Abstract
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.
Publication types
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Research Support, Non-U.S. Gov't
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Research Support, U.S. Gov't, P.H.S.
MeSH terms
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14-3-3 Proteins
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Adaptor Proteins, Signal Transducing
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Alanine / chemistry
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Alkaline Phosphatase / pharmacology
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Amino Acid Motifs
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Amino Acid Sequence
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Animals
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Calcium / metabolism
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Calcium-Calmodulin-Dependent Protein Kinase Type 2
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Calcium-Calmodulin-Dependent Protein Kinases / metabolism
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Cell Line
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Chromaffin Cells / metabolism
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Cross-Linking Reagents / pharmacology
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Cysteine / chemistry
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Dose-Response Relationship, Drug
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Exocytosis
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GTP-Binding Proteins*
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Glutathione Transferase / metabolism
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Humans
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Models, Genetic
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Molecular Sequence Data
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Mutation
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Nerve Tissue Proteins / chemistry*
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Nerve Tissue Proteins / physiology*
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Neurons / metabolism
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PC12 Cells
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Phosphoric Monoester Hydrolases / metabolism
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Phosphorylation
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Plasmids / metabolism
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Precipitin Tests
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Protein Binding
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Protein Structure, Tertiary
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Rabphilin-3A
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Rats
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Recombinant Fusion Proteins / metabolism
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Sequence Homology, Amino Acid
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Serine / chemistry
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Temperature
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Transfection
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Two-Hybrid System Techniques
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Tyrosine 3-Monooxygenase / chemistry*
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Tyrosine 3-Monooxygenase / metabolism
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Vesicular Transport Proteins
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Zinc Fingers
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rab GTP-Binding Proteins / chemistry
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rab3 GTP-Binding Proteins / metabolism
Substances
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14-3-3 Proteins
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Adaptor Proteins, Signal Transducing
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Cross-Linking Reagents
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Nerve Tissue Proteins
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RIMS1 protein, human
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Recombinant Fusion Proteins
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Rim protein, mammalian
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Rims1 protein, mouse
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Rims1 protein, rat
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Vesicular Transport Proteins
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Serine
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Tyrosine 3-Monooxygenase
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Glutathione Transferase
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Calcium-Calmodulin-Dependent Protein Kinase Type 2
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Calcium-Calmodulin-Dependent Protein Kinases
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Alkaline Phosphatase
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Phosphoric Monoester Hydrolases
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GTP-Binding Proteins
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Rim2 protein, mouse
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rab GTP-Binding Proteins
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rab3 GTP-Binding Proteins
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Cysteine
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Alanine
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Calcium