Focal adhesion kinase is a recently characterized tyrosine kinase that is concentrated at focal contacts in cultured cells. It is thought to play an important role in the regulation of the integrin-based signal transduction mechanism involved in the assembly of this membrane specialization. In this study, we examined the immunocytochemical distribution of focal adhesion kinase in Xenopus skeletal muscle and its role in the formation of two sarcolemmal specializations, the myotendinous junction and the neuromuscular junction, using a monoclonal antibody (2A7) against this protein. Immunoprecipitation of Xenopus embryonic tissues with this antibody demonstrated a single band at a relative molecular mass of 116 kDa. A distinct concentration of immunolabeling for focal adhesion kinase was observed at the myotendinous junction of muscle fibers in vivo. At this site, the labeling for this protein is correlated with an accumulation of phosphotyrosine immunolabeling. Focal adhesion kinase was not concentrated at the neuromuscular junction in muscle cells either in vivo or in vitro. However, it was localized at spontaneously formed acetylcholine receptor clusters in cultured Xenopus myotomal muscle cells, although its distribution was not exactly congruent with that of the receptors. In these cells, the accumulation focal adhesion kinase was induced by polystyrene microbeads. In addition, beads also induce the formation of acetylcholine receptor clusters and myotendinous junction-like specializations. By following the appearance of the focal adhesion kinase relative to the formation of these sarcolemmal specializations at bead-muscle contacts in cultured muscle cells, we conclude that the accumulation of this protein was in pace with the development of the myotendinous junction, but occurred well after the clustering of acetylcholine receptors. These results suggest that focal adhesion kinase may be involved in the development and/or maintenance of the myotendinous junction through an integrin-based signaling system. Although it can accumulate at acetylcholine receptor clusters formed in culture, it does not appear to be involved in the development of the neuromuscular junction.

REFERENCES

Baker
L. P.
,
Chen
Q.
,
Peng
H. B.
(
1992
).
Induction of acetylcholine receptor clustering by native polystyrene beads: implication of an endogenous muscle-derived signalling system.
J. Cell Sci
102
,
543
555
Baker
L. P.
,
Peng
H. B.
(
1993
).
Tyrosine phosphorylation and acetylcholine receptor cluster formation in cultured Xenopus muscle cells.
J. Cell Biol
120
,
185
195
Bloch
R. J.
,
Hall
Z. W.
(
1983
).
Cytoskeletal components of the vertebrate neuromuscular junction: vinculin,-actinin, and filamin.
J. Cell Biol
97
,
217
223
Bloch
R. J.
,
Pumplin
D. W.
(
1988
).
Molecular events in synaptogenesis: nerve-muscle adhesion and postsynaptic differentiation.
Amer. J. Physiol
254
,
345
–.
Bockholt
S. M.
,
Otey
C. A.
,
Glenney
J. R.
Jr.
,
Burridge
K.
(
1992
).
Localization of a 215-kDa tyrosine-phosphorylated protein that cross-reacts with tensin antibodies.
Exp. Cell Res
203
,
39
46
Bockholt
S. M.
,
Burridge
K.
(
1993
).
Cell spreading on extracellular matrix proteins induces tyrosine phosphorylation of tensin.
J. Biol. Chem
268
,
14565
14567
Bozyczko
D.
,
Decker
C.
,
Muschler
J.
,
Horwitz
A. F.
(
1989
).
Integrin on developing and adult skeletal muscle.
Exp. Cell Res
183
,
72
91
Burridge
K.
,
Fath
K.
,
Kelly
T.
,
Nuckolls
G.
,
Turner
C.
(
1988
).
Focal adhesions: transmembrane junctions between the extracellular matrix and the cytoskeleton.
Annu. Rev. Cell Biol
4
,
487
525
Burridge
K.
,
Turner
C. E.
,
Romer
L. H.
(
1992
).
