Rim, a component of the presynaptic active zone and modulator of exocytosis, binds 14-3-3 through its N terminus.
Rim1, a brain-specific Rab3a-binding protein, localizes to the presynaptic cytomatrix and plays an important role in synaptic transmission and synaptic plasticity. Rim2, a homologous protein, is more ubiquitously expressed and is found in neuroendocrine cells as well as in brain. Both Rim1 and Rim2 contain multiple domains, including an N-terminal ... zinc finger, which in Rim1 strongly enhances secretion in chromaffin and PC12 cells. The yeast two-hybrid technique identified 14-3-3 proteins as ligands of the N-terminal domain. In vitro protein binding experiments confirmed a high-affinity interaction between the N terminus of Rim1 and 14-3-3. The N-terminal domain of Rim2 also bound 14-3-3. The binding domains were localized to a short segment just C-terminal to the zinc finger. 14-3-3 proteins bind to specific phosphoserine residues. Alkaline phosphatase treatment of N-terminal domains of Rim1 and Rim2 almost completely inhibited the binding of 14-3-3. Two serine residues in Rim1 (Ser-241 and Ser-287) and one serine residue in Rim2 (Ser-335) were required for 14-3-3 binding. Incubation with Ca2+/calmodulin-dependent protein kinase II greatly stimulated the interaction of recombinant N-terminal Rim but not the S241/287A mutant with 14-3-3, again indicating the importance of the phosphorylation of these residues for the binding. Rabphilin3, another Rab3a effector, also bound 14-3-3. Serine-to-alanine mutations identified Ser-274 as the likely phosphorylated residue to which 14-3-3 binds. Because the phosphorylation of this residue had been shown to be stimulated upon depolarization in brain slices, the interaction of 14-3-3 with Rabphilin3 may be important in the dynamic function of central nervous system neurons.
Mesh Terms:
14-3-3 Proteins, Adaptor Proteins, Signal Transducing, Alanine, Alkaline Phosphatase, Amino Acid Motifs, Amino Acid Sequence, Animals, Calcium, Calcium-Calmodulin-Dependent Protein Kinase Type 2, Calcium-Calmodulin-Dependent Protein Kinases, Cell Line, Chromaffin Cells, Cross-Linking Reagents, Cysteine, Dose-Response Relationship, Drug, Exocytosis, GTP-Binding Proteins, Glutathione Transferase, Humans, Models, Genetic, Molecular Sequence Data, Mutation, Nerve Tissue Proteins, Neurons, PC12 Cells, Phosphoric Monoester Hydrolases, Phosphorylation, Plasmids, Precipitin Tests, Protein Binding, Protein Structure, Tertiary, Rats, Recombinant Fusion Proteins, Sequence Homology, Amino Acid, Serine, Temperature, Transfection, Two-Hybrid System Techniques, Tyrosine 3-Monooxygenase, Vesicular Transport Proteins, Zinc Fingers, rab GTP-Binding Proteins, rab3 GTP-Binding Proteins
14-3-3 Proteins, Adaptor Proteins, Signal Transducing, Alanine, Alkaline Phosphatase, Amino Acid Motifs, Amino Acid Sequence, Animals, Calcium, Calcium-Calmodulin-Dependent Protein Kinase Type 2, Calcium-Calmodulin-Dependent Protein Kinases, Cell Line, Chromaffin Cells, Cross-Linking Reagents, Cysteine, Dose-Response Relationship, Drug, Exocytosis, GTP-Binding Proteins, Glutathione Transferase, Humans, Models, Genetic, Molecular Sequence Data, Mutation, Nerve Tissue Proteins, Neurons, PC12 Cells, Phosphoric Monoester Hydrolases, Phosphorylation, Plasmids, Precipitin Tests, Protein Binding, Protein Structure, Tertiary, Rats, Recombinant Fusion Proteins, Sequence Homology, Amino Acid, Serine, Temperature, Transfection, Two-Hybrid System Techniques, Tyrosine 3-Monooxygenase, Vesicular Transport Proteins, Zinc Fingers, rab GTP-Binding Proteins, rab3 GTP-Binding Proteins
J. Biol. Chem.
Date: Oct. 03, 2003
PubMed ID: 12871946
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