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The Journal of Neuroscience, June 1, 2001, 21(11):3806-3818
Src-Class Kinases Act within the Agrin/MuSK Pathway to Regulate
Acetylcholine Receptor Phosphorylation, Cytoskeletal Anchoring, and
Clustering
Ali S.
Mohamed,
Kimberly A.
Rivas-Plata,
Jonathan R.
Kraas,
Suha M.
Saleh, and
Sheridan L.
Swope
Department of Neuroscience, Georgetown University Medical Center,
Washington DC 20007-2197
 |
ABSTRACT |
Synaptogenesis at the neuromuscular junction requires agrin-induced
stable localization of acetylcholine receptors (AChRs) at the endplate.
The effects of agrin are transduced by the muscle-specific receptor
tyrosine kinase (MuSK). This study provides evidence that Src-class
protein tyrosine kinases mediate the effects of agrin-activated MuSK to
regulate clustering and anchoring of AChRs in skeletal muscle. MuSK was
complexed with both Src and Fyn in the C2 mouse muscle cell line. These
associations were enhanced by agrin and by increasing protein tyrosine
phosphorylation with pervanadate. Coupling between MuSK and the
Src-class kinases in vivo appeared to be caused by a
phosphotyrosine-SH2 domain interaction because binding of MuSK to the
SH2 domains of Fyn and Src in vitro was specific,
enhanced by phosphorylation, and dependent on MuSK autophosphorylation.
In addition, Src and Fyn phosphorylated MuSK. AChR phosphorylation,
stimulated by agrin or pervanadate, was inhibited by blocking Src-class
kinases with PP1. Furthermore, agrin-induced clustering and
cytoskeletal anchoring of AChRs was dependent on Src-family kinases.
These data support the conclusion that Fyn and Src act downstream of
MuSK to regulate the stable localization of AChRs at the neuromuscular
endplate during agrin-induced synaptogenesis.
Key words:
synaptogenesis; neuromuscular junction; Src; Fyn; skeletal muscle; cytoskeleton; phosphorylation; MuSK; agrin; PP1; Src
homology 2 domain; pervanadate
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INTRODUCTION |
Synaptogenesis is best understood at
the neuromuscular junction (NMJ), where many of the molecular players
have been identified (Hall and Sanes, 1993
; Sanes and Lichtman, 1999).
In the immature myotube, nicotinic acetylcholine receptors (AChRs) are
dispersed throughout the membrane. During innervation, the receptors
become stably localized to the site of nerve-muscle contact. One of
the major goals in studying the NMJ is to determine the mechanism by
which AChRs are concentrated underneath the motor nerve terminal. During synaptogenesis, mobile AChRs are likely to diffuse to the neuron
contact site and become anchored there. This process, known as
"diffusion trap," is a model originally proposed by Edwards and
Frisch (1976).
Three proteins critical to the formation of the NMJ endplate are agrin,
the muscle-specific receptor tyrosine kinase (MuSK), and rapsyn.
Agrin is a neuron-derived factor that induces postsynaptic differentiation events, including clustering, cytoskeletal anchoring, and phosphorylation of AChRs (Godfrey et al., 1984; Wallace et al.,
1991; Wallace, 1995; Gautam et al., 1996; Cohen et al., 1997; Jones et
al., 1997; Meier et al., 1997; Rimer et al., 1997). Previous studies
indicate that the receptor for agrin includes MuSK (DeChiara et al.,
1996; Glass et al., 1996). For example, MuSK-deficient myotubes fail to
cluster AChRs in response to agrin (Glass et al., 1996). Rapsyn is a
cytoskeletal, peripheral membrane protein crucial for AChR clustering
and anchoring at the postsynaptic endplate (Barrantes et al., 1980;
Burden et al., 1983; Walker et al., 1984; Froehner et al., 1990;
Phillips et al., 1993; Gautam et al., 1995). In rapsyn minus myotubes,
agrin activates MuSK but does not induce phosphorylation or clustering
of AChRs, indicating that rapsyn transduces the action of
agrin-activated MuSK (Gautam et al., 1995; Apel et al., 1997). Although
insights into the mechanism for synaptogenesis at the NMJ have been
gained, the exact relationships among agrin, MuSK, and rapsyn remain to
be determined.
Protein tyrosine phosphorylation is important for AChR clustering and
cytoskeletal anchoring (Meier et al., 1995; Wallace, 1995; Ferns et
al., 1996; Fuhrer et al., 1997; Swope et al., 1999). It has been
suggested that a kinase downstream of MuSK directly phosphorylates
AChRs (Apel et al., 1997; Fuhrer et al., 1997). Src-class kinases
represent the predominant protein tyrosine kinase activity in
AChR-enriched postsynaptic membranes (Swope and Huganir, 1993).
Src-family kinases complex with the AChR and phosphorylate the receptor
in vitro (Swope and Huganir, 1993, 1994; Fuhrer and Hall,
1996). The functional significance of AChR phosphorylation by Src-class
kinases may be in anchoring the receptor to the cytoskeleton. For
example, Src-family kinases are complexed with rapsyn, and activation
of Src-class kinases by rapsyn in heterologous cells results in AChR
phosphorylation and cytoskeletal binding (Mohamed and Swope, 1999). The
goal of the present study was to further clarify the signal
transduction cascade by which Src-family kinases may mediate
synaptogenesis at the NMJ. The relationship between MuSK and Src-class
kinases was explored. In addition, we examined whether the effects of
agrin to stimulate AChR phosphorylation, cytoskeletal anchoring,
and clustering are dependent on Src-class kinases.
| |
MATERIALS AND METHODS |
Constructs. The full-length coding sequence of
Torpedo Fyn in pBK-CMV
lac (pBK-CMV) was described
previously (Mohamed and Swope, 1999). Src and the dominant negative Src
in pCDNA3 (Biscardi et al., 1999) were gifts from Dr. S. Parsons
(University of Virginia, Charlottesville, VA). Mouse rapsyn in
pGW1-CMV, myc-tagged Torpedo MuSK, catalytically inactive
myc-tagged MuSK (MuSK[K686R]), and empty vector pBK-CMV
(Gillespie et al., 1996) were generous gifts from Dr. R. L. Huganir (Johns Hopkins University). The Bruton's tyrosine kinase (Btk)
cDNA in pCIS-2 (Yang et al., 1995) was a gift from Dr. S. Desiderio
(Johns Hopkins University). The construct encoding neural-specific
agrin (C-agrin 4,8) (Ferns et al., 1993) was a gift from Dr. M. Ferns
(McGill University, Montreal, Canada). pGEX2T Fyn and Src SH2
domain fusion proteins were prepared as described previously (Swope and
Huganir, 1993). Src SH2 domain fusion protein construct was prepared by
PCR using a proviral pRSV-vSrc (DeLorbe et al., 1980) as a template
followed by subcloning into pGEX-2T as described (Swope and Huganir,
1994). The proviral pRSV-vSrc was a gift from Dr. J. Brugge, originally
from Dr. J. M. Bishop (University of California, San
Francisco). pGEX Grb2 SH2 construct (Oligino et al., 1997) was a
gift from Dr. C. R. King (Georgetown University, Washington, DC).
