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.

The Rab3 family of low molecular weight guanosine triphosphatases associates with secretory granules and synaptic vesicles and plays a role in exocytosis. Rim1 was originally identified in brain as a Rab3-binding protein that is localized to the cytomatrix of the active zone in presynaptic nerve terminals (1). It is a large protein (ϳ1553 amino acids) containing a zinc finger domain, a PDZ domain, and two C2 domains (Fig. 1B). It has similarities to several related proteins, which are also localized to the presynaptic cytomatrix, e.g. Piccolo, Aczonin (2,3), and Bassoon (4) (reviewed in Refs. 5 and 6). Whereas Rim1 is primarily expressed in brain, Rim2, a homologue of Rim1, is more widely expressed (7,8).
Because of its many motifs, Rim may act as a common protein scaffold for modulators of presynaptic function. Indeed, gene knock-out experiments in Caenorhabditis elegans (9) and in mice (10,11) indicate that Rim plays an important regulatory role in neurotransmitter release and synaptic plasticity. The ability of Rim1 to modulate secretion has been investigated in neuroendocrine cells, which, like neurons, have a highly differentiated, regulated exocytotic pathway. Transient expression of full length Rim1 (12) or its N-terminal domain (1,12,13) enhances secretion in chromaffin, PC12, and insulin-secreting cells.
Rim binds a variety of proteins through its multiple domains: Rab3a-GTP 1 binds to a ϳ30-amino acid peptide immediately N-terminal to the zinc finger (12,14); Munc13-1 binds to the zinc finger (15); Rim-binding proteins bind to a conserved proline-rich sequence between the two C2 domains (7); cyclic AMP-GEFII binds to a region between the zinc finger and the first C2 domain (8); ␣1B Ca 2ϩ channel subunit, synaptotagmin, and SNAP-25 each bind to the two C2 domains (16).
The functional implications of some of these interactions have been investigated. The interaction of Rim1 with Rab3a-GTP is unnecessary for the ability of Rim to enhance secretion from neuroendocrine cells because Rim1 mutants without the ability to bind Rab3a-GTP enhance secretion (12). Munc13 proteins regulate neurotransmitter release at the active zone and strongly enhance primed secretion from chromaffin cells (17), perhaps through interaction with syntaxin (18). Although the N-terminal domain of Munc13 interacts with Rim1, this interaction in chromaffin cells is not important for the enhancement of secretion by Munc13 (15). In neurons, the interaction of Rim with Munc13 speeds the ability of transiently expressed Munc13-1 to restore function in mice lacking Munc13-1 (15). Cyclic AMP-GEFII enhances secretion from PC12 and insulinsecreting cells through a mechanism that requires both interaction with Rim2 and the direct binding of cyclic AMP.
In a previous study we had localized the region of Rim1 necessary for the enhancement of secretion to a 90-amino acid segment, Rim1(51-190), which encompasses the zinc finger domain (the amino acid numbering reflects splice variants). We demonstrated that the intact zinc finger is necessary for the enhancement of secretion (12). The segment did not include the N-terminal peptide that binds Rab3-GTP. The zinc finger domain probably acts at a relatively late step in the exocytotic pathway because direct addition to permeabilized chromaffin cells of a peptide encompassing the domain enhances Ca 2ϩ -de-pendent catecholamine secretion. 2 These results provided the rationale for the yeast two-hybrid analysis used in the present study to identify potential protein ligands of the zinc finger and nearby domains. We found that the N-terminal domain of Rim interacts with the 14-3-3 family of proteins, identified the sites of interaction, and investigated the functional effects of the interaction.

