Phosphorylation of Rabphilin-3A, a Putative Target Protein for Rab3A, by Cyclic AMP-Dependent Protein Kinase

doi:10.1006/bbrc.1994.2413

Biochemical and Biophysical Research Communications

Volume 203, Issue 3, 30 September 1994, Pages 1927-1934

https://doi.org/10.1006/bbrc.1994.2413 Get rights and content

Abstract

Rab3A small GTP-binding protein and its associated proteins, such as Rabphilin-3A, a putative target protein for Rab3A, MSS4, a stimulatory GDP/GTP exchange protein for Rab3A, and Rab GDI, a translocator for the Rab family members including Rab3A, are implicated in neurotransmitter release. We have investigated here the phosphorylation of these proteins by cyclic AMP-dependent protein kinase (protein kinase A). Neither Rab3A, MSS4, nor Rab GDI was phosphorylated, but Rabphilin-3A was phosphorylated at its N-terminal region. About 0.8 mol of phosphate was maximally incorporated into one mol of Rabphilin-3A. These results suggest that Rabphilin-3A is one of the targets for protein kinase A which modulates neurotransmitter release.

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Ca<sup>2+</sup> sensor proteins in spontaneous release and synaptic plasticity: Limited contribution of Doc2c, rabphilin-3a and synaptotagmin 7 in hippocampal glutamatergic neurons
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A null model for Doc2c has not yet been described. Rabphilin-3A, encoded by the gene Rph3a, was first identified and isolated as a putative target of Rab3a (Shirataki et al., 1993), a small G-protein involved in vesicle trafficking and neurotransmitter release (Numata et al., 1994; Shirataki et al., 1994; Takahashi et al., 1995). Electron microscopy, subcellular fractionation and imaging revealed that rabphilin-3a is transiently recruited to presynaptic vesicles (Mizoguchi et al., 1994) where it reversibly interacts with Rab3a (Stahl et al., 1996).
Presynaptic neurotransmitter release is strictly regulated by SNARE proteins, Ca²⁺ and a number of Ca²⁺ sensors including synaptotagmins (Syts) and Double C₂ domain proteins (Doc2s). More than seventy years after the original description of spontaneous release, the mechanism that regulates this process is still poorly understood. Syt-1, Syt7 and Doc2 proteins contribute predominantly, but not exclusively, to synchronous, asynchronous and spontaneous phases of release. The proteins share a conserved tandem C₂ domain architecture, but are functionally diverse in their subcellular location, Ca²⁺-binding properties and protein interactions. In absence of Syt-1, Doc2a and -b, neurons still exhibit spontaneous vesicle fusion which remains Ca²⁺-sensitive, suggesting the existence of additional sensors. Here, we selected Doc2c, rabphilin-3a and Syt-7 as three potential Ca²⁺ sensors for their sequence homology with Syt-1 and Doc2b. We genetically ablated each candidate gene in absence of Doc2a and -b and investigated spontaneous and evoked release in glutamatergic hippocampal neurons, cultured either in networks or on microglial islands (autapses). The removal of Doc2c had no effect on spontaneous or evoked release. Syt-7 removal also did not affect spontaneous release, although it altered short-term plasticity by accentuating short-term depression. The removal of rabphilin caused an increased spontaneous release frequency in network cultures, an effect that was not observed in autapses. Taken together, we conclude that Doc2c and Syt-7 do not affect spontaneous release of glutamate in hippocampal neurons, while our results suggest a possible regulatory role of rabphilin-3a in neuronal networks. These findings importantly narrow down the repertoire of synaptic Ca²⁺ sensors that may be implicated in the spontaneous release of glutamate.
Deletion of synapsins I and II genes alters the size of vesicular pools and rabphilin phosphorylation
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Citation Excerpt :
One such specificity concern is whether ablation of a major phosphoprotein of the synaptic vesicle changes the phosphorylation profile of other synaptic vesicle proteins. Thus, we assessed the phosphorylation of the 234S residue of rabphilin, a protein that associates with synaptic vesicles through its binding to Rab3, and like synapsins, it is a target for protein kinase A (Numata et al., 1994; Fykse et al., 1995; Lonart and Südhof, 2001; Lonart et al., 2003; Foletti et al., 2001) and possibly for CaMKII-dependent phosphorylation (Kato et al., 1994; Foletti et al., 2001). We confirmed previous findings that rabphilin level was slightly reduced in DKO brains (Rosahl et al., 1995).
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Rab3A is a GTP-binding protein of synaptic vesicles that regulates neurotransmitter release and cycles on and off synaptic vesicles as a function of exocytosis. Rab3A presumably functions via GTP-dependent interactions with effectors. Two putative rab3A effectors have been described in neurons, rabphilin which is a soluble protein that moves onto and off synaptic vesicles in concert with rab3A, and RIM which is an active zone protein that only binds to rab3A on docked vesicles. Rabphilin is an abundant, evolutionarily conserved protein whose function has remained enigmatic since a knockout of rabphilin does not display the functional deficiencies observed in the rab3A knockout. However, previous studies have shown that rabphilin is phosphorylated by protein kinase A and CaM Kinase II, suggesting that it may have a regulatory role. In the present study, we have examined the site and regulation of rabphilin phosphorylation in living nerve terminals using phospho-specific antibodies raised against phospho-serine²³⁴ of rabphilin. With these antibodies, we demonstrate that rabphilin is physiologically phosphorylated on serine²³⁴, and that soluble rabphilin which is not bound to rab3A on synaptic vesicles is the primary target. However, different from synapsins which are induced to dissociate from synaptic vesicles by PKA phosphorylation, phosphorylation of rabphilin is not instrumental for dissociating rabphilin from synaptic vesicles. Our data support the notion that dissociated rabphilin is a synaptic phosphoprotein in vivo that may play a role in the regulation of nerve terminal protein-protein interactions.
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Regular ArticlePhosphorylation of Rabphilin-3A, a Putative Target Protein for Rab3A, by Cyclic AMP-Dependent Protein Kinase

Abstract

Regular Article
Phosphorylation of Rabphilin-3A, a Putative Target Protein for Rab3A, by Cyclic AMP-Dependent Protein Kinase