Antibodies. The anti-myc rabbit polyclonal (JH2235) raised
against the peptide EQKLISQQDL and anti-rat agrin rabbit polyclonal (JH1037) raised against the peptide sequence (K)LEDAVTKPELRPCPT corresponding to amino acids 1922-1936 in the C terminus of rat agrin
were gifts from Dr. R. Huganir (Johns Hopkins University). The rat
monoclonal antibody (mAb) 148 against the AChR 
(Ratnam et al.,
1986) was a gift from Dr. J. Lindstrom (University of Pennsylvania,
Philadelphia, PA). The C-terminal rabbit anti-Src antibody that
recognizes all Src kinases (src2), the N-terminal Fyn-specific antibody (FYN 3), the N-terminal Fyn-specific
mouse monoclonal (FYN 15), the N-terminal MuSK-specific goat polyclonal antibody (N19), the C-terminal MuSK-specific goat polyclonal antibody (C19), and the monoclonal anti-phosphotyrosine antibody (PY99) were
purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The
affinity-purified anti-MuSK rabbit polyclonal antibody, 
-MuSK-ZH (Fuhrer et al., 1997), was a gift from Dr. Z. W. Hall [National Institute of Neurological Disorders and Stroke (NINDS), National Institutes of Health (NIH), Bethesda, MD]. The anti-myc 9E10 ascites were purchased from Sigma (St. Louis, MO). The monoclonal
anti-phosphotyrosine antibody, 4G10, and the Src-specific monoclonal
antibody, GD11, were purchased from Upstate Biotechnology (Lake Placid,
NY). The Src-specific mouse monoclonal antibody, Ab-1, was
purchased from Oncogene-Calbiochem (Cambridge, MA). The
phosphorylation-state-specific anti-Src-family kinase antibody, GW001,
was raised against the phosphopeptide
KRLIEDNEpY416TARQG corresponding to the
amino acids 409-421 in the catalytic domain of avian c-Src, to which
an amino terminal lysine was added. The antiserum was affinity purified
by applying the flow-through of a column containing nonphosphorylated
peptide to an affinity column derived from the phosphopeptide. The
purified anti-Src-class kinase phosphorylation state-specific
antibodies were then eluted from the
KRLIEDNEpY416TARQG column. A MuSK-specific
rabbit antiserum, GW002, was made against the peptide
KPSFCSIHRILQRMCERAEGTVGV corresponding to the C-terminal 23 amino acids
of mouse MuSK with an amino terminal lysine.
Cell culture and transfection. Cell culture reagents were
purchased from Life Technologies (Gaithersburg, MD). The mouse muscle cell line C2C12 (C2), obtained from Dr. E. Ralston (NINDS, NIH), was
grown and maintained in growth media composed of DMEM, 20% FBS, and
0.5% chick embryo extract. For induction of differentiation, myoblasts
at 60-80% confluence were switched to a low serum differentiation media (DM) composed of DMEM plus 2% horse serum. For experiments examining the effect of pervanadate and/or PP1 treatments on
phosphorylation, cytoskeletal anchoring, or clustering, cells were
grown on collagen- or gelatin-coated dishes. The quail fibroblast cell
line, QT-6 (Moscovici et al., 1977), was maintained as described
(Blount and Merlie, 1988). COS-7 cells were grown and maintained as
described (Gluzman, 1981). QT-6 or COS-7 cell cultures at ~50%
confluence were transfected using the calcium phosphate method of Chen
and Okayama (1987). For QT-6 transfection, 2 µg each of inactive
MuSK[K686R], MuSK, rapsyn, Fyn, Src, Btk, or pBK-CMV plasmid
constructs was used per 60 mm dish, and pBK-CMV was added, if
necessary, to bring the total amount of plasmid DNA to 12 µg per
dish. For transfection of COS-7 cells, 30 µg of agrin 4,8 was used
per 100 mm dish. In each case, transfection efficiency was typically
50%, as determined by the Green Lantern vector (Life Technologies).
For transfection of C2 cells, 2 µg dominant negative Src or empty
plasmid was mixed with 3 µl of FuGene (Roche) and 100 µl of
serum-free DMEM and pipetted directly onto 35 mm wells containing
60-80% confluent myoblasts. After 1-2 min, 2 ml of DM was added, and
the cells were returned to the incubator. After 48 hr, the medium was
replaced with fresh DM, and the cells were treated with or without
agrin 10 hr later. Transfection efficiency was typically ~20%, as
determined by the Green Lantern vector.
Phosphorylation of MuSK in QT-6 cells. Transfected QT-6
cells expressing MuSK or MuSK[K686R] were solubilized in 2%
SDS, 50 mM Tris, pH 7.4, 150 mM NaCl, 2 mM EDTA, 4 mM EGTA, 2 mM sodium orthovanadate (SDS-lysis buffer) for 10 min at 22°C. The lysates were
diluted to a final concentration of 0.4% SDS and 2% Triton X-100 at 1 mg/ml protein. Lysates were sonicated for 30 sec on ice and spun at
225,000 × g for 10 min at 4°C. Solubilized proteins after centrifugation were immunoprecipitated with the polyclonal anti-myc antibody (JH2235). Immunoprecipitates were analyzed by Western
blotting using a mixture of anti-phosphotyrosine monoclonal antibodies
4G10 and PY99.
Phosphorylation of MuSK and AChR in C2 myotubes. C2 myotubes
were preincubated for 1 hr in DMEM supplemented with 0.1% horse serum
followed by pretreatment with or without 10 or 20 µM PP1 for 1 hr. Myotubes were then incubated
with or without 100 pM agrin or 30 µM pervanadate. Treated myotubes were rinsed
twice with ice-cold PBS and solubilized on ice for 10 min using 50 mM Tris, pH 7.4, 1% Triton X-100, 1% sodium
deoxycholate, 0.1% SDS, 150 mM NaCl, 2 mM EDTA, 4 mM EGTA, and 1 mM sodium vanadate (RIPA buffer) supplemented
with the protease inhibitors, 0.1 mM
phenylmethylsulfonylfluoride, 10 µg/ml pepstatin, 10 µg/ml
chymostatin, 10 mg/µl antipain, and 10 U/µl Trasylol. Lysates were
centrifuged at 225,000 × g for 10 min, and the
supernatants were immunoprecipitated with antibodies specific for MuSK
(GW002) or AChR (88b). Phosphorylation of MuSK was analyzed by Western
blotting with a mixture of the anti-phosphotyrosine antibodies, 4G10
and PY99. Phosphorylation of AChR was detected with a mixture of the
phosphorylation state-specific anti-AChR subunit, JH1360, and
anti-AChR 
subunit, JH1358. AChR blots were stripped and reprobed
with AChR subunit mAb 148.
Binding of MuSK to SH2 fusion proteins. SH2 domain fusion
proteins were prepared as described previously (Swope and Huganir, 1994). In brief, the cDNAs for SH2 domains from Src-class kinases and
Grb2 were inserted into a bacterial expression vector containing the
sequence for glutathione S-transferase (GST). The SH2-GST fusion protein was grown in BL21 bacteria and purified on glutathione agarose (Sigma). The fusion protein still bound to the agarose was
subsequently used as an affinity reagent. Transfected QT-6 cells
expressing MuSK or MuSK[K686R] were solubilized in SDS-lysis buffer
for 10 min at 22°C. The lysates were diluted to a final concentration
of 0.4% SDS plus 2% Triton X-100, sonicated for 30 sec on ice, and
spun at 225,000 × g for 10 min at 4°C. Solubilized proteins, after centrifugation, were incubated with the SH2 domain fusion protein affinity resin for 2 hr, and nonspecific proteins were
washed away as described previously (Swope and Huganir, 1994). Binding of MuSK was detected by Western analysis using anti-myc mouse
monoclonal 9E10.
Immunoprecipitation and coimmunoprecipitation. C2 myotubes
were rinsed with ice-cold PBS and solubilized for 15 min either in 50 mM Tris, pH 7.4, 150 mM
NaCl, 4 mM EGTA, 4 mM EDTA,
1% Triton X-100, and 2 mM Na-orthovanadate
(Triton lysis buffer) or in RIPA buffer supplemented with the protease
inhibitors, 0.1 mM phenylmethylsulfonylfluoride, 10 µg/ml pepstatin, 10 µg/ml chymostatin, 10 mg/µl antipain, and 10 U/µl Trasylol. Lysates were centrifuged at 225,000 × g for 10 min, and the supernatants were immunoprecipitated
with antibodies specific for MuSK (a mixture of N19 and C19 or,
alternatively, GW002), AChR (88b), Fyn (Fyn 15), or Src (Ab-1).
Immunoprecipitates were analyzed for precipitated or coprecipitated
proteins by immunoblotting using specific antibodies for MuSK
(-MuSK-ZH), a mixture of the phosphorylation state-specific
anti-AChR subunit (JH1360), and anti-AChR subunit (JH1358)
antibodies, AChR subunit (mAb 148), Fyn (Fyn 3), Src (GD11), or Fyn
and/or Src (Src2).