MATERIALS AND METHODS
Yeast Two-hybrid Library Screen-Residues 28 -399 of Rim1 (pG-BKT7-Rim1 (28 -399)) was constructed in-frame with the GAL4 DNA binding domain of bait vector pGBKT7 (Matchmaker Gal4 Two-Hybrid System 3, Clontech). Yeast two-hybrid screening was carried out according to the standard protocol of this system by using a rat brain cDNA library (Clontech). Approximately 2 ϫ 10 6 yeast cells were screened, and positive clones were selected for their ability to grow on plates lacking leucine, tryptophan, histidine, and adenine hemisulfate salt and assayed for ␣-galactosidase activity on media supplemented with X-␣-gal (Clontech). Prey DNA was amplified from the positive clones by using the Expand Long Template PCR system (Roche, Mannheim, Germany) and sequenced.
Glutathione S-Transferase (GST) Fusion Protein Expression and in Vitro Binding Assays-GST-14-3-3 fusion proteins were expressed in Escherichia coli HB101 cells by isopropyl-␤-D-thiogalactoside induction. Bacterial lysates were incubated with glutathione-Sepharose 4B beads (Pharmacia) for 1 h at 4°C and washed three times with 1ϫ phosphatebuffered saline.
Phosphatase Treatment-2 ϫ 10 6 cells transfected with HA-tagged Rim or Rabphilin3 constructs were lysed in 600 l of Nonidet P-40 lysis buffer lacking sodium vanadate. One-fourth to one-half of the lysates was adjusted to 1% Nonidet P-40, 10 mM HEPES, pH 8.0, 150 mM NaCl, 0.1 mM dithiothreitol, 5 mM MgCl 2 , 10 g/l of aprotinin, 10 g/l of leupeptin, and 1 mM PMSF. Alkaline phosphatase (Roche) was added to a final concentration of 100 units/ml, and the samples were incubated at 37°C for 4 h (Rim1) or 1 h (Rim2). After phosphatase treatment, the samples were placed on ice and incubated with GST-14-3-3 immobilized on glutathione-Sepharose beads for 2 h at 4°C. Bound proteins were detected by immunoblotting. Cell lysates without alkaline phosphatase treatment were used as positive control.
Chromaffin Cell Preparation, Transfection, and Secretion Experiments-Chromaffin cell preparation, transient transfection, and secretion experiments were performed as described previously (30,31). Calcium phosphate precipitation was used for transfections according to Wilson et al. (32) in 12-well plates (22.6-mm well diameter). Secretion experiments were performed 4 -5 days after transfection at 30°C in a physiological salt solution containing 145 mM NaCl, 5.6 mM KCl, 2.2 mM CaCl 2 , 0.5 mM MgCl 2 , 5.6 mM glucose, 15 mM HEPES (pH 7.4), and 0.5 mM ascorbate. Permeabilized cell experiments were performed in potassium glutamate solution (KGEP) containing 139 mM potassium glutamate, 20 mM 1,4-piperazinediethanesulfonic acid (pH 6.6), 2 mM ATP/ Mg 2ϩ , and 5 mM EGTA with or without 30 M Ca 2ϩ (33). Human growth hormone (hGH) was measured with a high-sensitivity chemiluminescence assay from Nichols Institute (San Juan Capistrano, CA). Endogenous catecholamine secretion was measured with a fluorescence assay. Stimulated release was calculated as the fraction of total of hGH or catecholamine released into the incubation medium. Data were expressed as mean Ϯ S.E. There was usually 0.5-1.0 ng of hGH and 30 -60 nmol of catecholamine/22.6-mm diameter well. 2 L. Sun, M. A. Bittner, and R. W. Holz, unpublished observations.

14-3-3 Interacts with the N-terminal Rim1 in a Yeast
Twohybrid Screen-N-terminal Rim1(28 -399), including the zinc finger and the high-charged region, was used as bait in a yeast two-hybrid screen. It was selected because it includes the zinc finger that alone enhances secretion as well as nearby domains that could modulate zinc finger function. It did not include the domain that is required for Rab3a binding to avoid colonies expressing Rab proteins. Rim1(28 -399) was well expressed in lysates from yeast transformed with the plasmid encoding the bait (pGBKRim1 (28 -399)) (data not shown). A rat brain cDNA prey library was used to transform the bait-expressing yeast cells. Approximately 2 ϫ 10 6 transformants were screened, and a total of 50 positive prey clones were isolated and sequenced. Twenty-two of the clones encoded four types of 14-3-3 isoforms, , ␤, ␥, and ␦. These clones contained slightly different cDNA sizes, but all had the full-length cDNA of 14-3-3. Because 14-3-3 was the most frequent isoform, it was chosen for further characterization.
