Mizoguchi A, Yano Y, Hamaguchi H, Yanagida H, Ide C, Zahraoui A, Shirataki H, Sasaki T, Takai Y. approximately postnatal day 16, a period critical for the activity-dependent pruning of supernumerary climbing fibers in the cerebellum. We propose that the phosphorylation of rabphilin regulates neuronal activity through development and in a synapse-specific manner. Keywords: rabphilin, phosphospecific antibodies, protein kinases, development, climbing fibers, immunohistochemistry Synaptic transmission, the main form of cell to cell communication in the nervous system, is triggered by Ca2+ and initiated by synaptic vesicle Y16 exocytosis and secretion of neurotransmitters. Many of the proteins that regulate the targeting, docking, priming, and fusion of synaptic vesicles with the plasma membrane have been identified. These proteins belong to families of molecules with homologs that mediate intracellular vesicle transport and include solubleexperiments, our understanding of the regulation and functional significance of most of these phosphorylation events remains fragmentary. To study the physiological relevance of phosphosynaptic proteins, we have generated a panel of antibodies that recognize synaptic proteins only in their phosphorylated states. In this report, we describe the results obtained with two phosphospecific antibodies directed against phosphorylated rabphilin. Rabphilin, originally identified on the basis of its GTP-dependent interaction Y16 with the small GTPase Rab3A (Shirataki et al., 1993), has been localized to synaptic vesicles (Mizoguchi et al., 1994; Li, 1996), from which it dissociates together with Rab3A during or after exocytosis (Stahl et al., 1996). In addition to Rab3A, Y16 several potential interacting molecules have been suggested for rabphilin, including phosphoinositides (Chung et al., 1998), rabaptin 5 (Ohya et al., 1998), -actinin (Kato et al., 1996), and -adducin (Miyazaki et al., 1994). Whereas these multiple binding partners have implicated rabphilin in exocytosis, endocytosis, and in interactions with the cytoskeleton, its true function remains controversial. In fact, overexpression of full-length rabphilin stimulated exocytosis in pheochromocytoma 12 (PC12) cells, chromaffin cells, and pancreatic cells (Chung et al., 1995; Komuro et al., 1996; Arribas et al., 1997), but its microinjection inhibited neurotransmitter release in squid nerve terminals (Burns et al., 1998). Furthermore, the rabphilin knock-out (KO) displayed no obvious impairments in synaptic transmission (Schluter et al., 1999). Phosphorylation of rabphilin occurs within its central domain on serine-234 primarily by protein kinase A (PKA) and on serine-274 mainly by Ca2+/calmodulin kinase II (CaMKII) (Fykse et al., 1995). Studies with hippocampal synaptosomes and cultured cerebellar granule cells have indicated that rabphilin can be phosphorylated in a stimulation-dependent manner (Fykse, 1998; Lonart and Sdhof, 1998). In this report, we have analyzed the individual contributions of the two phosphorylation sites on rabphilin. We have identified the regions of the brain that have high levels of phosphorabphilin, localized it to a specific subset of synapses, and observed a striking developmental regulation of this modification. MATERIALS AND METHODS The mouse monoclonal antibodies used in this study were: anti-rabphilin from Transduction Laboratories (Lexington, KY), anti-synaptophysin from Boehringer Mannheim (Indianapolis, IN), anti-calbindin from Swant (Bellinzona, Switzerland), and anti-Rab3a from Synaptic Systems (Goettingen, Germany). The nuclear marker Toto-3 was purchased from Molecular Probes (Eugene, OR). Secondary antibodies for immunostaining were from Jackson ImmunoResearch (West Grove, PA) and included fluorescein isothiocyanate-conjugated AffiniPure goat anti-rabbit IgG and Texas Red-conjugated AffiniPure goat anti-mouse IgG. Secondary antibodies for quantitative Western blot analysis were obtained from Amersham Pharmacia Biotech (Arlington, IL) and included anti-rabbit Ig from donkey [125I-labeled F(ab)2 fragment] and anti-mouse Ig from sheep [125I-labeled F(ab)2 fragment]. Casein kinase II (CKII; recombinant from Two peptides corresponding to amino acids 230C239 (TRRASEARMS) and 270C279 (RRANSVQASR) of rabphilin (Li et al., 1994; Fykse et al., 1995were synthesized with a phosphoserine at position 234 or 274, respectively. An additional cysteine residue was introduced at the C terminus for coupling purposes. The peptides were coupled to Imject maleimide-activated keyhole limpet hemocyanin (Pierce, Rockford, IL) and used as immunogen in rabbit. The polyclonal antisera were affinity purified as follows. A peptide with unrelated sequence, a peptide with the same sequence but Foxo1 with unphosphorylated serine (related nonphosphopeptide), and the peptide used as immunogen (phosphopeptide) were coupled to Imject maleimide-activated bovine serum albumin (BSA; Pierce). The conjugates were then linked to cyanogen bromide-activated Sepharose 4B (Sigma). The polyclonal antisera were first sequentially passed over columns carrying the peptide with unrelated sequence and the related nonphosphopeptide to remove nonspecific antibodies. Finally, the antisera were affinity purified by binding and elution from a column carrying the phosphopeptide. in vitroA recombinant fragment of rat rabphilin encompassing amino acids 1C361 [wild-type (WT); a gift from Dr. Sdhof, University of Texas Southwestern Medical Center, Dallas, TX] as well as single serine to alanine mutants at the phosphorylation sites (S234A and S274A) were expressed and purified as described (Li et.
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