Detergent extraction. C2 myotubes were preincubated with
DME-F12 (Sigma) supplemented with 1 mg/ml bovine serum albumin (BSA) (RIA Grade, Sigma) and pretreated with or without 10-20
µM PP1 followed by treatment with or without
100 pM agrin. Treated myotubes were rinsed twice
with ice-cold PBS and extracted on ice with 10 mM
HEPES, pH 7.4, 50 mM NaCl, 300 mM sucrose, 1 mM
MgCl2, and 1 mM
Na-orthovanadate (Prives buffer) (Prives et al., 1982) containing 0.5%
Triton X-100 and the protease inhibitors 0.1 mM
phenylmethylsulfonylfluoride, 10 µg/ml pepstatin, 10 µg/ml
chymostatin, 10 mg/µl antipain, and 10 U/µl Trasylol. Treatment of
cultured myotubes with Prives buffer rapidly extracts the plasma
membrane and other membranous organelles but leaves the cytoskeleton
intact (Prives et al., 1982). After the desired time, the buffer was
removed, and the cells were rinsed once with Prives buffer with 0.1%
Triton X-100. Residual cytoskeletal proteins were solubilized and
scraped from the dish with SDS-PAGE sample buffer, sonicated for 30 sec, and spun at 225,000 × g for 10 min at 4°C.
Solubilized cytoskeletal proteins were analyzed by SDS-PAGE and Western
blotting using rat mAb 148, specific for the AChR subunit. For
determining the effects of PP1 on the amount of AChRs, sister C2
cultures were treated and processed as above, except the Prives buffer
contained no Triton X-100.
In vitro kinase assays. C2 myotubes were washed two
times in ice-cold PBS and then solubilized in RIPA buffer plus the
protease inhibitors 0.1 mM phenylmethylsulfonylfluoride,
leupeptin 10 µg/ml, and 10 U/µl Trasylol. Lysates were spun at
225,000 × g for 10 min at 4°C. Supernatants from
each 60 mm dish of C2 myotubes were divided into three equal portions.
One-third of each supernatant was used for immunoprecipitation of Fyn,
using mAb Fyn 15, Src using mAb Ab-1, or MuSK using GW002. The
immunocomplexes were immobilized using protein G- or protein
A-Sepharose and washed three times in RIPA buffer, then twice in kinase
reaction buffer without ATP. The immunoprecipitates were used in an
in vitro phosphorylation reaction with 20 µM ATP and 7.5 µg acid-denatured enolase as
described previously (Feder and Bishop, 1990). The reactions were
analyzed by Western blotting with a mixture of the anti-phosphotyrosine antibodies, 4G10 and PY99.
Treatments with PP1, agrin, or pervanadate. For biochemical
analysis, C2 myotubes were preincubated in DMEM supplemented with 0.1%
horse serum for 1 hr, then PP1, agrin, or pervanadate were added
directly to the medium. For detergent extraction experiments, C2
myotubes were preincubated for 1 hr in DME-F12 medium supplemented with
0.1% RIA grade BSA before agrin or PP1 treatments. Q-T6 cells were
treated with pervanadate in growth medium containing Medium 199 (Life
Technologies) supplemented with 1% DMSO, 5% FBS, 10% tryptose
phosphate broth, and 1% penicillin-streptomycin. For microscopic
analysis of clustering, myotubes in DM were treated overnight with
agrin. The concentrations of agrin, pervanadate, and PP1 were 100 pM, 30 µM, and 10-20
µM, respectively.
Immunocytochemistry. Myotubes in DM were incubated at
37°C/8% CO2 for 1 hr with 80 nM tetramethyl(rhodamine)--bungarotoxin or
Texas Red--bungarotoxin (Molecular Probes, Eugene, OR). Cells at
room temperature were washed with PBS, fixed for 15 min with 3%
paraformaldehyde/4% sucrose, and then permeabilized with 0.1% Triton
X-100 for 5 min. Myotubes expressing the dominant negative Src were
identified by double labeling with the pan-Src kinase antibody,
src2, followed by FITC-goat-anti-rabbit secondary antibody (Molecular Probes). Cells were visualized by epifluorescence on a Nikon
Eclipse TE300 inverted microscope using a 20× objective. Images were
captured with a Magnafire digital camera and software. Cluster sizes
were analyzed with Adobe Photoshop.
Immunoblotting. Proteins were resolved by SDS-PAGE and
transferred to polyvinylidene difluoride (PVDF) membranes (Immobilon, Millipore, Bedford, MA) by electroblotting. The PVDF membranes were
blocked in 5% nonfat milk in Tris-buffered saline (50 mM Tris, pH 7.4, 200 mM
NaCl) containing 0.05% Tween 20. For phosphotyrosine immunoblotting,
the PVDF membranes were blocked in 3-5% BSA. The dilutions for
primary antibodies were as follows: anti-MuSK antibody (-MuSK-ZH), anti-phosphotyrosine 4G10, anti-phosphotyrosine
PY99, and anti-AChR subunit mAb 148, each at 1:1000; anti-Fyn
(Fyn3) and Src2 (Pan-Src), each at 1:500; anti-myc mouse monoclonal
(9E10), anti-active state Src kinase-specific rabbit antibody (GW001), and anti-Src mouse monoclonal (GD11), each at 1:200. Horseradish peroxidase-conjugated secondary antibodies were from Jackson
ImmunoResearch Laboratories (West Grove, PA) and were used at dilutions
of 1:50,000. Horseradish peroxidase-bound signal was detected using an
enhanced chemiluminescence (ECL) (Pierce, Rockford, IL) followed by
exposure of BioMax MR-1 film (Eastman Kodak, Rochester, NY) or, for
greater sensitivity, HyperFilm ECL (Amersham, Arlington Heights, IL). For some experiments, blots were stripped by incubation at 55°C for
30 min in 62.5 mM Tris, pH 6.8, 2% SDS, and 100 mM -mercaptoethanol and reanalyzed by Western blotting.
Agrin production. Medium from COS-7 cells expressing a
C-agrin 4,8 or transfected with empty plasmid pBK-CMV was collected and
replaced each day for 2 d. The concentration of agrin in the collected medium was determined by Western blotting (Sugiyama et al.,
1994) using agrin-specific polyclonal antiserum (JH1037) and standard
purified agrin of known concentration provided by Dr. J. E. Sugiyama (NINDS, NIH).
Fusion protein production. SH2 domain fusion proteins
derived from Fyn, Src, and Grb2 as well as backbone GST protein were prepared as described previously (Swope and Huganir, 1993).
Pervanadate preparation. Sodium pervanadate was prepared as
described previously (Meier et al., 1995).
Quantification of Western analysis. Analysis of Western
blots was performed by scanning exposed films in transmittance mode and
quantifying the digital data with a GS-710 Imaging Densitometer and
Multianalyst software (Bio-Rad, Hercules, CA). The integrated optical
density (O.D.) for the area of each band, stated as volume or
O.D. × area and here abbreviated as O.D., was determined using Multianalyst software. To determine the linear range of Biomax film and
HyperFilm ECL, serial dilutions of several proteins, including MuSK,
Src, Fyn, and AchR, were transferred to PVDF, probed with an
appropriate antibody, reacted with ECL reagent, and exposed to film.
The O.D. versus dilution factor was plotted; O.D. values between 0.2 and 10 were linear for both Biomax film and HyperFilm ECL. Thus,
quantification was performed using bands that were not saturated and
had O.D. values within this range. All data for coimmunoprecipitation
and phosphorylation experiments were normalized for random variability
in the level of recovered protein. To analyze changes in
coimmunoprecipitation, the O.D. for the coprecipitated protein was
normalized for the immunoprecipitated protein. For example,
coprecipitation of Src with MuSK was corrected for the amount of MuSK
immunoprecipitated (see Fig. 1A). Normalization for
all of the coimmunoprecipitations of Figures 1 and 2 was performed in
the same manner. Likewise, analysis of phosphorylation was corrected
for any variability in isolation of the phosphorylated protein or
kinase. Thus, the O.D. from the anti-phosphotyrosine signal was divided
by the anti-protein O.D. for MuSK and AChR, whereas enolase
phosphorylation was normalized for the amount of kinase isolated. For
Src and Fyn autophosphorylation, the O.D. from the
anti-PY416Src antibody was divided by the
anti-Src or anti-Fyn O.D. This normalization corrected for random
variability in the expression and immunoprecipitation of each specific
protein. Each experiment was performed three times, unless indicated
otherwise. Data are expressed as the mean ± SEM for
n 3. Except for Figures 8C and 9E, all Figures represent the data of a single
representative experiment.
Protein determinations. Protein concentrations were
determined by the method of Lowry using BSA as a standard (Lowry et
al., 1951).