14-3-3 Directly Interacts with the High-charged Region of Rim-To investigate the in vitro interaction between 14-3-3 and N-terminal Rim1, HEK293 cells that had been transfected with plasmids encoding HA-tagged Rim1 constructs were lysed in 1% Nonidet P-40 buffer. Cell lysates were incubated with GST-14-3-3 fusion proteins immobilized on glutathione-Sepharose beads. Proteins bound to the beads were subjected to 10% SDS-PAGE, and the bound HA-tagged Rim1 was detected with anti-HA antibody.
Rim Interaction with 14-3-3 Requires Rim Phosphorylation-The interaction of 14-3-3 with target proteins is often mediated by the recognition of a phosphoserine found in a consensus binding motif (see the "Introduction"). To determine whether phosphorylated residues are involved in the Rim interaction with 14-3-3, lysates from HEK293 cells transiently expressing various Rim constructs were treated with alkaline phosphatase and then incubated with GST-14-3-3 bound to glutathione-Sepharose 4B beads. The association of Rim with GST-14-3-3 was detected by immunoblotting. Phosphatase treatment greatly reduced the association of both Rim1 and Rim2 with 14-3-3 (Fig. 3A). The decrease in association was not attributable to loss of Rim1 or Rim2 during the incubations, because equivalent amounts of Rim proteins were present in the samples after phosphatase treatment (Fig. 3A, bottom).
The experiments revealed two differences between the interaction of Rim1 and Rim2 with 14-3-3. First, whereas the interaction of Rim1 with 14-3-3 remained stable during the 4-h incubation at 37°C in the absence of phosphatase, the association of Rim2 with 14-3-3 was greatly reduced after a 1-h incubation at 37°C (in the absence of phosphatase) compared with the samples left on ice (Fig. 3A). Second, the binding of Rim2(1-396) to GST-14-3-3 was reversible during extensive washing in contrast to the stable binding of Rim1(1-399) to GST-14-3-3 (Fig. 3B). The stronger binding to Rim1 may reflect the presence of two critical phosphorylated 14-3-3-binding domains in Rim1 and only one in Rim2 (see below).
Rim2 contains a potential consensus site for 14-3-3 binding, RYRS 335 DP, and another site that has similarities to a consensus site, RSPS 238 VS. Binding to 14-3-3 was strongly reduced by the S335A mutation, whereas no change was observed for the S238A mutation (Fig. 4B). The construct with the double mutation Rim1(1-396)S238A/S335A lost interaction to the same degree as Rim2(1-396)S335A. The data suggest that phosphorylated Ser-335, but not Ser-238, forms a functional 14-3-3 binding site.
The ability of CaMKII to stimulate Myc-14-3-3 binding to GST-Rim1 S241/287A was investigated. The Ser 3 Ala mutations virtually completely inhibited the ability of CaMKII to stimulate Myc-14-3-3 binding (Fig. 5, bottom row), indicating that phosphorylation of one or both of the residues is required for CaMKII-stimulated binding.
Zinc Finger of Rim2 Enhances Secretion-The zinc finger domains of Rim1 and Rim2 are highly homologous. We had demonstrated previously (12) that the Rim1 zinc finger strongly enhances secretion in adrenal chromaffin cells when cells were stimulated with the nicotinic agonist dimethylphenylpiperazinium or with Ca 2ϩ (in permeabilized cells). Constructs containing the zinc finger of Rim2 also enhanced secretion (Fig. 7A, right panel). The enhancing effects of Rim2 were reproducible in five independent experiments. The enhancement induced by transiently expressed Rim2 was somewhat less than comparable Rim1 constructs.
