Abstract

The nicotinic acetylcholine receptor (AChR) is phosphorylated on tyrosine residues both in vitro and in vivo. This phosphorylation appears to regulate the rate of receptor desensitization and is associated with AChR clustering induced by the neuronal extracellular matrix protein agrin. To identify the protein tyrosine kinase(s) that phosphorylates the nicotinic receptor, we have used molecular cloning techniques to identify and characterize two protein tyrosine kinases that are highly expressed in Torpedo electric organ, a tissue enriched in synaptic components including the AChR. One of the kinases was identified as the Torpedo homolog of neuronal fyn, whereas the other was a novel kinase we have named fyk due to its homology to both fyn and yes protein tyrosine kinases. Using antibodies to fyn and fyk, Fyn was shown to be a 55-kDa protein phosphorylated on tyrosine residues, whereas Fyk was a 56-kDa/53-kDa doublet phosphorylated on serine and tyrosine residues. At the mRNA and/or protein level, fyn and fyk were present in Torpedo electric organ, skeletal muscle, and brain. Both kinases were detected in the membrane fractions enriched in the AChR, with Fyn and Fyk representing 36 and 8%, respectively, of the protein tyrosine kinase activity in these postsynaptic membranes. In addition, Fyn and Fyk were shown by coimmunoprecipitation to be specifically associated with the AChR. Furthermore, the AChR was phosphorylated in Fyn and Fyk immunoprecipitates. These results indicate that Fyn and Fyk are involved in the regulation of postsynaptic membrane function and suggest that these protein tyrosine kinases may phosphorylate the AChR.

Highlights

  • Molecular Cloning of Two Abundant Protein Tyrosine Kinases in Torpedo Electric Organ That Associate with the Acetylcholine Receptor*

  • The nicotinic acetylcholine receptor (AChR)is phos- Protein tyrosine kinases were originally identified as retrophorylated on tyrosine residues both in vitro and in viral oncogene products and subsequently shown to have uiuo

  • These results indicate that Fyn and Fyk are involved in the regulation of postsynaptic membrane function and suggest that these protein tyrosine kinases may phosphorylate the AChR

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Summary

RESULTS

Between members of the src family of Identification of Torpedo Electric Organ Protein !&rosine protein tyrosine kinases, the amino acid sequence between. Kinase Clones-Our initial goal was to identify protein tyro- the myristylation motif and the SH3 domain is the region sine kinases enriched in T. californ~aelectric organ by mo- that is most unique (Cooper, 1990). Protein tyrosinekinases contain several identity of our two clones, we compared the unique domain highly conserved subdomains withinthe catalytic region mak- of each clone to known protein tyrosine kinases. The upper ing them particularlyamenable to analysis by thePCR clone in Fig. 2 was still most homologous to fyn and showed (Wilks, 1991). Otides based on theconserved subdomains VI, VII, VIII, and identity with fyn in this N-terminal region. The second clone was almost equafly homologous, 32% identity, R.

LV LG
Liver Muscle Blood Brain Organ Electric
Protein stain
Findings
DISCUSSION
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