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.
Highlights
The Rab[3] family of low molecular weight guanosine triphosphatases associates with secretory granules and synaptic vesicles and plays a role in exocytosis
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
Rim binds a variety of proteins through its multiple domains: Rab3a-GTP1 binds to a ϳ30-amino acid peptide immediately N-terminal to the zinc finger (12, 14); Munc[] binds to the zinc finger (15); Rim-binding proteins bind to a conserved proline-rich sequence between the two C2 domains (7); cyclic AMPGEFII binds to a region between the zinc finger and the first C2 domain (8); ␣1B Ca2ϩ channel subunit, synaptotagmin, and SNAP-25 each bind to the two C2 domains (16)
Summary
Yeast Two-hybrid Library Screen—Residues 28 –399 of Rim[1] (pGBKT7-Rim[1] (28 –399)) was constructed in-frame with the GAL4 DNA binding domain of bait vector pGBKT7 (Matchmaker Gal[4] Two-Hybrid System 3, Clontech). GST-Rim1(1–399) or GST-Rim1(1–399)S241/ 287A (2.5–20 g of protein) bound to beads was incubated with 20 units/l of CaMKII (preactivated by autophosphorylation with ATP/ Mg2ϩ in the presence of CaCl2 and calmodulin) in 50 l of kinase reaction buffer containing 50 mM Tris-HCl, pH 7.5, 0.1 mM ATP, 10 mM MgCl2, 2 mM dithiothreitol, and 0.1 mM EDTA for 30 min at 30 °C. Beads were washed and incubated with Myc-14-3-3-transfected HEK293 cell lysates (in Nonidet P-40 lysis buffer) for 3 h at 4 °C. Beads were washed three times in lysis buffer and boiled in 1ϫ SDS sample buffer and subjected to 10% SDS-PAGE followed by immunoblotting with the anti-Myc antibody (1:1000 dilution) and a secondary antibody for enhanced chemiluminescence (Amersham Biosciences). There was usually 0.5–1.0 ng of hGH and 30 – 60 nmol of catecholamine/22.6-mm diameter well
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