Effects of Serine Mutations on the Ability of Rim to Enhance Secretion-14-3-3 plays an important role in synaptic transmission (26), and addition of exogenous 14-3-3 has been reported to enhance secretion in chromaffin cells (27,34). To determine whether decreasing the interaction between Rim1 or Rim2 and 14-3-3 alters the effects of Rim on secretion, we cotransfected plasmids encoding hGH with plasmids encoding either wt Rim or Ser 3 Ala mutants defective in 14-3-3 binding and measured stimulated hGH secretion. All the mutants enhanced secretion similarly to wt in digitonin-permeabilized cells (Fig. 7A), suggesting that interaction of 14-3-3 with transfected Rim does not significantly alter the ability of transfected Rim to enhance secretion. Consistent with these results, purified recombinant GST-14-3-3 protein (3 M) that was added directly to chromaffin cells during both a 4-min permeabilization and 2-min stimulation with 30 M Ca 2ϩ did not alter the ability of transiently expressed Rim1 or Rim2 constructs to enhance secretion (Fig. 7B). In another experiment with 6-min permeabilization and 15 min of stimulation with 30 M Ca 2ϩ , recombinant 14-3-3 (8 M) without fused GST also did not enhance the stimulatory effect of Rim1(1-399) (data not shown). 3 Serine-to-aspartate mutations were constructed to mimic phosphorylation and possibly create the equivalent of a constitutively phosphorylated Rim. Rim(1-399)S241/287D did not support binding to 14-3-3 (data not shown). This result was not unexpected because 14-3-3 often does not accept aspartate as a substitute ligand for phosphate (35). Nevertheless, we investigated the effect of the aspartate mutations on the ability of Rim to enhance secretion. The mutant enhanced secretion similarly to Rim(1-399) (data not shown).

DISCUSSION
Rim1 is a component of the presynaptic active zone and is required for normal synaptic transmission and synaptic plasticity. 14-3-3 proteins are a ubiquitous family of phosphoserinebinding proteins. They are expressed in nerve terminals and are also required for normal synaptic transmission and synaptic plasticity. This study provides the first evidence for the convergence of these two regulatory pathways.
Full-length Rim1 strongly enhances secretion when transiently expressed in adrenal chromaffin cells (12). We had localized the region necessary for the enhancement to a 90amino acid segment Rim1(51-190) that encompasses the zinc finger domain and demonstrated that the structural integrity of the zinc finger is necessary for the enhancement of secretion (12). These results provided the rationale for the yeast twohybrid analysis used in the present study. Rim1(28 -399), FIG. 7. Ser 3 Ala mutation of Rim1 and Rim2 did not change their enhancing effects on secretion. A, chromaffin cells were cotransfected with plasmids encoding hGH and N-terminal Rim1, N-terminal Rim2, and the indicated serine to alanine of these constructs, or pCMVneo. Four to 5 days after transfection, cells were permeabilized with 20 M digitonin in KGEP buffer without Ca 2ϩ and with 2 mM ATP/Mg 2ϩ for 4 min, followed by incubation with or without 30 M Ca 2ϩ for 2 min. Stimulated secretion was calculated as the fraction of total hGH released into the incubation medium. There were four wells/group. B, addition of 14-3-3 recombinant protein to permeabilized chromaffin cells did not affect the enhancing effects of Rim1 and Rim2 on secretion. Cells were transfected and permeabilized as above. 3 M GST-14-3-3 fusion protein was added during the permeabilization together with 2 mM ATP/Mg 2ϩ for 4 min. Cells were then stimulated with 30 M Ca 2ϩ in the continuing presence of 3 M 14-3-3 fusion protein for 2 min. which includes the zinc finger and nearby regions which could modulate its function, was used as bait. Almost half of the colonies identified with Rim1(28 -399) as bait expressed an isoform of 14-3-3. In vitro binding studies demonstrated the direct interaction of 14-3-3 with N-terminal Rim1 and Rim2 and localized the interaction to a region just C-terminal to the zinc finger domain (Rim1(226 -399)). The K m for 14-3-3 binding to Rim1 was ϳ0.1 M, which is similar to the K m for binding of 14-3-3 to other proteins (25). This binding is likely to be of physiological importance because immunoprecipitation of transiently expressed Rim1 constructs from HEK293 cell lysates pulled down endogenous 14-3-3 (Fig. 4A), and the transiently expressed proteins could be cross-linked within cells (Fig. 6).