Tyrosine phosphorylation of paxillin and pp125FAKaccompanies cell adhesion to extracellular matrix: A role in cytoskeletal assembly.
J. Cell Biol
119
,
893
903
Chen
Q.
,
Sealock
R.
,
Peng
H. B.
(
1990
).
A protein homologous to the Torpedo postsynaptic 58K protein is present at the myotendinous junction.
J. Cell Biol
110
,
2061
2071
Chiquet
M.
,
Fambrough
D. M.
(
1984
).
Chick myotendinous antigen. I. A monoclonal antibody as a marker for tendon and muscle morphogenesis.
J. Cell Biol
98
,
1926
1936
Chiquet
M.
,
Fambrough
D. M.
(
1984
).
Chick myotendinous antigen. II. A novel extracellular glycoprotein complex consisting of large disulfide-linked subunits.
J. Cell Biol
98
,
1937
1946
Fertuck
H. C.
,
Salpeter
M. M.
(
1976
).
Quantitation of junctional and extrajunctional acetylcholine receptors by electron microscope autoradiography after 125I--bungarotoxin binding at mouse neuromuscular junctions.
J. Cell Biol
69
,
144
158
Guan
J.-L.
,
Trevithick
J. E.
,
Hynes
R. O.
(
1991
).
Fibronection/integrin interaction induces tyrosine phosphorylation of a 120 kDa protein.
Cell Regul
2
,
951
964
Guan
J.-L.
,
Shalloway
D.
(
1992
).
Regulation of focal adhesion-associated protein tyrosine kinase by both cellular adhesion and oncogenic transformation.
Nature
358
,
690
692
Hanks
S. K.
,
Calalb
M. B.
,
Harper
M. C.
,
Patel
S. K.
(
1992
).
Focal adhesion protein-tyrosine kinase phosphorylated in response to cell attachment to fibronectin.
Proc. Nat. Acad. Sci. USA
89
,
8487
8491
Hynes
R. O.
(
1992
).
Integrins: Versatility, modulation, and signaling in cell adhesion.
Cell
69
,
11
25
Juliano
R. L.
,
Haskill
S.
(
1993
).
Signal transduction from the extracellular matrix.
J. Cell Biol
120
,
577
585
Kanner
S. B.
,
Reynolds
A. B.
,
Vines
R. R.
,
Parsons
J. T.
(
1990
).
Monoclonal antibodies to individual tyrosine-phosphorylated protein substrates of oncogene-encoded tyrosine kinases.
Proc. Nat. Acad. Sci. USA
87
,
3328
3332
Khurana
T. S.
,
Watkins
S. C.
,
Chafey
P.
,
Chelly
J.
,
Tome
F. M. S.
,
Fardeau
M.
,
Kaplan
J.-C.
,
Kunkel
L. M.
) (
1991
).
Immunolocalization and developmental expression of dystrophin-related protein in skeletal muscle.
Neuromusc. Disorders
3
,
185
194
Kornberg
L. J.
,
Earp
H. S.
,
Turner
C. E.
,
Prockop
C.
,
Juliano
R. L.
(
1991
).
Signal transduction by integrins: Increased protein tyrosine phosphorylation caused by clustering of1integrins.
Proc. Nat. Acad. Sci. USA
88
,
8392
8396
Law
D. J.
,
Lightner
V. A.
(
1993
).
Divalent cation-dependent adhesion at the myotendinous junction: Ultrastructure and mechanics of failure.
J. Muscle Res. Cell Motil
14
,
173
185
Luther
P. W.
,
Bloch
R. J.
(
1989
).
Formaldehyde-amine fixatives forimmunocytochemistry of cultured Xenopus myocytes.
J. Histochem. Cytochem
37
,
75
82
Peng
H. B.
,
Baker
L. P.
,
Chen
Q.
(
1991
).
Induction of synaptic development in cultured muscle cells by basic fibroblast growth factor.