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RESULTS |
Association of Src-class kinases with MuSK
To test for a relationship between Src-class kinases and MuSK,
complex formation between these two types of kinases was examined initially. C2 myotube proteins were solubilized under mild conditions of 1% Triton lysis buffer to maintain protein-protein interactions and immunoprecipitated with MuSK-specific antibodies. When the precipitates were analyzed for Src by Western blotting, there was
specific coimmunoprecipitation of Src with MuSK compared with empty
beads (Fig. 1A,
lane 2 vs 4). In a similar manner,
Fyn was specifically coimmunoprecipitated with MuSK as demonstrated
with a Fyn-specific antibody in Western analysis of the anti-MuSK
immunoprecipitates and control beads (Fig. 1B,
lane 2 vs 4). Activation of other receptor tyrosine kinases, such as the PDGF receptor, results in
binding of Src-class kinases (Claesson-Welsh, 1994). In fact, the trkB
receptor kinase, which is closely related to MuSK, binds Fyn (Iwasaki
et al., 1998). Therefore, whether activation of MuSK by agrin enhanced
association of MuSK with Src and Fyn was tested. Treatment of C2 cells
for 1 hr with agrin increased complex formation between MuSK and both
Fyn and Src (Fig. 1A,B, lane
1 vs 2). The percentage of each kinase complexed
with MuSK was determined by comparing the Src and Fyn signal in the
MuSK immunoprecipitate with an aliquot of lysed cells. Densitometry
scanning quantification, with normalization for recovery of MuSK, was
performed to determine the percentage of Src and Fyn complexed with
MuSK. In the absence of agrin, the percentage of total Src and Fyn
precipitated with MuSK was 1.7 ± 0.2 and 2.10 ± 0.08%,
respectively. After agrin treatment, the percentage of Src
coimmunoprecipitated was increased to 3.26 ± 0.09% over
nontreated cells (p < 0.05), whereas the percentage of Fyn was increased to 3.8 ± 0.3%
(p < 0.025). For both kinases, an enhancement
of association with MuSK in response to agrin occurred in every
experiment. The average percentage increase in association of Fyn and
Src with MuSK caused by agrin was 80 ± 20 and 110 ± 50%,
respectively. These data indicated that Src and Fyn were in a complex
with MuSK and that this association was stimulated by agrin.
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Figure 1.
Effect of agrin on coimmunoprecipitation of MuSK
with Fyn and Src. A, C2 myotubes were treated with 100 pM agrin (Agrin +) or an
equal volume of conditioned medium vehicle (Agrin  )
for 1 hr. Myotubes were solubilized in lysis buffer and centrifuged,
and supernatants representing 90% of a 100 mm plate were
immunoprecipitated with a mixture of goat polyclonal anti-MuSK
antibodies, N19 and C19 (MuSK), or empty protein
A-Sepharose (PAS). The immunoprecipitates and 100 µg
of lysate supernatant after centrifugation (Lys) were
resolved by 8% SDS-PAGE and analyzed by Western blotting using
anti-Src antibody GD11 (-Src). B, The
blot from A was stripped and reanalyzed by Western
blotting using anti-Fyn antibody Fyn3 (-Fyn).
C, The blot from A was stripped and
reanalyzed by Western blotting using anti-MuSK-ZH antibody
(-MuSK). D, C2 myotubes were
treated with agrin or conditioned medium vehicle and solubilized as in
A. The supernatants after centrifugation were
immunoprecipitated with anti-Fyn antibody Fyn15 (Fyn),
anti-Src antibody Ab-1 (Src), or empty protein
G-Sepharose (PGS). The immunoprecipitates and 100 µg
of lysate supernatant after centrifugation (Lys) were
resolved by 7% SDS-PAGE and analyzed by Western blotting using
anti-MuSK-ZH antibody (-MuSK).
E, The blot from D was stripped and
reanalyzed by Western blotting with anti-Src-family kinase antibody
Src2 (-pan-Srcks). In
A-E, an arrowhead
indicates the position of Src, Fyn, MuSK, MuSK, and Src-class kinases,
respectively.
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The existence of this complex was further tested by the reciprocal
experiment. Fyn and Src immunoprecipitates were isolated from C2
myotubes and analyzed for MuSK. MuSK was coimmunoprecipitated with both
Fyn and Src but not precipitated by empty beads (Fig. 1D, lanes 2 and 4 vs
6). By comparing with an aliquot of lysate (Fig.
1D, lane 7), the percentage of MuSK
complexed with Fyn and Src was calculated to be 1.6 ± 0.3 and
1.3 ± 0.3%, respectively. Agrin also affected the
coimmunoprecipitation of MuSK with Fyn and Src (Fig.
1D, lane 1 vs 2 and lane
3 vs 4). After treatment of C2 cells with agrin,
the percentage of MuSK precipitated with Fyn rose to 2.8 ± 0.6%
(p < 0.05), and the percentage precipitated with Src rose to 2.0 ± 0.4% (p < 0.025).
In three experiments, agrin enhanced association of MuSK with Fyn and
Src, 80 ± 20 and 57 ± 5%, respectively. These data
provided strong evidence that in C2 myotubes, Fyn and Src were
complexed with MuSK under control conditions and that association was
stimulated further by agrin.
Because Fyn, Src, and MuSK are all protein tyrosine kinases, the effect
of increasing phosphorylation on complex formation was examined.
Treatment of C2 cells with pervanadate, an inhibitor of protein
tyrosine phosphatases (Huyer et al., 1997), resulted in a dramatic
increase in protein tyrosine phosphorylation, indicating constitutive
phosphorylation and dephosphorylation in C2 myotubes (A. S. Mohamed and S. L. Swope, unpublished results). To examine the
effect of phosphorylation on association of MuSK and Src-class kinases,
myotubes were treated under control conditions or with pervanadate, and
MuSK was immunoprecipitated from the lysates. Src-class kinase(s) was
specifically coprecipitated with MuSK as demonstrated by analysis of
the MuSK precipitates by Western blotting using a pan-Src-class kinase
antibody (Fig. 2A,
lane 2 vs 8). Coimmunoprecipitation of the
Src-class kinase(s) with MuSK was enhanced by treatment with
pervanadate (Fig. 2A, lane 1 vs
2). In two experiments, inhibition of protein tyrosine
dephosphorylation increased the coimmunoprecipitation of Src-class
kinases with MuSK from 1.35 ± 0.03 to 2.8 ± 0.1%, and in a
third experiment the increase was from 3.8 to 7.3%. Thus, all three
experiments showed an increase in complex formation between Src kinases
and MuSK by pervanadate treatment, with an average increase of 117 ± 6%. The reverse analysis was also performed. Fyn and Src were immunoprecipitated from control and pervanadate-treated myotubes, and
the precipitates were analyzed for MuSK. Pervanadate treatment increased coimmunoprecipitation of MuSK with Fyn (Fig.
2B, lane 1 vs 2). The
percentage of MuSK associated with Fyn in control and
pervanadate-treated cells was 1.4 ± 0.2 and 2.6 ± 0.4%
(p < 0.025), respectively. Stimulation of
complex formation by pervanadate occurred in every experiment, and the
mean increase was 88 ± 9%. The association of MuSK with Src was
also reproducibly increased by a pervanadate treatment (Fig.
2B, lane 3 vs 4). The
percentage of MuSK precipitated with Src increased from 1.01 ± 0.06 to 2.4 ±+ 0.2% (p < 0.025),
with an average increase of 140 ± 20%. These data suggested that
in C2 cells, association of MuSK with Src and Fyn was being repressed
by a constitutively active protein tyrosine phosphatase and was
enhanced by increased phosphorylation when the phosphatase was
inhibited.
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Figure 2.
Effect of pervanadate on coimmunoprecipitation of
MuSK with Fyn and Src. A, C2 myotubes were treated with
(PV +) or without (PV )
30 µM pervanadate for 30 min. Myotubes were solubilized
in lysis buffer and centrifuged, and supernatants representing 90% of
a 100 mm plate were immunoprecipitated with a mixture of anti-MuSK
antibodies N19 and C19 (MuSK), anti-Fyn antibody
Fyn15 (Fyn), anti-Src antibody Ab-1
(Src), or empty protein A-Sepharose
(PAS). The immunoprecipitates and 100 µg of lysate
supernatant after centrifugation (Lys) were resolved by
8% SDS-PAGE and analyzed by Western blotting using anti-Src-family
kinase antibody Src2 (pan-Srcks).