14-3-3 proteins typically bind to specific phosphoserine residues. Indeed, alkaline phosphatase treatment of N-terminal domains of Rim1 and Rim2 almost completely inhibited the binding of 14-3-3. An analysis of Ser 3 Ala mutations in Rim identified two serine residues in Rim1 (Ser-241 and Ser-287) and one serine residue in Rim2 (Ser-335) that were required for 14-3-3 binding. These are consensus sites for CaMKII phosphorylation. The ability of CaMKII in vitro to stimulate 14-3-3 binding to Rim(1-399) and but not to Rim(1-399 S241/287A) (Fig. 5) indicates the importance of Ser-241 and/or Ser-287 phosphorylation for binding.
Because N-terminal constructs of Rim1 and Rim2 containing these mutations enhanced secretion similarly to constructs without the mutations, it is unlikely that phosphorylation and subsequent 14-3-3 interaction are important for the effects on secretion of these constructs in our transfection assays. How-ever, Rim2 is endogenously expressed in bovine chromaffin cells. 2 It is possible that the normal physiological modulation by 14-3-3 of Rim function is masked in transfection assays because of the high levels of Rim expression.
Possible consequences of the interaction of Rim with 14-3-3 other than effects on secretion were also investigated. Because 14-3-3 can regulate the subcellular localization of proteins with which it interacts (25), it was possible that 14-3-3 regulates the location of Rim1 in the cells. Transiently expressed N-terminal Rim1 accumulates in the nucleus of chromaffin cells because of an exposed nuclear localization signal in the high-charged region immediately C-terminal to the zinc finger (12). Because the phosphorylated serines to which 14-3-3 binds are located in this region, we investigated whether disruption of the 14-3-3 interaction altered the subcellular localization of N-terminal Rim. Mutation with Ser 3 Ala mutations that prevent 14-3-3 interactions did not alter the nuclear accumulation of N-terminal Rim1 (data not shown), indicating that 14-3-3 binding does not regulate the nuclear accumulation of the constructs.
Rim1 interacts with Rab3a through a 50-amino acid peptide just N-terminal to the zinc finger (12,14). Munc13 binding to Rim1 requires an intact zinc finger (15). Disruption of the ability of the nearby high-charged region of Rim1 to bind 14-3-3 did not alter the in vitro binding of either Rab3a-GTP or Munc13 to Rim1 (data not shown), suggesting that 14-3-3 does not regulate these interactions.
Although the mutations in Rim1 that prevent its interaction with 14-3-3 had no apparent effect on the function of transiently expressed Rim1 in chromaffin cells, the interaction of 14-3-3 with Rim1 may, nevertheless, be important in the organization and function of the presynaptic nerve terminal. This is supported by the finding that CaMKII, which functions in the nerve terminal (40,41), phosphorylates the residues responsible for 14-3-3 binding. The interaction of Rim1 with 14-3-3, for example, may contribute to the reduced presynaptic function in Drosophila mutants lacking 14-3-3 (26).
In summary, Rim family members are multidomain proteins that likely have multiple functions in neurons and secreting cells. A theme that is developing for these proteins is that they serve as a protein scaffold that interacts with other proteins implicated in nerve terminal and neuroendocrine cell function. The ability of 14-3-3 proteins to strongly interact with Rim raises the possibility that these ubiquitous phosphoprotein ligands are modulating the protein interactions and function of Rim in the presynaptic cytomatrix and in neuroendocrine cells.
In the course of this study, we discovered that 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. The phosphorylation of this residue is stimulated upon depolarization in brain slices (39), suggesting that its interaction with 14-3-3 may be important in the dynamic function of central nervous system neurons.