
and S27 myotubes in which AChRs are not clustered by agrin.
As it reaches a peak after 40 min of agrin treatment, AChR
phosphorylation may play a role early in agrin-induced clustering and
compensate for phosphorylation of AChR
subunits, allowing
agrin-induced clustering of mutated AChRs lacking
cytoplasmic
tyrosine residues (14). Because phosphorylated AChR
subunits bind
to the adaptor molecule Grb2 in the Torpedo electric organ
(44), one role of
phosphorylation in the agrin signaling pathway in
myotubes may be to recruit Grb2 to AChRs, thereby linking further
signaling pathways to the AChR. Colledge and Froehner (44) have
speculated that such an interaction may have to be of low affinity,
which fits our observed low relative extent of
phosphorylation. In
fact, one intriguing possibility is that agrin-induced
phosphorylation, which is of high relative stoichiometry, regulates the
channel properties of the AChR such that clustered receptors have
accurate desensitization kinetics, whereas agrin-induced
phosphorylation regulates the association of AChRs with signaling
pathways, thereby facilitating postsynaptic assembly.
and
subunits are direct substrates for agrin-activated Src members bound to
the AChR but not for MuSK. First, phosphorylation of Src members and of
AChRs are strictly correlated over a wide range of agrin concentrations
and incubation times. Second, staurosporine inhibits Src family
activation, which intimately correlates with reduced AChR
and
phosphorylation, whereas MuSK is unaffected. Third, staurosporine
is known to inhibit both tyrosine kinase autophosphorylation and
phosphorylation of their direct substrates in parallel, as shown in the
case of the insulin receptor and its substrate IRS-1 and TrkA and its
substrate phospholipase C-
1 (45, 46). Finally, the differential
effect of herbimycin, which also inhibits MuSK, versus
staurosporine suggests a hierarchy of phosphorylation, where MuSK
activation is upstream of the activation of AChR-bound Src kinases,
which phosphorylate the AChR
and
subunits.
and
phosphorylation (29, 47). These previous findings and our present
studies suggest a complex of the AChR in association with rapsyn, MuSK,
and Src family kinases, in which rapsyn holds AChR-bound MuSK and Src
members in a conformation that allows activation of Src kinases by
MuSK. The latter scenario could occur via an interaction of MuSK
sequences with the Src family SH2 or SH3 domains as mentioned earlier.
Src members thus appear to phosphorylate the AChR in response to agrin,
a process that occurs downstream of MuSK and requires rapsyn.
phosphorylation, which, in turn, highly
correlates with AChR aggregation (12). Together, these striking
correlations suggest that AChR-associated Src family kinases play a
role in agrin-induced postsynaptic differentiation, to which AChR
phosphorylation itself may contribute.
Activation of AChR-bound Src family kinases by agrin correlates with
AChR clustering
/
, and S27 myotubes were
incubated for 40 min with neural (4,8) or muscle (0,0) agrin at
concentrations of up to 100 nM. In the case of
staurosporine and herbimycin, inhibitors were added to cells 5-6 h
prior to agrin treatment. AChRs were isolated from cell extracts by
-BT precipitation, and phosphorylation of receptor-associated
proteins (MuSK, Src-related kinases) and AChR subunits was assayed by
phosphotyrosine immunoblotting. For AChR clustering, myotubes were
treated for 15 h with up to 100 nM agrin followed by
staining with rhodamine-conjugated
-BT and analysis by fluorescence
microscopy (see also Refs. 12 and 21).
-fodrin (1, 48), are known substrates for Src class kinases (30). Talin and paxillin are also found at focal adhesion sites, which have similarities to the postsynaptic apparatus. In both
cases, large protein complexes mediating cell-cell contact are
assembled by tyrosine kinase signaling (49). Moreover, integrins, which
modulate agrin signaling, are found at both structures (50). Together,
these similarities suggest that AChR-associated Src family kinases may
contribute to postsynaptic assembly in a way analogous to their
proposed role in focal adhesion site assembly, by phosphorylating
additional proteins, including cytoskeletal elements, and thereby
stabilizing growing AChR aggregates.
and
subunits and possibly further proteins in the vicinity. This AChR-bound signaling cascade causes preassembled AChR protein complexes containing rapsyn, tyrosine
kinases, and postsynaptic proteins to be linked to a primary synaptic
scaffold formed by MuSK (20), thereby providing a stepwise assembly
mechanism of the postsynaptic apparatus. In addition, Src kinases may
contribute to the stabilization and further differentiation of the
postsynaptic apparatus, by phosphorylating cytoskeletal proteins,
thereby modifying associations between the postsynaptic membrane and
the cytoskeleton. Under several different experimental conditions,
therefore, Src phosphorylation and activation are correlated with AChR
clustering, and the precise role of Src family kinases in postsynaptic
assembly remains to be investigated.
![]() |
ACKNOWLEDGEMENTS |
---|
We are grateful to Drs. Dominique Di
Scala-Guenot, Matthias Gesemann, and Anne McKinney and to the members
of the Fuhrer laboratory for valuable comments on this manuscript. We
also thank Roland Schoeb and Eva Hochreutener for excellent help with
the photography and Dr. Medha Gautam for preparation of rapsyn /
cells.
![]() |
FOOTNOTES |
---|
* This work was supported by the Kanton Zürich and the Dr. Eric Slack-Gyr Foundation and by grants from the Swiss National Science Foundation and the Swiss Foundation for Research on Muscle Diseases (to C. F.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed. Tel.: +41-1-635-33-10; Fax: +41-1-635-33-03; E-mail: chfuhrer@hifo.unizh.ch.
Published, JBC Papers in Press, January 31, 2001, DOI 10.1074/jbc.M007024200
2 C. Fuhrer, unpublished observations.
3 M. Moransard and C. Fuhrer, unpublished observations.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
AChR, acetylcholine
receptor;
-BT,
-bungarotoxin;
AMPA,
-Amino-3-hydroxy-5-methylisoxazole-4-propionate;
CT, C-terminal;
BAPTA-AM, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic
acid tetrakis(acetoxymethyl ester);
MuSK, muscle-specific kinase;
PAGE, polyacrylamide gel electrophoresis;
SH2 and SH3, Src homology 2 and 3, respectively;
ANOVA, analysis of variance.
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