Abstract
In order to study the role of tyrosine kinase signaling in the post-synaptic density (PSD), tyrosine-phosphorylated proteins associated with the PSD-95/NMDA receptor complex were analyzed. The NMDA receptor complex from the mouse brain was successfully solubilized with deoxycholate and immunopurified with anti-PSD-95 or anti-phosphotyrosine antibody. Immunoblot analyses revealed that the predominantly tyrosine-phosphorylated proteins in the NMDA receptor complex are the NR2A/B subunits and a novel 120 kDa protein. Purification and microsequencing analysis showed that the 120 kDa protein is mouse PSD-93/Chapsyn-110. Recombinant PSD-93 was phosphorylated by Fyn in vitro, and Tyr-384 was identified as a major phosphorylation site. Tyrosine phosphorylation of PSD-93 was greatly reduced in brain tissue from Fyn-deficient mice compared with wild-type mice. Furthermore, an N-terminal palmitoylation signal of PSD-93 was found to be essential for its anchoring to the membrane, where Fyn is also localized. In COS7 cells, exogenously expressed PSD-93 was phosphorylated, dependent on its membrane localization. In addition, tyrosine-phosphorylated PSD-93 was able to bind to Csk, a negative regulator of Src family kinases, in vitro as well as in a brain lysate. These results suggest that PSD-93 serves as a membrane-anchored substrate of Fyn and plays a role in the regulation of Fyn-mediated modification of NMDA receptor function.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Amino Acid Sequence
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Animals
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Brain / metabolism
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COS Cells
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Cell Membrane / metabolism
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Disks Large Homolog 4 Protein
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Electrophoresis, Polyacrylamide Gel
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Escherichia coli / metabolism
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Focal Adhesion Kinase 1
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Focal Adhesion Protein-Tyrosine Kinases
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Genetic Vectors
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Glutathione Transferase / metabolism
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Green Fluorescent Proteins
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Guanylate Kinases
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Immunoblotting
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Intracellular Signaling Peptides and Proteins
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Luminescent Proteins / chemistry
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Luminescent Proteins / metabolism
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Membrane Proteins
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Mice
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Models, Genetic
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Molecular Sequence Data
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Nerve Tissue Proteins / chemistry*
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Nerve Tissue Proteins / physiology*
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Peptides / chemistry
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Phosphorylation
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Phosphotyrosine / chemistry
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Precipitin Tests
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Prosencephalon / metabolism
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Protein Binding
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Protein Processing, Post-Translational
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Protein Structure, Tertiary
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Protein-Tyrosine Kinases / chemistry
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Proto-Oncogene Proteins / chemistry*
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Proto-Oncogene Proteins / metabolism
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Proto-Oncogene Proteins c-fyn
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Recombinant Proteins / chemistry
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Sequence Homology, Amino Acid
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Silver Staining
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Tyrosine / chemistry
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Tyrosine / metabolism
Substances
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Disks Large Homolog 4 Protein
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Dlg4 protein, mouse
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Intracellular Signaling Peptides and Proteins
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Luminescent Proteins
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Membrane Proteins
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Nerve Tissue Proteins
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Peptides
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Proto-Oncogene Proteins
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Recombinant Proteins
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postsynaptic density proteins
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Green Fluorescent Proteins
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Phosphotyrosine
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Tyrosine
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Glutathione Transferase
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Protein-Tyrosine Kinases
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Focal Adhesion Kinase 1
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Focal Adhesion Protein-Tyrosine Kinases
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Fyn protein, mouse
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Proto-Oncogene Proteins c-fyn
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Ptk2 protein, mouse
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Dlg2 protein, mouse
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Guanylate Kinases