B, C2 myotubes were treated with (PV +)
or without (PV ) pervanadate and solubilized as in
A. The supernatants after centrifugation were
immunoprecipitated with anti-Fyn antibody Fyn15 (Fyn),
anti-Src antibody Ab-1 (Src), a mixture of anti-MuSK
antibodies N19 and C19 (MuSK), or empty protein
G-Sepharose beads (PGS). The immunoprecipitates and 100 µg of lysate supernatant after centrifugation (Lys)
were resolved by 7% SDS-PAGE and analyzed by Western blotting using
anti-MuSK-ZH antibody (-MuSK). In
A and B, an arrowhead
indicates the position of Src-class kinases and MuSK,
respectively.
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Association between receptor kinases, such as the PDGF
receptor, and Src-class kinases is mediated by a phosphotyrosine-SH2 domain interaction. Because association of MuSK and Src-class kinases
was enhanced by increased tyrosine phosphorylation, it was predicted
that MuSK might bind with the SH2 domains of Fyn and Src.
To test this possibility, fusion protein affinity chromatography was
used. SH2 domain-containing fusion proteins derived from Fyn, Src, and
Grb2 were incubated with myc-tagged MuSK solubilized from
heterologous transfected QT-6 fibroblasts. MuSK bound to the Fyn and
Src SH2 domain fusion proteins as detected by anti-myc Western analysis
(Fig. 3A). The binding was
specific in that MuSK did not bind to backbone fusion protein or the
Grb2 SH2 fusion protein. In addition, MuSK did not bind to the SH3
domains of Fyn or Src (J. R. Kraas and S. L. Swope,
unpublished results). Because MuSK was solubilized under extremely
stringent conditions of 2% SDS lysis buffer, which promotes
dissociation of protein complexes, these data suggested that MuSK bound
directly to the Fyn and Src SH2 domain fusion proteins. However, an
indirect interaction cannot be ruled out.
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Figure 3.
Direct binding of MuSK to the SH2 domains of Fyn
and Src. QT-6 cells were transfected with pBK-CMV-mycMuSK
[A-C
(WT)] or pBK-CMV-mycMuSK[K686R]
[C (K686R)]. Forty-eight hours
after transfection, cells were treated for 10 min with 20 µM pervanadate (A, B), then
solubilized with 2% SDS lysis buffer and diluted to 0.4% SDS/2%
Triton X-100 as described in Materials and Methods. A,
Lysates representing cells from 100% of a 100 mm dish were incubated
at 4°C for 3 hr with 10 µg of a Fyn (Fyn), Src
(Src), or Grb2 (Grb2) fusion protein or
backbone (GST) protein. B, Lysates
representing cells from 100% of a 100 mm dish were incubated at 4°C
for 3 hr with 0.3-30 µg of Fyn (Fyn) or Src
(Src) SH2 domain fusion protein or 30 µg of backbone
protein (GST). C, Before
solubilization, cells were treated with (+PV) or
without (PV) pervanadate as in A
and B. Lysates representing cells from 100% of a 100 mm
dish transfected with pBK-CMV-mycMuSK (WT) or
pBK-CMV-mycMuSK[K686R] (K686R) were incubated at 4°C
for 3 hr with 10 µg of a Src SH2 fusion protein (Src)
or backbone protein (GST). In
A-C, bound proteins and an aliquot of
the lysate representing 1.5% of a 100 mm dish (Lys)
were resolved by 7% SDS-PAGE gel and analyzed by Western blotting
using 9E10 anti-myc antibody. Molecular weight markers, in kilodaltons,
are indicated on the left. In
A-C, an arrowhead
indicates myc-MuSK.
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The concentration-dependence for binding of MuSK to the Fyn and Src SH2
domains was analyzed. Increasing concentrations of Fyn and Src SH2
domain fusion proteins, 0.3-30 µg, were incubated with solubilized
myc-tagged MuSK. MuSK bound slightly better to the Src SH2 domain (Fig.
3B). These data suggested that association of MuSK with Fyn and Src was
mediated by binding of the Src-class kinases' SH2 domains to the
receptor tyrosine kinase and that MuSK bound with a slightly higher
affinity to Src.
Receptor tyrosine kinases bind Src-class kinases via phosphotyrosine
motifs on the receptors. In vivo, these tyrosines are phosphorylated by ligand-induced dimerization and
auto/transphosphorylation of the receptor tyrosine kinases
(Claesson-Welsh, 1994; Thomas and Brugge, 1997). Whether the binding of
MuSK to the SH2 domain fusion protein of Src-class kinases was
dependent on MuSK transphosphorylation was tested using a catalytically
inactive MuSK construct containing a lysine to arginine point mutation
(MuSK[K686R]) (Gillespie et al., 1996). First, whether MuSK
autophosphorylation in the QT6 cells occurred was determined by testing
whether MuSK[K686R] could be a substrate for wild-type MuSK. In fact,
myc-tagged MuSK[K686R] was phosphorylated when coexpressed with
nontagged wild-type mouse MuSK, but not when expressed alone (data not
shown). Thus, wild-type MuSK was capable of auto/transphosphorylation
in this system.
The catalytically inactive MuSK[K686R] was used to test
whether binding to the SH2 domain of Src was dependent on
autophosphorylation. Heterologous cells expressing the wild type or
MuSK[K686R] were treated without or with pervanadate to inhibit
dephosphorylation and then solubilized. Lysates were incubated with the
Src SH2 domain fusion protein and binding of myc-tagged wild type or
MuSK[K686R] was determined by anti-myc Western analysis. In the
absence of pervanadate treatment, wild-type MuSK bound to the Src SH2
domain but MuSK[K686R] did not (Fig. 3C, lane 7 vs 8). This absence of binding was not caused by altered
expression of the inactive MuSK because analysis of the lysates
demonstrated that both wild type and MuSK[K686R] were expressed (Fig.
3C, lanes 11 and 12). Treatment of the
cells expressing wild-type MuSK with pervanadate to block dephosphorylation caused an increase in the binding of wild-type MuSK
to the Src SH2 domain fusion protein (Fig. 3C, lane
1 vs 7). However, after treatment with
pervanadate, binding of MuSK[K686R] was still not detected (Fig.
3C, lane 2). Analysis of the lysates demonstrated
that the expression levels of wild type and MuSK[K686R] were
unaffected by pervanadate (Fig. 3C, lanes 5 and
6 vs 11 and 12). These data indicated
that binding of the wild-type MuSK to the SH2 domain fusion protein was
dependent on tyrosine phosphorylation of MuSK. In addition, the
phosphorylation was attributable to autophosphorylation of MuSK
because catalytically inactive MuSK was unable to bind to the SH2
domain fusion protein.
Phosphorylation of MuSK and the AChR by Src-class kinases
A mechanism for reciprocal regulation between receptor and
Src-class kinases has been elucidated in other systems.
Ligand-activated autophosphorylation of receptor tyrosine kinases
results in binding of Src-class kinases via their SH2 domains. This
binding leads to phosphorylation of the receptor kinase by Src-class
kinases and thus creates binding sites on the receptor for an
additional SH2 domain containing enzymes and adapter proteins
(Claesson-Welsh, 1994). As described above and by others, MuSK does
autophosphorylate (Gillespie et al., 1996). Whether MuSK could be a
substrate for Src and Fyn was tested next. For this analysis, the
myc-tagged MuSK[K686R] was coexpressed with Src and Fyn in a
heterologous cell line. Because MuSK was not catalytically active, any
MuSK phosphorylation could not be caused by autophosphorylation. In fact, when MuSK[K686R] was expressed alone and then
immunoprecipitated with anti-myc antibody, no phosphorylation was
observed, as demonstrated by phosphotyrosine Western blotting (Fig.
4A, lane
7); however, when coexpressed with Src, MuSK[K686R] was
phosphorylated (Fig. 4A, lane 1). Some
random variability in the isolation of MuSK occurred (Fig.
4B, lanes 1-8); however, changes in the
amounts of MuSK precipitated could not account for the "all" or
nothing phosphorylation of MuSK[K686R] in the presence or absence of
Src, respectively (Fig. 4A,B,
lane 1 vs 7). Isolation of MuSK was
dependent on transfection with the MuSK[K686R] plasmid because MuSK
could not be precipitated from control cells transfected with empty plasmid (Fig. 4B, lane 10). Thus,
catalytically inactive MuSK could be phosphorylated upon coexpression
with Src.
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Figure 4.