Neuron
6
,
237
246
Peng
H. B.
,
Chen
Q.
(
1992
).
Induction of dystrophin localization in cultured Xenopus muscle cells by latex beads.
J. Cell Sci
103
,
551
563
Peng
H. B.
,
Baker
L. P.
,
Dai
Z.
(
1993
).
A role of tyrosine phosphorylation in the formation of acetylcholine receptor clusters induced by electric fields in cultured Xenopus muscle cells.
J. Cell Biol
120
,
197
204
Qu
Z.
,
Moritz
E.
,
Huganir
R. L.
(
1990
).
Regulation of tyrosine phosphorylation of the nicotinic acetylcholine receptor at the rat neuromuscular junction.
Neuron
4
,
367
378
Rochlin
M. W.
,
Chen
Q.
,
Tobler
M.
,
Turner
C. E.
,
Burridge
K.
,
Peng
H. B.
(
1989
).
The relationship between talin and acetylcholine receptor clusters in Xenopus muscle cells.
J. Cell Sci
92
,
461
472
Ruoslahti
E.
,
Pierschbacher
M. D.
(
1987
).
New perspectives in cell adhesion: RGD and integrins.
Science
238
,
491
497
Schaller
M. D.
,
Borgman
C. A.
,
Cobb
B. S.
,
Vines
R. R.
,
Reynolds
A. B.
,
Parsons
J. T.
(
1992
).
pp125FAK a structurally distinctive protein-tyrosine kinase associated with focal adhesions.
Proc. Nat. Acad. Sci. USA
89
,
5192
5196
Sealock
R.
,
Paschal
B.
,
Beckerle
M.
,
Burridge
K.
(
1986
).
Talin is a post-synaptic component of the rat neuromuscular junction.
Exp. Cell Res
163
,
143
150
Shear
C. R.
,
Bloch
R. J.
(
1985
).
Vinculin in subsarcolemmal densities in chicken skeletal muscle: localization and relationship to intracellular and extracellular structures.
J. Cell Biol
101
,
240
256
Swasdison
S.
,
Mayne
R.
(
1989
).
Location of the integrin complex and extracellular matrix molecules at the chicken myotendinous junction.
Cell Tiss. Res
257
,
537
543
Tidball
J. G.
,
Daniel
T. L.
(
1986
).
Myotendinous junctions of tonic muscle cells: structure and loading.
Cell Tiss. Res
245
,
315
322
Tidball
J. G.
,
O'Halloran
T.
,
Burridge
K.
(
1986
).
Talin at myotendinous junctions.
J. Cell Biol
103
,
1465
1472
Tidball
J. G.
,
Lin
C.
(
1989
).
Structural changes at the myogenic cell surface during the formation of myotendinous junctions.
Cell Tiss. Res
257
,
77
84
Trotter
J. A.
(
1985
).
A morphometric analysis of the muscle-tendon junction.
Anat. Rec
213
,
26
32
Turner
C. E.
,
Kramarcy
N.
,
Sealock
R.
,
Burridge
K.
(
1991
).
Localization of paxillin, a focal adhesion protein, to smooth muscle dense plaques, and the myotendinous and neuromuscular junctions of skeletal muscle.
Exp. Cell Res
192
,
651
655
Turner
C. E.
,
Schaller
M. D.
,
Parsons
J. T.
(
1993
).
Tyrosine phosphorylation of the focal adhesion kinase pp125FAKduring development: Relation to paxillin.
J. Cell Sci
105
,
637
645
Wallace
B. G.
,
Qu
Z.
,
Huganir
R. L.
(
1991
).
Agrin induces phosphorylation of the nicotinic acetylcholine receptor.
Neuron
6
,
869
878
Zachary
I.
,
Rozengurt
E.
(
1992
).
Focal adhesion kinase (p125FAK): A point of convergence in the action of neuropeptides, integrins, and oncogenes.
Cell
71
,
891
894
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