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.
Sun
Lei
L
Department of Pharmacology, University of Michigan Medical School, Ann Arbor, Michigan 48109-0632, USA.
Bittner
Mary A
MA
Holz
Ronald W
RW
D048948:14-3-3 Proteins; D048868:Adaptor Proteins, Signal Transducing; D000409:Alanine / Q000737:chemistry; D000469:Alkaline Phosphatase / Q000494:pharmacology; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000378:metabolism; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D002460:Cell Line; D019439:Chromaffin Cells / Q000378:metabolism; D003432:Cross-Linking Reagents / Q000494:pharmacology; D003545:Cysteine / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D005089:Exocytosis; D019204:GTP-Binding Proteins; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009154:Mutation; D009419:Nerve Tissue Proteins / Q000737:chemistry* / Q000502:physiology*; D009474:Neurons / Q000378:metabolism; D016716:PC12 Cells; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D012694:Serine / Q000737:chemistry; D013696:Temperature; D014162:Transfection; D020798:Two-Hybrid System Techniques; D014446:Tyrosine 3-Monooxygenase / Q000737:chemistry* / Q000378:metabolism; D033921:Vesicular Transport Proteins; D016335:Zinc Fingers; D020691:rab GTP-Binding Proteins / Q000737:chemistry; D020692:rab3 GTP-Binding Proteins / Q000378:metabolism; D000097624:Rabphilin-3A
D048948:14-3-3 Proteins; D048868:Adaptor Proteins, Signal Transducing; D000409:Alanine; D000469:Alkaline Phosphatase; D002118:Calcium; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D003432:Cross-Linking Reagents; D003545:Cysteine; D019204:GTP-Binding Proteins; D005982:Glutathione Transferase; D009419:Nerve Tissue Proteins; D010744:Phosphoric Monoester Hydrolases; D011993:Recombinant Fusion Proteins; D012694:Serine; D014446:Tyrosine 3-Monooxygenase; D033921:Vesicular Transport Proteins; D020691:rab GTP-Binding Proteins; D020692:rab3 GTP-Binding Proteins; D000097624:Rabphilin-3A; C491492:RIMS1 protein, human; C107293:Rim protein, mammalian; C491490:Rims1 protein, mouse; C491489:Rims1 protein, rat; C475524:Rim2 protein, mouse