Phosphorylation of MuSK by Src and Fyn. QT-6 cells
were transfected with pBK-CMV-mycMuSK[K686R] and Src
(Src), Fyn (Fyn), or Bruton's tyrosine
kinase (Btk) in the absence or presence of rapsyn
(Rap). Replicate control cultures were transfected with
rapsyn or empty vector (pCMV) alone. After
40 hr, the cells were solubilized with 2% SDS lysis buffer, diluted to
0.4% SDS/2% Triton X-100, and MuSK immunoprecipitated from lysate
representing 90% of a 100 mm plate using an anti-myc antibody JH2235.
Immunoprecipitates were resolved by 8% SDS-PAGE and analyzed by
immunoblotting with (A) anti-phosphotyrosine
antibodies 4G10 and PY99 (-PY) and
(B) anti-myc antibody 9E10
(-myc).
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Phosphorylation of MuSK by Fyn was also tested. In this transfection
system, the activity of expressed Fyn is low, but Fyn activity can be
increased by rapsyn (Mohamed and Swope, 1999). In fact, phosphorylation
of MuSK via Fyn was observed only after coexpression of rapsyn (Fig.
4A, lane 3 vs 4). In two
of three experiments, phosphorylation of MuSK by Src was also
potentiated by rapsyn. We tested whether the phosphorylation of MuSK
was specific for Src-family kinases. Btk, a non-receptor kinase
distinct from but related to the Src-family, mediated little or no
phosphorylation of MuSK, demonstrating the specificity of the effect of
Src and Fyn (Fig. 4A, lanes 5 and 6).
These data supported the idea that MuSK can be phosphorylated by Src
and Fyn and in addition suggested that MuSK is a substrate for these
kinases in muscle.
Whether Fyn and Src phosphorylate MuSK in muscle cells was examined
pharmacologically. PP1 is a highly selective Src-class kinase
inhibitor. Depending on the specific Src family kinase and the cell
type examined, PP1 has ID50 values of 10-100
nM in vitro and 0.5-30
µM in intact cells (Hanke et al., 1996; Liu et al., 1999). First, inhibition of Fyn and Src by PP1 in C2 myotubes was
demonstrated. Activation of Src-class kinases results in
auto/transphosphorylation at Y416 (chicken
Src numbering) within the catalytic domain (Thomas and Brugge, 1997).
Thus, phosphorylation at this site can be used as a measure of
activation. We developed an anti-Y416
phosphorylation state-specific antibody, GW001, to examine the activity
of Src-class kinases by Western blotting, as described in Materials and
Methods. The ability of PP1 to inhibit Src and Fyn activities in C2
cells was analyzed using the anti-PY416
Src antibodies. The Y416 phosphorylation
of Fyn and Src immunoprecipitated from control C2 myotubes was low
(Fig. 5A, lanes 1 and 5). Treatment of C2 myotubes with pervanadate increased
the phosphorylation of Fyn and Src on Y416
(Fig. 5A, top panel, lane 1 vs
2 and lane 5 vs 6). Thus,
pervanadate revealed constitutive activity and autophosphorylation of
Fyn and Src. Pretreatment of myotubes with 10 or 20 µM PP1 inhibited the pervanadate-induced
increase in Y416 phosphorylation on Fyn
and Src (Fig. 5A, lane 2 vs 3 and
4 and lane 6 vs 7 and 8).
The changes in anti-PY416 Src signal could
not be explained by alterations in the isolation of Fyn and Src (Fig.
5A, bottom panel). Upon
normalization for the amount of kinase isolated, Fyn
autophosphorylation/activity was found to be 75 ± 7 and 90 ± 2% with 10 and 20 µM PP1, respectively. Src
activity was inhibited by 60 ± 10 and 60 ± 10% with 10 and 20 µM PP1, respectively. The higher sensitivity
of Fyn to PP1, compared with Src, agreed with a previous report (Hanke
et al., 1996). These data demonstrated that as measured by
autophosphorylation, PP1 blocked the activity of Fyn and Src in C2
myotubes.
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Figure 5.
Effect of inhibiting Src-class kinases on
pervanadate-induced MuSK and AChR phosphorylation in
vivo. A, C2 myotubes were treated with 10 or 20 µM PP1 or vehicle for 1 hr followed by treatment with
(PV +) or without (PV ) 30 µM pervanadate for 10 min. Myotubes were solubilized in
RIPA buffer and centrifuged, and supernatants representing 20% of a 60 mm dish were immunoprecipitated with Fyn antibody Fyn15
(Fyn) or Src antibody Ab-1 (Src). The
immunoprecipitates were resolved by 8% SDS-PAGE and analyzed by
Western blotting using phosphorylation state-specific anti-Src-class
kinase antibody GW001 (-PY 416) (top
panel). The PY416 blot was stripped and reanalyzed by
Western blotting using anti-Src-class kinase antibody Src2
(pan Srcks) (bottom panel).
B, Supernatants representing 20% of a 60 mm dish as in
A were immunoprecipitated with anti-MuSK antibody GW002
(MuSK). The immunoprecipitates were resolved by
7% SDS-PAGE and analyzed by Western blotting using a mixture of
anti-phosphotyrosine antibodies 4G10 and PY99
(-PY) (top panel).
Supernatants representing 1% of a 60 mm dish were resolved by 7%
SDS-PAGE and analyzed by Western blotting using anti-MuSK antibody
-MuSK-ZH (-MuSK) (bottom
panel). C, Supernatants representing 20%
of a 60 mm dish as in A were immunoprecipitated with
anti-AChR antibody 88b (AChR). The immunoprecipitates
were resolved by 8% SDS-PAGE and analyzed by Western blotting using a
mixture of the phosphorylation state-specific anti-AChR subunit
(JH1360) and anti-AChR subunit (JH1358) antibodies
(-AChR-PY) (top panel).
The top blot from C was stripped and
reanalyzed by Western blotting using the AChR subunit antibody mAb
148 (-AChR) (bottom panel). In
A-C, an arrowhead
indicates the position of Src-class kinases, MuSK, and AChR and subunits, respectively.
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To reveal constitutive phosphorylation of MuSK by Src-class kinases,
pervanadate and PP1 were also used. C2 myotubes were treated with
pervanadate, and endogenous MuSK phosphorylation was examined by
immunoprecipitation and Western blotting. Tyrosine phosphorylation of
MuSK was essentially undetectable in control cells, whereas pervanadate
treatment resulted in a striking increase in MuSK phosphorylation (Fig.
5B, lane 1 vs 2). Incubation of C2 cells with PP1 inhibited pervanadate-induced MuSK phosphorylation (Fig. 5B, lane 2 vs 3 and
4). In each of three experiments, PP1 at 20 µM was slightly more effective than at 10 µM. Inhibition of pervanadate-induced MuSK
phosphorylation by 10 and 20 µM PP1 was 50 ± 10 and 60 ± 10%, respectively. Neither pervanadate nor PP1
affected MuSK expression (Fig. 5B, bottom
panel). The effect of PP1 suggested that approximately half
of the induced phosphorylation of MuSK was caused by Src-class kinases.
One of the major goals of this study was to determine whether Src-class
kinases phosphorylate and regulate AChRs of skeletal muscle.
Phosphorylation of the AChR by Src and Fyn was also tested pharmacologically. Once again, C2 cells were treated with or without pervanadate and PP1. AChRs were immunoprecipitated and tyrosine phosphorylation was examined using phosphorylation state-specific anti- and anti- subunit antibodies. AChR phosphorylation was low
in control cells, whereas pervanadate dramatically increased receptor
phosphorylation (Fig. 5C, lane 1 vs
2). When we corrected for recovery of AChRs (Fig.
5C, bottom panel), a 22- to 26-fold increase in phosphorylation of the subunit and a 10- to 130-fold increase in phosphorylation of the subunit occurred. This
phosphorylation appeared to be attributable, in part, to Src-class
kinases, as demonstrated by inhibition with PP1 (Fig. 5C,
lane 2 vs 3 and 4).
Cells pretreated with 10 and 20 µM PP1 showed a
60 ± 7 and 70 ± 10% inhibition of pervanadate-induced subunit phosphorylation, respectively. Inhibition of subunit
phosphorylation was 54 ± 4 and 72 ± 7% by 10 and 20 µM PP1, respectively. Therefore,
phosphorylation of AChRs by Src-class kinases occurred in C2 cells.
