Functional Interaction of Src Family Kinases with the Acetylcholine Receptor in C2 Myotubes

doi:10.1074/jbc.271.50.32474

Functional Interaction of Src Family Kinases with the Acetylcholine Receptor in C2 Myotubes*

From the Department of Physiology, University of California, San Francisco, California 94143 and the Section on Synaptic Mechanisms, Laboratory of Cell Biology, National Institute of Mental Health, Bethesda, Maryland 20892

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Abstract

Tyrosine phosphorylation of the β subunit of the acetylcholine receptor (AChR) has been postulated to play a role in AChR clustering during development of the neuromuscular junction. We have investigated the mechanism of this phosphorylation in mammalian C2 myotubes and report that the tyrosine kinase Src binds and phosphorylates glutathione S-transferase fusion proteins containing the N-terminal half of the cytoplasmic loop of the β subunit. No binding occurs to the related kinases Fyn or Yes or to the corresponding regions from the γ and δ subunits. Furthermore, AChRs affinity-isolated from C2 myotubes using α-bungarotoxin-Sepharose were specifically associated with Src and Fyn and had tyrosine-phosphorylated β subunits. We suggest that AChRs are initially phosphorylated by Src and subsequently bind Fyn in a phosphotyrosine-dependent manner. These interactions are likely to play an important role in construction of the specialized postsynaptic membrane during synaptogenesis.

Footnotes

↵^¶ Supported by postdoctoral fellowships from the Swiss National Science Foundation, the Ciba-Geigy Jubilaeumsstiftung, and the Human Frontier Science Program.
↵* The work was supported in part by grants from the National Institutes of Health and the Muscular Dystrophy Association and then intramurally by the National Institute of Mental Health. The experiments reported in this article were initiated at the Dept. of Physiology, University of California, San Francisco, and completed at the National Institutes of Health. 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.
↵¹ The abbreviations used are:

AChR

acetylcholine receptor

GST

glutathione S-transferase

PAGE

polyacrylamide gel electrophoresis

ECL

enhanced chemiluminescence

PGS

protein G-Sepharose.
- Received August 6, 1996.
- Revision received September 23, 1996.