MuSK phosphorylation in C2 myotubes was inhibited by PP1 (Fig.
5B). These data suggested that Src-class kinases
phosphorylated MuSK in muscle cells. However, because MuSK
autophosphorylates, the effect of PP1 on MuSK might have been to
inhibit autophosphorylation. Whether PP1 could block MuSK kinase
activity in vitro was used to further test the conclusion
that MuSK was phosphorylated by Src kinases in C2 cells. C2 myotubes
were treated with or without pervanadate, MuSK was immunoprecipitated,
and MuSK activity was analyzed in vitro using enolase as a
substrate (Fig. 6A).
Phosphorylation of both MuSK and enolase was detected by
anti-phosphotyrosine immunoblotting. In control cells, no
phosphorylation of MuSK or enolase was observed (Fig.
6A, top and bottom panels,
lane 2). Pervanadate treatment caused a striking increase in
both MuSK and enolase phosphorylation (Fig. 6A,
top and bottom panels, lane 1).
Phosphorylation of enolase occurred in vitro because it was dependent on ATP in the reaction (Fig. 6A,
bottom panel, lane 3). In contrast, MuSK
phosphorylation was independent of ATP in the in vitro
reaction, demonstrating that MuSK phosphorylation occurred in the
intact myotubes (Fig. 6A, top panel,
lane 1 vs 3). As expected, inclusion of 1 or 5 µM PP1 in the in vitro reaction did
not affect the in vivo phosphorylation of MuSK (Fig.
6A, top panel, lane 1 vs
4 and 6). In addition, enolase
phosphorylation by MuSK was not affected by 1 or 5 µM PP1 added to the in vitro reaction (Fig. 6A, bottom panel,
lane 1 vs 4 and 6). The
phosphorylation of enolase in the presence of 10 and 20 µM PP1 was 96 ± 4 and 104 ± 5%,
respectively, of the level in the absence of PP1. These data
demonstrated that PP1 did not directly inhibit the enzymatic activity
of MuSK.
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Figure 6.
Effect of inhibiting Src-class kinases on Fyn,
Src, and MuSK kinase activities in vitro.
A, C2 myotubes were treated with pervanadate at 30 µM (PV +) or vehicle (PV
) for 30 min. Myotubes were solubilized in RIPA buffer and
centrifuged, and supernatants representing 30% of a 60 mm dish were
immunoprecipitated with anti-MuSK antibody GW002
(MuSK). The kinase activity of immunoprecipitated
MuSK was determined with an in vitro kinase assay using
enolase as a substrate in the presence of 1 or 5 µM PP1
(PP1 +) or vehicle (PP1 ) and the
presence (ATP +) or absence (ATP ) of
ATP. For examining MuSK phosphorylation, kinase reactions were resolved
by 7% SDS-PAGE and analyzed by Western blotting using
anti-phosphotyrosine antibodies 4G10 and PY99
(-PY) (top panel).
Enolase was resolved by 8% SDS-PAGE and analyzed by Western blotting
using anti-phosphotyrosine antibodies 4G10 and PY99
(-PY) (bottom panel).
Arrowheads indicate the positions of MuSK
(MuSK) and enolase (ENO).
B, Supernatants from A representing 30%
of a 60 mm dish were immunoprecipitated with anti-Fyn antibody Fyn 15 (Fyn) or anti-Src antibody Ab-1 (Src).
The kinase activities of immunoprecipitated Fyn and Src were determined
with an in vitro kinase assay using enolase as substrate
as described in A. Enolase and Src-class kinases were
resolved by 8% SDS-PAGE and analyzed by Western blotting using
anti-phosphotyrosine antibodies 4G10 and PY99
(-PY) (top panel). The
blots were stripped and reanalyzed by Western blotting using the
pan-Src kinase antibody Src2 (pan-Srcks)
(bottom panel). Arrowheads
indicate the positions of enolase (ENO) and Src-class
kinases (SrcKs).
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In contrast, PP1 did block the enzymatic activity of Fyn and Src
in vitro. Using the same paradigm, myotubes were treated with or without pervanadate, Fyn and Src were isolated, and in vitro phosphorylation of enolase in the presence or absence of PP1
was determined. Pervanadate treatment of the myotubes stimulated in vitro phosphorylation of enolase by both Fyn and Src
(Fig. 6B, top panel, lane 1 vs
2 and lane 8 vs 9). The
phosphorylation of enolase by Fyn and Src occurred in vitro
because it was dependent on ATP in the reaction (Fig.
6B, lane 1 vs 3 and lane
8 vs 10). Fyn-mediated phosphorylation of enolase was
inhibited by PP1 (Fig. 6B, lane 1 vs
4 and 6). Fyn activity was inhibited
in vitro by 69 ± 5 and 96 ± 4% with 1 and 5 µM PP1, respectively. Similarly, Src activity
was inhibited by 91 ± 1 and 100 ± 1% with 1 and 5 µM PP1, respectively (Fig.
6B, lane 8 vs 11 and
13). These data demonstrated that the enzymatic activity of
Src and Fyn kinases from C2 myotubes was inhibited by PP1, whereas the
activity of MuSK was not. Furthermore, these data support the idea that
phosphorylation of MuSK and the AChR in C2 myotubes was attributable to
Src-class kinases (Fig. 5B,C).
Agrin activates MuSK and induces MuSK phosphorylation (Glass et al.,
1996). Whether agrin-stimulated phosphorylation of MuSK was dependent
on Src-class kinases was tested. For this experiment, C2 myotubes were
treated with agrin, MuSK was immunoprecipitated, and phosphorylation
was examined by Western blotting. Agrin induced MuSK phosphorylation
(Fig. 7A, lane 1 vs
2). The effect of agrin on MuSK phosphorylation was
reduced by PP1 (Fig. 7A, lane 2 vs 3 and 4). At 10 and 20 µM, PP1 blocked agrin-stimulated MuSK
phosphorylation by 40 ± 10 and 69 ± 6%. These data argue
that phosphorylation of MuSK upon agrin treatment was attributable, in
part, to Src-class kinases.
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Figure 7.
Effect of inhibiting Src-class kinases on
agrin-induced MuSK and AChR phosphorylation in vivo.
A, C2 myotubes were treated with 10 or 20 µM PP1 (PP1 +) or vehicle
(PP1 ) for 1 hr followed by treatment with 100 pM agrin (+) or conditioned medium vehicle () for 10 min.
Myotubes were solubilized in RIPA buffer and centrifuged, and
supernatants representing 40% of each 60 mm dish were
immunoprecipitated with anti-MuSK antibody GW002
(MuSK). The immunoprecipitates were resolved by
7% SDS-PAGE and analyzed by Western blotting using a mixture of
anti-phosphotyrosine antibodies 4G10 and PY99
(-PY) (top panel).
Supernatants representing 1% of each 60 mm dish were resolved by 7%
SDS-PAGE and analyzed by Western blotting with anti-MuSK antibody
MuSK-ZH (-MuSK) (bottom
panel). Arrowheads indicate the position
of MuSK. B, Supernatants representing 40% of a 60 mm
dish, as in A, were used for immunoprecipitation with
anti-AChR antibody 88b (AChR). The immunoprecipitates
were resolved by 8% SDS-PAGE and analyzed by Western blotting using a
mixture of the phosphorylation state-specific anti-AChR subunit
(JH1360) and anti-AChR subunit (JH1358) antibodies
(AChR) (top panel). The blot was
stripped and reanalyzed by Western blotting using anti-AChR subunit
antibody mAb 148 (AChR) (bottom
panel). Arrowheads indicate the positions
of the AChR and subunits.
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Phosphorylation of the AChR in response to agrin does not occur in
myotubes derived from a MuSK knock-out mouse (Glass et al., 1997).
Thus, agrin-stimulated phosphorylation of the AChR is dependent on
MuSK. Whether agrin-stimulated/MuSK-mediated AChR phosphorylation was
dependent on Src-family kinases was examined. C2 myotubes were treated
with agrin, AChRs were isolated, and phosphorylation was analyzed by
Western blotting using the phosphorylation state anti- and anti-
subunit antibodies. Agrin induced AChR phosphorylation (Fig.
7B, lane 1 vs 2). The
agrin-stimulated phosphorylation of the AChR was inhibited by PP1 (Fig.
7B, lane 2 vs 3 and
4). The AChR subunit phosphorylation was
blocked by 51 ± 7 and 69.5 ± 0.7% with 10 and 20 µM PP1, respectively. The subunit
phosphorylation was blocked by 60 ± 10 and 74 ± 8% with 10 and 20 µM PP1, respectively. These data
indicated that agrin activated Src-class kinases, resulting in
phosphorylation of the AChR in C2 myotubes.
Cytoskeletal anchoring of the AChR by Src-class kinases
Agrin stimulates AChR anchoring to the cytoskeleton in muscle
cells (Wallace, 1992). Anchoring of AChRs is thought to be regulated by
tyrosine phosphorylation (Wallace, 1992; Mohamed and Swope, 1999).
Because Src-class kinases appeared to mediate the effect of agrin to
stimulate AChR phosphorylation (Fig. 7B), a role for these
kinases to regulate receptor anchoring was tested. The levels of
anchored AChRs were determined by examining the resistance of the
receptors to extraction with 0.5% Triton X-100, an approach previously demonstrated to be useful for analyzing interaction of AChRs
with the cytoskeleton (Prives et al., 1982). For this experiment,
myotubes were treated with agrin, agrin plus PP1, or vehicle. The cells
were then extracted with 0.5% Triton X-100 over a time course of 0-20
min. The detergent-resistant cytoskeletal material remaining on the
plate was eluted with SDS-PAGE sample buffer and analyzed for AChRs by
Western blotting. With no extraction, the levels of total AChRs in the
myotubes were similar for all treatments (Fig.
8A,B,
lanes 1-3). Thus, neither agrin nor PP1 affected the total
amount of receptor. After 5 min of Triton X-100 extraction, little AChR
was nonextractable in the control cells (Fig. 8A,
lane 6). Thus, most of the AChRs were soluble and not linked to the cytoskeleton in control cells. However, after agrin treatment, the level of nonextractable AChRs was increased, reflecting agrin-induced cytoskeletal anchoring (Fig. 8A,
lane 4 vs 6). In addition, PP1 partially
reversed the action of agrin (Fig. 8A, lane
4 vs 5). The effect of PP1 could not be explained by an
enhanced loss of cells (Fig. 8B). The same
effects of agrin and PP1 were seen at 10 min of Triton X-100
extraction. As expected, most AChRs in control cells were solubilized
by 10 min of extraction with Triton X-100 (Fig. 8A,
lane 9). In cells treated with agrin, a pool of AChRs
continued to be resistant to 10 min of detergent extraction (Fig.
8A, lane 7). The pool of receptors
resistant to 10 min of extraction was smaller in cells treated with PP1 (Fig. 8A, lane 8). The same trend was
observed with 20 min of extraction (Fig. 8A,
lanes 11-12). The data in Figure 8C show the
reproducibility of this effect. In four experiments, at all time points
of Triton X-100 extraction, the levels of AChRs in the nonextractable
fraction were enhanced by agrin, and the effect of agrin was
significantly reversed by PP1 (Fig. 8C). These data indicated that Src-class kinases mediate at least part of the effect of
agrin to induce cytoskeletal anchoring of the AChR in muscle cells.
Furthermore, these results support the importance of Src-class kinases
in the stabilization of AChRs at the endplate during formation of the
NMJ.
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Figure 8.
Effect of inhibiting Src-class kinases on
agrin-induced AChR cytoskeletal anchoring. A, C2
myotubes were treated with 20 µM PP1 (PP1
+) or DMSO vehicle (PP1 ) for 1 hr followed by
treatment with 100 pM agrin (Agrin +) or
conditioned medium vehicle (Agrin ) for 1 hr as
indicated. Treated myotubes were extracted with Prives buffer
containing 0.5% Triton X-100 for the indicated times and rinsed once
with Prives buffer containing 0.1% Triton X-100. The proteins
remaining on the dish were extracted using SDS-PAGE sample buffer,
sonicated, and centrifuged. Aliquots of the SDS-extracted proteins
representing 10% of a 35 mm dish were resolved by 8% SDS-PAGE and
analyzed by Western blotting using anti-AChR subunit antibody mAb
148. B, Sister C2 cultures were treated with or without
agrin, with or without PP1 as in A, and treated with
Prives buffer without Triton X-100. Dishes were rinsed once with Prives
buffer without Triton X-100. The proteins on the dish were extracted
using SDS-PAGE sample buffer, sonicated, and centrifuged. SDS-extracted
proteins representing 10% of 35 mm dishes were resolved by 8%
SDS-PAGE and analyzed by Western blotting using anti-AChR subunit
antibody mAb 148. C, Four independent experiments as in
A were analyzed. C2 myotubes were treated with agrin
( ), agrin + 20 µM PP1 ( ), or vehicle control ( ).
The data were quantified by densitometric scanning and represent the
mean ± SEM of the percentage of the total level of AChRs that
remained on the dishes after 0.5% Triton X-100 extraction. In
A and B, arrowheads
indicate the position of the AChR subunit.
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Maturation of AChR clustering by Src-class kinases
Whether the effect of agrin to induce AChR clustering was mediated
by Src-class kinases was also examined. Two approaches were used:
inhibition of Src kinases with PP1 or expression of a dominant negative
Src. C2 myotubes were treated with agrin in the presence or absence of
PP1, and AChR clustering was analyzed using
tetramethyl(rhodamine)--bungarotoxin or Texas Red--bungarotoxin labeling and epifluorescence microscopy. Agrin-induced AChR clustering was still apparent in the presence of PP1 (Fig. 9, A vs
B). However, the size of receptor aggregates was altered by
inhibition of Src-class kinases. The size distribution of receptor
clusters was analyzed by binning into categories of <15 µm, 15-25
µm, and >25 µm (Fig. 9E, +PP1 and
PP1). The most obvious effect was a loss of large clusters and a dramatic shifting of clusters to sizes <15 µm. The
percentage of clusters >25 µm in agrin-treated cultures was 57 ± 3%, n = 5, whereas after inhibition of
Src-class kinases the number was reduced to 4.0 ± 0.1%,
n = 5 (Fig. 9E, PP1 vs +PP1 clear bars). Conversely, the percentage of clusters
<15 µm was 13 ± 3% in control cells and 86 ± 6%, n = 5, in PP1-treated cells (Fig.
9E, PP1 vs +PP1,
hatched bars). These data indicated that inhibition
of Src-class kinases with PP1 blocked the formation of large AChR
clusters in response to agrin.
The role of Src-class kinases in the maturation of large AChR clusters
was corroborated using a dominant negative approach. C2 cells were
transfected to express a dominant negative Src and treated with agrin,
and AChR clustering was analyzed. Cells expressing the dominant
negative Src were identified by staining with a pan-Src kinase
antibody, src2, whereas AChRs were identified with Texas Red--bungarotoxin. Again, agrin-induced clustering occurred in cells
expressing the dominant negative kinase; however, compared with control
cultures transfected with empty plasmid, the size of receptor clusters
was reduced by expression of dominant negative Src (Fig. 9,
C vs D). In contrast, expression of GFP
did not affect AChR clustering (data not shown). For control cells,
37 ± 2% of the clusters were 15-25 µm and 33 ± 3% were >25 µm, whereas 31 ± 4% of the clusters were <15
µm (see Fig. 9E, pBK). In
contrast, in the cells expressing the dominant negative Src, 91 ± 2% of the clusters were <15 µm, whereas only 3 ± 1% were
>25 µm (Fig. 9E, DN Src). These
results provide strong evidence that Src-class kinases are critical for
maturation of AChR clustering.
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DISCUSSION |
It has been predicted that a protein tyrosine kinase downstream of
MuSK directly phosphorylates the AChR (Burden, 1998; Sanes et al.,
1998; Fuhrer et al., 1999; Sanes and Lichtman, 1999). In this study,
MuSK was found to be complexed with the Src-class kinases Fyn and Src
in the C2 mouse muscle cell line. These associations were enhanced by
agrin and by increasing protein tyrosine phosphorylation. MuSK bound
specifically to the SH2 domains of Fyn and Src, and this in
vitro binding was dependent on autophosphorylation of MuSK. In
addition, Fyn and Src phosphorylated MuSK in transfected cells. A
highly selective inhibitor of Src-family kinases, PP1, blocked the
enzymatic activity of Fyn and Src from C2 myotubes but did not affect
the kinase activity of Mu
TOP
ABSTRACT
INTRODUCTION
